Nature-based tourism (NBT) is vulnerable to a rapid increase in visitors because natural resources are often open access. Market failure caused by over-exploitation of natural resources is an example of social dilemmas in common-pool resource systems. Game theory, which describes people’s decision making under conflicts, has been applied to the analysis of social dilemmas in NBT. However, previous studies use non-cooperative games assuming individualistic players and discuss the emergence of social dilemmas only in a limited situation. Here, we demonstrate, by developing a two-player non-cooperative game of wildlife viewing, that the traditional game-theoretic approach fails to find social dilemmas. By analysing the competition between tour operators (players) with different social value orientations (SVOs), we found that concentration of tours becomes a Pareto-inefficient Nash equilibrium (PINE) when both players are competitive. Whether the wildlife-viewing market is a Prisoner’s dilemma depends on players’ SVOs. Furthermore, we found that fair punishment on competitive players promotes rather than suppresses the emergence of PINE. Our results suggest that the diversity of SVOs is an essential factor in understanding social dilemmas in NBT. The amount is expected to reach USD 12,450 billion in 2028. Meanwhile, many tourism destinations face a rapid increase in visitors with the associated problems: degraded tourist experiences, damage to nature, and threats to culture and heritage2. Nature-based tourism (NBT) is vulnerable to overcrowding because natural resources are often open access. On an analogy with the tragedy of the commons4, we can imagine the tourism dilemma as follows: (1) Self-interested tourism firms increase tour supply to maximise their benefits. (2) Over-exploitation of natural resources happens, undermining the basis of NBT. (3) The economic value of NBT decreases, and the payoff allocation to tourism firms becomes inefficient. The tourism dilemma is an example of social dilemmas in common-pool resource systems7.Tourism demand has been rapidly increasing in recent years. According to the World Travel and Tourism Council8. There are many game-theoretic studies in the fields of fisheries, water resource management, and climate change11. Game theory is not widely applied to NBT, but several studies discuss tourism dilemmas based on non-cooperative games. For example, Sobhee and co-workers12 use the Prisoner’s dilemma to represent the conflict between fishers and water-sport operators in a marine park. Blanco and co-workers13 propose a strategic-form game structurally different from the Prisoner’s dilemma, pointing out that many tourism firms voluntarily engage in resource conservation. In another paper14, they demonstrate, in an evolutionary extension of the above game, that sustainable and non-sustainable firms can coexist in a sufficiently large market. He and co-workers15 also use an evolutionary game to investigate how government supervision affects the behaviour of tourism firms and visitors. Bimonte16 represents the conflict between residents and visitors using a Bayesian game and concludes that incomplete information causes social dilemmas. Pirotta and Lusseau17 develop an agent-based model of wildlife viewing and analyse the interactions of tour operators, visitors, and animals. By numerical simulation, they evaluate the impacts of resource conservation policies  on the profits of tour operators.Game theory, which describes people’s decision making under conflicts, is a useful tool to analyse social dilemmas in common-pool resource systemsTheory of Games and Economic Behavior in 194418, the individualistic player assumption has been widely applied to non-cooperative games. On the other hand, psychological experiments demonstrated that individuals have social preferences and make decisions by comparing the payoffs for themselves and others27. To examine the relationships between personality traits and human behaviour, we need to consider utility functions which include information about opponents’ payoffs. A generalised model of social preferences is social value orientation (SVO)26. Mathematically, SVO is expressed as a function that calculates each person’s utility from the payoffs for self and others. The SVO model can describe the behaviour of players with different personality traits . Despite the achievements of psychological studies, the diversity of personality traits gathers little attention in game-theoretic studies on tourism dilemmas.Game-theoretic studies on NBT aim to evaluate the effects of resource conservation policies by developing mathematical models of tourism dilemmas. However, most previous studies discuss tourism dilemmas in limited situations, assuming individualistic players. The individualistic player maximises his or her own but does not consider others’ payoffs. Since the publication of 31, which is a popular form of NBT. We represent the competition between tour operators by a two-player non-cooperative game and identify mathematical conditions under which the market falls into social dilemmas. In other words, we identify the combinations of parameters that produce Pareto-inefficient Nash equilibria (PINE) as the solutions in the Wildlife-Viewing game (WVG). Furthermore, we impose a punishment on tour concentration and examine its impact on the emergence of PINE. To overcome the limitation of previous studies, we assume three types of players with different SVOs  and solve the WVG for all SVO combinations. The concept of our game originates with wildlife-viewing tours conducted in the Amami Oshima of Japan. One of the target animals is the Amami rabbit , which is an endangered endemic species33. The concentration of tours hinders the activities of the Amami rabbit and harms the environment suitable for rabbit viewing. According to Kubo and co-workers31, tour operators in the Amami Oshima use money-back guarantee systems to attract visitors. If a visitor fails to view the Amami rabbit in a tour, the tour operator returns a part of the tour fee to the visitor. The WVG includes parameters corresponding to these characteristics.This paper focuses on the tourism dilemma in wildlife viewingThis paper is constructed as follows. In the Methods section, we give the mathematical expressions of the WVG and PINE. In the Results section, we calculate the Nash equilibria, Pareto-efficient solutions, and PINE of the game. Moreover, we calculate the solutions of the WVG with punishment for tour concentration. In the Discussion section, we summarise the results and conclude the paper by pointing out the limitations of our approach. Supplementary Information provides details of mathematical calculation.Before introducing a mathematical expression of the WVG, we provide an informal description of the game. The WVG represents the competition between two operators who are running tours for viewing an endemic rabbit. An area inhabited by the rabbit is open to people, and no one has the right to own the area. Anyone can visit the viewing area by paying a cost. However, information about the viewing area is not open to visitors, and they cannot encounter the rabbit without an operator’s guidance. The operators supply wildlife-viewing tours to visitors and receive fees from them. This paper considers wildlife viewing as a common-pool resource consumed by individuals. The rabbit is sensitive to human-generated noise and leaves the viewing area if two operators conduct tours simultaneously. If an operator fails to encounter the rabbit during a tour, he or she returns a part of the tour fee to the visitor (money-back guarantee). As each operator’s net profit can be negative, he or she needs to consider the other operator’s action in conducting a tour. In the WVG, the operators make decisions based on utility rather than monetary payoffs. Each operator evaluates the utility of action by comparing monetary payoffs for the self and the other. The operator’s personality trait (SVO) influences the evaluation of utility. For simplicity, we do not consider the process in which the operators gather visitors. Moreover, we assume that the tour fee, cost, and money-back rate are constant regardless of the operators. Under these conditions, we examine whether the wildlife-viewing market falls into social dilemmas.N = {1, 2} be the set of players. Each player can conduct a tour by paying a cost C > 0. If a player encounters the rabbit during a tour, it receives a tour fee B > 0 from the visitor. If a player fails to find the rabbit, it returns a part of the tour fee to the visitor and receives δB. δ ∈  is called the guarantee coefficient. Each player encounters the rabbit with a probability P ∈ . However, if the two players conduct tours simultaneously, the noise they generate threatens the rabbit, and the encounter probability decreases to θP. θ ∈  is called the vigilance coefficient. Let S = {T, F} be the set of pure strategies. A player with strategy T conducts a tour, while a player with strategy F does not. When each player i ∈ N chooses strategy si ∈ S, his or her expected payoff is given by m ∈ {1, 2} is the number of players with strategy T. Using equation  indicates player i’s SVO. The set of all weight vectors draws the SVO ring on the two-dimensional space αi: individualistic, competitive, and prosocial. The individualistic player, who is characterised by the weight vector , maximises his or her own but does not consider the other’s objective payoff. Non-cooperative games usually assume individualistic players. The competitive and prosocial players are characterised by the weight vectors Each player determines his or her strategy based on the subjective payoff bimatrix (SPBM) rather than the OPBM. Psychological studies propose mathematical functions for payoff transformationmentclass2pt{minim35. In the WVG, players make decisions based on the SPBM. Hence, the Nash equilibrium is Vi denotes the subjective payoff for player i.Here, we show the mathematical expression of PINE. The Nash equilibrium is the set of strategy profiles where every player shows the best response to the other’s strategy36. Let s ∈ S2. We define the region Pareto superior to us as us to any point of P(us), at least one player can improve his or her objective payoff without decreasing the other’s. The complement of the Pareto-superior region, denoted by σ ∈ S2 is a Pareto-efficient solution if all possible objective payoff allocations are Pareto inferior to uσ. The set of Pareto-efficient solutions is An objective payoff allocation is Pareto efficient if no player can increase his or her objective payoff without decreasing the other’sus} Fig. . By moviion Fig. . A stratquations  and 6),,36. Let D = { ∣ δ ∈ , P ∈ } be a two-dimensional space spanned by the guarantee coefficient and the initial encounter probability. Figure D. i of type τi ∈ {I, C, P}. The symbols I, C, and P are abbreviations for individualistic, competitive, and prosocial types, respectively. The dashed line indicates δ = C/B. The result is summarised in the following two theorems:First, we identified the conditions under which the SPBM (equation ) has NasConsider a two-player WVG. Each player is individualistic, competitive, or prosocial. The