Ice accretion causes problems in vital industries and has been addressed over the past decades with either passive or active de-icing systems. This work presents a smart, hybrid (passive and active) de-icing system through the combination of a low interfacial toughness coating, printed circuit board heaters, and an ice-detecting microwave sensor. The coating’s interfacial toughness with ice is found to be temperature dependent and can be modulated using the embedded heaters. Accordingly, de-icing is realized without melting the interface. The synergistic combination of the low interfacial toughness coating and periodic heaters results in a greater de-icing power density than a full-coverage heater system. The hybrid de-icing system also shows durability towards repeated icing/de-icing, mechanical abrasion, outdoor exposure, and chemical contamination. A non-contact planar microwave resonator sensor is additionally designed and implemented to precisely detect the presence or absence of water or ice on the surface while operating beneath the coating, further enhancing the system’s energy efficiency. Scalability of the smart coating is demonstrated using large (up to 1 m) iced interfaces. Overall, the smart hybrid system designed here offers a paradigm shift in de-icing that can efficiently render a surface ice-free without the need for energetically expensive interface melting. Removing ice from surfaces typically requires melting. Nonetheless, authors demonstrate a smart low interfacial toughness coating integrated with underlying heaters and ice-detecting microwave sensors that enable energy efficient smart large-scale de-icing without melting. Ice mitigation strategies can be divided into either active or passive methods. Active de-icing involves an external energy input used to remove the ice, typically through thermal, chemical, or mechanical methods. In contrast, passive de-icing either reduces the accretion rate of ice, lowers the adhesion strength between ice and the surface, or both. Neither route towards an ice-free surface is seen as a cure-all today, as active de-icing methods utilize substantial energy but passive de-icing coatings cannot keep a surface ice-free indefinitely. A hybrid system that synergistically combines passive and active de-icing technologies may be an attractive solution to the ice accretion paradigm.Undesired ice accumulation is problematic in industries such as renewable energy  are known for their excellent water repellency with high water contact angle and low contact angle hysteresis26. SHS have demonstrated good laboratory scale de-icing in terms of icing delay, removal of supercooled water droplets, and droplet freezing delay due to their low thermal conductivity and minimal surface/droplet contact area29. However, the icing delay of SHS is typically measured on the scale of minutes, still necessitating a method of ice removal once it has accreted.As an alternative, passive de-icing methods utilize coatings with specific surface properties to lessen the ice accretion rate or reduce ice’s adhesion to the surface so it can be removed by its own weight, wind, or other aerodynamic/environmental forces. In their recent review, Dhyani et al. detail the many surface design strategies for passive de-icing30. Due their weak bond with ice, such materials have shown exceptionally low ice adhesion strengths in various coating configurations including thin films31, self-assembled monolayers32, and lubricant infused surfaces34. Zhao et al. fabricated silicon-oil infused icephobic coatings that demonstrated a low shear ice adhesion strength for cylindrical ice at −10 °C33. Similarly, Liu et al. presented fluorinated PDMS films for significant ice adhesion reduction and delayed icing35. High surface energy amphiphilic materials can also reduce a surface’s ice adhesion strength substantially by creating a nanometer-scale liquid water surface layer, as the bond between liquid water and solid ice is much weaker than a solid-solid bond34. For example, the absorption of water vapor has been demonstrated for poly(ethylene glycol) blended with PDMS, resulting in a thin water layer that enhances passive de-icing36.Passive de-icing coatings can also reduce the adhesive bond between ice and the coated substrate without necessarily reducing the ice accretion rate. Polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE) are two materials known for their low surface energy that frequently have been used for passive de-icing37. Ma et al. introduced a titanium nitride/polytetrafluoroethylene composite SHS coating as a photothermal de-icing approach38. The designed photothermal superhydrophobic surface not only delayed ice formation but also converted absorbed light to heat energy and melted the surface ice. Additionally, Gao et al. demonstrated the use of a hybrid SHS coating and electrical heating for wind turbine de-icing39. They showed significant energy savings (90%) when de-icing the whole turbine blade by only coating the leading edge with their SHS coating and electrical heaters. Many other works have demonstrated hybrid ice mitigation combining a SHS and active heating43. Hybrid de-icing methods can also employ lubricant infusion. Jamil et al. used silicone lubrication on a candle soot coating as a natural light absorber44. In their work, conductive iron oxide nano particles served as a heat dissipator, eventually melting the iced interface. However, an intractable issue remains with previous hybrid de-icing approaches. Because hydrophobic coatings repel only liquid water, melting the ice is required for this strategy to be effective. And so, while the energy consumption in these studies was reduced in comparison to a purely active de-icing method, the required energy was still substantial and would scale with the size of the iced interface. Considering that the latent heat of melting ice (334 J/g) is about 160x greater than ice’s specific heat capacity (2.09 J/g °C), a hybrid de-icing system that could avoid melting would provide substantial energy efficiency benefits.Hybrid methods that combine active de-icing and SHS coatings have recently been explored. Cheng et al. fabricated a SHS coating using magnetic particles for hybrid de-icing and showed that increasing the temperature above 0 °C allowed the coating to exhibit excellent ice/water removal46. LIT materials minimize the strain energy necessary to propagate an interfacial crack between the ice and surface, enabling size-independent de-icing, i.e. requiring a constant applied force for ice removal irrespective of the size of the iced interface. To-date, various LIT materials have been reported, including polymers such as polypropylene, PTFE, and ultra-high molecular polyethylene (UHMW-PE)46, as well as aluminum-based quasicrystalline coatings45. Zeng et al. introduced a LIT coating comprised of porous PDMS that exhibited lower interfacial toughness and hydrophobicity with increasing porosity47. Dhyani et al. fabricated transparent LIT PDMS and polyvinylchloride (PVC) coatings for photovoltaic applications, simultaneously demonstrating both a low interfacial toughness and ice adhesion strength48. Yu et al. fabricated robust LIT coatings based on PTFE particle assemblies, where the interfacial toughness was maintained after repeated icing and de-icing cycles49. And yet, to-date LIT materials have only been used as passive de-icing coatings.Materials exhibiting low interfacial toughness (LIT) with ice represent a paradigm shift in how the adhesion between ice and a surface may be reduced, especially large (>cm) iced interfaces52. At the applied optimal voltage, the sensor’s response to the presence and absence of ice is also recorded.In this work we develop hybrid LIT de-icing coatings based on UHMW-PE. Whereas all previous hybrid de-icing technologies have required energy-intensive ice melting, incorporating LIT materials enables mechanical de-icing that circumvents the melting step. The mechanical properties of both the LIT coating and ice determine the toughness and strength of their adhesive interface. Accordingly, the influence of the elastic modulus on ice adhesion strength and interfacial toughness is first measured at varying temperatures (−5 °C to −60 °C) for both the LIT material and ice. Next, we study the effect of thermal loading on the interfacial toughness using miniature printed circuit board resistive heaters. A comprehensive study using several lengths of ice is carried out to optimize the voltage required to increase the surface temperature to −5 °C, where the lowest interfacial toughness with ice was observed. The effect of the supplied heat on the coating’s interfacial toughness with ice is investigated by applying the optimal voltage. The coatings are further rendered ‘smart’ through the inclusion of an embedded microwave resonator sensor, enabling on-demand de-icing where the active system can be shut off immediately once the sensor detects that the surface has been de-iced. The microwave sensor consists of a split ring resonator and transmission lines and operates by exploiting the large difference in dielectric properties between water and ice, as previously demonstrated53,E is the elastic modulus of ice in GPa and T is the temperature in °C. Based on this equation, the modulus of ice decreases about 5% as the temperature increases from −40 °C to −5 °C. The elastic modulus of the coating was investigated using Dynamic Mechanical Analysis (Methods). The loss modulus was statistically constant between 25 °C and −60 °C . Both the interfacial toughness and ice adhesion strength depend on the mechanical properties of the coating and ice. The effect of temperature on the dynamic elastic modulus of polycrystalline ice has been previously measured to follow °C Fig. , whereasFc = 145 N/cm at −5 °C, to Fc = 237 N/cm at −30 °C. This corresponds to an increasing interfacial toughness with ice