This quantitative study explores the significant psychological and behavioral dynamics in coworking spaces. We collected data from a sample of 175 people working in Italian coworking spaces and found that a more cooperative organizational climate increases coworking space users’ happiness. We also found that this relationship is positively moderated by several job crafting behaviors. More specifically, when workers are proactive in the work environment, they are more likely to benefit from the potential advantages  that cooperation-oriented work settings provide, which, in turn, amplifies the positive effect of cooperative work settings on individual happiness. These findings make a useful contribution to both the growing literature on coworking spaces and the more general job crafting literature. Indeed, the previous research on both behavioral dynamics that are specific to coworking spaces and on the role played by job crafting in influencing workers’ happiness remains limited. The study’s managerial implications concern the relevance of establishing a cooperative climate and encouraging workers’ proactivity to promote their happiness. In the last few years, work organization has changed significantly in many different contexts  and the perceived happiness of coworking users. However, since users’ participation in these new collaborative spaces  worldwide was 13%  monthly fee, the more advanced coworking spaces provide users with both office facilities  and amenities purely for relaxation and recreation , and even well-being services and social experiences   and conducting daily “curation” processes  through their community managers to give users a fruitful, rich, and smooth coworking experience  from the more “community-led” spaces  model and the conservation of resources theory  increasing structural resources, (H2b) increasing social resources, (H2c) increasing challenging demands, and (H2d) decreasing hindering demands is higher.The first step of our analysis involved contacting the promoters of the Italian national conference of coworking spaces. This official event gathers together the majority of Italian coworking spaces to share ideas, discuss trends, and find common pathways to enhance the “coworking movement” as a whole. Thanks to the partnership we established with the conference board, we were able to verify our key methodological choices, starting from the sample selection; test and refine our survey questions through a focus session with a significant sample  and, to a lesser extent, in the southern regions .As we were interested in selecting only coworking spaces with either a formal or “de facto” community manager, we carefully examined each space’s website to ensure sure it fit our sample selection criteria. We contacted the community managers of each of the selected coworking spaces by e-mail and explained the study’s goals to them in detail, as well as the type of coworking space we were interested in and the content of our survey. Ultimately, about 18% of the coworking spaces we approached (42 out of 239) agreed to participate in the study. Each community manager sent the survey to all the coworking members of the community, inviting them to respond to the survey while ensuring their anonymity. We did not find significant differences in the participation rates of coworking spaces according to their regional location.Overall, we received 190 surveys, but 15 could not be used because incomplete. Our final sample therefore included 175 coworking users , 58.3% of whom being female, with an average age of 40.61 years (SD 8.19). 12% have a high school diploma, 58.9% a bachelor degree, 29.1% a master degree or a Phd. These variables do not reveal significant differences among the different coworking spaces. Coworking users were entrepreneurs, consultants, professionals from a wide variety of sectors. Most respondents were web designers (31%), entrepreneurs (23%), writers (7%), management consultants  project managers (10%), and marketing/sales technicians (12%). About 15% of the marketing/sales technicians and about 12% of all project managers worked steadily for an organization. All the other respondents were self-employed. Considering our respondents’ average age , education level (medium-high), occupation , and self-employment status shows that the general profile seems to be consistent with that found in other previous studies on coworking spaces ; “I ask colleagues for advice” ; “When an interesting project comes along in my coworking space, I offer myself proactively as project coworker” ; and “I make sure that my work is mentally less intense” (decreasing hindering demands). The Cronbach’s alpha estimates for these scales are 0.84, 0.72, 0.85, and 0.74, respectively.To rule out the potential confounding effects of socio-demographic variables, we controlled for age, gender, education level, and job tenure. Specifically, participants’ gender and age were used as control variables because they have been suggested to influence people’s subjective level of happiness . Thus, to prevent the potential problems associated with CMV, we took several precautions. We used well-established and reliable scales with varying scale formats. Further, to decrease evaluation apprehension, we also specified that there were no right or wrong answers. We guaranteed respondents’ confidentiality to reduce socially desirable responses  for all the items. We then compared the model with the CLF to the model minus the CLF by using standardized regression weights , χ2/df = 1.95, CFI = 0.92, IFI = 0.92, TLI = 0.91, RMSEA = 0.06]. We compared the hypothesized model with several other models, although none of them produced a better fit than the hypothesized model: a five-factor model factor model in which increasing structural resources and increasing social resources were combined into one factor  and a three-factor model in which all job crafting variables were combined into one factor . We also tested the single-factor model with all the items loaded on a common factor, which showed a very poor fit .Second, before testing our hypotheses, we studied the structural validity of the scales used by performing a confirmatory factor analysis in AMOS. The hypothesized six-factor model  exhibited an acceptable fit [χNext, we calculated the composite construct reliability (CR) coefficients and average variance extracted (AVE) Table . The CR Finally, because some employees were nested within the same coworking space, we sought to verify that our observations could be treated independently. First, we calculated two kinds of intra-class correlation coefficients (ICC): ICC(1) and ICC(2)  upon which the current literature on coworking spaces has mostly focused. More specifically, our results on the moderating role of job crafting behaviors provide a possible explanation of First, we found that what are commonly called “approach” job crafting behaviors  positively moderate the relationship between the collaborative climate and happiness. In other words, when coworkers’ attitudes are not to “passively” take advantage of the opportunities that a collaborative work setting provides but to “customize” and even generate such opportunities in a proactive, personal way, consistent with their needs, aspirations, and inclinations, a positive effect on their happiness can be observed. This result is in line with classic literature on the relationship between autonomy and intrinsic motivation , a behavior such as “decreasing hindering job demands” has a “negative” nature, whereby workers try to reduce or avoid specific tasks , as suggested by our findings, implies that managerial initiatives aimed at encouraging proactivity may play a crucial role in generating coworker happiness. The job crafting literature suggests that contextual elements may facilitate or hinder job crafting behavior  which, on the one hand, may provide unique interpretative opportunities in relation to our study’s goals but, on the other hand, may be somewhat limiting. Indeed, one may wonder about the nature of work activities in coworking spaces and whether they can be conceptualized as job crafting since there is no formal managerial job design. However, the concept of job crafting, as originally proposed in the seminal contribution by Wrzesniewski and Dutton , encapsuWhile our specific research setting was coworking spaces, future research may explore whether the same results hold true in traditional companies and workplaces. We believe that this is likely the case because though co-working spaces have specific features that create an ideal context for encouraging and facilitating cooperation and proactivity, there is nothing preventing traditional workplaces from providing similar opportunities to their employees in various ways and to different degrees. We believe that the positive relationship between climate for cooperation and happiness, moderated by proactive behaviors such as job crafting, is likely a general phenomenon, which can be positively triggered by favorable work arrangements and circumstances (such as coworking spaces) or constrained and limited in other work situations, depending on the availability of cooperation opportunities. Thus, future research may explore in more detail the significance of contextual variables in different kinds of workplaces and organizational cultures.A possible limitation of our study concerns the research setting. By utilizing heterogeneous sources, we included some of the most representative coworking spaces in Italy in our study. We are fairly certain that our results are relevant for the Italian context. However, cultural elements may have influenced these results; therefore, caution should be exercised in generalizing them to other national cultures. Such cultural differences could be explored in future studies.Finally, our study was conducted before the outbreak of the COVID-19 pandemic. Consequently, our data and results refer to a “normal” (pre-pandemic) situation, where physical proximity represents an extremely relevant aspect of work in coworking spaces. Future research may attempt to understand whether the relationship between climate for cooperation and happiness still holds true in work situations in which remote working is prevalent or in which social distancing significantly constrains the possibility for direct interaction and collaboration. We believe that, in their essence, our results should have a general quality as they could hold true not only in coworking spaces but also in traditional workplaces . However, it is clear that the very meaning of “climate for cooperation” may change significantly depending on the modalities and tools through which people may (or may not) establish cooperative relations. Co-working itself may indeed evolve in ways that are hard to predict within a post-pandemic world. A reflection on this topic clearly goes well beyond the scope of our study. Nevertheless, we believe that our results may inspire future research to explore if and how constrained modalities of interaction and communication may change the relationships between cooperation, job crafting, and happiness that we have illustrated.In summary, this study indicates a positive relationship between climate for cooperation and happiness in coworking spaces. It also shows that this relationship is stronger when coworkers act as job crafters, especially when they proactively reshape their work by increasing their job resources and challenging job demands.We believe that this line of research on behavioral dynamics in coworking spaces is important for several reasons. First, most previous studies on coworking spaces are either theoretical or conceptual in nature. With some exceptions (Avila et al. Second, our study is one of the first to focus on happiness as a relevant outcome of work in coworking spaces. Coworking is a relatively new phenomenon and, thus far, much attention has been devoted to knowledge creation (Bouncken and Aslam Overall, we believe that this line of research should be extended, not only due to the inherent interest in coworking spaces as a relatively new organizational phenomenon but also because increasing our knowledge on the internal dynamics of these new organizational contexts may also inform new research and reflection on more traditional companies (Blagoev et al.