SPBM of the game always has at least one pure-strategy Nash equilibrium.Proof. The conditions for Nash equilibria are shown in Tables S1–S3 of Supplementary Information. The union of the conditions for -Nash, -Nash, -Nash, and -Nash is P ∈  regardless of players’ SVOs. Hence, the SPBM always has at least one of the Nash equilibria.Assume that the guarantee coefficient (δ) and the initial encounter probability (P) are randomly determined. The probability of tour concentration -Nash) in the competitive market (GCC) is equal to or higher than that in the individualistic market (GII). In contrast, the probability of tour concentration in the prosocial market (GPP) is equal to or lower than that in the individualistic market.Proof. Let δ, P) is randomly chosen from D, the area of the parameter region, denoted by δ < C/B. From Table S1, we obtain P0 = (C − δB)/(B(1 − δ)). δ, P) ∣ δ ∈ , P ∈ } under θ > 1/2 and is the empty set under θ ≤ 1/2. We have θ ∈ . Moreover, we have  ⊇  ⊇  when θ > 1/2. Hence, we obtain δ ≥ C/B (Tables S2 and S3). Thus, Theorem 2 is proved.C/B (the left side of the dashed line), the failure of a tour gives a negative profit (δB − C < 0). In this case, the distribution of Nash equilibria changes depending on players’ SVOs. The game GII yields the solution -Nash when P ∈ . In the game GCC, -Nash emerges when P ∈ . Since θ ∈ , the parameter region of -Nash is broader in GCC than in GII. The competitive player aggressively conducts a tour even with low initial encounter probability. Therefore, tour concentration frequently occurs in the competitive market. In the game GPP, -Nash emerges only in limited conditions: θ > 1/2 and P ∈ . Since P0/θ < P0/(2θ − 1) under the constraint of θ > 1/2, the parameter region of -Nash is narrower in GPP than in GII. Unlike the competitive player, the prosocial player avoids tour concentration even with high initial encounter probability. However, the prosocial player is vulnerable to competition with the individual and competitive players. The games GIP and GCP can have -Nash as the solutions. Under this equilibrium, the prosocial player stops the supply of tours, and the individualistic or competitive player monopolises the market. Similarly, the individualistic player is also vulnerable to competition with the competitive player. The game GIC can lead to the solution -Nash. When the guarantee coefficient is higher than or equal to C/B (the right side of the dashed line), a tour failure results in a non-negative profit (δB − C ≥ 0). In this case, -Nash is dominant in most of the games.Here, we focus on the emergence of -Nash, which implies concentration of tours. When the guarantee coefficient is lower than D. We have the following theorem:Second, we identified the conditions under which the OPBM (equation ) has ParThe OPBM of the WVG always has at least one Pareto-efficient solution.Proof. The conditions for Pareto-efficient solutions are shown in Table S4 of Supplementary Information. The union of the conditions for -Pareto, -Pareto, -Pareto, and -Pareto is P ∈ . Hence, the OPBM always has at least one of the Pareto-efficient solutions.Finally, we identified the conditions under which the WVG has PINE as the solutions. Tables SAssume that the guarantee coefficient (δ) and the initial encounter probability (P) are randomly determined. (1) Consider the case where at least one player is individualistic or prosocial. The objective payoff allocation given by a Nash equilibrium of the SPBM is almost surely Pareto efficient. Non-competitive markets fall into social dilemmas with probability zero. (2) Consider the case where both players are competitive. If the guarantee coefficient is lower thanC/Band the initial encounter probability lies in the interval , the objective payoff allocation given by a Nash equilibrium of the SPBM is not Pareto efficient. IfB > C, the competitive market falls into a social dilemma with a positive probability.Proof. Let δ, P) is randomly chosen from D, GII, GCC, and GIC with δ < C/B. From Table S5, we obtain ∣DPINE(GII)∣ = ∣{∣δ ∈ , P = P0/θ}∣ = 0, ∣DPINE(GCC)∣ = ∣{∣δ ∈ , P ∈ }∣≥0, and ∣DPINE(GIC)∣ = ∣DPINE(GII)∣ = 0. Under the constraint of B > C, we have P0 < 1, which results in ∣DPINE(GCC)∣ > 0. Thus, Theorem 2 is proved.□GCC. In the blue region, the Nash equilibrium is , but the Pareto-efficient solutions are  and . Here, we provide a numerical example of PINE. Consider the case where B = 1.5, C = 1.0, δ = 0.3, P = 0.6, and θ = 0.6. By equations ,,GCC. In GCC. Competitive players cannot escape from PINE without an incentive to change their strategies. Here, we impose punishment on the objective payoff allocation from  and examine its impact on the emergence of PINE.We demonstrated that the strategy profile , which implies tour concentration, becomes a PINE in the game ε > 0 is a penalty on tour concentration. The SPBM is GCC (Section 4 of Supplementary Information). In the game GII, for example, the border between -Nash and other equilibria shifts from P = P0/θ to P = (P0 + ε0)/θ ) > 0 and θ ∈ , the new border is located on the right side of the original border. In the game GPP with θ > 1/2, similarly, the border between -Nash and other equilibria shifts from P = P0/(2θ − 1) to P = (P0 + 2ε0)/(2θ − 1) /θ. The parameter region of -Pareto under punishment is narrower than the original region  reduces each player’s objective payoff byϵ > 0. When both players are competitive, the parameter region of -PINE over the parameter spaceDis equal to or broader than that in the original game without punishment. If the guarantee coefficient (δ) and the initial encounter probability (P) are randomly determined, the punishment monotonically increases the probability that the competitive market falls into a social dilemma.Proof. Let P ∈  (Section 4.2 of Supplementary Information) and has -Pareto under P ∈ ((P0 + ε0)/θ, 1] (Section 5.2 of Supplementary Information). As the inequality P0 < (P0 + ε0)/θ holds over D, the condition for -PINE is P ∈ . The parameter region of -PINE is expressed as P0 + ε0)/θ > P0/θ, we have  ⊇ . Hence, we obtain δ, P) is randomly chosen from D, the areas of the parameter regions give the probabilities of -PINE in the games GCC. Thus, Theorem 5 is proved.□B = 1.5, C = 1.0, δ = 0.5, P = 0.8, θ = 0.6, and ϵ = 0.8. The OPBM without punishment is GII). In this case, the SPBM is equal to the OPBM (V = U and Vε = Uε). In the original game, strategy T is the best response for both players, and the SPBM has the unique Nash equilibrium . The objective payoff allocation  given by the equilibrium is Pareto efficient. With the introduction of fair punishment into the OPBM, the Nash equilibrium shifts to  or , and the concentration of tours is suppressed. The objective payoff allocation  or  given by the new equilibrium is also Pareto efficient. The total objective payoff increases from 0.22 to 0.35. Second, we consider the competitive market (GCC). In this case, the SPBM is Here, we provide a numerical example of the failure of fair punishment. Assume that In this paper, we developed a non-cooperative game of wildlife viewing and demonstrated that the SVOs of tour operators (players) are associated with the emergence of PINE. The WVG describes the competition between individualistic, competitive, and prosocial players in the market. Compared to the individualistic player, the competitive player aggressively conducts a tour even if the probability of encountering the target animal is low (Theorem 2). In contrast, the prosocial player avoids the concentration of tours, which causes a decrease in the tour profit (Theorem 2). The WVG always has at least one pure-strategy Nash equilibrium and has at least one Pareto-efficient solution (Theorems 1 and 3). From the distributions of Nash equilibria and Pareto-efficient solutions over the parameter space, we found that PINE emerge when both players are competitive (Theorem 4). In the competitive market, the concentration of tours becomes a Nash equilibrium, but the objective payoff allocation given by the solution is not always Pareto efficient. If at least one player is individualistic or prosocial, the objective payoff allocation given by the Nash equilibrium is almost surely Pareto efficient. Whether the wildlife-viewing market is a Prisoner’s dilemma depends on players’ SVOs.To remove PINE from the competitive market, we need to provide players with an incentive to stop tour supply when the encounter probability is low. We imposed fair punishment on tour concentration and examined its impact on the emergence of PINE. If at least one player is individualistic or competitive, the punishment decreases the parameter region where tour concentration is a Nash equilibrium. However, the punishment loses its effect in the competitive market and does not change the distribution of Nash equilibria. Unlike the individualistic player, the competitive player makes decisions based on the difference between the objective payoffs for self and the other. As fair punishment maintains the objective-payoff difference, it does not influence the behaviour of competitive players. On the other hand, the punishment decreases the parameter region where tour concentration is a Pareto-efficient solution. From these results, we immediately find that the punishment increases the parameter region of PINE when both players are competitive (Theorem 5). In other words, fair punishment promotes rather than suppresses the emergence of PINE in the competitive market. Our results suggest the possibility that the traditional game-theoretic approach assuming individualistic players fails to find social dilemmas in NBT.30). Furthermore, the SVO-based approach adopted in the WVG is widely applicable to various non-cooperative games. We provide three examples in Section 6 of Supplementary Information: the Hawk-Dove game, Battle of the Sexes, and Public Goods game37. In each of the games, individualistic, competitive, and prosocial players show different behaviours even though they have the same OPBM. Like the WVG, the Hawk-Dove game and Battle of the Sexes are potential social dilemmas in which only competitive players are attracted to PINE. In the Public Goods game, the total objective payoff is maximised when both players are prosocial. The SVO-based approach enables us to find hidden theoretical solutions of non-cooperative games.By assuming players with different SVOs, we demonstrated that the WVG is a potential social dilemma. Although this paper was inspired by the rabbit-viewing tour conducted in the Amami Oshima of Japan, the WVG can represent wildlife-viewing tours not only in Japan but also around the world , which are likely to contribute to further research on the tourism dilemma. Some studies report that equity and efficiency preferences do not always explain people’s decision making. Capraro and co-workers52 highlight the importance of normative motives  for altruistic behaviour in social dilemmas.We conclude this paper by discussing two limitations of our approach. First, our results do not fully reflect the diversity of personality traits. As illustrated by the SVO ring Fig. , there e37. If the relationships between players form a social network, local interactions of players influence the state of the whole market53. Results from evolutionary game theory and psychological experiments suggest that reputation and voluntary punishment promote altruistic behaviour in repeated games on social networks58. As shown by this paper, tour concentration can be a PINE in the competitive market, and the solution is resistant to fair punishment introduced by an external force (e.g. the government). Unfortunately, we could not find a way to remove the PINE from the competitive market. To overcome this problem, we need to analyse the dynamic interactions of players in an extended WVG.Second, the WVG developed in this paper is a single-shot game with two players and assumes a highly simplified tourism market. We can consider various extensions of the WVG. For example, an evolutionary extension of the game enables us to analyse the dynamics of players’ behaviourSupplementary Information


Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases.Activity‐based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS‐CoV‐2 as proof of concept. The resulting probes were specific for the active site labeling of 3CL Tuning the ligand space allows to rapidly customize chemical probes for efficacy and active site specificity for a protease of interest. The application of this ligand selection approach is exemplified by generating probes for the two non‐homologous yet essential proteases of the new corona virus SARS‐CoV‐2 providing complementary chemical tools for proteomic labeling and inhibitor discovery. Both 3CLpro and PLpro liberate themselves from the polyprotein by cleaving their respective N‐ and C‐terminal sequences as described for the homologous sequences of SARS‐CoV‐1.[pro by N‐terminal self‐cleavage is required for full activation of the protease and the ability of trans‐processing of other non‐structural proteins.pro show that the N‐terminus of one 3CLpro monomer is in close proximity to the active site pocket of the other monomer and the N‐terminus is cleaved between the short consensus sequence Leu–Gln and Ser already during expression.pro of SARS‐CoV.pro version fused with a non‐cleavable N‐terminal Strep‐tag II (t3CLpro). Modeling predicted an identical behavior of the fusion peptide at the active site compared to the native sequence of wild type prior to cleavage . Codon‐optimized sequences of the 3CLpro and PLpro domains of nsp3 and nsp5 of SARS‐CoV‐2 were ultimately cloned into an IPTG inducible vector and heterologously expressed in Escherichia coli. Proteins were either purified via affinity chromatography or used in situ in the native cell of the expression system. Confirming our predictions regarding the N‐terminal modification, the purified t3CLpro version indeed was inactive in a protease assay using oligopeptide substrates linked to a fluorogenic 7‐amino‐4‐methylcoumarin group . This represented the unique opportunity to validate the utility of the LS‐ABPP strategy against PLpro and the pre‐activation stage of 3CLpro of SARS‐CoV‐2 and demonstrate its versatility for customizing probes to different targets.Hereby dimerization and maturation of 3CLLS‐ABPP allows to rapidly screen the ligand space. We designed LS‐ABPP to rapidly tailor a probe scaffold to the active site pocket of an enzyme of interest and demonstrate its potential for the development of specific probes against t3CLpro and PLpro of the pandemic corona virus SARS‐CoV‐2. We synthesized a chemical LS probe based on the core of tyrosine. The probe was equipped with a chloroacetamide warhead conferring reactivity towards the protein's active site and a terminal alkyne group for fluorescent labeling of probe‐bound enzyme by bioorthogonal click chemistry via the 1,3‐dipolar Huisgen alkyne–azide cycloaddition  and Cys114Ala (PLpro). We then screened the probes in situ against t3CLpro and PLpro expressed in intact E. coli cells and their corresponding active site mutants  for one hour  versus active site mutant (m) proteins .LS18 gave similar results for 3CLpro in its native form with truncated N‐terminus as well as with our structural models of the extended native N‐terminus prior to self‐cleavage of 3CLpro and the Strep‐tag II‐modified non‐cleavable N‐terminus . To exclude an influence of the N‐terminal Strep‐tag II for probe binding, we used an enterokinase cleavage site to remove the tag, yielding a version of 3CLpro only two amino acids longer than the native N‐terminus after self‐cleavage. Successful labeling of this truncated version of t3CLpro with probe LS18 confirmed that the small size of the probe allowed to profile the active site of the protease independent from its activation stage .Building on these results we performed covalent molecular docking of proComparing homologs of 3CL. In order to investigate if our LS probe strategy also allows to label 3CLpro of the closely related SARS‐CoV‐1, another member of the Sarbecovirus subgenus, we also expressed its wild type and mutant sequences and performed LS probe labeling experiments in situ.pro homologs were virtually identical , demonstrating robustness even across different viral strains. However, probes LS06 and LS17 displayed concentration‐dependent specificity for t3CLpro of SARS‐CoV‐1 while being unspecific for SARS‐CoV‐2 . These minor yet important differences highlight the possibility of using the LS probe strategy for the fine tuning of chemical probes to the active site properties of an enzyme of interest.For most ligand‐modified LS probes the labeling intensity and specificity for the active site of both t3CLCompetitive screening identifies enzyme inhibitors. Since our probes exhibited sensitive and active site‐specific labeling of PLpro as well as of the pre‐activation stage of 3CLpro, we aimed to exploit their potential as chemical tools for the competitive screening of enzyme inhibitors. We searched over 1000 structures of commercially available food grade additives, natural products, and protease inhibitors, most of which were approved for human use, and manually selected 44 compounds with electrophilic motifs such as aldehydes, Michael acceptors, epoxides, and esters that could potentially react covalently with the nucleophilic active site cysteine of 3CLpro and PLpro (Supporting Information Table S2). The electrophilic compound library was prepared in form of DMSO stocks and individually pre‐incubated for an initial screening with the purified proteases followed by labeling with the probes (LS18 for t3CLpro and LS24 for PLpro). Successful inhibitors would block the active site and thereby prevent subsequent probe labeling, which could be read out by lowered in‐gel fluorescence  were calculated from curve fittings of concentration‐dependent quantitative competitive labeling . In contrast, curcumin (M03) almost completely inhibited labeling of PLpro with an IC50 of 26 μm and was inactive against t3CLpro . However, curcumin is a well‐known pan‐assay interference (PAIN) compound and as such of no particular interest.X05 and M03 inhibited enzyme activity of PLpro with a fluorogenic peptide substrate, confirming the validity of the competitive screening approach . Of greater interest was the activity of salvianolic acid B , which inhibited labeling of t3CLpro with an IC50 of 12 μm  and salvianolic acid A  were the most active with IC50 values of 10 and 4.8 μm, respectively. Interestingly, salvianolic acid C , which differs from SalA only by a hydroxy group locked into a benzofuran ring, was considerably less active with an IC50 of 91 μm  only exhibited an IC50 of 32 μm. These results demonstrate a fine‐tuned structure–activity relationship of salvianolic acid derivatives for the inhibition of t3CLpro. To achieve a better understanding of the molecular basis of protease inhibition, we performed molecular docking of SalA into the active site of t3CLpro , we finally prepared and tested extracts of its dried roots in a competitive labeling assay with probe LS18 against t3CLpro and observed potent inhibition down to 1 mg mL−1  comprising a cleaved N‐ and C‐terminus. Probe LS18 labeled n3CLpro with great sensitivity at probe concentrations down to 0.2 μm . Consequently, we employed substrate cleavage assays to determine the IC50 values for the inhibitors identified for the pre‐activation stage of 3CLpro. All salvianolic acid‐related compounds completely inhibited the enzymatic activity of n3CLpro albeit at comparably high concentrations .Phenethyl isothiocyanate, which is produced from its precursor gluconasturtiin by vegetables of the m Figure –f. We thm Figure –g. Also,o Figure . All of 1 Figure . To invem Figure . In contpro encompass somewhat different structural requirements than inhibitors of mature 3CLpro and may be missed in standard screening assays.Our results suggest that inhibitors of the pre‐activation stage of 3CLProtease labeling in the human proteome. We next aimed to investigate a potential application of the probes to label the proteases in the background of a native human proteome. We used lysates of human hepatocellular carcinoma (HepG2) cells and supplemented them with different concentrations of purified t3CLpro and PLpro. Applying probe LS18 for t3CLpro and LS24 for PLpro at 20 μm followed by click chemistry resulted in the detection of each of the proteases as strong bands at protease concentrations down to 77 μg mL−1 . Finally, we compared the labeling of t3CLpro and n3CLpro, titrated into the proteome of human alveolar epithelial cells (A549) that had been used previously in infection models of SARS‐CoV‐2.pro was labeled by probe LS18 with equal sensitivity as in the HepG2 proteome  activity of PLpro that modulates proteostasis of the host cell during the infection process.pro and PLpro by competitive profiling. A great amount of work has been dedicated to finding inhibitors of the mature stage of 3CLpro using virtualpro inhibitors of SARS‐CoV.