from Γ = 2.2 ± 0.5 J/m2 to Γ = 5.6 ± 1.2 J/m2 over the same thermal range. However, at −40 °C cohesive detachment was observed where a portion of the ice remained on the surface after partial interfacial fracture. While the critical ice removal force decreased to 207 N/cm , with cohesive fracture this is no longer a true interfacial property. As discussed above, ice is at most 5% stiffer at the lower temperatures investigated here. This should have decreased the interfacial toughness, which was instead 2.2 times larger at −30 °C than at −5 °C. The ice adhesion strength was similarly 1.8 times greater  heaters . Two other temperature probes both measured the temperature inside the ice adhered to the LIT coating (Tice), either directly above the heater or 5 cm away along the length direction  decreased, the required de-icing force increased , a maximum decrease in the de-icing force was observed  and heater resistance (R = 5.9 Ω) were constant, resulting in Q = 3.28 tD. The tD for each length of ice was measured and statistically analyzed (t-test), which revealed that for interfacial lengths between 50 mm and 150 mm, the de-icing time remained constant  is low and would arise naturally from environmental forces such as wind, drag, or centripetal acceleration (in the case of wind turbines).The heat flux required for modulating the interfacial temperature would be ant Fig. . These r2 to achieve ice-free surfaces, with heaters covering the entire iced area65. For our designed de-icing system the heaters cover only 10% of the surface, which decreases the power consumption by an order of magnitude. Moreover, the heater’s resistivity increases with the length of the printed copper on the substrate as, R = l/σa . Using the 4.4 V de-icing supply voltage, the power density of our heater is W = U2R−1A−1 = 2 kW/m2 (A is the area covered by the heater). Accordingly, not only do the heaters cover just 10% of the total area, their resistivity is 10× lower, overall resulting in heaters with a power density 100× higher than the same heaters fully covering the surface. However, this is still an underestimate in the total efficiency gains as our heaters raise 10% of the interface to a subzero temperature, which would not de-ice even for complete heater coverage as it would need to bring the surface to at least 0 °C, and typically much higher65.The areal power density is a commonly used metric to compare the efficacy of de-icing systems. Previously reported aircraft de-icing systems have required 10–25 kW/mL > Lc) was measured while the heater locally raised the interfacial temperature from −20 °C to −5 °C  to its initial value. The surface roughness was also unaffected , demonstrating the environmental durability of the hybrid de-icing system. Only the harsh abrasion increased the de-icing force statistically significantly  was still substantially less than that of the unabraded UHMW-PE film without heaters , confirming the scalability of the developed system. Scattering of the light between the LIT material and ice during interfacial separation also allowed us to monitor the crack propagation in real time , based on the previously reported work by Kozak et al.The optimized sensor was used to successfully monitor the entire icing and de-icing process through changes in the sensor’s electrical characteristics, such as the resonant frequency Fig. . InitialTH = −5 °C. Due to the horizontal configuration of our icing setup (see Methods), even after interfacial fracture the detached ice sits on the surface unadhered, with a small gap in between the ice and LIT coating remaining due to the intrinsic roughness of the UHMW-PE and the imperfect fracture surface. The difference between adhered and unadhered ice was also detectable, as evidenced by the sudden change in the sensor’s measured resonant amplitude (−10.61 dB to −14.43 dB) and frequency (1.734 GHz to 1.872 GHz). In more realistic de-icing scenarios environmental forces such as gravity, wind shear, drag, etc. would completely remove the ice from the surface, and even more precise detection would be possible as the sensor would return to its baseline profile. Finally, the system was thawed, and the sensor’s resonant profile began to disappear, indicating the presence of liquid water on the surface  to obtain a local temperature of ace Fig. . The speWhile the energy efficiency of a de-icing system is highly specific to the application and environmental conditions, here our smart hybrid LIT de-icing system reduces energy consumption in four synergistic ways. First, the use of LIT materials enables mechanical de-icing and therefore eliminates the de-icing step requiring the largest energy input: the phase change of solid ice to liquid water. Second, the mechanism of LIT materials allows for our resistive heaters to be sparsely placed on the surface, requiring only ~10% of total areal coverage to be effective. Third, the meandering traces of copper used to construct the heaters display an order of magnitude improvement in resistance over current heating systems, leading to faster de-icing at smaller applied voltages. And finally, the inclusion of an ice sensor enables the smart system to only be activated when ice is truly adhered to the surface, and also allows the system to be turned off immediately following de-icing.In this work, we explored a hybrid de-icing system utilizing LIT coatings, where thermal modulation substantially improved the de-icing performance without melting the interface. The interfacial toughness between ice and UHMW-PE was found to be 2.2 times larger at −30 °C than at −5 °C. Accordingly, resistive heaters were patterned periodically underneath the LIT material and were optimized to locally raise the temperature to a warmer but still sub-zero value. Heaters underneath only ~10% of the total ice length were effective at reducing the interfacial toughness, as if the entire surface were held at this temperature even though 90% of the surface wasn’t heated. The hybrid LIT de-icing system was also rendered smart through the addition of a microwave resonator sensor. The sensor operated at a resonant frequency, a resonant amplitude, and a quality factor of 2.005 GHz, −12.95 dB, and 205, respectively, taking advantage of the significant differences in dielectric properties between ice and water at the resonant frequency. This planar, contactless device embedded underneath the LIT material was able to monitor and detect ice formation and removal in real-time. Given the fatal consequences of aircraft icing, and the strong push towards renewables such as wind power, our smart hybrid LIT de-icing system may find immediate usage in multiple ice-prone sectors worldwide, especially considering its energy efficiency, scalability, and durability.The LIT film was ultra-high molecular weight polyethylene (UHMW-PE) with an adhesive backing and a thickness of 0.127 mm , used as received. The planar microwave sensor and the PCB resistive heaters were fabricated on Rogers RT/Duroid® 5880 Laminates, with permittivity of 2.2, loss tangent of 0.0009, dielectric thickness of 0.79 mm, and copper cladding thickness of 35 µm .The storage and loss moduli of the UHMW-PE were measured using a Dynamic Mechanical Analyzer (TA Instruments) at an applied frequency of 1 Hz using a temperature sweep from −60 °C to +30 °C. The strain was not constant during the temperature sweep as the instrument was setup in auto strain adjustment mode. However, the strain variation measured was negligible (0.05% to 0.03%) and within the linear viscoelastic range.For experiments where the heater was not involved, the UHMW-PE film was adhered uniformly to an aluminum (Al) sheet with a thickness of 0.254 mm  using the adhesive backing. For experiments involving the heater, the UHMW-PE film was adhered directly to the fabricated device (heat and sensor), again using the adhesive backing. The topography of the UHMW-PE surfaces on the Al or the heater/sensor was measured using a LEXT™ OLS5100 3D Laser Scanning Microscope  molds of various lengths (Lice = 5–200 mm). First, the molds were filled with deionized water at room temperature. Next, the temperature of the surface was decreased to the target temperature using the Peltier stage, and the water was allowed sufficient time to fully freeze (minimum 1 h). Once frozen, a moving probe with a motorized linear stage was connected to a force gauge . The force gauge probe with cross section of 5 mm × 10 mm impacted the ice’s mold at a constant speed of 100 µm/s, and the detachment force was measured with 0.1 N accuracy. The ice adhesion measurements were conducted at varying temperatures (−40 °C to −5 °C). The temperature of the ice and surface of the coating was monitored using a BK Precision 725 thermocouple with an accuracy of ±0.7 °C. After each measurement, the coating was cleaned with isopropyl alcohol  using a Kim wipe (KimTech).The force necessary to de-ice the LIT surface was measured using a custom push-off method described elsewhereτice) and interfacial toughness with ice (Γ) are important parameters measured to fully characterize the interface between a surface and ice46. In the strength-controlled regime of fracture, the force to dislodge ice (Fice) is used to measure τice using the interfacial area, A, or τice = Fice/A. In the toughness-controlled regime of fracture that occurs for longer interfaces, this force plateaus at some critical value, Fc. One can calculate the toughness of the ice/coating interface using the measured Fc value, the modulus of ice, E, and the ice thickness, Hice, as 46. The interfacial length where the fracture transitions from strength- to toughness-mediated fracture is commonly referred to as the critical length, Lc. All of these parameters may directly depend on temperature, or indirectly due to temperature-dependent material properties. Accordingly, the ice adhesion strength and interfacial toughness