The total weight of the epoxy resin in the fiber/epoxy composites was 22%, and the 11% weight loss was achieved at around 350 °C, regardless of the presence of oxygen; however, the features of heat–treated fibers/epoxy were associated with the pyrolysis atmosphere at a higher temperature. The pyrolysis products in inert atmospheres, with water contact angles of 58.8°, can considerably improve the tensile properties of flexible composites at the elastic stage. Furthermore, the flexible composite granules were prepared to plug large channels in sand–filled pipes, and the plugging rate had the potential to reach 81.1% with an injection volume of 5.0 PV. The plugging performance was essentially unaffected by water salinity, owing to the high stability of flexible composite granules in mineralized water. The findings of this study present a realistic route to the industrial application of fiber/epoxy, as well as a novel approach for encouraging the efficient use of scrap wind turbines on a large scale.The number of scrap wind turbines is expanding globally as the wind power industry develops rapidly. Zero–waste recycling of scrap wind turbine blades (WTB) is the key for wind power firms to achieve green and sustainable development on the premise of satisfying environmental protection criteria. In this work, the pyrolysis of fiber/epoxy composites obtained from scrap WTB in oxidizing inert atmospheres was investigated. Various characterization methods were employed to characterize the microstructure and chemical characteristics of the heat–treated fiber/epoxy and to reveal the pyrolysis mechanism. In addition, the heat–treated fibers/epoxy were used as reinforcing agents to investigate their impact on the elastic deformation of butadiene styrene rubber–based flexible composites, and the reinforcing mechanism was revealed. The results revealed that the constituents of fiber/epoxy composites were mostly fiberglass (SiO Wind power generation is a type of green power generation that helps to promote the global clean energy transition, reduce carbon dioxide emissions, and solve the climate change challenge ,2. Due tAt present, the chemical recovery of waste fiber/epoxy composites is mostly focused on the recovery of reinforcing materials  from composites due to the high cost of recycling resin ,11,12. TPyrolysis has long been seen as a potential technology for the clean and effective exploitation of high–quality organic solid waste, capable of converting organic solid waste into carbon material or liquid for chemical raw materials ,17. A nuDue to the substantial disorder within the pulverized structure after mechanical pulverization, scrap WTB may not be acceptable for high–end application in industry. As the global yearly scrap volume of WTB is so great, it is better suited to more practical industrial uses. Scrap WTB powder is most likely to be used as cement filler in the building sector, but this application has several limitations ,23. The Given the global need for energy and governments’ concerns about energy security, the development of oil and gas resources is a field requiring long–term study ,26. The In this study, the waste fiber/epoxy composites were ground and then thermally treated to investigate the pyrolysis process of waste fibers/epoxy in oxidizing inert atmospheres. Thereafter, the pyrolysis products were used as reinforcement agents to improve the elastic deformation of flexible materials. In addition, the application potential of flexible composite granules as plugging agents of large channels was studied via granulation with a twin screw extruder in order to expand the possibility of their large–scale application. This study proposes a novel method for recycling scrap WTB in its entirety instead of recycling the resin or glass fiber separately.n: 100000) was purchased from the China Petrochemical Corporation. Lightweight liquid paraffin and NaCl (AR) were purchased from Shanghai Aladdin Biochemical Technology Co., LTD. .Scrap bulk WTB  were obt−1. After pyrolysis for 1 h, the obtained sample was labeled as fiber/C1 . The mixture was placed in a high temperature, high pressure reaction kettle after being uniformly agitated with a glass rod. The flexible composite was produced after stirring at 180 °C for 2 h. To produce the molded flexible composite for mechanic performance testing, the flexible composite was cut into pieces, put into a dumbbell mold (50 × 8.5mm), and extruded at 180 °C on a high temperature hot table. After compaction and chilling, dumbbell–shaped samples were produced. The flexible composites that were made with fibers/epoxy, fiber/C1 and fiber/C2, were reinforcing agents that were labeled fiber/epoxy@FC, fiber/C1@FC, and fiber/C2@FC, respectively.The flexible composite granules have the same composition as the flexible composite; however, their form and size were different. To prepare flexible composite granules using fiber/C2 as reinforcing agent, the SBR, liquid paraffin, and fiber/C2 with a mass fraction of 1:10:8 were added into the barrel containing the twin screw extruder . The reaction temperature of the twin screw, the grinding head temperature, and the underwater cutting temperature were 180, 140, and 60 °C, respectively. After dehumidifying using a vibrating screen and fan, the flexible composite granules were obtained. The average size of the flexible composite granules was controlled by the size of the grinding head.−1) for 3 months.An X–ray diffractometer  was used to characterize the components of the fibers/epoxy before and after pyrolysis. To analyze the elements and chemical states of the fibers/epoxy, X–ray photoelectron spectroscopy  was utilized. A field emission scanning electron microscope  was used to analyze the microstructure of the fibers/epoxy before and after pyrolysis. A synchronous thermal analyzer  was used to evaluate the thermal characteristics of the fibers/epoxy under various heat treatment settings. The tensile characteristics of the flexible composites at various temperatures were characterized using a universal testing machine . The digital rubber hardness tester was used to assess the hardness of the flexible composite granules. The aging properties of the flexible composite granules were studied after soaking in a NaCl solution . Flexible composite granules (0.5 wt%) were injected into the simulated mineralized water with a NaCl concentration of 50~200 g L−1; the injected rate was 1 mL min−1. The flow and pressure changes were measured during the injection process. After the pressure remained constant, the two ends of the sand–filled pipe were sealed and aged for 3 days in a 120 °C oven. Then, DI water was injected, and the breakthrough pressure, flow rate, and pressure change were recorded, as was the permeability k2 (mD) of the sand–filled pipe.A sand–filled pipe simulation system  was usedD, %) was determined in accordance with the following equation (Equation (1)) [The plugging rate (ion (1)) :2, and CaCO3, respectively , and Di is the length of the determined fibers; the Nano Measurer 1.2.5 software determined the values of Di and ni. It was found that the fibers which had an average length of less than 96 μm accounted for 85.1% of the total fiber content  granules on the surface d. The va content .The surface elements of the fibers were mostly O, Si, and Ca, whereas the debris on the glass fiber surface was mostly composed of C and O e, thus i2 atmospheres were shown in 0.05, T0.11, and T0.22 denoted thermal mass losses of 5%, 11%, and 22%, respectively, whereas Tmax represented the temperature at which the greatest decomposition rate was derived from the DTG curve. The pyrolysis of the fibers/epoxy in the N2 atmosphere resulted in a single weight loss, which occurred mostly at 350 °C. The TG curve, however, altered substantially when exposed to air in an oxygenated environment. In the air atmosphere, the initial decomposition of the epoxy resin in the fiber/epoxy composite happened earlier. Interestingly, an 11% mass loss of fibers/epoxy was attained at roughly 354~357 °C, without the presence of oxygen. Ma et al. attributed the first degradation step to the release of ester groups because the monomer and curing agent of the resin were methylhexahydrophthalic anhydride and N,N–Dimethylbenzylamine, respectively [The TG and DTG curves of fiber/epoxy degradation in air and in Nectively ; howeverectively ,38. The ectively . The fir2 . Peaks at 1606 and 1507 cm−1 were linked to aromatic ring C=C stretching vibrations, and the absorption peak in the 1300–1100 cm−1 range was related to ether’s C–O stretching vibration. Moreover, the peak at 830 cm−1 was attributed to aromatic C–H out–of–plane deformation vibrations and the precise peak variations of fiber/C1 and fiber/C2 in the 915 cm−1 epoxy ring were not discernible. Fiber/C1 showed a typical C=O peak at 1723 cm−1, and the spectral band of 1300~1100 cm−1 widened and intensified, thus showing that additional oxygen–containing compounds were formed during the degradation of epoxy resin in an O2 environment. The intensity of the C–H stretching vibration in the range of 2800–3000 cm−1 of fiber/C1 considerably decreased or almost vanished; however, the peak in the fiber/C2 range remained, thus showing that carbon compounds were lost under aerobic circumstances and retained in the absence of O2.FTIR was used to further analyze the chemical structure of pyrolysis products b. C–H st2 remained  The glass fibers in the fiber/epoxy composite remained relatively stable throughout the pyrolysis process, and the pyrolysis of epoxy resin was connected to the pyrolysis atmosphere. The early degradation of epoxy resin in air was substantially faster than in nitrogen, and the pyrolysis of epoxy resin in air cannot be described simply through pyrolysis or combustion models.(2) The water contact angles of the fibers/epoxy after heat–treatment in the oxidizing and inert atmospheres were 18.1 and 58.8°, respectively. The pyrolysis of epoxy resin in an oxidizing atmosphere could result in more oxygen–containing sites, but pyrolysis in an inert atmosphere would result in the removal of most of the O and the formation of an amorphous carbon structure.(3) SBR and liquid paraffin were more compatible with fiber/C2 than fiber/epoxy and fiber/C1; therefore, fiber/C2@FC demonstrated the greatest tensile performance in the temperature range of −20 to 60 °C, with the tensile performance in the first elastic stage reaching 200% at 60 °C.(4) Fiber/C2 composite granules could achieve an 81.1% plugging rate at 5.0 PV, and the plugging performance was essentially unaffected by water salinity, thus indicating that they have a high application potential in plugging large channels during oil and gas extraction.(5) Pyrolysis offers a novel approach for the value–added exploitation of fibers/epoxy from scrap WTB, as well as a new method for large–scale fiber/epoxy applications in flexible composite materials.


Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana (FWB), which devastates banana production worldwide. Biocontrol is considered to be the most efficient approach to reducing FWB. Here we introduce an approach that spatiotemporally applies Piriformospore indica and Streptomyces morookaensis strains according to their respective strength to increase biocontrol efficacy of FWB. P. indica successfully colonizes banana roots, promotes lateral root formation, inhibits Foc TR4 growth inside the banana plants and reduces FWB. S. morookaensis strain Sm4-1986 secretes different secondary compounds, of which xerucitrinin A (XcA) and 6-pentyl-α-pyrone (6-PP) show the strongest anti-Foc TR4 activity. XcA chelates iron, an essential nutrient in pathogen-plant interaction that determines the output of FWB. 6-PP, a volatile organic compound, inhibits Foc TR4 germination and promotes banana growth. Biocontrol trials in the field demonstrated that application of S. morookaensis lead to improvement of soil properties and increase of rhizosphere-associated microbes that are beneficial to banana growth, which significantly reduces disease incidence of FWB. Our study suggests that optimal utilization of the two biocontrol strains increases efficacy of biocontrol and that regulating iron accessibility in the rhizosphere is a promising strategy to control FWB.The soil-borne fungus  Piriformospore indica and Streptomyces morookaensis, inhibits the soil-borne fungus Foc TR4 growth and reduces Fusarium wilt of banana.Spatiotemporal application of two biocontrol strains,  Musa spp.), originated in Southeast Asia and the Western Pacific2, are now widely distributed throughout the humid tropics and sub-tropics where they provide a staple food for about 400 million people in the developing countries in Africa, Asia, and Latin America3. Bananas are the most exported fruit in the world, having a production of 119 million tons and export trade value of 13.3 billion dollars in 20204.Bananas (Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive diseases in banana production worldwide. FWB has restricted banana production for more than a century5. The epidemic of FWB led to the replacement of Foc race 1 susceptible Gros Michel with resistant Cavendish, which currently covers about 40% of the global production and is likely only banana present on supermarket shelves for non-producing countries6. However, a newly emerged race of Foc, tropical race 4 (TR4), is virulent not only on Cavendish but also on almost all other banana cultivars. Foc TR4 caused serious losses in banana plantation worldwide, which resulted in the abandonment of thousands of hectares of banana orchards7. Currently, there are no effective methods to control FWB caused by Foc TR4.Fusarium wilt of banana (FWB) caused by the soil-borne fungus Foc pathogen can linger in soil for up to 30 years even in the absence of plant hosts, which makes it particularly difficult to be eliminated from the infected soil9. Being a vascular pathogen, Foc colonizes banana roots and reaches the vascular bundles3, leading to the ineffectiveness of chemical control. Repeated use of chemical fungicides has raised great concern for environmental pollution and human health. Breeding resistant cultivars is thought to be the most effective way to control FWB, but all commercial banana varieties tested are susceptible to Foc TR4, and they are propagated by cloning due to the nature of sterile triploid10. Thus, biological control of FWB has gained great interest11.The Piriformospora indica is a well-known endophytic fungus that colonizes roots of a broad spectrum of plant species and confers diverse beneficial effects on host plants14. P. indica colonization of banana significantly alleviated disease symptoms caused by Foc TR415, and if the colonization was combined with the addition of Dictyophorae echinovolvata culture substrates, the best disease resistance enhancement to Foc TR4 infection was achieved16. Streptomyces are naturally abundant in soils, and it is likely that they will cause less damage to the surrounding ecosystem when applied. Many Streptomyces species have been used as biocontrol agents to protect plants against various diseases due to their ability to produce a range of secondary metabolites that can either inhibit the growth of phytopathogens or promote plant growth19. Streptomyces malaysiensis 8ZJF-21 was isolated from medicinal plants in Hainan of China. The extracts of S. malaysiensis 8ZJF-21 showed strong inhibitory effects on the mycelial growth of Foc TR4 and when the strain was used as a biocontrol agent, it significantly reduced disease symptoms of the banana plantlets infected by Foc TR420. Streptomyces sp. H3-2 was isolated from the rhizosphere of banana plantations. The extracts of the strain H3-2 suppressed the growth and spore germination of Foc TR4, and pot experiment showed that Streptomyces sp. H3-2 promoted the vegetative growth of banana plantlets and significantly inhibited the Fusarium wilt disease symptom caused by Foc TR421. Additionally, the application of Streptomyces improved the soil microbial communities and enhanced plant resistance to pathogens22. Rhizosphere is an important interface involved in the exchange of resources including nutrients, compounds, etc. between the plants, the soil environment, and the microbes. It has been known that competition for essential nutrients such as iron in the rhizosphere is a crucial factor that determines the survival of microbes23. Rhizosphere microorganisms that produce growth-inhibitory siderophores could suppress the pathogen growth and thus protect plants against pathogen infection24. The approach that takes advantage of the ability of microbes in restricting pathogens access to the essential nutrients is an effective strategy for biocontrol.Piriformospore indica in the endophytic compartments and Streptomyces morookaensis in the rhizosphere to control FWB. S. morookaensis strain 4-1986 (Sm4-1986) improves soil properties, shifts rhizosphere microbial structures, and suppresses Foc TR4 growth by secreting active compounds when applied to the field. P. indica colonizes and proliferates in the intracellular spaces within the banana roots to promote lateral root formation and restrict the growth and extension of Foc TR4 inside the banana roots. In addition, soil properties such as pH and iron content are also important factors affecting the control of FWB.Here, we present a spatiotemporal approach using S. morookaensis Sm4-1986 displayed antifungal activity against Foc TR425. We reasoned that the anti-Foc TR4 activity of Sm4-1986 might be due to the secreted compounds. In PDB medium, GFP-labeled Foc TR4 grew well showing strong fluorescence   agar assay showed that there was a clear yellow zone around the Oxford cup, indicating a strong iron-chelating ability of XcA  Fig.  and 6-pecA) Fig.  are partXcA Fig. .Foc TR4 grew well in PDB medium as indicated by the consistently increasing optical density, however, the optical density of Foc TR4 culture decreased when 6-PP  were added  and transmission electron microscope (TEM) to observe the shape of FocTR4 spores. The surface of Foc TR4 spores became wrinkled when treated with XcA  to analyze the interaction between XcA and 6-PP and to examine how these two compounds synergistically affect Foc TR4 germination at different concentrations. RSM analyses revealed the coexistence of XcA and 6-PP at lower concentrations was able to suppress Foc TR4 spore germination, indicating a synergistic effect of these two compounds  promoted banana plantlet growth although higher concentrations (>200 μΜ) inhibited banana plantlet growth and caused brown color on the rhizomes  increased the growth of banana plantlets .The field trials were carried out for two consecutive years in a land that had been abandoned due to the severe infection of S. morookaensis strain Sm4-1986 again in the following year. After growing for 8 months, Fusarium wilt disease incidence of the treated banana plants was 9.1% (15 of the 164 diseased plants still remained diseased), and most of the P. indica-treated banana plants grown in the Sm4-1986 treated sites did not show Fusarium wilt symptoms  method was used to identify differential biomarkers in the healthy and diseased rhizosphere soils. In bacterial community, the uncultured bacteria belonging to ity Fig.  togetherity Fig.  were ide3+, TF), and pH values of the soils from the healthy and diseased plants , total phosphorus (TP), total ferrum Fe+, TF, anO-hydroxyphenylacetic acid (EDDHA) compound, one of the most efficient iron-chelating agents, to reduce available iron in the medium31. Addition of EDDHA  completely suppressed the growth of Foc TR4 in comparison with the control on PDA . A mycelial plug (5 mm in diameter) was placed in the center of a PDA plate and cultured in an incubator at 28 °C in the dark for a week. P. indica on PDA plate was refreshed once a month. For liquid culture, a small mycelial plug (5 mm in diameter) was punched from mycelium margin of the stock plate and cultured in a 250 ml Erlenmeyer flask containing 100 mL PDB (potato dextrose broth) at 28 °C with a rotation speed of 200 rpm for 7 days. The culture was harvested and homogenized in a blender, and then filtered with cheese cloth. Spore number was determined using a Malassez hemocytometer and the filtrate solution was adjusted to 1 ×106 chlamydospores/ml for use. To observe lateral root formation, we grew banana plantlets in glass bottles containing 1/2 Hoagland medium40 for a month. P. indica inoculum of 1 ×106 chlamydospores/ml was used to inoculate the banana plants. To observe P. indica colonization patterns in banana roots, we used P. indica solution of 1 ×105 chlamydospores/ml. For field trials, five mycelium plugs were cultured in a 1000 ml Erlenmeyer flask containing 500 ml PDB for 10 days, and then the whole culture was collected and diluted by 5 times with water for use. Banana plantlets were placed into the solution by dipping