[pro‐specific probes provide the unique opportunity to study this protease prior to N‐terminal self‐cleavage and discover inhibitors of a pre‐maturation stage of the enzyme that cannot be assessed using conventional substrate cleavage assays. We identified salvianolic acid derivatives as potent inhibitors of 3CLpro, with salvianolic acid A effectively blocking probe labeling at single‐digit micromolar concentrations. Also, extracts of roots of Salvia miltiorrhiza (red sage), the natural producer of salvianolic acids, confirmed a strong inhibitory activity. Extracts of S. miltiorrhiza and SalA are known for having anti‐thrombotic effectsSalB) have been clinically approved and are widely used in China for the treatment of coronary heart disease.The proteases of the SARS‐CoV‐2 are currently in the focus of research for drug development against COVID‐19.9b, 15pro and PLpro proteases and highlighted their value as chemical tools for the active site‐specific labeling of these proteases in complex proteomes as well as for the screening of inhibitors for drug development against COVID‐19.We report the development of a highly adaptable ligand selection strategy that allows to rapidly customize chemical probe scaffolds in order to achieve active site specificity for a protease of interest. As proof of concept we targeted the two cysteine proteases of the corona virus SARS‐CoV‐2, demonstrating that selectivity and efficacy can be indeed controlled and fine‐tuned via the ligand structure. We found probes with single‐digit micromolar activity for corona virus 3CLThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Hep+/+Tgr5 and Hep−/−Tgr5 mice were fed a high fat diet (HFD) for 7 weeks and then orally gavaged with three doses of a highly potent, TGR5-specific agonist, Compound 18 (10 mg/kg), or vehicle, over 72 h and underwent an oral glucose tolerance test (OGTT) after the last dose. Herein, we report that TGR5 mRNA and protein is present in mouse hepatocytes. Cumulative food intake, body weight, and adiposity do not differ between Hep+/+Tgr5 and Hep−/−Tgr5 mice with or without treatment with Compound 18. However, administration of Compound 18 improves glucose tolerance in HEP+/+Tgr5 mice, but not in Hep−/−Tgr5 mice. Further, this effect occurred independent of body weight and GLP-1 secretion. Together, these data demonstrate that TGR5 is expressed in hepatocytes, where it functions as a key regulator of whole-body glucose homeostasis.The bile acid receptor, TGR5, is a key regulator of glucose homeostasis, but the mechanisms by which TGR5 signaling improves glucose regulation are incompletely defined. In particular, TGR5 has an increasingly appreciated role in liver physiology and pathobiology; however, whether TGR5 signaling within the liver contributes to its glucoregulatory effects is unknown. Therefore, we investigated the role of hepatocyte TGR5 signaling on glucose regulation using a hepatocyte-specific TGR5 knockout mouse model. Hepatocyte-specific  Bile acids are amphipathic steroid molecules that activate the nuclear receptor, farnesoid X receptor (FXR), and the transmembrane G-protein coupled receptor, TGR5, to integrate lipid, glucose, and energy metabolism and maintain metabolic homeostasis ,2,3. DysTGR5 is expressed in many tissues including endocrine glands, adipocytes, muscle, liver, brain, and the gastrointestinal tract ,10,11,12TGR5 is also robustly expressed in the liver, but the role of hepatic TGR5 signaling in glucose regulation remains poorly understood. Various studies have identified anti-inflammatory, anti-apoptotic, choleretic, and proliferative effects of TGR5 signaling in nonparenchymal cell types of the liver. Within the liver, TGR5 is highly expressed on Kupffer cells , sinusoiTgr5−/− mice are more susceptible to liver injury than littermate controls as evidenced by an elevation of serum liver enzymes due to increased cytokine mRNA levels, more pronounced inflammatory infiltrates, and increased liver necrosis [Previous studies highlight a role for TGR5 in liver biology and pathobiology. For example, the genetic ablation of TGR5 or the inactivation of TGR5 signaling has been shown to make mice more susceptible of cholestatic liver injuries ,33,34,35necrosis , highligDespite the growing body of literature regarding the role of TGR5 in liver health, it is unknown if TGR5 signaling within the liver contributes to TGR5′s role in glucose regulation, largely due to a lack of cell-type specific in vivo studies of liver TGR5 function. As the hepatocyte is the predominant cell type in the liver, here, we tested the hypothesis that TGR5 signaling in the hepatocyte improves glucose regulation. It is thought that TGR5 is, at most, lowly expressed on hepatocytes; however, this has not been extensively studied. TGR5 expression has been identified in a human hepatocellular carcinoma cell line , in canitm1(KOMP)Vlcg)  were used for immunofluorescence analysis of hepatocyte TGR5 expression. Hepatocyte-specific TGR5 knockout mice were generated by crossing a TGR5 floxed mouse line (B6.Gpbar1 < tm1.1Auw/J) [21Mgn/J). To validate this model, hepatocytes were isolated from HEP+/+Tgr5 and HEPTgr5−/− mice and analyzed by RT-PCR to confirm the presence of Tgr5 mRNA in HEP+/+Tgr5 mice and loss of Tgr5 mRNA in the hepatocytes of HEPTgr5−/− mice  supplemented with 3.4% butter fat, 8.5% tallow, 13.1% soybean oil, 3.5% mineral mix, and 1% vitamin mix  by weight for 7 weeks to produce an obese, insulin resistant phenotype. Mice were matched for baseline body weight at the start of the HFD. Food intake and body weight were measured once per week . In a separate cohort of mice, following 7 weeks of the HFD, mice received 3 consecutive, daily doses of either vehicle  or Compound 18  by oral gavage. Compound 18 was formulated in 20% Captisol w/v with water, as previously described [HEP+/+Tgr5 , Compound 18 HEP+/+Tgr5 , Vehicle HEPTgr5−/− , and Compound 18 HEPTgr5−/− .All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Cornell University . Study mice were individually housed and maintained in a temperature and humidity-controlled room, with a 14:10 h light-dark cycle. Whole body TGR5 knockout mice (B6.Gpbar1 .1Auw/J)  with hep−/− mice . Startinescribed . An oralescribed . To miniescribed . Blood gThe HOMA-IR (homeostasis model assessment of insulin resistance) index was calculated as (fasting serum glucose × fasting serum insulin/22.5) to assess insulin resistance . Log (HOTgr5+/+) and knockout (Tgr5−/−) mice were used for immunofluorescence analysis, as previously described [Tgr5+/+) and knockout (Tgr5−/−) (Liver samples from whole body TGR5 wildtype (escribed . BrieflyTgr5−/−) . Detectit-test, as indicated. Differences were considered significant at p < 0.05.Data are presented as mean ± SEM. The main effect of sex was not significant so the data for males and females were combined. All statistical analyses were performed using GraphPad Prism 8.00 for Mac . Data were analyzed by two-factor ANOVA with Bonferroni’s post-test or Student’s −/−Tgr5 mice, confirming antibody specificity. TGR5 expression was also detected in some, but not all, hepatocytes in +/+Tgr5, but not −/−Tgr5 mice . These data demonstrate that Compound 18 promotes gallbladder filling independently of hepatocyte TGR5 signaling.TGR5 is highly enriched in the biliary tract ,25 and iHEP+/+Tgr5 and HEPTgr5−/− mice with and without Compound 18 treatment. Compound 18 improved glucose tolerance compared with vehicle-treated controls in HEP+/+Tgr5 mice, but not HEP−/−Tgr5 mice (p < 0.05). Furthermore, Compound 18-treated HEP+/+Tgr5 mice exhibited lower blood glucose excursions compared with Compound 18-treated HEP−/−Tgr5 mice (p < 0.05).To assess the role of hepatocyte TGR5 on glucose regulation, we performed an OGTT in HFD-fed HEP+/+Tgr5 and HEPTgr5−/− mice compared to vehicle controls (p < 0.05). While this Compound 18-induced increase in serum GLP-1 levels was diminished by 15 min post-glucose gavage, there remained an elevation of serum GLP-1 levels in Compound 18-treated mice compared with control (p < 0.05 by 2-factor ANOVA in the HEP+/+Tgr5 mice and p < 0.05 by Student’s t-test in the HEP−/−Tgr5 mice). Nevertheless, fasting serum insulin concentrations and serum insulin concentrations at 15 min after the glucose gavage did not differ between the genotype or treatment condition (A key mechanism by which TGR5 agonists improve glucose tolerance is through induction of GLP-1 secretion and subsequent enhancement of glucose-stimulated insulin secretion . Previouondition C. As expHEP+/+Tgr5 and HEPTgr5−/− mice with and without Compound 18 treatment. We chose the log(HOMA-IR) as a surrogate index of insulin sensitivity as it has been shown to have improved predictive accuracy compared with other indices [As expected, we did not observe alterations in insulin secretion during the OGTT between genotype or between treatment. It has been previously shown that enhanced TGR5 signaling improves insulin resistance in various tissues by decreasing inflammation  and lipo indices .HEP+/+Tgr5 mice exhibited a lower log(HOMA-IR) compared to Compound 18-treated HEP−/−Tgr5 mice (p < 0.05 by Student’s t-test). Together, with a lack of a difference in glucose-stimulated insulin secretion, these data suggest that hepatocyte TGR5 signaling may improve glucose homeostasis, in part, through an improvement in insulin sensitivity. Nevertheless, further work is needed to define the impact of hepatocyte TGR5 signaling on insulin sensitivity.There was no significant difference in fasting blood glucose A or fastThe TGR5 receptor is ubiquitously expressed throughout the body and has a well-defined role in many regulatory functions that affect hepatic metabolism and extrahepatic glucose homeostasis ,9,13,21;HEP+/+Tgr5 and HEPTgr5−/− mice with or without treatment with Compound 18, demonstrating that hepatocyte TGR5 signaling does not regulate body weight, food intake, or adiposity. While Briere et al. reported that 14 days of Compound 18 administration at 60 mg/kg reduced body weight and fat mass gain in HFD-fed mice [TGR5 has been implicated in body weight regulation through its role in energy metabolism. Specifically, TGR5 regulates energy expenditure by inducing mitochondrial biogenesis and thereby increasing basal metabolism in thermogenically competent tissues, such as brown adipose tissue and skeletal muscle and through the beiging of white adipose tissue ,18,19,48fed mice , it is lWhile our data are consistent with previous work demonstrating that TGR5 ablation contributes to metabolic dysregulation in mouse models ,34,37,4914C-2-deoxy-glucose tracers [HEP+/+Tgr5 mice, as compared to HEPTgr5−/− mice, following treatment with Compound 18 is mediated by reduced inflammation and decreased ectopic triglyceride deposition, characteristic of enhanced liver TGR5 signaling [The hepatocyte TGR5-dependent improvement in glucose tolerance occurred in the absence of a difference in insulin secretion, suggesting that hepatocyte TGR5 signaling regulates hepatic glucose metabolism and/or insulin sensitivity. Hepatic glucose metabolism is dictated predominantly by hepatic glucose output and insulin sensitivity . Using a tracers . It is aignaling ,24. Neve−/−Tgr5 mice have documented decreased bile flow from the liver [−/−Tgr5 mice [HEP+/+Tgr5 and HEPTgr5−/− mice in the absence of Compound 18. However, in response to Compound 18 administration, our data demonstrate a significant increase in gallbladder weight in both HEP+/+Tgr5 and HEPTgr5−/− mice. Together, these results suggest that hepatocyte TGR5 does not contribute to the role of TGR5 in gallbladder filling under basal conditions or in the presence of a TGR5 agonist. Further, our results highlight the need to identify cell-type specific effects and downstream signaling targets of TGR5 signaling in order to develop better bile acid-based therapeutics to treat diabetes and metabolic disease.A growing body of literature has highlighted the potential value of TGR5 agonists in the treatment of various metabolic and liver diseases ,40,41,52he liver  and reduhe liver ,32, whiche liver ,47,50. S−/− mice ,32,40. AIn conclusion, TGR5 is a promising therapeutic target, but its breadth of actions, particularly in the gallbladder, complicates this task. As such, there is a growing need to identify cell-type specific effects of TGR5 signaling in order to begin to identify and target the downstream effectors of TGR5 signaling. Our findings provide additional insight into the underlying mechanisms by which TGR5 agonists improve whole body glucose homeostasis. Importantly, our data demonstrate that hepatocyte TGR5 signaling regulates whole body glucose homeostasis.