of UHMW-PE with ice were measured from −5 °C to −40 °C and using ice lengths from 5 to 200 mm . If the two populations were statistically similar , the datapoint was included in the toughness regime, and the Fice value of the next shortest length of ice was considered. This procedure was repeated until the Fice value from the longest piece of ice in the strength-controlled regime was statistically different  from the Fice value of the shortest length of ice in the toughness-controlled regime. The adhesion strength was then determined from the slope of best linear fit in the strength regime. The interfacial toughness was calculated using 46. Lc was then determined by the intersection of these two lines. Note that, for some experiments the measurement of Fice for longer lengths of ice served as a substitute for directly measuring Γ, and for such cases we assume Fice = Fc.The calculation of 51. The microwave split ring resonator (SRR) sensor was designed in Ansys High-Frequency Structure Simulator  as the amount of time required for the heaters to increase the surface temperature to a target temperature, either −5 °C or −10 °C in this work. Note, however, that without the application of the external mechanical load, simply raising the temperature to −5 °C does not de-ice the surface. Once at the target temperature, the force gauge probe impacted the adhered ice within the mold, and the detachment force was measured. The temperature of the ice 0.5 mm above the heater was also measured as −25 °C throughout the experiment, confirming that the heating was indeed localized to the surface. While finding the correct operating voltage, the force probe was not engaged, and the ice filled mold was left intact.The sensor response was monitored with a Keysight Technologies N9918A Vector Network Analyzer . The de-icing force was again measured at −20 °C using a force gauge with accuracy of 0.1 N. At least five repeats were performed for each ice length. Following this, 13 additional icing/de-icing cycles were performed on this same sample using 150 mm lengths of ice. Finally, the five repeats of the 60, 80, and 100 mm pieces of ice were cyclically iced/de-iced, for a total of 43 repeated measurements on the same LIT/heater sample.The hybrid de-icing system was placed outdoors for 3 weeks in Toronto, ON, Canada, which included daily thermal fluctuations and one heavy rainstorm event on May 21, 2022. Afterwards, the de-icing force was measured using several lengths of ice at −20 °C.The LIT coating was contaminated by pipetting acetone onto its surface and allowing it to evaporate . A power sander  was used to constantly abrade the material at 12,000 rpm for 15 min. The roughness and topography of the coating before and after abrasion were measured using a LEXT™ OLS5100 3D Laser Scanning Microscope Fig. . After aTo evaluate the scalability of our hybrid de-icing system, a scaled-up version was designed (see supporting information, Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


Their inhibition sequence on PP2A was detected as follows: MCLR > MCLW > MCLA > MCLF > MCLY. Combined with homology modeling and molecular docking technology, the major interaction parameters between the MCs and PP2A were obtained. The correlation analysis for the major interaction parameters and inhibition effects showed that the hydrophobicity of Z4 had an important influence on the interaction of the MCs to PP2A. The introduction of hydrophobic Z4 directly weakened hydrogen bonds Z4→Pro213 and Z4←Arg214, indirectly weakened hydrogen bonds Adda5←Asn117, Glu6←Arg89, and MeAsp3←Arg89, but indirectly enhanced ionic bonds Glu6←Arg89, Glu6-Mn12+, and Glu6-Mn22+. In this way, the combination of the MCs with PP2A was blocked, and thus, the interactions between PP2A and the Mn2+ ions  were further affected; metal bonds Asp85-Mn12+ and Asp85-Mn22+ were weakened, while metal bond His241-Mn12+ was enhanced. As a result, the interactions in the catalytic center were inhibited to varying degrees, resulting in the reduced toxicity of MCs.Microcystins (MCs) exhibit diversified inhibition effects on protein phosphatases (PPs) due to their structural differences. To fully evaluate the potential mechanism for the discrepant inhibition effects, the five most frequent MCs with varying residues at position Z Toxic microcystins (MCs), produced by blue-green cyanobacterial, pose a worldwide threat to humans and wildlife ,2. When d-Ala1-L-X2-d-isoAsp3-l-Z4-Adda5-d-isoGlu6-Mdha7) [5 is rapidly wrapped in the hydrophobic cage structures of PP1/PP2A; for the irreversible step, the C=C double bond of Mdha7 undergoes an electrophilic addition reaction with the nucleophilic sites (Cys residue) in PP1/PP2A [MCs are a class of monocyclic heptapeptides that share a common structure of cyclo-(6-Mdha7) . Due to 6-Mdha7) . Among M6-Mdha7) . In cons6-Mdha7) . Based oPP1/PP2A ,14,15. B5/Mdha7 residues  were selected as the typical variants, and their inhibition effects on PP2A were explored by a colorimetric protein phosphatase inhibition assay [Based on the homology modeling strategy, the interactions between MCs and PP1/PP2A can be simulated, and the molecular mechanism for the discrepant inhibition effects of MCs can be evaluated more in-depth. The five most frequent MCs with the changed Zon assay ,23. With4, it was found that the electropositive Arginine (R) is more hydrophilic than the other four amino acids. When Arg4 was replaced by Trp4/Tyr4/Ala4/Phe4 (with increased hydrophobicity), the corresponding inhibition effect generally decreased. The lower inhibition of MCLY might be attributed to the electronegative OH in the side-chain of Tyr4. Unfortunately, the crystal structures for most MC-PP2A complexes (except for MCLR-PP2A) have not been prepared and solved. It would be difficult to elucidate the relationship between structural differences (changed Z4) and the discrepant inhibition of MCs on PP2A.A traditional colorimetric PP inhibition assay was carried out to evaluate the inhibition effect of MCs on PP2A. As shown in 4 residue in MCLR with Ala4, Phe4, Trp4, and Tyr4. With the help of molecular docking, the major interaction parameters for MC-PP2A complexes were obtained and are listed in 2+ ions, the interaction parameters related to the Mn2+ ions were obtained simultaneously. Besides, the basic parameters (logP and logS) for typical MCs were also calculated.The homology modeling strategy has been widely used to evaluate the interactions between structural analogues and proteins ,25. BaseTo evaluate the discrepant inhibition mechanism of MCs on PP2A, the correlation between inhibition data and the major interaction parameters was further evaluated by the Pearson correlation analysis. Regression analysis was not used to avoid deleting valid parameters associated with a few finite amino acid residues.p < 0.01 level, the combination areas for Adda5→PP2A and Glu6→PP2A, the hydrogen bonds for Z4←Arg214 and MeAsp3←Arg89, as well as the ionic bond for Glu6←Arg89 were significantly correlated with the inhibition effect at the three test concentrations. The metal bond for Asp85-Mn12+ was significantly correlated with the inhibition effect at 1 nM and 10 nM. Total hydrogen bonds, the combination area for Mdha7→PP2A, and the positive accessible surface area for Adda5→PP2A were significantly correlated with the inhibition effect at 1 nM. LogS, the combination area for MeAsp3→PP2A, and the hydrogen bond for Glu6←Arg89 were significantly correlated with the inhibition effect at 100 nM. At the p < 0.05 level, combination energy change, logP, the hydrophobic surface area for toxin→PP2A, the positive accessible surface area for Glu6→PP2A, the negative accessible surface area for Ala1→PP2A, the hydrophobic surface areas for Adda5→PP2A/Mdha7→PP2A, the hydrogen bond for Adda5←Asn117, the ionic bond for Glu6-Mn12+, the metal bonds for Asp85-Mn22+, His241-Mn12+, and combination energy change were significantly correlated with the inhibition effect at three test concentrations. LogS, the combination area for MeAsp3→PP2A, the negative accessible surface area for MeAsp3→PP2A, the hydrogen bond for Glu6←Arg89, the catalytic center exposure area for Asp85 + Mn12+, and the ionic bond for Glu6-Mn22+ were significantly correlated with the inhibition effect at 1 nM and 10 nM. The combination area for Mdha7→PP2A and the positive accessible surface area for Adda5→PP2A were significantly correlated with the inhibition effect at 1 nM and 100 nM. Total hydrogen bonds were significantly correlated with the inhibition effect at 10 nM and 100 nM. The hydrogen bond for Z4→Pro213 and the catalytic center exposure area for His241 + Mn12+ were significantly correlated with the inhibition effect at 1 nM. The metal bond for Asp85-Mn12+ was significantly correlated with the inhibition effect at 100 nM. Obviously, the above interaction parameters  were important for the combination of the MCs to PP2A.4 was substituted with Trp4/Ala4/Phe4/Tyr4, logP and hydrophobic surface areas (ASA-H) for MCs gradually increased, while logS showed a downward trend. In view of this, the hydrophobicity should have an important influence on the combination of MCs to PP2A; the increased hydrophobicity of Z4 would hinder the combination of MCs to PP2A by weakening the total hydrogen bonds (negative correlated with inhibition effect) and by acting on specific important interactions. Statistical frequency analysis of the key sites associated with the important interaction parameters , Glu6←Arg89, MeAsp3←Arg89, and enhancing ionic bonds Glu6←Arg89, Glu6-Mn12+, and Glu6-Mn22+. Correspondingly, the combination areas of MeAsp3, Adda5, and Glu6 with PP2A all decreased. As a result, the combination of the MCs with PP2A was blocked to certain degrees. Subsequently, the interactions between PP2A and the Mn2+ ions in the catalytic center were further affected; the metal bonds Asp85-Mn12+ and Asp85-Mn22+ were weakened, while