the roots for 5 sec, and then transplanted 7 days later.S. morookaensis strain Sm4-1986 was propagated on PDA medium at 28 °C in the dark for a week. A mycelium plug (5 mm in diameter) from the margin of a growing colony of S. morookaensis strain Sm4-1986 was inoculated into 200 ml PDB in a 500 ml Erlenmeyer flask and cultured at 28 °C in a rotary shaker at 200 rpm for 10 days. The fermentation liquid was collected for banana plantlet inoculation. For field trials, five mycelium plugs (1 cm in diameter) were inoculated into 500 ml PDB in a 1000 ml Erlenmeyer flask and cultured at 28 °C in a rotary shaker at 200 rpm for 10 days. The whole fermentation broth was collected and diluted by 3 times with water and then applied to the field.Foc TR4 and a GFP-tagged Foc TR4 displayed similar growth characteristics and virulence to bananas were used41. Foc TR4 was used for inoculating banana plantlets and GFP-tagged Foc TR4 for observing the fluorescence. Foc TR4 or GFP-tagged Foc TR4 was cultured on PDA at 28 °C in the dark for 7 days. A mycelium plug (5 mm in diameter) from the margin of a growing colony of Foc TR4 or GFP-tagged Foc TR4 was put in a 250 ml Erlenmeyer flask containing 100 mL PDB and cultured at 28 °C with a rotation speed of 200 rpm for 5 days. The culture was filtered with cheese cloth, and the number of conidia in the filtrate was counted using a Malassez hemocytometer and adjusted to 1 ×106 conidia/ml for use.Musa acuminate L. AAA group)42, which were purchased from the Center of Tissue Culture, South China Botanical Garden, Guangzhou, China. Banana plantlets at different growth stages were used in different experiments.Cavendish cultivar ‘Brazilian’ banana plants  by silica gel column chromatography. TLC analysis separated twenty fractions, and different fractions were then subjected to different column chromatography elution. The purified fractions were determined by UV, IR, NMR, and mass spectroscopic analyses, and compound classification and structure elucidation were determined26.P. indica and Foc TR4 were co-cultured on a glass slide, which was first covered by a thin layer of PDA. A mycelium plug of P. indica was placed on one end of the glass slide that was covered by a petri dish which was then cultured in an incubator at 28 °C for 3 days. After that, a Foc TR4 plug was placed on the other end of the glass slide, and the petri dish was covered and incubated at 28 °C for another 4 days. During the period of cultivation, the leading hyphae of P. indica and Foc TR4 could meet somewhere between the two plugs. A small disc containing the overlapped hyphae of P. indica and Foc TR4 was sliced out from the place where they met and then subjected to scanning electron microscope observation43. Hyphae from the individually cultured P. indica and Foc TR4 were used as controls.Foc TR4 mycelia was cultured in 50 ml PDB in a 100 ml Erlenmeyer flask shaken in a rotary shaker with 200 rpm at 28 °C for 5 days. The culture was filtered using sterile gauze, and the filtrate was centrifuged at 1000 rpm for 5 min to collect conidia, which were then resuspended in PDB and adjusted to 1×107 conidia/ml. Aliquot 200 μl of the conidial suspension into the cells of a 96-well plate. XcA or 6-PP was added to the cells containing Foc TR4 conidia to make a final concentration of 3 mM for XcA and 0.96 mM for 6-PP. Each treatment was repeated three times. After 72 h incubation at 28 °C in darkness; conidia were subjected to scanning electron microscope observation44.A plug of Foc TR4 mycelia were cultured in 1000 ml PDB at 28 °C for 5 days. The culture was filtrated with sterile gauze, and the filtrate centrifuged at 1000 rpm for 5 min to collect conidia, which were resuspended in 1000 ml PDB and adjusted to 1×107 conidia/ml. 6-PP was added to the culture making a final concentration of 6-PP at 0.96 mM. The conidial culture was incubated at 28 °C for 24 h in darkness; centrifuged at 3000 rpm for 10 min at 4 °C to collect spores45. The collected conidia were subjected to transmission electron microscope observation.Micro-propagated banana plantlets were grown on 1/2 MS in glass tubes containing either 3 mM XcA or different concentrations of 6-PP. The glass tubes were put in the tissue culture room at 26 °C with a photoperiod of 14/10 h light/dark. Banana plantlets were observed and photographed at the indicated time.2HPO4·12H2O, and 4.5 g NaH2PO4·2H2O in 1000 ml ddH2O, pH 7.2) at 4 °C; 40 min per washing. Washed samples were vapor-fixed with 1% (w/v) aqueous osmium tetroxide for 2.5 h at room temperature, followed by washing three times with 0.1 M phosphate buffer at 4 °C, and each time for 5 min. After washing, samples were subjected to gradient dehydration in different concentrations of ethanol as in the order of 30%, 50%, 70%, 80%, and 90% at 4 °C; each for 10 min, and finally kept in 100% ethanol for 50 min. At the end of dehydration, samples were dried in a critical point dryer . The dried samples were sputter-coated with gold palladium in a sputter coaster 45. Conidial or hyphae morphology of Foc TR4 or P. indica was observed by SEM .Conidial samples or mycelial discs were fixed in a solution containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 h, and then subjected to vacuum pumping to let samples sink to the bottom of tube. After that, the samples were stored in a refrigerator for 12 h. Pouring off the solution and washed samples three times with 0.1 M phosphate buffer ; and washed twice with 0.1 M phosphate buffer; 30 min for each. Washed samples were post-fixed in 1.0% osmium tetraoxide (in 0.1 M phosphate buffer) for 4 h. The fixed samples were washed six times again with 0.1 M phosphate buffer for a period of 2 h. During this period, the washing buffer was changed every 15 min for the first four washing times and 30 min for the last two times. After washing, samples were pre-stained with 0.5% uranyl acetate (in 0.1 M phosphate buffer) for overnight. All the above steps were carried out at 4 °C.6-PP-treated 46, which were observed under a transmission electron microscope (Tecnai G2 SpiriBio TWIN).On the next day, spore samples were washed six times with 0.1 M phosphate buffer at 4 °C; 15 min for the first 4 times and 30 min for the last two times. After washing, samples were dehydrated in gradient ethanol solutions  at room temperature (RT) for 20 min each, and final in 100% ethanol for 30 min with one change. After dehydration, samples were passed in three changes of epoxypropane: epikote 812  at RT, and then in epikote 812 at RT for 3 h. Finally, samples were kept in fresh epikote 812 for overnight. In next day, samples were kept in fresh epikote 812 at RT for 7 h, and then were picked up to put in a small plastic box containing epikote 812. Samples were hardened in an oven at 60 °C for 12 h. Finally, the hardened blocks containing samples were sectioned into ultrathin sections of about 70 nm40 containing P. indica at the concentration of 1 × 105 chlamydospores/ml for two weeks. Banana roots were harvested, washed thoroughly with running tap water, and cut into 1 cm segments, which were stained with 0.01% acid fuchsin-lactic acid for 5 min and destained in lactic acid for 1 min. These were sectioned with an automatic vibrating slicing machine ;47 and sections were observed under an optical microscope  and images were taken.Banana plantlets were cultured in 1/2 Hoagland mediumP. indica of 1 × 106 chlamydospores/ml or Foc TR4 of 1 × 106 conidia/ml was used for inoculation. 8QH was used at the concentration of 200 μM. After inoculation, banana plantlets were observed at the indicated time.Experiments with banana plantlets in pots were carried out in greenhouse at 25 °C/18 °C (day/night) with a photoperiod of 14 h /10 h (light/dark). Prior to inoculation, all plantlets were maintained in greenhouse for a week to adapt environments. Plantlets with similar size were selected for inoculation. Each treatment contained 15 plantlets and was repeated three times. To inoculate plantlets, 50 ml inoculum were used for each pot. Foc TR4. Fifteen plants in a line were treated as a block, and the treatment and control blocks were arranged alternately in field. Banana transplantation holes of the treatment block were treated with S. morookaensis strain 4-1986 a week before transplantation. On the other hand, banana plants at the seven-leaf stage were treated with P. indica by submerging the roots into the inoculation solution. Banana transplantation was carried out in late February 2019. In the following days, standard irrigation and fertilization practices were applied to banana plants, and other managements were followed the normal farm operations in banana orchard. At the end of the year, external symptoms of Fusarium wilt disease were investigated48. Plants showing external symptom of leaf chlorosis were cut down, and the pseudostems were chopped off to examine the internal symptoms49. The sites were treated with S. morookaensis strain 4-1986 again. New banana plants were treated with P. indica and transplanted into the treated hotspots in 2020. Fusarium wilt disease incidence of bananas plants was investigated at the end of the year. External and internal symptoms were examined to determine whether the plants were actually infected by Foc TR4.The field trials were carried out in a land of 0.8 hectares in Longmen County , Huizhou City, Guangdong Province, China. The small farmer had planted banana (Brazilian variety) in the land for more than 10 years and recently abandoned it because of the high incidence of Fusarium wilt caused by Soil samples were collected from the evenly distributed four sites around a plant. pH values of each sample  were measured three times using a pH meter . Total nitrogen (TN), total phosphorus (TP), and total ferrum (TF) were detected using an Elemental analyzer .50. The PCR reactions were carried out in a 50 μL reaction mixture containing 1.5 μL each primer, 1 μL dNTP, 10 μL Buffer, 0.2 μL Q5 High Fidelity DNA Polymerase, 10 μL High GC Enhancer, and 40 ng