Inactivation of pure PTP1B was about three times more efficient with polysulfides as compared to the combination of bicarbonate and H2O2. Bicarbonate alone had no effect on PTP1B, neither with nor without a combination with polysulfides, thus strengthening the notion that bicarbonate-assisted H2O2-mediated inactivation of PTP1B involves formation of peroxymonocarbonate. Furthermore, PTP1B was potently protected from polysulfide-mediated inactivation by either TRP14 or Trx1, in contrast to the inactivation by bicarbonate and H2O2. Comparing reductive activation of polysulfide-inactivated PTP1B with that of bicarbonate- and H2O2-treated enzyme, we found Trx1 to be more potent in reactivation than TRP14. Altogether we conclude that inactivation of PTP1B by polysulfides displays striking qualitative differences compared to that by H2O2 together with bicarbonate, also with regard to maintenance of PTP1B activity by either Trx1 or TRP14.Protein tyrosine phosphatases (PTPs) can be regulated by several redox-dependent mechanisms and control growth factor-activated receptor tyrosine kinase phosphorylation cascades. Reversible oxidation of PTPs is counteracted by reductive enzymes, including thioredoxin (Trx) and Trx-related protein of 14 kDa (TRP14), keeping PTPs in their reduced active states. Different modes of oxidative inactivation of PTPs concomitant with assessment of activating reduction have been little studied in direct comparative analyses. Determining PTP1B activities, we here compared the potency of inactivation by bicarbonate-assisted oxidation using H Proteins (NOXs) ,6 leads s (NOXs) ,7. The muaporins ,9. In adignaling . Recentlecessity . Previou species . Indeed,arbonate ,14 and Parbonate .2O2 and HCO3− however need further characterization, and should also be considered in view of the reduction and reactivation of oxidized PTP1B by the reductive enzyme systems. It is well known that different oxidized forms of the PTP1B can be reduced by different components of the thioredoxin (Trx) system. For example, the sulfenic acid form is potently reduced directly by Trx reductase 1 (TrxR1), but this enzyme cannot directly reduce the sulfenylamide form of PTP1B; that reduction requires either Trx1 or Trx-related protein of 14 kDa (TRP14) [2O2 and HCO3− on PTP1B activity in the presence of a complete Trx system coupled with peroxiredoxin 2 (Prx2) using pure recombinant proteins, showing that addition of HCO3− could potently overcome the complete reductive protection of PTP1B by the Trx system against the addition of H2O2 alone [2O2 and HCO3−, also comparing the protective effects of TRP14 in this context in relation to those exerted by Trx1. We found that both Trx1 and TRP14 potently protect PTP1B from inactivation by polysulfides, but less so from H2O2 in combination with HCO3−, and we found that Trx1 is more potent than TRP14 in reactivating oxidized PTP1B, especially upon prior inactivation by polysulfides.Several different forms of oxidative modifications of the active site Cys of PTP1B have been described, including a sulfenic acid (-SOH) ,7,10,15  (TRP14) ,24,25,26 (TRP14) . We receO2 alone ,26. In tA construct encoding the catalytic active site of human PTP1B (residues 1–322 of NCBI Reference Sequence NP_002818.1) was subcloned into a pD441–H6 vector, with expression of PTP1B, purification, and tag removal performed under reducing conditions as described before ,26,27. P2S3 from Dojindo Laboratories, Kumamoto, Japan, from a stock solution of 10 mM prepared freshly in degassed water and with the final concentrations in the experiments referring to those calculated for the Na2S3 salt) or 100 μM H2O2 together with 25 mM HCO3− in assay buffer , 1 mM sodium azide). After this treatment, PTP1B was again desalted using Zeba Spin Desalting Columns to remove oxidants from the buffer, with subsequent determination of protein concentration using Bradford reagent. Different preparations of PTP1B can display different basal activities but are nonetheless here given in absolute turnover numbers and within each experiment, the treated samples should be compared to the controls in the same experiment. For protection or reactivation experiments, either pre-reduced or pre-oxidized PTP1B was treated or incubated together with the combinations of TrxR1/Trx1/TRP14 and NADPH  as described in the text, with PTP1B activity finally determined in aliquots taken at 5, 15, and 60 min.Reduced recombinant PTP1B was desalted and then exposed for 30 min to either 100 μM polysulfide (NaThe activity of PTP1B was measured using a chromogenic substrate 4-nitrophenyl phosphate (pNPP)  (15 mM) as reported previously . The inc2O2 in comparison to the inactivation by polysulfides, with or without addition of HCO3−. Confirming what we [3− strongly potentiates the otherwise rather slow H2O2-dependent inactivation of PTP1B, but HCO3− is not inactivating PTP1B in the absence of H2O2 as here easily demonstrated by removal of H2O2 using addition of catalase  two minutes prior to activity measurements at either 5, 15, or 30 min of incubation with polysulfide. This revealed that the TRP14 system could almost completely block further time-dependent polysulfide-mediated inactivation of PTP1B when added to the system, and some reactivation of activity was also found when TRP14 was added at the earlier time points .2O2 produced by activated NOX enzymes needs to overcome the antioxidant defense systems and reductive enzymes that protect PTPs, including the activities of TrxR1-dependent Trx, TRP14, and Peroxiredoxins [3− to be available during EGFR signaling in cells [3− and H2O2 generates peroxymonocarbonate (HCO4−) that potently inactivates PTP1B [2O2 + HCO3− or by polysulfide treatment, and we evaluated how either Trx1 or TRP14 (both coupled with TrxR1 and NADPH) could maintain PTP1B in an active state when challenged with these different oxidation routes. With HCO3− concentrations being affected by pH alterations and thereby indirectly linking PTP1B regulation to physiological acid-base balance disturbances, as discussed previously [Growth factor-induced reversible oxidation and thereby inhibition of PTPs is a necessary prerequisite for downstream phosphorylation cascades to take place . To enabredoxins ,11,23. Res PTP1B . Recentles PTP1B . In the 3− enabled more efficient inhibition of PTP1B by H2O2, as found earlier [3−-potentiation was not seen with polysulfide-mediated PTP1B inactivation. Both with and without addition of HCO3−, as well as upon addition of catalase quenching any possible presence of H2O2, the inactivation of pure and pre-reduced PTP1B by polysulfides was very efficient. This clearly showed that polysulfide-mediated PTP1B inactivation is qualitatively different from that of H2O2-mediated PTP1B inactivation. It was previously shown that hydrogen sulfide (H2S) or polysulfide-treatment of PTP1B leads to inhibitory persulfidation of its catalytic Cys residue [2S3 as our polysulfide source. This will in solution easily autoxidize and disproportionate, resulting in a mixture of polysulfide compounds with different sulfur chain lengths that will be able to provoke persulfidation with addition of one or several sulfur atoms on reactive cysteine residues [1–3H species [2S3-derived polysulfides occurs primarily due to persulfidation of the active site Cys residue, upon its reaction with polysulfides . One can however also speculate that the active site Cys residue that has first been persulfidated with a single extra sulfur atom could possibly facilitate the formation of a sulfenylamide motif, in analogy to the reaction of a sulfenic acid form of the catalytic Cys as triggered upon exposure to H2O2 [We first validated that addition of HCO earlier ,12, but, residue ,22. This residue . It shouresidues ,33. Sincresidues . In addiresidues , which m to H2O2 ,18. FinaThese examples of qualitative differences in the oxidative inactivation of PTP1B, and its reactivation by either Trx1 or TRP14, should be considered when PTP1B activities are to be studied in a cellular context, where all these enzymes and oxidants will be present at different levels in a cell type- and growth context-dependent manner. The findings of our present study with the pure enzyme systems are summarized in 2O2 with HCO3− or upon polysulfide treatment [2O2, (ii) reducing and thus removing polysulfides as oxidative species, and, perhaps only to smaller extent, (iii) reactivating oxidatively inactivated PTP1B. These different regulatory pathways for PTP1B regulation are schematically summarized in 2O2 and HCO3−, forming peroxymonocarbonate as the PTP1B-inhibiting species. This was suggested earlier [2O2 and HCO3− but not upon a combination of polysulfides and HCO3−, the notion that peroxymonocarbonate is the inhibitory species in the former case becomes further supported. It is interesting to note that two recent reports also showed that formation of peroxymonocarbonate may promote inhibitory overoxidation of Prxs [2O2 levels, also indirectly could lead to further PTP1B inhibition in a cellular context.Based upon our findings using recombinant enzyme systems, and considering our prior observations with regards to regulation of PTP1B by either Hreatment ,22,23,34 earlier  and with of Prxs ,36, whic


Top-down control of Inhibitory Neuron Gamma (TING). Collectively, these functions arose from a codimension-2 bifurcation in the dynamical system. Our results highlight a key role for this top-down feedback, gating inhibition to facilitate often diametrically different computations.Growing evidence shows that top-down projections from excitatory neurons in piriform cortex selectively synapse onto local inhibitory granule cells in the main olfactory bulb, effectively gating their own inputs by controlling inhibition. An open question in olfaction is the role this feedback plays in shaping the dynamics of local circuits, and the resultant computational benefits it provides. Using rate models of neuronal firing in a network consisting of excitatory mitral and tufted cells, inhibitory granule cells and top-down piriform cortical neurons, we found that changes in the weight of feedback to inhibitory neurons generated diverse network dynamics and complex transitions between these dynamics. Changes in the weight of top-down feedback supported a number of computations, including both pattern separation and oscillatory synchrony. Additionally, the network could generate gamma oscillations though a mechanism we termed  Growing evidence suggests that top-down centrifugal feedback from higher cortical areas specifically target inhibitory interneurons in primary sensory regions. In the olfactory system, axons from excitatory neurons in the piriform cortex (PCx) synapse onto the inhibitory granule cells in olfactory bulb (OB), whereby they can modulate the function of the mitral/tufted cells (M/T), the principal relays of olfactory information from the bulb to the brain  and inhibitory (I) neurons ]. Finally, the mechanism underlying the dynamics, as well as the functional roles played by top-down control of inhibition occurred via a codimension-2 bifurcation in the dynamical system. By gating the weight of connections from piriform cortex to the inhibitory neurons in the bulb, a number of seemingly disparate computations could be supported by a single circuit, providing an additional framework for the diversity of inhibitory interneuron function in the olfactory bulb.To address this, we built a three-node network model consisting of an excitatory population of mitral/tufted cells (M), an inhibitory population of granule cells (G) and a top-down population of pyramidal/semilunar cells (P) in PCx, and studied how firing rate dynamics were influenced by top-down weights onto inhibition. Changing the weight of the top-down connections to local inhibitory neurons reshaped the dynamics of the local E-I circuit in a way that enhanced sensory discrimination as well as generated oscillatory synchrony including entraining gamma oscillations in the local circuit }. For