the metal bond His241-Mn12+ was enhanced. As a result, there was an increase in the exposure area of the catalytic center , resulting in lower inhibition effects of the MCs. A two-dimensional ligand-receptor interaction diagram for the combination of MCs with PP2A illustrated the key interactions, including hydrogen bonds MeAsp41-Mn12+ . The Pea4 residues  were selected as the typical variants. A protein phosphatase inhibition assay showed that their inhibition effects on PP2A were in the sequence of MCLR > MCLW > MCLA > MCLF > MCLY. With the assistance of molecular modeling, the interaction models for MC-PP2A were constructed by homology modeling, and the major interaction parameters between MCs and PP2A were obtained by molecular docking. The Pearson correlation analysis for the major interaction parameters and inhibition data verified the important influence of the hydrophobicity of Z4 on MC toxicity. The increased hydrophobicity of Z4 directly promoted the hydrophobic combination of Z4 to PP2A and weakened the hydrogen bonds Z4←Arg214 and Z4→Pro213. The hydrophobic combination of Z4 with PP2A could intervene with the interactions between other residues of the MCs and PP2A by weakening the hydrogen bonds MeAsp3←Arg89, Adda5←Asn117, and Glu6←Arg89 and by enhancing the ionic bonds Glu6←Arg89, Glu6-Mn12+ and Glu6-Mn22+. The above key interactions between the MCs and PP2A further influenced the interactions between PP2A and the Mn2+ ions . The eventual result was the increased exposure area of the catalytic center  and the lower inhibition effect on PP2A.To obtain a better understanding of the discrepant inhibition effect of the MCs on PP2A, the five most frequent MCs with the changed Z2, P-nitrobenzene disodium orthophosphate, sodium thiosulfate, tris(hydroxymethyl) aminomethane, and other reagents were purchased from Sinopharm .MCLR, MCLF, MCLA, MCLY, and MCLW were purchased from Sigma . PP2A was purchased from New England Biolabs Inc . Bovine serum albumin, dithiothreitol, MnCl2) and 1.0 g/L bovine serum albumin. Then, 10 μL of PP2A and 100 μL of test samples were mixed in a 96-well polystyrene microplate. With gentle shaking, the microtiter plates were kept at 25.0 °C for 15 min, and 90 μL p-nitrophenyl disodium orthophosphate (5 mM) was added. After 1 h, the absorbances at 405 nm were measured in a microplate reader. The inhibition of the test samples on PP2A was calculated by the formula of IPP2A (%) = (Acontrol − Asample)/Acontrol × 100%. Acontrol and Asample were the absorbance of the reference sample (without PP2A) and the test sample at 405 nm, respectively.The biological toxicity of the typical MCs was evaluated by a colorimetric protein phosphatase inhibition assay modified by Zong et al. ,23. Firs4 (in the revised model of MCLR-PP2A) with Ala4, Phe4, Trp4, and Tyr4, respectively [The molecular docking simulation was performed with MOE software . The original model for the MCLR-PP2A complex was obtained from the Protein Data Bank (PDB code 2IE3). Hydrogen atoms and charges were supplemented to PP2A to obtain the revised model. Based on the homology modeling strategy, models for MCLA-PP2A, MCLF-PP2A, MCLY-PP2A, and MCLW-PP2A complexes were obtained by substituting Argectively ,26. All The Pearson correlation analysis was used to analyze the correlation between the inhibition data and the major interaction parameters by IBM SPSS statistics .


Retinal neurovascular injuries are a leading cause of vision loss in young adults presenting unmet therapeutic needs. Neurovascular injuries damage homeostatic communication between endothelial, pericyte, glial, and neuronal cells through signaling pathways that remain to be established. To understand the mechanisms that contribute to neuronal death, we use a mouse model of retinal vein occlusion (RVO). Using this model, we previously discovered that after vascular damage, there was non-apoptotic activation of endothelial caspase-9 (EC Casp9); knock-out of EC Casp9 led to a decrease in retinal edema, capillary ischemia, and neuronal death. In this study, we aimed to explore the role of EC Casp9 in vision loss and inflammation. We found that EC Casp9 is implicated in contrast sensitivity decline, induction of inflammatory cytokines, and glial reactivity. One of the noted glial changes was increased levels of astroglial cl-caspase-6, which we found to be activated cell intrinsically by astroglial caspase-9 (Astro Casp9). Lastly, we discovered that Astro Casp9 contributes to capillary ischemia and contrast sensitivity decline after RVO (P-RVO). These findings reveal specific endothelial and astroglial non-apoptotic caspase-9 roles in inflammation and neurovascular injury respectively; and concomitant relevancy to contrast sensitivity decline. Neurovascular hypoxic-ischemic injuries in the retina are a leading cause of degenerative vision loss  characteNon-invasive in vivo visualization and manipulation of the NVU can be done in the retina. To study cell-specific roles of the components of the NVU in the context of neurovascular injury, we used a characterized mouse model of Retinal Vein Occlusion (RVO). RVO is the second most common cause of blindness after diabetic retinopathy amongst young adults  and currCaspases are a group of cysteine-dependent aspartate-directed proteases that can activate cell death and inflammatory signaling. Over recent years their roles in the brain have been further characterized as relevant promoters of synaptic pruning, axon guidance, differentiation and in glial cells, as mediators of inflammation and filament aggregation . Cell-spRetinal resident glial cells  provide neuronal and vascular support and participate in neuropathology –21. DuriMany studies assessing vitreous samples of RVO patients have identified increased levels of pro-inflammatory cytokines and chemokines –28; and Here, we aimed to evaluate the contribution of endothelial and astroglial caspase-9 (Astro Casp9) to vision dysfunction, inflammation and RVO pathology. We found that deletion of EC Casp9 contributed to a decline in contrast sensitivity and modulated the levels of inflammatory cytokines and glial responses in a time-dependent manner in RVO. Additionally, we discovered that knock-out of Astro Casp9 reduced capillary ischemia and contrast sensitivity decline.All animals were handled in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research and monitored by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.Animals used in this study were 2-month-old males and females. The tamoxifen-inducible EC Casp9 mouse line was generated by crossing Cdh5(Pac)-Cre/ERT2  with casCre recombination was induced in both colonies after five consecutive days of 2 mg tamoxifen intraperitoneal injections (100 μL of 20 mg/mL). Tamoxifen was dissolved in corn oil (20 mg/mL), then 2 mg of tamoxifen was administered intraperitoneally (IP) for five consecutive days when animals were eight weeks old Fig. .Fig. 1ECn = 2–3 veins occluded/eye) ten minutes after tail vein injection of the photoactivatable dye, Rose Bengal (37.5 mg/kg). Eight minutes after injection, eyes were dilated with tropicamide, and phenylephrine chloride eye drops, and mice were anesthetized with IP administration of a cocktail of ketamine (80–100 mg/kg) and xylazine (5–10 mg/kg). Two minutes after injection, depth of anesthesia was confirmed by toe-pinch. Afterwards, retinal veins were irradiated with the Micron IV image guided laser (532 nm) from Phoenix Research labs by delivering three adjacent laser pulses  to each vein at a distance of 375 μM from the optic nerve head. RVO occlusions were examined by fundus imaging after irradiation and one day after RVO (P-RVO). Occluded veins were identified by a whitening of the vessel at the site of the occlusion, dilation distal to the occlusion, and constriction of vessel diameter proximal to the occlusion. Persistent occlusions from the time of irradiation up to one day P-RVO, were classified as successful occlusions. Retinas with detachment, subretinal hemorrhage, or full ischemia one day P-RVO were excluded from further analysis. A detailed description of the RVO mouse model can be found in [RVO was induced in major retinal veins  and the outer plexiform layer (OPL).In vivo examination of the mouse optokinetic response was performed using the Striatech Optodrum. The awake mouse was placed on the platform inside the arena where the Optodrum presented a rotating stripe pattern to the animals at a rotation speed of 12°/second. Contrast sensitivity (1/contrast threshold) was determined by assessing the contrast threshold after presenting incremental visual acuity patterns at 0.05, 0.15 and 0.25 cycles/°. Readings were taken one-day pre-RVO, one and two days P-RVO.Mice were perfused with sterile saline and then with 4% paraformaldehyde (PFA). Eyes were enucleated and immersed in 4% PFA overnight at 4 °C, they were then washed three times for 10 min with 1X PBS and immersed in 30% sucrose for three nights at 4 °C. Eyes were embedded in optimal cutting temperature solution and kept at −80 °C. Embedded eyes were sectioned at 20 µm thickness. Retinal slides were chosen to match approximate location of OCT scans and then were washed with 1X PBS for 5 min. Sections were permeabilized with 0.1% Triton X-100 for two hours and blocked with blocking buffer , 1% Bovine Serum Albumin (BSA) (dissolved in 1X PBS and filtered) overnight at 4 °C. Primary antibodies for Iba-1 , CD68 (1:100 BioRad MCA1957GA), cl-caspase-6 , GFAP , nestin , and AQP-4  were prepared with blocking buffer and sections were incubated overnight at 4 °C. Afterwards, sections were washed four times with 1X PBS for 5 min, secondary antibodies were diluted in blocking buffer  and sections incubated for two hours at room temperature. Then, sections were washed four times with 1X PBS for five minutes and then stained with nuclear staining  for 5 min. After one wash with 1X PBS