soil DNA template. The PCR conditions for bacteria were initiated at 95 °C for 3 min, followed by 25 cycles of denaturation at 95 °C for 45 s, annealing at 50 °C for 45 s, and extension at 68 °C for 90 s, followed by a final elongation at 68 °C for 7 min, and then held at 4 °C. The PCR conditions for fungi were initiated at 98 °C for 2 min, followed by 30 cycles of denaturation at 98 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 1 min, followed by a final elongation at 72 °C for 5 min, and then held at 4 °C. The PCR products were pooled and visualized on 1.8% agarose gels, purified using a MinElute® PCR Purification Kit according to the manufacturer’s instructions, and quantified using QuantiFluorTM-ST .Soils were collected from four locations evenly distributed around the plants. The bacterial community composition of the rhizosphere was assessed by sequencing the V3-V4 region of the 16 S rRNA gene using the universal primers 338 F/806 R (forward primers 5′-ACTCCTACGGGAGGCAGCA-3′ and reverse primers 5′-GGACTACGCGGTATCTAAT-3′). The fungal ITS1 region was amplified using primer set ITS1 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′)51. Using Usearch with a cut-off of 97% similarity, the OTUs were clustered and the taxonomic classification was performed using RDP Classifier  with the classification threshold set at 0.8. The sequences were taxonomically identified by a BLASTn search of a curated NCBI database.High-throughput sequencing was carried out on the Illumina MiSeq platform . After pyrosequencing, raw sequences were processed with Prinseq (PRINSEQlite 0.19.5) to remove low-quality data and improve the syncretic rates of the subsequent sequence. Split sequences for each sample were merged using FLASH V1.2.7Experiments with banana plantlets in pots were carried out in greenhouse at 25 °C/18 °C (day/night) with a photoperiod of 14 h /10 h (light/dark). Prior to inoculation, all plantlets were maintained in greenhouse for a week to adapt to environments. Plantlets with similar size were selected for inoculation. Each treatment contained 15 plantlets and was repeated three times. The field trials were carried out in a land of 0.8 hectares in Longmen County , Huizhou City, Guangdong Province, China. Fifteen plants in a line were treated as a block, and the treatment and control blocks were arranged alternately in field.P < 0.05 using a one-way analysis of variance (ANOVA) with LSD test. Alpha diversity indices, including Chao 1, ACE, and Shannon, were calculated using the OTU table in QIIME51.All data analyses were analyzed using the SPSS 20.0 program , and the significance between control and treatments was assigned at 52. R package (v3.2.0) was used to draw the graph of redundancy analysis (RDA).The soil microbiome composition was ordinated by principal coordinates analysis (PCoA) using bray-curtis distance. Differences between microbiome composition of healthy and diseased plants were calculated by using PERMANOVA and ANOSIM. Bray–Curtis distances are sensitive to rare OTUs and thus emphasize differences in the presence or absence of taxa53. Two screening criteria were used: (1) linear discriminant analysis with a score of ≥ 3.0 ; (2) significance test with P < 0.05.Linear discriminant analysis effect size (LEfSe) was used to explore the most differentiated OTUs between health and diseased conditionshttps://web.rniapps.net/netshift)54.We also used the NetShift method to identify potential keystone driver taxa based on differences in network interactions between healthy and diseased plant microbiome (Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


E)-7-Phenyl-2-hepten-4,6-diyn-1-ol (1) and (Z)-7-Phenyl-2-hepten-4,6-diyn-1-ol (2) are isomeric natural polyacetylenes isolated from the Chinese medicinal plant Bidens pilosa L. This study first revealed the excellent anti-metastasis potential of these two polyacetylenes on human gastric cancer HGC-27 cells and the distinctive molecular mechanisms underlying their activities. Polyacetylenes 1 and 2 significantly inhibited the migration, invasion, and adhesion of HGC-27 cells at their non-toxic concentrations in a dose-dependent manner. The results of a further mechanism investigation showed that polyacetylene 1 inhibited the expressions of Vimentin, Snail, β-catenin, GSK3β, MST1, YAP, YAP/TAZ, and their phosphorylation, and upregulated the expression of E-cadherin and p-LATS1. In addition, the expressions of various downstream metastasis-related proteins, such as MMP2/7/9/14, c-Myc, ICAM-1, VCAM-1, MAPK, p-MAPK, Sox2, Cox2, and Cyr61, were also suppressed in a dose-dependent manner. These findings suggested that polyacetylene 1 exhibited its anti-metastasis activities on HGC-27 cells through the reversal of the EMT process and the suppression of the Wnt/β-catenin and Hippo/YAP signaling pathways.( Gastric cancer (GC) is the third leading cause of cancer-associated mortality in the world . With thβ-catenin signaling pathway is mostly considered to be closely related to tumor cell migration and invasion [β-catenin signaling pathway can promote tumor cell adhesion by forming a cadherin–catenin complex, enhancing the epithelial–mesenchymal transition (EMT) process, promoting clone initiation and invasion, and thus, aggravating tumor metastasis [β-catenin and YAP/TAZ) to the nucleus, binding with transcription factors, and then transcribing downstream target genes [β-catenin and Hippo/YAP signaling pathways. Phosphorylation YAP/TAZ can integrate with β-catenin, causing β-catenin to be retained in the cytoplasm, thus inhibiting the transcriptional activity of β-catenin [β, which, in turn, stabilizes cytosolic β-catenin and promoted its nuclear translocation of β-catenin [β-catenin and Hippo/YAP signaling pathways may be the key to producing promising results in the field of tumor control.Tumor metastasis is a complex, multifactorial dynamic process that mainly depends on the potential of tumor cell migration and invasion . The caninvasion . The acttastasis . In additastasis ,8,9. Thetastasis . Intereset genes . Further-catenin ,13. YAP -catenin . Given tE)-7-Phenyl-2-hepten-4,6-diyn-1-ol (polyacetylene 1) and (Z)-7-Phenyl-2-hepten-4,6-diyn-1-ol (polyacetylene 2)   are a paotection . For exaectively . In addito 29 μM . It is wβ-catenin and Hippo/YAP signaling pathways to deeply understand the molecular mechanisms. Overall, our research provided new insight for developing novel, secure, and effective anti-GC metastasis drugs from natural polyacetylenes compounds.Therefore, this study focused on the influence of two polyacetylene isomers on tumor metastasis, as well as the Wnt/1 displayed relatively obvious proliferation inhibitory activity against undifferentiated gastric cancer HGC-27 cells with an IC50 value of 52.83 μM, followed by human breast cancer MDA-MB-231 cells with an IC50 value of 73.92 μM. While other tested cells were all insensitive to polyacetylene 1 . In addition, polyacetylene 2 was inactive in all tested cells , including HepG2 cells, NCI-N87 cells, GES-1 cells, and MDCK cells. Based on the above results, the HGC-27 cells were used for further investigation in the anti-migration and invasion activities of polyacetylenes 1 and 2.As shown in 1 and 2 on the viability and proliferative capacity of HGC-27 cells and tried to find suitable non-cytotoxic concentrations for subsequent experiments. The viability of HGC-27 cells was analyzed using an MTT assay, as shown in 1 or 2 for 48 h, no significant difference (p > 0.05) in cell viability was observed compared with the control group. Furthermore, the proliferative potential of HGC-27 cells was evaluated by a colony formation assay, as shown in 1 or 2 treatment also had no substantial difference (p > 0.05) from that of the control group. These results indicated that 25 µM and below could be the non-cytotoxic concentrations of polyacetylenes 1 and 2 and were used in the following experiments.Conventional drugs that inhibit the migration and invasion of cells are often achieved by restraining proliferation, which cannot purely reflect the anti-migration and invasion activity of drugs ,19. Ther1 and 2 on the migratory viability of HGC-27 cells in vitro were evaluated using a wound-healing assay. As shown in 1 or 2 at 6.25, 12.5, and 25 µM for 24 h. Compared with the original (0 h) wound width, both polyacetylenes 1 and 2 could significantly inhibit the motility of HGC-27 cells in a concentration-dependent manner, while polyacetylene 1 demonstrated a greater potential for inhibiting cell migration (p < 0.05).A wound-healing assay is a simple and highly reproducible method to appraise the mass movement of cancer cells. In this study, the effects of polyacetylenes 1 and 2 on the migration and invasiveness of HGC-27 cells. The results demonstrated that polyacetylenes 1 and 2 could inhibit the migration and invasiveness of HGC-27 cells in a dose-dependent manner (p < 0.05). Furthermore, the inhibitory effect of the trans structure polyacetylene 1 was even better.Cell migration and invasion are pivotal steps in tumor cells as they disseminate from the primary tumor, invade across the basement membranes and endothelial walls, and, finally, colonize distant organs . A transt manner  (p < 0.01 and 2 significantly decreased the number of adherent cells (p < 0.05), while the inhibition effect of polyacetylene 1 visibly exceeded that of polyacetylene 2  and upregulating the expression of mesenchymal markers , thereby remolding the cytoskeleton and cell membrane, endowing cancer cells with increased motility and invasiveness, and causing the metastasis and spread of lesions . Therefotylene 1 . Simultaβ-catenin signaling pathway plays a vital role in regulating the migration and invasion of tumor cells. The key molecule of the Wnt signaling pathway β-catenin is involved in cell adhesion through forming cadherin–catenin complexes, as well as in gene transcription through interacting with transcription factors. The nuclear accumulation of β-catenin, which is a hallmark of Wnt