a given Δμ > 0, distinguishing the pair  was the same as distinguishing  in terms of pattern separation. In this framework two different distances, for instance, Δμ = 0.1 or μ = 0.5 | μ1 ∈ , Δμ is given} was correlated with the transition boundary ΓΔ of the same value μ . Furthermore, these computations could be accomplished even when the stimuli were periodic, fluctuating at the frequency of sniffing (Neimark-Sacker bifurcation), suggesting that these findings are a general feature of this network. Our results provide both a mathematical framework for how top-down control of inhibition shapes the dynamics of a network, and a link between such dynamics and the computations that neural circuits can perform.An important point to consider is how changes in top-down weights may be instantiated biologically? This point depends on the timescale of weight changes. On short time scales, changes in inhibitory drive to granule cells can facilitate olfactory discrimination . Local excitatory mitral and tufted cells broadcast activity patterns to a pyramidal/semilunar cell population in piriform cortex, that then synapses back onto inhibitory granule cells.In parallel, changing top-down weights onto inhibitory neurons can increase the synchrony between two stimuli that were initially asynchronous. A number of experimental and theoretical studies have explored the privileged role that inhibitory interneurons play in generating gamma oscillations (Whittington et al., Studies on dynamics of local excitatory and inhibitory neurons in the olfactory system both experimentally and mathematically are extensive (Wilson and Cowan, Finally, we found that the firing rate representations of mitral/tufted cells, granule cells, and piriform neurons resided within distinct domains on a manifold defined by the stimulus and the weight of feedback. These domains corresponded to transitions in the dynamics of the system. Changes in the top-down weights moved a transition boundary that delineating these domains across different stimuli. When two stimuli were on opposites sides of this transition boundary, their dynamics operated under two different regimes, and their representations were pushed further apart. By contrast, when the stimuli were both on the same side of the transition boundary, within regimes corresponding to similar dynamics, their activity became more synchronous; effectively binding those stimuli together. Changes in top-down weight were therefore changes in the location of the transition boundary that could either marshal the representations of two stimuli together or push them apart. In conclusion, we identified a model that links the dynamics of neural systems with the computations they are hypothesized to perform and may be used as a generalized framework to study the diverse effects of feedback onto inhibitory populations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.KP conceived and supervised the project. ZC performed all the experiments and analysis. ZC and KP made the figures and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N+1” array architecture for the in-situ detection of icing in cold climates. T-type thermoelements were used to fabricate a sensor with six independent temperature sensing points, capable of two-dimensional temperature mapping. The sensor was intended to detect the high latent heat of fusion of water (334 J/g) which is released to the environment during ice formation. The sensor was embedded on a plywood board and an aluminium plate, respectively by an epoxy resin. Three different ice accretion cases were considered. Ice accretion for all cases was achieved on the surface of the resin layer. In order to analyse the temperature variation for all three cases, the first 20 s response for each case was averaged between three cases. A temperature increase of (1.0 ± 0.1) °C and (0.9 ± 0.1) °C was detected by the sensors 20 s after the onset of icing, attributed to the latent heat of fusion of water. The results indicate that the sensor design is well-suited to cold temperature applications and that detection of the latent heat of fusion could provide a rapid and robust means of icing detection.A test was performed to determine the efficacy of a novel multi-channel thermocouple temperature sensor employing “ Aircraft and wind turbines encounter the icing phenomenon when they operate under high humidity and low temperature. Water droplets in the air impinge on the surface of these machines. If convective and/or conductive cooling is sufficient to remove the latent heat of fusion of the water, the water will freeze on the surface. This icing is broadly categorized as rime or glaze ice according to the rate of convective and/or conductive cooling. Rime ice occurs under low-temperature and humidity conditions. The impinged liquid water droplets freeze immediately on contact with a surface with a sufficient cooling rate . On the In-flight icing usually occurs when the aircraft passes through clouds containing supercooled liquid water droplets, which can freeze on surfaces such as the wings, fuselage, and engine inlet. Ice accretion can change the shape of the leading edge of the aerofoil, resulting in significant degradation of the aerodynamic performance. When aircraft are exposed to severe icing conditions for a long time, ice threatens the controllability and flight safety, as it reduces the stall margin and increases the stall speed and causes an abrupt drop in pitching moment. The icing on the engine intake deteriorates the performance of the engine due to the distorted flow of intake air. If ice projectiles, which are shed from the surface, are sucked into the engine, it can cause engine failure. Forty aviation accidents from the period 2006 to 2010 were directly related to in-flight icing occurring on the wings, fuselage, or control surfaces The installation of wind turbines in cold climates or at high elevations is often desirable due to the availability of land, low population density, increased power due to higher air density, and greater wind speeds with increasing elevation ,5. Such To mitigate the effects of icing and to ensure aviation safety, ice protection systems have been equipped to aircraft and wind turbines. Ice protection systems require ice detection systems and anti-/de-icing systems with logical algorithms. Several methods for anti-/de-icing systems have been suggested: fluid application, thermal melting, mechanical removal, and hydro-/ice-phobic coatings. The fluid application method prevents icing by applying a chemical solution such as ethylene glycol. The chemical solution lowers the freezing point of the impinged water . With thAnti-/de-icing systems are controlled based on measured data from the ice detection systems to determine the operating time, intervals, and intensity. A reliable ice detection system is therefore essential, and two or more ice detection systems have been applied in aviation and wind energy fields. In the aviation field, ice detectors can be classified into two categories: icing condition detectors and ice accretion detectors. Icing condition detectors monitor the ambient liquid water contents (LWC) and temperature ,19. DualCurrently adopted ice detection systems in the wind energy industry are also icing condition detectors and ice accretion detectors . Wind tuAlthough a lot of devices were developed and demonstrated as listed above, important issues were reported. Richards, T.  reportedFor these reasons, military aircraft mainly adopt the electro-thermal ice detector . Thin wiInfrared cameras  are usedN+1”, where “N” is the desired number of TC junctions or “sensor sensing points” (SSPs) in the array. In comparison, conventional TC architecture requires “2N” thermoelements for “N” TC junctions. Therefore, the benefit of the “N+1” architecture from space and material savings scales significantly with the number of SSPs. Thermocouple technology is robust and well-understood, and “N+1” MCTCA architecture represents a versatile, low-cost method for fast, accurate temperature mapping over a wide range of operating temperatures [To this end, this study suggests a novel ice detection sensor by using a proprietary multi-channel thermocouple array (MCTCA) technology ,48,49,50eratures . N+1” architecture is governed by the same variables as conventional “2N” architecture. Most notably, the Seebeck coefficient of each thermoelement should be uniform throughout its length. The introduction of additional junctions in the “N+1” architecture necessarily creates “dissimilar regions” where the Seebeck coefficient is altered across the width of the junction by the presence of the second thermoelement material. It has been shown that where the junction size is small relative to the junction spacing, the accuracy of the “N+1” architecture is very close to that of conventional TCs [The accuracy of the “onal TCs .An ice detection sensor using the MCTCA can expect the following advantages. First, the MCTCA detects ice events with relative temperature changes between structure surface temperature and the latent heat of fusion due to the icing events. Therefore, it is not affected by different temperature distributions along the considered structures and/or ambient temperature conditions due to convection, rotation, etc. Second, the thin and flexible MCTCA is simple in structure and easily implemented on a curved surface such as the leading edge of turbine blades, aircraft wings, and engine intakes without any disturbance of the flow field. To create a smooth surface, the thin wire array can be covered by resin or insulted between the surface layers, unlike the electro-thermal sensors which require protection grooves and infrared cameras attached on the outer surfaces. Third, the MCTCA is able to detect the presence or absence of icing as well as the distribution of ice over a wide area of the wing/blade. Therefore, an ideal power distribution can be suggested for the anti-/de-icing systems. Fourth, the rate of false-positives is intrinsically lower with this detection method because it can only detect the formation of ice; liquid water, dust, insects, etc., produce no signal. Finally, the MCTCA can be designed as an independent active ice detector. The MCTCA can accurately measure the surface temperature to detect the latent heat of fusion  like theThis study was performed to verify the feasibility of the MCTCA as an ice detection sensor. The accuracy and response time of the sensor was investigated at low temperatures such as rime ice conditions in a climate chamber. To consider the attachment on various surfaces, the dependency of the surface material was analysed. The performance of the sensor when embedded in resin to create a smooth surface was also evaluated.Vemf’ created between its ends when a temperature difference exists between them is given by:hotT’ and ‘coldT’ are the temperatures at either end of the thermoelement and ‘S(T)’ is the Seebeck coefficient, defined as:MCTCAs obey Seebeck theory, as do conventional TCs, and their output voltages can be reconciled to infer the temperature at each SSP. T0' is the ‘cold junction’ temperature at the data logger and ‘T1′ is the ‘hot junction’ temperature at the sensing point.When two different thermoelements are combined to form a TC, the temperature-dependent voltage as measured at the ‘cold junction’ of the TC is given by:When the Seebeck coefficient of a material is known over its operational temperature range, the integral in Equation (1) can be approximated as:abV’ measured between ‘a’ and ‘b’ due to a temperature gradient with ‘d’ is given by Equation (6):ad’‘V and bd’‘V are the thermoelement’s individual electromotive forces (EMF) between ‘a’-‘d’ and ‘b’-‘d’ respectively (see e’ at temperature ‘T2’:Equation (3) then simplifies to:vely see . Similar‘N+1′ configured sensor was studied in the authors’ previously published works [Equations (6) and (7) demonstrate how each junction of the TC array behaves as if it were a separate TC. A detailed explanation about the characteristics/working principles of the ed works .ISO standard T-type copper (0.51 mm) and constantan (0.51 mm) (Cu:Ni/55:45 by wt.) thermoelements were chosen, as these offer high accuracy in the −40 to 125 °C temperature range over other commercial types, e.g., a sensitivity of ± 0.5 °C compared to ± 1.1 °C for K-type TCs . All extThe sensor architecture employed in this study comprised a single copper ‘common line’ thermoelement, to which 6 constantan thermoelements were soldered with a typical separation of 35 mm see a,b. WhilTo assess the impact of the underlying material on the sensor response, particularly in relation to thermal inertia and conductivity, a MCTCA of similar architecture was mounted atop each of a 195 × 100 × 10 mm plywood board and a 212 × 102 × 3 mm aluminium plate. Each SSP was labelled S1-S6 (wood sensor) and SA1-SA6 , as shown in An operator’s finger was used as a heat source to apply localised heating to each of the SSP’s in order to test their individual responses. Detailed numerical calibration via mathematical simulation and experimental calibration using calibrated thermocouples (which are certificated by United Kingdom Accreditation Service) were carried out to analyse the potential error across the MCTCA. As a result of these calibration studies, the size of the “dissimilar region” and changes in Seebeck coefficient of the material were found as the two most critical parameters that can cause error. More details about calibration methods applied in this paper are described in Ref. . The expMCTCA and commercial TCs measurements were recorded with a mean frequency of 0.320 Hz using an NI 9213 data logger and a proprietary LabVIEW based data logging software developed by J.