for five minutes, retinal sections were mounted with Fluoromount-G. Retinas were imaged using a spinning disk confocal microscope (BioVision Technologies) in which z-stack images were taken.Sections were first permeabilized with 0.1% Triton X-100 for two hours. TUNEL was then done following the manufacturer’s protocol (Promega G3250). Sections were then processed for IHC as described above.Images were first processed using FIJI and brightness and contrast settings were adjusted to appropriate parameters based on uninjured (no RVO) or positive control (TUNEL) retinal tissue and background. Then, the number of cells were quantified based on colocalization with cellular marker or nuclei marker using the multi-point function in FIJI. Analysis was performed by an experimenter who was blinded to genotype and treatment. Values were averaged by images per section and then averaged values of sections per eye. More details about IHC quantification of cells can be found in .Images were first processed using FIJI by adjusting the scale first based on the magnification at which images were taken. Area of interest was selected with the polygon setting. Then, the thresholding setting was set up based on levels of expression of uninjured retinal tissue. The same thresholding parameter was then used to quantify the percent area of expression in all retinal images by an experimenter that was blinded to genotype and treatment. Values were averaged by images per section and then averaged values of sections per eye.Images were processed using FIJI by adjusting the scale, then applying the thresholding setting that was based on uninjured retinal tissue. Afterwards, the analyze particle function was applied to identify particles bigger than 49 µm and measurements settings were adjusted to determine microglia area, shape description, integrated density, centroid, perimeter, and Ferret’s diameter. Values were averaged by images per section and then averaged values of sections per eye.Images were processed using FIJI and after setting up the scale and selecting the brightness and contrast parameters of expression for Iba-1 and CD68, images were converted to RGB color. Then, color threshold function was applied and used to measure the area of colocalization. Values were averaged by images per section and then averaged values of sections per eye.Animals were perfused with saline, and retinas were then dissected, minced, and placed in RIPA buffer with protease (Fisher) and phosphatase inhibitors II and III (Sigma). Tissue was then sonicated on ice with a Sonic Dismembrator  for one minute (1 second on/ 1 second off). Retinas were matched one-day P-RVO based on the fraction of veins occluded and disorganization of inner retinal layers (DRIL) percentages, two matched retinas were then combined. Protein concentrations were determined using a BCA assay. A total of 100 µg of retinal protein was loaded per sample. Total protein transfer was determined using REVERT (LI-COR). Protein blots were blocked with LI-COR Blocking Buffer for one hour at room temperature and then incubated for two nights with primary antibody (GFAP GA5 Sigma 63893) at 4 °C. Blots were washed three times with 0.1% TBS-Tween 20 and then incubated with secondary antibodies at 1:5,000 overnight at 4 °C. After three ten-minute washes with 0.1% TBS-Tween 20, blots were imaged using the LI-COR Odyssey Imaging System. Blots were analyzed using Image Studio Lite Software; all signals were first normalized to total protein loaded and then to the average of uninjured littermate control values.Retinal tissue was prepared the same way as for Western Blot, except for the replacement of RIPA buffer with the manufacturer’s cell lysis buffer. Cytokine arrays were performed according to manufacturer’s protocol  and blots were imaged using the LI-COR Odyssey Imaging System and analyzed with Image Studio Lite Software. Analysis of the cytokine array was done following recommendation of the protocol . Averaged relative expression of each cytokine was normalized to each membrane’s positive control. Data is presented as relative expression levels.p < 0.05.The RVO model, in vivo and in vitro measures and analysis were done by investigators blinded to animal genotype. Eyes that presented with retinal detachment, cataract, excessive edema or intraretinal hemorrhage were excluded from all analyses and only eyes that had one or more occlusion by one day P-RVO were considered viable. GraphPad Prism and Excel were used for statistical analysis. One-way and Two-way ANOVA were used to determined statistical differences followed by Fisher’s LSD test; data are presented as mean ± SEM. Significance was set to be One of the effects of RVO is damage to visual function, including a significant decline in contrast sensitivity threshold , 38. We After we found that EC Casp9 promoted a decline in vision function, we sought to evaluate the EC Casp9 downstream signaling pathway which mediates loss of vision. Inflammatory cytokines are known to be a main contributor of neovascularization, and correlate with hypoxic-ischemic injury and retinal edema , and areWe then assessed potential protein-protein interactions of caspase-9 with RVO-induced and EC Casp9-regulated cytokines by performing STRING . The STR+ cells increase significantly two days P-RVO when comparing one-day P-RVO to two-days P-RVO retinas, independent of EC Casp9 expression  death , 8. In tion Fig. . We thenRVO Fig. . To furtRVO Fig. . FurtherRVO Fig. . At two RVO Fig. . Taken tBecause other retinal glial cells, Müller glia and astrocytes, are also involved in the release of inflammatory cytokines, we looked at markers of macrogliosis including nestin and aquaporin-4 (AQP-4). Nestin is an intermediate filament whose expression is downregulated in mature macroglia , but in Next, we sought to determine if RVO and EC Casp9 induced changes in GFAP, as increased levels of GFAP are often associated with an inflammatory response of Müller glia and astrocytes . IHC anaPrior work showed that pharmacological inhibition of caspase-9 decreased the levels of its downstream target caspase-6 in astrocytes P-RVO . AblatioTo evaluate if EC Casp9 signaling was linked to cl-caspase-6 in astrocytes, we stained uninjured, one and two days P-RVO sectioned retinas from iEC Casp9 KO and WT littermates and then evaluated the level of cl-caspase-6 by quantifying the percent area of expression in the RGL, where astrocytes are located in the retina. The analysis revealed that RVO induces an increase in cl-caspase-6 two days after injury and that EC Casp9 deletion significantly blocks the change in cl-caspase-6 levels in the RGL one and two days P-RVO Fig. . SimilarTo test the potential role of cl-caspase-6 in astrocytes, we assessed the only currently known function of astroglial cl-caspase-6; cleavage of GFAP , 59, 61.Our current and previous results suggest that caspase-9 is upstream of the activation of astroglial caspase-6. To evaluate whether Astro Casp9 is activating astroglial caspase-6 in RVO and neurodegeneration, we generated a tamoxifen-inducible Astro Casp9 knockout by crossing a caspase-9 flox/flox mice with a characterized astroglial promoter hGFAP-Cre ERT2 .We first assessed the levels of astroglial cl-caspase-6 P-RVO by performing IHC staining of cl-caspase-6 and quantifying the levels measuring the percent area of expression in uninjured and injured iAstro Casp9 WT and KO Fig. . The ana+ nuclei of the different retinal layers suggest that there are not any significant differences between injured iAstro Casp9 WT and iAstro Casp9 KO  which allows for immunodetection of cells with DNA degradation. Quantification of TUNEL KO Fig. . These dMeasurement of the disorganization of the inner retinal layers (DRIL) is used as a marker of capillary non-perfusion and correlates with visual acuity decline in RVO , 63. We ).We previously found that inhibition of caspase-9 or ablation of EC Casp9 protected from retinal edema at early points P-RVO . To evalS5To investigate the role of Astro Casp9 in vision function we evaluated visual acuity and contrast sensitivity in uninjured iAstro Casp9 WT and KO which revealed that there were no significant differences between genotypes Fig. . InjuredWe have previously shown that non-apoptotic EC Casp9 regulates many of the retinal changes linked to retinal vascular injury, and now queried whether EC Casp9 was also required for vision impairment and inflammation. In this study, we showed that deleting caspase-9 in hypoxic endothelial cells regulates changes in visual function and inflammation in glial cells and cytokines. By using an established mouse model of RVO, we found that deleting EC Casp9 causes a decrease in: (1) contrast sensitivity decline, (2) inflammatory cytokines, and (3) glial cell reactivity. One of the observed EC Casp9 glial changes was increased levels of astroglial cl-caspase-6. We then were able to establish that Astro Casp9 was upstream of astroglial cl-caspase-6 and when Astro Casp9 was knocked-out, there was decreased capillary ischemia and contrast sensitivity decline P-RVO Fig. .RVO impacts contrast sensitivity, which has prospective use as indicator of disease progression , 64. ConPro-inflammatory cytokines are known to play an important role in RVO as they correlate with macular edema and hypoxic-ischemic injury . Here, whttps://www.bu.edu/nf-kb/gene-resources/target-genes/). A recent in vitro enzymatic study suggested that NFκ-B is a potential substrate of caspase-6 [While caspase-9 has not been shown to directly activate cytokines, downstream caspase-9 targets such as caspase-6, caspase-7 and caspase-3 are known to cleave cytokine proforms and increase overall cytokine levels . Many ofaspase-6 . These dMost of the cytokines are expressed in microglia, and in RVO a recent study suggested that microglia are a main source of cytokines . In a moWe also found that Astro Casp9 deletion led to a significant decrease in astroglial caspase-6, suggesting that activation of astroglial caspase-6 occurs in a cell-intrinsic manner. It is important to mention that the levels of astroglial caspase-6 in the iAstro Casp9 KO were not entirely eliminated. This could indicate that some activation of astroglial caspase-6 is due to EC Casp9-astroglial signaling or that the promoter used to generate our mouse model (GCE) does not target all astrocytes in the retina. It is known that GFAP is not expressed in all astrocytes and not all cells that express GFAP are astrocytes . In caseOur results suggest that Astro Casp9 loss did not ameliorate retinal swelling, indicating that Astro Casp9 is not an active participant in the development of retinal edema. However, Astro Casp9 loss protected the retina from capillary ischemia as measured by DRIL. DRIL was characterized by Sun and others as a predictor of decline in visual acuity  and capiWe demonstrate an important role for EC Casp9 and Astro Casp9 in RVO-induced inflammation, visual dysfunction, and capillary ischemia Fig. . These fSupplementary MaterialWestern blotsChecklist


Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control. Ticks are blood-sucking ectoparasites that can transmit etiologic agents of human and animal diseases and cause enormous loss to global livestock production . To contRhipicephalus (Boophilus) microplus, the southern cattle tick, is one of the most widespread invasive ticks worldwide [R. (B.) microplus tick is a pathogen vector that causes babesiosis (Babesia bovis and Babesia bigemina) and anaplasmosis , which are the most impactful tick-borne diseases of cattle, globally [R. (B.) microplus control; however, rapid resistance to such chemicals in R. (B.) microplus has been reported [R. (B.) microplus was reported to be resistant to many common pesticides such as organophosphate, pyrethroid, fipronil, amitraz, and macrocyclic lactone [R. microplus [R. microplus glutathione S-transferase (RmGST) receives more attention due to its involvement in tick resistance [R. microplus tick, it is necessary to unravel the binding mechanism of GTX to RmGST. orldwide . R. (B.)globally . Pesticireported . R. (B.) lactone . Enhanciicroplus ,11,12,13sistance . RmGST wsistance . RmGSTs sistance ,16. To dsistance ,18. Thussistance ,20,21. IR. (B.) microplus GST (RmGST) was reported to be closely related to the μ-class GST [R. microplus, RmGST contains 223 amino acids. The N-terminus displays higher sequence similarity than the C-terminus, which is common throughout GST families [In mammals, GSTs are classified into five families, namely alpha  A. Each s (β1−β4) B. The N- (β1−β4) B. RmGST  (β1−β4) B [8,22,2To date, several GST-GSH/GTX crystal structures have been solved ,30,31,32To explore how the ligand binding influences the RmGST flexibility, the C-alpha root mean square deviations (RMSDs) and root mean square fluctuation (RMSFs) were computed. The C-alpha RMSDs of whole RmGST were in the range of ~0.2–0.35 nm A. In sin3) is smaller than the H-site (~17–20 nm3). The binding of ligand results in the consistent size of the G-site pocket. Nonetheless, the significant expansion of the G-site was found in chain B of GTX(B)  at the dimer interface A and theT dimers . Herein,To investigate the behavior of all substrates, the average number of hydrogen bonds between each substrate and its environment were computed . SeemingIn Furthermore, the key interaction networks for ligand binding were investigated . Consideass GSTs ,36. Althll cases . This hell cases . This prll cases . Unlike ll cases . For doull cases . No GTX-ll cases . Each chIn the case of GSH, it is interesting that the presence of two GSHs induced the tighter binding to RmGST. Although a small dissimilar number of RmGST-GSH interactions were spotted, GSH seemed to be more rigid inside a pocket when compared with GTX. For GSH, the E2 tail directs to the tip of the mu loop and mainly interacts with residues in the G-site . When GSH moves towards the H-site, this allows the E2 terminus to hydrogen bond with Q102, R113, Y116, N209, G210 . In the 3) and solvated with TIP3P water molecules . The protonation states of charged amino acids were set at physiologic pH. Counter ions were added to neutralize the system. The energy minimization of 50,000 steps was performed to remove close contacts, using steepest descent algorithm followed by 1-ns NVT and 10-ns NPT runs. To explore the effect of single and double ligand binding, the systems with one ligand in one chain (GSH(A), GTX(A), GSH(B), and GTX(B)) and with one ligand in each chain (GSH(AB) and GTX(AB)) were set. The prefixes of “GSH” and “GTX” were used to represent the systems with GSH and GTX, respectively. The letter in a bracket ((A) and (B)) indicates the ligand-containing chain. Two copies with different random seeds of 1000 ns production runs were performed. The endings of “1” and “2” refer to simulations 1 and 2. In sum, 12 systems were set here ) [p = 1 ps was used for pressure coupling. The temperature of the protein, ligand, and solution were coupled separately using the v-rescale thermostat [t = 0.1 ps. The time step of 2 fs was used for integration. The coordinates were recorded every 2 ps. The GROMACS 5 package (e 2021))  was emple 2021))  with a Fermostat  at 300 KAll data were analyzed by GROMACS tools and in-house codes. “Gmx hbond” was used to compute all hydrogen bonds where the hydrogen-donor-acceptor cutoff angle was set to 30° and the cutoff radius (X-acceptor) was 0.35 nm. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were calculated using an initial structure at 0 ns from each production run as a reference. A principal component analysis (PCA) was calculated using “gmx covar” and “gmx anaeig”. VMD was used for visualization and graphic images .In this study, the interaction modes between RmGST and its substrate (GSH) and inhibitor (GTX) were revealed through molecular dynamics simulations. GSH was more rigid inside the active site, while GTX was flexible. GTX occupied both active sites (H- and G- sites), while GSH mostly aligned in the G-site and left the free H-site for further binding of co-substrate. The also suggests the binding order where GTX/GSH acts as the first substrate and xenobiotic or insecticide comes second. Further experiment is needed. Comparing the binding between GSH and GTX, GSH seemed to be more rigid inside the G-site which may facilitate further conjugation reaction. The presence of an s-hexyl chain on GTX seemed to significantly disrupt the interaction network inside a pocket. Although the hexyl moiety occupied the H-site close to the connecting loop between ructures ,36, thisructures . The ins


The brain contains a delicate and precise balance between excitatory and inhibitory neurons, which have different roles in the formation, development, and homeostasis of brain circuits. The balance between these populations is maintained by a number of local control mechanisms, regulating presynaptic efficacy as well as postsynaptic excitability. In pathology, an abnormal excitation-to-inhibition (E/I) ratio leads to neuronal hyperexcitability and network hyperactivity, constituting the basis for epilepsy and subsequent cognitive dysfunction, and is associated with many neurodevelopmental, neurodegenerative, and psychiatric diseases. Epileptic seizures can be induced by blocking GABAergic inhibition and repressed by enhancing inhibition, illustrating the role of E/I-ratio imbalance in neuronal pathology . These diseases all share phenotypic traits with the archetype of developmental disorders, fragile X syndrome (FXS)  progression, in both sporadic and familial AD , induced by chronic exposure to opioids, is mediated by specific alternations in excitatory and inhibitory spinal neurons. Chronic treatment with morphine was associated with both enhanced excitation of excitatory neurons, as well as with reduced excitation of inhibitory neurons. The effect seems to be mediated by changes in neurotransmitter-dependent mechanisms, as treatment with morphine did not alter most passive membrane properties in any of the tested cell populations. The study by Zhang et al. discusses hyperactivity in the context of locomotion and psychomotor behavior. Inhibition of 14-3-3 protein in the hippocampus induces locomotor hyperactivity that is mitigated by pharmacological blockade of dopamine and by inhibition of firing of dopamine neurons in the ventral tegmental area. This work also identifies the lateral septum as a relay station between the ventral tegmental area and the hippocampus.The study by This Frontiers Topic highlights the versatility of hyperactivity and hyperexcitability in neurological disease, observed in neurodevelopmental and neurodegenerative disorders; as a consequence of intrinsic changes in neuronal properties or due to circuit-wide changes affecting excitation and/or inhibition. The mechanisms underlying each case might differ, but the phenotypic outcome remains the same. Most importantly, for each individual case, it is still unclear whether hyperactivity is just a consequence of an imbalanced system, or is in itself the source of further dysfunctionality by amplifying the defects associated with the disease.MT and MS contributed equally to the writing and editing of this manuscript. Both authors contributed to the article and approved the submitted version.MT was supported by an unrestricted gift from the Research for Preventing Blindness Foundation to the Flaum Eye Institute.