signaling activation, is found in more than 50% of GC [1 on the nuclear localization proportion of β-catenin and the expression of Wnt signaling pathway molecules. Compared with the control group, the expression of β-catenin, p-β-catenin, GSK3β, and p-GSK3β in the polyacetylene 1 treatment groups was obviously decreased in a dose-dependent manner  DC., and Carthamus tinctorius L. Polyacetylene analogs have an alkyne-conjugated macrocyclic structure, which, upon activation, can form benzene-type free radicals that can easily enter the interior of tumor tissues, and upon targeting in tumor cells, rapidly seize the hydrogen atoms of DNA, cracking the DNA strands, and thus, producing remarkable anti-tumor activity. In addition, due to the carbon–carbon double bond and triple bond, polyacetylene compounds possess structural diversity and are easy-to-form isomers, which may exert different pharmacological effects. In the present study, we first revealed the excellent anti-metastasis potential of polyacetylenes 1 and 2 on human undifferentiated gastric cancer HGC-27 cells in vitro. Meanwhile, these two compounds also have other unique properties, such as low toxicity for normal gastric mucosal cells and kidney cells, small molecular weight for entering solid tumors expediently, and a succinct structure for easy chemical synthesis. With these characteristics, polyacetylenes 1 and 2 would be the high-potential candidate drugs for anti-gastric cancer metastasis.Naturally occurring polyacetylenes are characterized by the presence of two or more carbon–carbon triple bonds in their carbonic skeleton and are considered important active ingredients in many medicinal and edible plants, such as 1 significantly suppressed EMT markers, namely, Vimentin and Snail, while it enhanced the level of E-cadherin, indicating that polyacetylene 1 could reverse the EMT process. At the advanced stage of tumorigenesis, the expression of EMT-related transcription factor Snail is upregulated, which, in turn, inhibits the expression of E-cadherin and increases the expression of Vimentin [As shown in Vimentin . ChangesVimentin .β-catenin signaling is considered a regulator of EMT. Hence, the effects of polyacetylene 1 on the Wnt/β-catenin signaling pathway were subsequently revealed. The results showed that polyacetylene 1 inhibited β-catenin entry into the nucleus through two pathways: the inhibition of GSK3β expression and phosphorylation, and the promotion of ubiquitinated degradation of β-catenin phosphorylation, which, in turn, inhibited downstream transcriptional expressions of c-Myc, ICAM-1, VCAM-1, MMPs, etc. Our results suggested that polyacetylene 1 not only inhibits the Wnt/β-catenin signaling pathway but also accelerates the ubiquitination and degradation of β-catenin, thereby inhibiting EMT and metastasis of HGC-27 cells. In the cytoplasm, β-catenin binds to intracellular peptides in the cytoplasmic domain of E-cadherin to form the E-Cad/Cat complex, which mediates cell junctions and maintains cell polarity, while the phosphorylation of β-catenin inhibits the adhesion function of E-cadherin, resulting in unstable intercellular adhesion [β-catenin signaling plays a critical role in EMT regulation by downregulating the expression of E-cadherin, which subsequently leads to the release and activation of β-catenin. Wnt signaling enhances the expression of Snail by inhibiting its phosphorylation, thus inducing EMT in cancer cells [Wnt/adhesion . Wnt/β-cer cells .β-catenin. The overlaps between the biological processes controlled by YAP/TAZ and Wnt/β-catenin suggest that these factors do not act in isolation, but may interact with each other. Phosphorylated-β-catenin promotes TAZ degradation by acting as TAZ to b-TrCP presenters [β-catenin, Wnt also induces TAZ stabilization and TAZ nuclear entry in a manner that is independent of Hippo signaling. YAP and TAZ are integral components of the β-catenin destruction complex that serves as a cytoplasmic sink for YAP/TAZ. In addition, YAP can induce the inactivation of GSK3β, which, in turn, stabilizes cytoplasmic β-catenin and promotes the nuclear translocation of β-catenin [β-catenin, resulting in β-catenin retention in the cytoplasm, thereby inhibiting the transcriptional activity of β-catenin [1 prevented YAP/TAZ from entering the nucleus through multiple pathways, including the inhibition of MST1 phosphorylation, enhancement of LATS1 phosphorylation, and reduction of YAP expression. The reduction in YAP/TAZ in the nucleus leads to decreases in ICAM-1, VCAM-1, Cyr61, and Sox2. Then, the migration and invasion abilities of HGC-27 cells were significantly inhibited. This was the first report that demonstrated that polyacetylene 1 can regulate the Hippo–YAP signaling pathway.The Hippo–YAP pathway is also involved in the EMT process of gastric cancer cells. Several molecules were reported to promote EMT or enhance tumor metastasis via the Hippo–YAP pathway in gastric cancer . The groesenters . By cont-catenin . Phospho-catenin . In this1 and 2 exhibited anti-migration, invasion, and adhesion activity on HGC-27 cells at their nontoxic concentrations by reversing the EMT process and inhibiting Wnt/β-catenin and Hippo/YAP signaling pathways. Future research involving animal experiments is needed to comprehensively elucidate the stability, bioavailability, and metabolism of polyacetylenes 1 and 2. These findings will be helpful for the future applications of polyacetylenes 1 and 2 as promising therapeutic agents for gastric cancer.In conclusion, polyacetylenes 2 in a humidified incubator.Human gastric carcinoma HGC-27 cells, human gastric carcinoma BGC-823 cells, human gastric carcinoma NCI-N87 cells, human breast cancer MDA-MB-231 cells, human hepatocellular carcinoma HepG2 cells, human colon cancer HCT-116 cells, human cervical cancer Ca Ski cells, human pancreatic cancer PANC-1 cell, human lung cancer A549 cells, human nasopharyngeal carcinoma CNE-2 cells, human gastric epithelial GES-1 cells, and Madin–Darby canine kidney MDCK cells were purchased from the cell bank of the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences  and maintained in RPMI-1640  or DMEM  culture medium supplemented with suitable fetal bovine serum  at 37 °C with 5% COBidens pilosa L. were extracted three times in 95% ethanol for 7 d per extraction, then extracted with petroleum ether, ethyl acetate, and n-butanol in turn. The extraction solutions of different polarities were combined and concentrated under reduced pressure to obtain the petroleum ether extraction part, the ethyl acetate extraction part, and the n-butanol extraction part. The petroleum ether extraction site was separated into 17 sub-streams (Fr. A–Q) via column chromatography using a step-gradient eluent mixture of petroleum ether-ethyl acetate (from 100:0 to 0:100), of which Fr. A, Fr. B, Fr. F, Fr. H, Fr. I, Fr. K, Fr. N, and Fr. O had obvious characteristic absorption peaks of polyacetylene, and were the main peaks. The fragment Fr.F (357 mg) was purified using semi-preparative HPLC (acetonitrile:water = 55:45) to obtain the compound polyacetylene 2 (5.3 mg). Fr. H was purified using semi-preparative HPLC (acetonitrile:water = 55:45) to obtain the compound polyacetylene 1 (125.3 mg). To view the polyacetylenes 1 and 2 1H and 13C NMR data, see the The roots of β-catenin (D10A8), p-β-catenin (D2F1), GSK-3β (D5C5Z), p-GSK3β (D3A4), MST1 (D8B9Q), p-MST1 (E7U1D), LATS1 (C66B5), p-LATS1 (D57D3), YAP (D8H1X), p-YAP (D9W2I), YAP/TAZ (D24E4), MMP2 (D4M2N), MMP7 (D4H5), MMP9 (D6O3H), MMP14 (D1E4), c-Myc (D3N8F), ICAM-1 (E3Q9N), VCAM-1 (D8U5V), MAPK (137F5), p-MAPK (D13.14.4E), Sox2 (D6D9), Cox2 (D5H5), Cyr61 (D4H5D), GAPDH (D16H11), and Histone-H3 (D1H2) were used at a dilution of 1:1000. HRP-conjugated goat anti-rabbit IgG secondary antibody (98164) and FITC-conjugated goat anti-rabbit IgG secondary antibody (17A2) were used at dilutions of 1:5000 and 1:100, respectively. All of these were purchased from Cell Signaling Technology . Matrigel was purchased from BD Biosciences .Rabbit monoclonal antibodies against E-cadherin (24E10), Vimentin (D21H3), Snail (C15D3), 1 and 2. Cells in the logarithmic growth phase were inoculated into the 96-well plate at 1 × 105 cells/mL and treated with polyacetylene 1 or 2 at different concentrations for 48 h. Then, 20 µL 5 mg/mL MTT  was added to each well and the cells were incubated for 4 h. Then, 150 µL DMSO  was used to dissolve formazan. The absorbance of each well was measured using a microplate reader at 490 nm. Cytotoxicity was expressed as a 50% inhibition concentration (IC50).The MTT method was used for the cytotoxicity evaluation of polyacetylenes 1 or 2 for 24 h. The medium was replaced with fresh medium every 2 days. After 14 days of incubation, the colonies were fixed in 4% paraformaldehyde and stained in 0.1% crystal violet solution. Images were captured, colonies consisting of more than 50 cells were counted, and data analysis was performed.HGC-27 cells in the logarithmic growth phase were inoculated into a 6-well plate at 400 cells per well and then incubated with polyacetylene 5 cells/mL. A 200 μL pipette tip was used to draw a vertical line in the direction perpendicular to the parallel lines when HGC-27 cells were adherent to 90%. The cells were treated with polyacetylene 1 or 2 for 24 h separately. Migration and cell movement throughout the wound area was observed with an inverted optical microscope  and imaged using a camera attached to the microscope at 100× magnification at 6, 12, and 24 h. The percentage of wound closure was analyzed using ImageJ.HGC-27 cells in the logarithmic growth phase were inoculated into a 6-well plate at 5 × 105 cells/mL and treated with polyacetylene 1 or 2 for 24 h separately. The 96-well plates were precoated with PBS-diluted Matrigel and blocked in advance. The cells were inoculated into the Matrigel-coated wells for 2 h. After incubation, the cells were fixed and stained in DAPI staining solution. The average number of adhered cells in