-S. Kim et al. ,48,49,50The measurement error of T-type thermocouples with the NI 9213 data logger is less than ± 1 °C with the 0.02 °C measurement sensitivity at the temperature range of −40 to 70 °C . The LabTesting was performed within a Sanyo Gallenkamp HCC065.PF4.MOD environmental chamber. A sketch of the experimental set-up is illustrated in An insulating pad consisting of ~4 mm thickness cardboard wrapped in thin polythene sheet was applied to one half of each sensor apparatus, such that three thermocouple junctions  were exposed to direct ice accretion on the surface of the resin, while three were insulated from direct contact with the water droplets . The two sensors were placed in a mirror image configuration as shown in A fine water mist was generated using a spray bottle modified with an extended nozzle to allow injection through the upper port of the environmental chamber a. The waBefore the water was sprayed, ice cubes were placed inside the bottle to maintain the water temperature close to zero, and insulation was applied to the copper nozzle extension to minimise heat losses to the environment. A 10 s spray was discarded immediately prior to each test to clear the internal pipes and ensure that all water had come directly from the iced reservoir. Commercial TC measurements confirmed that the water exit temperature was (1.0 ± 0.5) °C. During each test, the spray nozzle was inserted a fixed distance into the chamber and held straight. Spraying in this way produced a wide (>0.5 m) coverage of consistent water droplet characteristics at the bottom of the chamber. The sensors were placed at the centre of this area to ensure the most even distribution of icing across the sensor areas c. Three Spraying was repeated for durations of up to 60 s with rest periods in between to allow heat to dissipate. Repeated spray events were chosen in favour of continuous spraying because the cooling power of the chamber was insufficient to support sustained ice formation with the considered water droplet characteristics. After closing the chamber, the temperature was set to −15 °C and the apparatus was allowed to equalise. SSP and TC measurements were monitored to ensure readings had settled to a steady state before commencing the spray injection. Following the first spray event, the temperature was allowed to return to an approximately steady state in order to fully characterise the sensor response (defined as a temperature change of < 0.01 °C/ min). For all subsequent spray events, the sensor was only partially allowed to return towards equilibrium. This was undertaken to reduce the time required between tests and because we expected the heat input from the latent heat of fusion to be independent of absolute temperature. The total ice mass accreted on each sensor during the full test duration ranged between 27 g and 54 g, dependent on which test case.wc’, is 4.187J/g∙K and the specific heat of ice, ‘ic’, is 2.108 J/g∙K. The maximum sensible heat input to the system due to the injection of water at (1.0 ± 0.5) °C when the chamber is −15 °C is given byQ/m’ is the sensible heat per unit mass, ‘ΔT1′ is the temperature change from 1 to 0 °C, and ‘ΔT2′ is the temperature change from 0 to −15 °C. This figure does not account for heat lost to the chamber interior. Comparatively, the latent heat of fusion for water is 334 J/g [The specific heat capacity of water, ‘ 334 J/g . Thus, wThe outputs of the two sensors were each divided into two “regions” as described in (b) of After spraying the water, it is clearly seen that the temperatures are increased rapidly. Based on the predicted heat inputs from both the sensible and latent heat, we believe that this increase is mainly due to ice accretion. The composite material using resin is widely applied for aircraft and wind turbines. This study confirmed that the MCTCA can be attached to such a composite surface or inserted between composite layers of the surface and thus not induce any disturbance of the flow fields. Although the insulating properties of the resin negatively impact sensitivity and response time, the MCTCA could still accurately predict the increase in the surface temperature due to latent heat. If the thermal conductivity and the thickness of resin can be correlated, the MCTCA can predict surface temperature more accurately. This will be further investigated in future work.As shown in The full response of the first spray event for each test case is shown in Since the rate of water deposition to the sensor surface(s) from the spray mist is considered to be constant, the first 20 s of spraying  of the first spray event (where no pre-existing ice is present) should be comparable in each test case. Therefore, the response of each region may be averaged between Case I, Case II and Case III as shown in This study also confirmed that the MCTCA showed a proper response time as an icing detector. Icing is a phenomenon that occurs over several minutes to hours. Even with a very small amount of spraying, the MCTCA could immediately detect the temperature changed due to the latent heat of fusion for icing accretion. The MCTCA can warn of icing on a specific surface in a very early time without the aid of other sensors.It is important to note that the convection effects due to ambient wind are ignored in this study. This is mainly because the fluid velocity within the chamber is almost 1 m/s in the test bed. Therefore, Reynold’s number should be small, and the primary heat transfer mechanism is conduction with only a minor convective contribution. Furthermore, the proposed sensor measures the relative temperature differences due to latent heat of fusion during the ice events. Nevertheless, testing within an icing wind tunnel might be needed to investigate how the sensor responds with more convection. It is likely that this increased convection will affect the temperature change detectable by the sensor. However, L. Gao et al.  recorded3 for LWC at 2 m/s freestream velocity. The considered icing condition is the rime ice condition. Three different cases are considered where water spraying times are different to consider the ice thickness effects. By the series of experimental studies, the following conclusions can be drawn.In this feasibility study, a novel MCTCA sensor utilising T-type thermoelements was manufactured and tested experimentally to assess its suitability for cold-temperature ice-detection applications and to evaluate it against current industry-adopted approaches. The capability of the sensor in mapping temperatures across a 2D surface was demonstrated. Temperatures measured by the sensor were consistent with those measured using commercially available T-type thermocouples within the ± 0.5 °C error range under the given test conditions. The capability of the sensor for ice-detection was evaluated using an environmental chamber at −15 °C. The considered water droplet characteristics generated by the spray system are approximately 36 μm for MVD and 2.54 g/cm(1)MCTCA can detect the surface temperate rise regardless of the surface material which has high  and low (wood) thermal conductivities. The temperature measured by the MCTCA for Case II (40 s spraying time) is rapidly increased due to the latent heat of fusion. In order to compare all three test cases, the response of each region is averaged. Temperature increases of (1.0 ± 0.1) °C for plywood board and (0.9 ± 0.1) °C for aluminium plate were detected, respectively, within 20 s after spraying the water droplets.(2)This study confirmed that the MCTCA can be used as an active sensor. Although this study measured the surface temperature in real time for four hours, the low-power MCTCA can be operated for a much longer time.(3)The MCTCA can be used as the ice detection system without making any disturbance to the flow field. Although the sensor was embedded in approximately 2 mm thickness epoxy resin, the MCTCA is able to predict the surface temperature increase sufficiently. If the thermal conductivity and the thickness of epoxy resin are correlated, the MCTCA is able to predict the surface temperature more accurately.(4)The MCTCA displayed a very quick response time as the ice detection system. The MCTCA reliably predicted the temperature increase even though resin was applied and very little ice accumulated. Although the spraying time was varied to 20, 40, and 60 s for this experiment under the specified rime ice condition, the MCTCA detected immediate temperature changes due to the latent heat of fusion. The response time of the MCTCA is much faster than currently employed approaches in industry, e.g., power curve monitoring for wind turbine application. This gives the sensor a huge advantage in applications where detection of ice formation is extremely time-sensitive.This method of detection is particularly robust compared to other methods, because it directly measures icing in the area of concern rather than at a nearby location which may experience different conditions. There is a low rate of false-positives due to the nature of the latent heat signature, which is only seen when ice forms and is easily distinguished from any other heat inputs. The authors highlight aviation and wind energy generation as two industries which may benefit from this technology. The proposed MCTCA sensor can be embedded on the aerofoil surface of an aircraft wing or a wind turbine blade to provide real-time predictions of temperature distribution and ice accretion. Furthermore, the proposed sensor technology might be applied to any structures operating at cold climatic areas, as this sensor measures only relative temperature changes due to the latent heat of fusion during icing events.For future work, more detailed sensitivity studies varying resin thickness will be conducted. A better understanding of how the resin thickness affects the signal in both latency and magnitude will allow for more accurate predictions of surface temperature changes due to latent heat. Additionally, a thin-film type sensor will be developed and tested at a cold climatic wind tunnel. The thin-film process allows for the fabrication of smaller junctions which should improve accuracy by reducing the size of “dissimilar regions”. It also lowers the thermal inertia of the sensor, which should reduce latency.