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Therefore, Atractyloside A may be an effective natural drug against B/Victoria influenza infection.Influenza viruses pose a serious threat to human health, infecting hundreds of millions of people worldwide each year, resulting in a significant increase in global morbidity and mortality. Influenza activity has declined at the onset of the COVID-19 pandemic, but the genetic diversity of B/Victoria lineage viruses has increased significantly during this period. Therefore, the prevention and treatment of the influenza B Victoria strain virus should continue to attract research attention. In this study, we found that Atractyloside A (AA), one of the effective components in Atractylodes lancea (Thunb.) DC shows potential antiviral properties. This study shows that AA not only possesses anti-influenza B virus infection effects  Influenza is an acute respiratory infection caused by the influenza virus  was stored in the Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Atractyloside A (AA) was purchased from MedChemExpress (Cat#HY-N0237). The C57BL/6 mice used in this study were purchased from Beijing Wei Tong Li Hua Laboratory Animal Technology Co., Ltd.4 cells/well) in an incubator at 37°C and 5% CO2 for 12 h and then treated with different concentrations of Atractyloside A (AA); four replicate wells were used for each test group. After treatment for 24 h and 48 h, CCK-8 was added and cells were further cultured for 2 h; the OD450 value was detected, and cell viability was calculated.A549 or MDCK cells were cultured in a 96-well plate  group, or a control group. Seventy-two hours after infection of the MDCK cells with 0.05 MOI IBV, half maximal inhibitory concentration assay (IC50) was obtained by colorimetric CCK-8 assay and calculated using non-linear regression analysis in GraphPad Prism 9 software.A549 or MDCK cells were plated in 12-well plates and assigned to an IBV group, an IBV+AA group, or a control group. Each experimental group was treated with the corresponding solution and placed in a cell incubator for 24 h. Immunofluorescence assays were then performed. After incubation, the culture mixture was discarded and cells were fixed with 4% paraformaldehyde for 30 min, treated with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA). After blocking, the cells were stained with primary antibody for 2 h at room temperature and then stained with fluorescent secondary antibody for 1 h in the dark. Finally, the samples were stained with DAPI solution for 10 min before observation. The antibody used in this study was the Anti-Influenza B Virus Nucleoprotein from Abcam (Cat#ab20711).2-109 copies as a standard to calculate the viral copy number after IBV infection. The primers used in this study were 5′-ATTTTGCAAAYCTCAAAGGAACA-3′ (Forward) and 5′-TTGTTCTRTCGTGCATTATAGG-3′ (Reverse). The IBV-Victoria probe used in this study was 5′-VIC-TGGGYAGACCAAAATGCACRG-BHQ1-3′.After the A549 or MDCK cells were plated in 12-well plates and incubated for 12 h, 0.1 MOI of IBV was added with different concentrations of AA. Twenty-four hours after IBV infection + treatment, cellular RNA was extracted and subjected to the quantitative real-time PCR (qPCR) utilizing a quantitative PCR instrument (ABI7500). The plasmid pUC57-HA (2896bp) was constructed based on the HA gene of IBV, and the plasmid was diluted to 10After the A549 or MDCK cells were plated in 12-well plates and incubated for 12 h, 0.1 MOI of IBV was added with different concentrations of AA. Twenty-four hours after IBV infection + treatment, the culture supernatant was collected for a hemagglutination assay (HA). In 12 wells of a 96-well V-shaped microplate, 25 μl of PBS was added to each well. Twenty-five microliters of cell culture solution were added to the first well and then serially diluted 10 times. After serial multiple dilutions, 25 μl of 1% chicken red cell suspension was added to each well, mixed well, and incubated for 40 min at room temperature for result determination.A549 cells were inoculated with 0.1 MOI of IBV, and the IBV+AA treatment group and control group were started simultaneously. Twenty-four hours after infection of the A549 cells with IBV, the cells were collected, and total proteins were extracted from the cells. Each group sample was loaded at 25 μg of total protein for detection. After SDS-PAGE was transferred to a PVDV membrane and blocked at room temperature for 2 h, the corresponding primary antibody was added for incubation at 4°C overnight. Then, the membrane was incubated with goat anti-rabbit or goat anti-mouse secondary antibody for 50 min. The primary antibodies used in this study were the Anti-Influenza B Virus Nucleoprotein from Abcam (Cat#ab20711) and the Rig-I Pathway Antibody Sampler kit from Cell Signaling Technology (Cat#8348).A total of 293 cells were transfected together with pIFN-β-Luc plasmids and Renilla plasmids (pRL-TK). Two hundred ninety-three cells were infected with 0.05 MOI of IBV before harvesting, together with the addition of AA (40 μM) as the intervention. The luciferase activity was tested using the Dual-Lumi™ Luciferase Assay kit  after IBV infection for 24 h. pIFN-β-Luc (Cat#ZT206) and pRL-TK (Cat#BR018) plasmids were from the Hunan Fenghui Biotechnology Co., Ltd.4.5 TCID50 of IBV. Animals were divided into four groups (six mice for each group): an IBV infection group, an IBV infection + AA treatment group (10 mg/kg and 30 mg/kg), and a control group. AA was administered orally over 14 consecutive days, starting 1 day before IBV inoculation. IBV infection and control groups were orally treated with a placebo solution  over 14 consecutive days. Body weight, food intake, and mortality were then monitored; death was defined at ≥35% weight loss. Fourteen days after the IBV infection, the animals were euthanized, and the lung tissue was collected and placed in tissue fixative for histopathological analysis. The fixed lung tissue was utilized for HE staining, immunohistochemical analysis using the nucleoprotein of the IBV antibody, and immunofluorescence analysis using the F4/80 and the nucleoprotein of the IBV antibodies. Analysis software  was used to analyze the average signal density of stained samples. The analysis software analyzes each image to derive the IOD  value for each area (pixel area of tissue). The average optical density value IOD/area (Mean Density) was also derived. The antibodies used in this study were the Anti-Influenza B Virus Nucleoprotein antibody from Abcam (Cat#ab20711) and the Anti-F4/80 antibody from Cell Signaling Technology (Cat# 70076S).Six-week-old female C57BL/6 SPF-grade mice were used for intranasal inoculation with 50 μl of PBS containing 1 × 10To test the ability of AA to activate macrophages, we used 6-week-old SPF-grade C57BL/6 mice to isolate and obtain primary bone marrow-derived cells. For the specific experimental methods, we refer to previous studies , Percp-CD11c , and FITC-CD206  flow antibodies for the flow cytometry analysis.To further analyze the effect of AA on macrophage polarization t-tests. Univariate and multivariate ANOVAs were used to calculate between-group differences. A p < 0.05 was used to determine significance; significance levels are presented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.All experiments were performed at least three times independently. Data are presented as mean ± standard deviation (SD) and compared between groups using 50) and cytotoxic concentration 90 (CC90) for each group were calculated using GraphPad Prism 9 software. At 24 h, the CC90 of A549 and MDCK cells were 829.9 μM and 265.9 μM, respectively. The CC50 of A549 and MDCK cells were 184.1 μM and 180.7 μM, respectively. MDCK and A549 cells were then plated on 12-well plates, infected with 0.1 MOI of IBV, and incubated with AA for 48 h. After incubation, crystal violet staining was performed . We found that after IBV infection, with increasing AA concentration, nucleoprotein immunofluorescence decreased significantly  with increasing AA concentrations. With increased AA concentrations, virus copy numbers in MDCK cells decreased by 1.88×, 885.9×, and 2.59× 103× compared with the IBV group. Compared with the IBV group, HA in A549 cells decreased by 1.28×, 1.5×, and 2× with increased AA concentrations. With increasing AA concentrations, HA in MDCK cells decreased by 1.31×, 1.89×, and 2.12× compared with the IBV group. Combined with the above results, AA could attenuate IBV infection in vitro.The results of HA detection and virus copy number detection are shown in p-IRF3 were increased (p-IRF3 in the absence of IBV infection (in vivo. We found that the IBV+AA-treated mouse group showed higher expression of IFN-β in the serum compared to the IBV-infected group (p < 0.05) and the control group (p < 0.001)  . This fiin vivo, challenge experiments were performed as shown in in vivo, conducted pathological analyses of the lung tissue of experimental animals. H&E staining results showed that the AA treatment group showed less tissue damage  were present in the lung tissue of the AA treatment group and this was associated with significant inhibition of IBV replication  .in vitro assays. The isolated bone marrow-derived macrophages (BMMs) were plated on 6-well plates in three groups: AA group, LPS group, and mock control group. Each experimental group was treated with the respective solution and incubated for 48 h. Cells were then collected and stained with F4/80, MHCII, CD40, CD80, and CD86 flow antibodies for flow analysis. Flow cytometry results are shown in p < 0.001). BMMs in the AA-induced group showed increased expression of CD40 (p < 0.0001), CD80 (p < 0.01), and CD86 (p < 0.0001) compared to the mock control group  compared to the IBV group (p < 0.001). The proportion of M2 macrophages (F4/80+CD11c-CD206+) was higher in AA compared to the IBV (p < 0.01) and mock groups (p < 0.001). We also analyzed the effect of AA on macrophage polarization in vitro  with AA treatment  of the Changchun University of Chinese Medicine.DL, WL, HL, and JH were responsible for the experiment design and drafting of the manuscript. JH, XZ, ZG, PW, JZ, YX, and YuL performed experiments. JH, JF, YiL, and YZ analyzed data. All authors contributed to the article and approved the submitted version.