five fields was counted using a fluorescence microscope .HGC-27 cells were seeded into a 96-well plate at 1 × 101 and 2 were assessed by using the Matrigel invasion system. Transwell chambers  in 24-well plates were coated with polymerized Matrigel . A total of 600 μL starved treated cells were placed in the upper chamber at 5 × 105 cells/mL. Medium containing 20% FBS and polyacetylene 1 or 2 were added to the lower chamber as the chemoattractant. After incubation for 24 h, the invaded and migrated cells in the lower chamber were obtained via staining and counted.The HGC-27 cell line possessed a high degree of metastatic potential. Cell migration and invasion treated with polyacetylenes 1 for 24 h. After incubation, the cells were fixed, permeated, blocked, and incubated with a specific primary antibody and a secondary antibody. The nuclei were counter-stained with DAPI staining solution. After mounting with an anti-fluorescence quencher, images were captured using a fluorescence microscope .Cells were inoculated on the chamber slides of 24-well culture plates and then treated with polyacetylene 1 treatment on protein expression in HGC-27 cells, total protein was extracted after treating the cells with polyacetylene 1 for 24 h. Total protein was separated using SDS-PAGE gel electrophoresis and transferred to PVDF membranes according to their molecular weight. After blocking and incubating with a specific primary antibody and a secondary antibody, the signal detection was performed using ECL  and observed with a Tanon 5200 luminescence imaging system . Grayscale analysis was performed using Image J, and the experiments were performed three times in parallel.To determine the effect of polyacetylene p < 0.05 was considered statistically significant.All the quantitative data were presented as mean ± SD and analyzed using ImageJ version 2.1  and SPSS standard version 20.0 software . The comparison of different experimental groups was performed using a t-test or one-way ANOVA analysis.


Mutations and truncations in APC, resulting in loss of APC function and hence elevated β-catenin levels and upregulation of Wnt signalling, are associated with numerous cancers including colorectal carcinomas. APC has a long intrinsically disordered region (IDR) that contains a series of 15-residue and 20-residue binding regions for β-catenin. Here we explore the multivalent nature of the interaction of β-catenin with the highest affinity APC repeat, both at equilibrium and under kinetic conditions. We use a combination of single-site substitutions, deletions and insertions to dissect the mechanism of molecular recognition and the roles of the three β-catenin-binding subdomains of APC.The Wnt signalling pathway plays key roles in cell proliferation, differentiation and fate decisions in embryonic development and maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between β-catenin’s ARM repeat domain and the intrinsically disordered protein adenomatous polyposis coli (APC). APC is a giant multivalent scaffold that brings together the different components of the so-called “β-catenin destruction complex”, which drives β-catenin degradation  The majority of PPIs events involve the interactions of 10 or fewer amino acid between two binding partners. However, there are exceptions to this rule; one example being the interaction between adenomatous polyposis coli (APC) and β-catenin, which forms an unusually large and elongated binding surface with approximately 5000 A˚a buried , 2  that pack in a linear fashion to produce elongated, one-dimensional architectures with a continuous hydrophobic core and extended solvent-accessible surfaces  and are β-catenin carries out two distinct functions in the cell, the first of which is as the signal transducer in the canonical Wnt signalling pathway  and resulting in the transcription of genes that are of developmental importance and those involved in tissue homeostasis. In the absence of a Wnt signal, cytosolic β-catenin is continuously synthesised and then sequestered and targeted for proteasomal degradation by a multi-protein complex called the β-catenin destruction complex (BDC) that forms biological condensates . The BDCThe behaviour of IDPs in complex with their binding partners can be divided into static complexes , in whicE. coli C41 cells (The plasmids encoding the armadillo (ARM) repeat domain of human β-catenin (residues 134–671) and APC were kind gifts from Prof. W. I. Weis and Dr. S. H. McLaughlin, respectively. The R3 region of APC and fragments thereof  and shor41 cells . Transfoβ-catenin was further purified using a Mono-Q column (Cytiva) equilibrated in 50 mM Tris-HCl buffer pH 8.9, 50 mM NaCl, 1 mM DTT and eluted with a linear NaCl gradient to 1M NaCl. Fractions containing greater than 95% β-catenin were pooled, flash frozen, lyophilised, and stored at −80°C . FurtherAPC constructs were labelled at the single cysteine residue located at position 1,501, which was retained in all of the fragments made here. Proteins were buffered exchanged into PBS containing 1 mM TCEP and labelled with either fluorescein-maleimide or Alexa Fluor™ 488 C5 maleimide. Fluorescein-maleimide was added at a 5-fold molar excess concentration to protein and incubated for 1 h at room temperature; excess dye was removed by acetone precipitation of the APC construct ; the labbc labelled at Cys 1501 with Alexa-488 was used in a competition fluorescence assay to measure the IC50 values of the phosphomimetic APCbc variants for β-catenin. In these experiments, a solution of 20 nM Alexa-488-APC and 200 nM β-catenin was incubated in the cuvette at 25°C for 30 min prior to titration of unlabelled APCbc variants using a Microlab 500C dispenser (Hamilton). After addition of each aliquot, the sample was stirred for 30 s and equilibrated for a further 60 s before measurement. The IC50 was calculated using the following equation:F is the measured fluorescence, Ff is the fluorescence of the free ligand, ΔF is the change in fluorescence between the free and bound form, and U is the concentration of the competing APC ligand.The binding of fluorescent labelled APC constructs to β-catenin was monitored either by fluorescence or by fluorescence anisotropy using a LS55 fluorimeter (Perkin Elmer). All proteins were buffer exchanged into PBS, 1 mM DTT, and measurements were made at 25°C. Excitation and emission wavelengths were 495 and 519 nm, respectively, and slit widths were 5 nm. APCQ is the measured fluorescence and R is the measured anisotropy. The subscripts f and b denote the unbound (free) and saturated bound forms of labelled APC. The adjusted anisotropy data were then fitted using a single-state Hill plot to derive the Kd and Hill coefficient, h:P is the APC concentrationAll other APC constructs were labelled with fluorescein, and the dissociation constants were measured by fluorescence anisotropy. In these experiments, aliquots of β-catenin were titrated into a cuvette containing 10 nM fluorescein-labelled APC, and the sample was stirred and equilibrated before measurement (as described above). Both fluorescence anisotropy and fluorescence intensity were measured at 15°C. As there was a difference in the fluorescence intensity of bound and unbound APC, the fluorescence anisotropy was adjusted using the following equation:Kd, R is the gas constant and T is the temperature in Kelvin.To calculate the Gibbs free energy of binding from the kobs, was plotted against the concentration of β-catenin, and kon was calculated from the linear fit:Stopped-flow fluorescence was used to measure the kinetics of binding with a SX-19 stopped-flow fluorimeter (Applied Photophysics). The excitation wavelength was 495 nm, and a cut-off filter of 515 nm was used to measure the emission. The slit widths were 2 nm for both excitation and emission. All proteins were prepared in PBS buffer, 1 mM DTT, and the experiments were performed at 15°C. The β-catenin concentrations used were at least ten times higher than that of the APC to ensure pseudo first-order conditions; a fixed concentration of fluorescein-labelled APC (25 nM) was rapidly mixed with varying concentrations of unlabelled β-catenin  in a 1:1 volume ratio, and the change in fluorescence intensity was recorded. For dissociation experiments, the complex of β-catenin and labelled APC was pre-formed by mixing the two proteins in a 1:1 molar ratio (200 nM) and incubated for 1 h at 15°C in the dark. The pre-formed complex was then mixed in a 1:1 volume ratio with 10 times molar excess of the same APC construct, and the change in fluorescence intensity was measured. A minimum of five traces was collected and averaged. The averaged trace was plotted using GraphPad Prism 5  and fitted to either a single exponential phase or the sum of two exponential phases. For the association experiments, the observed rate constant, The region of APC used in this work encompasses the third 20aa repeat (R3)  and flanking regions that contribute to the interaction with β-catenin . This rebc, the five phosphorylation sites were mutated to the phosphomimetic glutamate either individually or in combination , and the IC50s were measured by competition fluorescence assay using APCbc labelled with Alexa-488 at Cys 1,501  is tightly coordinated and fixed in a specific orientation by three β-catenin residues and therefore would be predicted to have the tightest binding affinity of the five phosphorylated residues. Phospho-S1505 (S2) does not appear to make any contacts with β-catenin in the crystal structure of the complex, but the phosphomimetic mutation at this site nevertheless increased the binding affinity 5-fold. This could be as result of the interaction of E1505 with R1523 within the short helix formed by this region of APC, which could stabilise its interaction with β-catenin. Alternatively, it may reflect a “fuzzy” behaviour of the APC-β-catenin interaction, in which the interface, and the interactions formed by specific residues, in solution is different from the static structure