Post-translational modification of proteins exerts essential roles in many biological processes in plants. The function of these chemical modifications has been extensively characterized in many physiological processes, but how these modifications regulate lignin biosynthesis for wood formation remained largely unknown. Over the past decade, post-translational modification of several proteins has been associated with lignification. Phosphorylation, ubiquitination, glycosylation, and S-nitrosylation of transcription factors, monolignol enzymes, and peroxidases were shown to have primordial roles in the regulation of lignin biosynthesis. The main discoveries of post-translational modifications in lignin biosynthesis are discussed in this review. Wood is a biological composite and a valuable source of feedstock for construction timber, pulp and paper, and biofuels . Wood isPinus , coniferyl alcohol (G-unit), and sinapyl alcohol (S-unit), also known as monolignols. The biosynthesis of lignin occurs in several consecutive reactions involving up to 11 different enzyme families and 24 metabolites . The patPinus , b, Eucacalyptus , and Pop suspension-cultured cells, a phosphopeptide derived from PAL2 was detected  is a family of enzymes that catalyzes the deamination of phenylalanine to cinnamic acid, representing the first reaction step in the phenylpropanoid pathway . PAL enzdetected . The autdetected . Consistbidopsis . In PAL on sites . The higon sites .max of PAL2 is slightly reduced. On the other hand, longer co-incubation of up to 4 h reduced the protein stability of the phosphorylated isoform of PAL2 and result in a 3-fold reduction in the Vmax when compared to the non-phosphorylated isoform  catalyzes the 3/5-methylation of caffeoyl- and 5-hydroxyferuloyl–containing acids, aldehydes, and alcohols for monolignol biosynthesis. In P. trichocarpa. PtrAldOMT2 is the highest transcribed gene in the monolignol biosynthetic pathway and the third-highest transcribed gene in stem differentiating xylem (SDX). The protein abundance of PtrAldOMT2 is also the highest of all monolignol enzymes, accounting for 5.9% of the SDX proteome  . Protein process .Pinus taeda SDX, In vitro phosphorylation assays demonstrated that PtMAPK6 could phosphorylate PtMYB1 and PtMYB4, transregulators of lignin biosynthesis in SDX. Replacement of Lysine68 for arginine abolished the kinase activity of PtMAPK6, demonstrating that the lysine is essential for mediating target phosphorylation. Serine236 was identified to be the phosphorylation site in PtMYB4. Replacement of Serine236 for a glutamate residue (mimicking constitutive phosphorylation of PtMYB4) did not interfere with the affinity for DNA binding but significantly reduced the transactivation of the target gene , and 3, 4 (SCW synthesis) of SDX, but its kinase activity for PtMYB4 phosphorylation was mainly observed in the initial stages of 1 and 2. The phosphorylated isoform of PtMAPK6, representing the active form of the enzyme, was only found in stages 1 and 2 , but the severity of dwarfism was reduced when the plants reached eight months old  and the similar phosphoregulation of MYB TFs suggest an important role of MAPK in the regulation of lignin biosynthesis in both angiosperm and gymnosperm species. Future studies focusing on MYB phosphorylation by MAPK6 and their interaction with external stimuli would better inform the phosphoregulation of lignin biosynthesis. For instance, is mechanical wounding the only stimulus of LTF1 phosphorylation, or are other signals such as ethylene response, cell wall biosynthesis, and cell remodeling also involved? PTMs are found in diverse classes of proteins, highlighting the regulatory interplay among PTMs as a common strategy to regulate protein functions . Other tPALs-GFP and KFBs in Arabidopsis reduced the conversion efficiency of phenylalanine to t-cinnamic acid by up to 80%  interacts with cinnamoyl-CoA reductase (OsCCR) to mediate its protein degradation via the proteasome pathway, thereby regulating lignin content in the cell walls  resulted in defects in Arabidopsis growth and vessel formation, mediated by repressed central metaxylem vessel formation and central protoxylem vessel formation  domain are key regulators of plant development, senescence, SCW formation, and biotic and abiotic stress responses . Severalll death , but itsbidopsis . VND7 haelopment . Repressormation . Inducedot cells .GSNOR1) in transgenic Arabidopsis expressing VND7 resulted in mutant seedlings lacking xylem vessel differentiation (mutant “suppressor of ectopic vessel cell differentiation induced by VND7” or seiv)  . Indeed,ntiation . VND7 dintiation . The expgulation . Biotin  of VND7 . Cystein of VND7 . Therefo of VND7 .VND7 had low levels of detected VND7 protein after 24 h, while the addition of proteasome inhibitor increased the abundance of VND7  nst1 double mutants rescued the pendent inflorescence stem phenotype and restored stem strength and deposition of secondary walls in interfascicular fibers. Overexpression of PtrWND2B and PtrWND6B  in Arabidopsis increased the transcript expression of cellulose synthases , xylan biosynthetic genes , and lignin biosynthetic genes (4CL1 and CCoAOMT1)  . PtrWND2CoAOMT1) . Overexpal cells . Express and TFs . Taken tormation . Ortholoormation . PTMs idP. tomentosa, PtoMYB156 and PtoMYB221 from the subgroup 4 of the R2R3-MYB TF family work as transrepressors of lignin genes ; followed by two one-electron reductions: (i) CoI + RH → CoII + R⋅; (k2) and (ii) CoII + RH → FeIII + R⋅; (k3), where FeIII, CoI, CoII, and RH are the native enzyme, compound I, compound II, and the monolignol, respectively  was higher in the ZePrx 34.70 compared to ZePrx 33.44 in the presence of either p-coumaryl alcohol or coniferyl alcohol substrates. On the other hand, the opposite effects were observed when coniferaldehyde, sinapyl alcohol, and sinapaldehyde were used as substrates. Moreover, the k3,  was higher for ZePrx 33.44 comparing to the ZePrx 34.70 in the presence of coniferyl alcohol. Again, the opposite effects were observed in the presence of sinapyl alcohol, where the reactivity was higher for the ZePrx 34.70  and ubiquitination  of monolignol enzymes decrease the enzymatic activity and protein stability  plays an essential role in regulating TFs for lignin biosynthesis . Howeverin vitro and in vivo and how they regulate lignin biosynthesis and wood formation. Comprehensive identification of PTMs in trees will be crucial to understanding the transduction of complex regulatory information that mediates wood formation.Much of the early work on plant PTMs have focused on model organisms such as Arabidopsis. These PTM in woody species, particularly in the regulation of lignin biosynthesis has remained largely unknown. Due to the large variation in lignin content, composition, and structure between Arabidopsis and perennial woody species, the extent of similarity or difference in the SCW-associated PTMs across species should be evaluated. Systematic co-expression analysis of SCW protein abundances with PTM enzymes during SDX formation, or genome-scale protein interactomics could reveal putative PTM regulatory interactions controlling lignin biosynthesis. The resulting interactions may guide future studies to verify whether these PTMs occur DS and JW conceptualized and literary reviewed and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.