captured in the crystallography. Constructs containing four or more phosphomimetics showed a ∼150-fold increase in affinity.abc containing all five phosphomimetics (referred to subsequently as pAPC), was also measured by fluorescence anisotropy, (see next section), and the Kd determined to be 5.3 nM , suggesting that this region only interacts with β-catenin in the cell when phosphorylated, which is consistent with what is observed in the crystal structures (as discussed above). The lysine-binding domain b has a dissociation constant of 246 nM, which is similar to that of p-c; when domain b is combined with unphosphorylated domain c (bc), the affinity shows only a small increase (196 nM) relative to b alone, again consistent with the weak interaction of unphosphorylated c with β-catenin. When domain b is combined with domain a (ab) the affinity increases approximately 2.5-fold relative to b alone (90 nM versus 246 nM). The entire unphosphorylated APC fragment comprising all three domains (abc) has a 12-fold higher affinity than bc and a 5-fold higher affinity than ab. The increase in affinity upon adding unphosphorylated c to ab is somewhat surprising given the very small increase in affinity observed upon adding unphosphorylated c to b. Adding p-c to ab  results in the largest increase in affinity of adding any single domain .We next looked at APC R3 constructs comprising each of the three different β-catenin binding interfaces either alone or in combination , and theDifferences were observed in the shapes of the binding curves, indicating that there is cooperative binding of the subdomains for some of the APC constructs but not others. Cooperativity was observed for pAPC, as indicated by a Hill coefficient of 2, but not for the unphosphorylated APC or for any of the phosphorylated or unphosphorylated fragments (Hill coefficient of 1 within error) . This reabc D1486S is higher than the affinity of p-c alone (160 nM), suggesting that some contacts of domain a with β-catenin are retained when domain c is bound; alternatively there may be some fraying of the β-catenin interface in the isolated p-c fragment, meaning that p-c does not effectively recapitulate the interactions present in the full p-abc and p-abc D1486S constructs.Because the lysine-binding domain b is the middle domain, it cannot be deleted in the same way as the helical domain a and phospho domain c. However, its affinity for β-catenin can be dramatically weakened by mutation of the lysine-interacting residues D1486 and E1494 . The mutWe also investigated the contributions of the three domains of APC to the kinetics of β-catenin binding by stopped-flow fluorescence measurements of the fluorescein-labelled APC . The ki. Only on4 linker between tandem immunoglobulin domains but also includes lysine and glutamate residues to aid solubility . The affinity was similar to that of unphosphorylated APCabc (16.5 nM) that has a weakened c domain and pAPCabc D1486S (30 nM) that has a weakened b domain, suggesting that the polyproline linker disrupts the interface and prevents domains b and c either side of it from binding to β-catenin simultaneously. Only the flexible linker was inserted at 1,478 (between domains a and b), and it did not change the binding affinity of APCabc but reduced the affinity of pAPCabc 5-fold, suggesting disruption of contacts in domains a and/or b.Insertion of either the rigid or the flexible linker at 1,500 between domains b and c into either APCisrupted . A reducabc and pAPCabc. There were also small increases (∼2.5-fold) in the dissociation rates. Insertion of the polyproline linker into pAPCabc had a larger effect on the dissociation kinetics, increasing the rate of the fast dissociation phase 6-fold to a value similar to that of APC; the rate of the slow dissociation phase was increased 9-fold but it was still much slower than constructs APCabc and pAPCabc D1486S with weakened c/b domain. Interestingly, insertion of the flexible linker at 1,478 (between a and b) increased the association rate of APCabc 2-fold. This result could be due to some steric stress between binding interfaces of the two domains that is relieved upon linker insertion.As observed at equilibrium, the linker insertions at 1,500 (between b and c) had only small effects on the association rate, decreasing it ∼2-fold for both APCabc with either the flexible or rigid linker inserted at bc, indicating a single binding event. In contrast, for pAPCabc with the flexible or rigid linker inserted at the same position the Hill coefficient was ∼2, indicating that cooperativity between b and c is maintained upon linker insertion. Insertion of the polyproline linker into bc in pAPCabc gave a Hill coefficient of 1, consistent with this linker preventing the binding of b and p-c simultaneously. From these results and the results for the truncations showing that the Hill coefficient is 1 for constructs ab and abc lacking the high-affinity p-c domain, we can conclude that a and b are not independent domains but rather form one contiguous binding surface.The Hill coefficient was ∼1 for APCβ−TrCP (Here we explore the molecular recognition of the third 20aa repeat region of APC as an example of the β-catenin binding partners that are phospho-regulated in the β-catenin destruction complex (BDC). In its unphosphorylated form, APC contacts are predominantly with β-catenin ARM5-9, thus allowing the interaction of β-catenin ARM1-4 with axin . The forβ−TrCP . Phosphoβ−TrCP . APC repβ−TrCP :1) In a multivalent system such as this, the strength of the interactions of the individual subdomains are context dependent, i.e., they are dependent on the presence or absence of the other subdomains. The most striking examples are the effects of a mutation that disrupts key contacts in domain b and the effects of mutations that mimic phosphorylated residues in domain c: The mutation D1486S in domain b causes a 100-fold decrease in the affinity of the unphosphorylated APC, whereas there is only a 4-fold decrease in the affinity when the mutation is made in the phosphomimetic pAPC. Likewise, the phosphomimetic mutations in domain c cause a 100-fold increase in the affinity of APC D1486S, whereas there is only a 3-fold increase in the affinity when the same phosphomimetic mutations are introduced in APC.2) This cooperativity of the interactions is evident also in the different shapes of the binding curves, and it can be quantified with the Hill coefficient. Cooperativity (Hill coefficient of 2) is only observed for pAPC and for loop insertions where b and c retain their ability to bind simultaneously. No cooperativity (Hill coefficient of 1) is observed for variants in which either b or c has a weakened affinity , for insertions such as polyproline linker where b and c cannot bind simultaneously, and for the fragment ab lacking domain c. The Hill coefficient of 1 observed for constructs ab and abc indicates that a and b effectively form a contiguous domain rather than two independent domains.3) The similar affinities of pAPC D1486S and APC, and of fragments p-c and b, suggest that the interactions made by these two domains with β-catenin are of similar strength . We expe−1) and pAPC (0.0021 s−1), and its association rate is in between those of APC (49 μM−1s−1) and pAPC (207 μM−1s−1). The observation that the mutations and insertions affect both dissociation phases rather than only one or other indicates that there is not a simple sequential unbinding mechanism and that the dissociation of b and c domains is tightly coupled.4) In a multivalent system, large effects of domain deletion/mutation on the dissociation kinetics are expected due to the probability of rebinding being much lower when one or other of the domains is absent or mutant. A single phase is observed for the association kinetics of APC and all of the variants, whereas the dissociation kinetics are biphasic. Several different kinetic mechanisms can be envisaged: Binding/unbinding of the different domains of APC could be sequential or concurrent. Further, given that the affinities of domains b and c are similar, there could also be parallel  binding/unbinding pathways of similar energies corresponding to different orders of domain binding/unbinding. This is analogous to the parallel (un)folding pathways observed for repeat proteins, which is a result of the different repeats having similar stabilities . The mutWe can compare these findings with those of another β-catenin ligand, TCF7L2 . Like APAbove, we compare the effects of the different APC variants on the binding affinity in terms of how they disrupt the interaction with β-catenin. However, these mutations (which include charge substitutions and linker insertions) could potentially alter the binding affinity by changing the conformational ensemble of the unbound APC. To address this point, we measured the CD spectra of a subset of the APC constructs and compared them with literature values for folded, (pre)molten globule and unfolded proteins from the Protein Circular Dichroism Data Bank (PCDDB) . We founIt is important to understand kinetic stability, as measured by dissociation rates, as well as thermodynamic stability in efforts to develop inhibitors of protein-protein interactions. β-catenin is upregulated in many cancers, but the identification of small molecule inhibitors is challenging given the very large, extended interaction surface. Moreover, positive and negative regulators share part of this interface, making selective inhibition even more difficult, and inhibitors must also avoid disrupting the interaction of β-catenin with E-cadherin at the cell membrane. An alternative approach is to direct the cytosolic pool of β-catenin for degradation using either small molecule PROTACs (proteolysis-targeting chimeras) or peptide-based molecules. To this end, dissecting the contributions of the different APC subdomains of APC to β-catenin binding will provide critical information for developing ligands capable of effectively engaging β-catenin.