Food allergy (FA) affects ∼10% of adults; however, little is known about the extent to which FA phenotypes and psychosocial burden vary depending on timing of allergy onset, whether in childhood or as an adult.This study explored FA characteristics according to timing of FA onset in US adults.Between 2015 and 2016, a cross-sectional survey was administered to 40,443 US adults. Complex survey–weighted results were tabulated across key demographic and clinical strata. Linear regression models explored covariate-adjusted variability in FA-related psychosocial burden across 3 groups: (1) adults solely with childhood-onset FA, (2) adults solely with adult-onset FA, and (3) adults with both childhood- and adult-onset FA.P < .001). Adults solely with childhood-onset FA, compared to adults solely with adult-onset FA, had significantly lower rates of environmental allergies  and medication allergies . Adults with both childhood- and adult-onset FAs reported highest rates of all comorbidities. Adults solely with adult-onset FA reported significantly lower FA-related psychosocial burden (P < .05).Adults with both childhood- and adult-onset FAs, compared to adults with solely childhood- or adult-onset FAs, are significantly more likely to have severe FAs , physician-diagnosed FAs , and multiple FAs  (Differences exist in reaction severity, health care utilization, atopic comorbidities, and psychosocial burden according to the timing of FA onset. Future research exploring the heterogeneity of phenotypic expressions of adult FA may inform underlying mechanisms. For example, the most common allergens among adults with childhood-onset IgE-FA include egg, peanut, and milk,,Differences in health care utilization between adults with childhood- and adult-onset IgE-FA have also not been well examined. While research has found the estimated prevalence of physician-confirmed IgE-FA, ED visits, and current epinephrine autoinjector (EAI) prescriptions in adults with IgE-FA, these data fail to differentiate by timing of IgE-FA development.Additionally, it is not yet known how quality of life (QoL) differs between adults with childhood-onset IgE-FA and adult-onset IgE-FA. A recent study characterized the psychosocial burden of IgE-FA among a population-based sample of US adults and found specific demographic characteristics and allergic conditions to be associated with increased psychosocial burden.To better understand the phenotypic characterization of childhood- and adult-onset IgE-FA among adults in the United States, we aimed to characterize the distribution and determinants of IgE-mediated FA in 3 US adult subpopulations: (1) adults with solely childhood-onset IgE-FA, (2) adults with solely adult-onset IgE-FA (over 18 years of age), and (3) adults with both childhood- and adult-onset IgE-FA. Utilizing convincing symptom reports, likely cases of IgE-mediated FA were identified in these 3 subpopulations. We sought to explore differences in sociodemographic predictors, IgE-FA severity, reaction characteristics, health care utilization, atopic comorbidities, and IgE-FA–related psychosocial burden among these 3 groups .Fig 1SumEligible study participants included adults (≥18 years old) who resided in a US household and were able to complete the survey in English or Spanish online or by telephone. Similar to the 2009-2010 national pediatric IgE-FA prevalence survey,www.jaci-global.org). The “convincing IgE-FA” definition is intended to identify cases of IgE-mediated FA. Timing of allergy onset and outgrowing an allergy (if applicable) were assessed for each specific allergy. Childhood-onset IgE-FA is defined as having onset of the first food allergen-specific reaction before age 18. Convincing IgE-FAs for which a doctor’s diagnosis was also reported were considered to be physician confirmed. A severe reaction history was indicated by report of multiple specific stringent symptoms occurring within 2 or more of 4 organ systems  in response to the question, “Think back to the most severe allergic reaction to . . . that you have ever had. What were your symptoms?”Reported FAs were only considered to be convincingly IgE mediated if the most severe reaction reported to that food included at least 1 symptom on the stringent symptom list developed by our expert panel, even if IgE-FAs were reported to be physician diagnosed  using ‘svy: commands.’ Relative proportions of demographic characteristics were compared using weighted Pearson chi-square statistics, which were corrected for the complex survey design with the second-order correction of Rao and Scott and converted into Surveys were completed by 40,443 adults living in the United States. A survey completion rate of 51.2%  was observed from the population-based AmeriSpeak panel, from which prevalence was estimated. As reported in www.jaci-global.org).These survey data indicate that although there is substantial heterogeneity among US adults with IgE-FA regarding the typical timing of IgE-FA onset, older people are more likely to report the onset of each IgE-FA during adulthood. However, some of these specific IgE-FAs are far more likely to start during adulthood , while others are more likely to start during childhood  (P < .001), physician-diagnosed IgE-FA , and multiple IgE-FAs  compared to adults with solely adult-onset IgE-FA. Adults with both childhood- and adult-onset IgE-FA were also more likely to report a history of ≥1 severe food-allergic reactions (57.3%), physician-diagnosed IgE-FA (54.2%), and multiple IgE-FAs (74.8%) than either group individually .Adults with solely childhood-onset IgE-FA were more likely to report a history of ≥1 severe food-allergic reactions , 1 or more lifetime ED visits , and 1 or more allergy-related ED visits in the past year  compared to adults with adult-onset IgE-FA. This trend also remained consistent across the 9 common food allergens, with childhood-onset rates being higher than adult-onset rates .Specific FA reaction symptoms respondents reported experiencing during their most severe food-allergic reaction differed greatly between adults with childhood-onset and adult-onset IgE-FA. Across the 9 most common food allergens, adults with childhood-onset IgE-FA reported a higher frequency of stringent skin, respiratory, gastrointestinal, and cardiovascular symptoms than the adults with the corresponding adult-onset IgE-FA. Even when analyzing the most prevalent IgE-FAs among adults , adults that developed the same IgE-FA during childhood were more likely to report severe reaction symptoms categorized  adults with solely childhood-onset IgE-FA, (2) adults with solely adult-onset IgE-FA, and (3) adults with both childhood- and adult-onset IgE-FA; as such, it provides data that help articulate differences in sociodemographic predictors, IgE-FA severity, reaction characteristics, health care utilization patterns, atopic comorbidities, and IgE-FA–related psychosocial burden according to timing of IgE-FA onset. The estimated 14 million US adults with solely childhood-onset IgE-FA reported higher frequency of severe symptoms than the estimated 5.8 million adults with only adult-onset IgE-FA. Adults with childhood-onset IgE-FA had higher rates of health care utilization, with more than half having a physician-confirmed IgE-FA diagnosis, compared to only 30% of adults with adult-onset IgE-FA. The adults with childhood-onset IgE-FA also reported higher QoL impairment than adults with solely adult-onset IgE-FA.,,,,,The demographic data from this study revealed sex differences in age-specific IgE-FA: men had a higher prevalence of IgE-FA in all 3 categories of adults with IgE-FA, but women had a higher risk of developing adult-onset IgE-FA. However, the demographic characteristics associated with pediatric IgE-FA, such as race and ethnicity, were not directly associated with the risk of adult-onset IgE-FA. The demographic characteristics of each group of adults with IgE-FA align with previous research indicating that childhood-onset IgE-FA is more prevalent among male subjects, and that female subjects have a higher risk of developing IgE-FA during adulthood.,Adults with solely childhood-onset IgE-FA were more likely to report experiencing severe symptoms, such as chest tightening or vomiting, compared to adults who developed the same IgE-FA during adulthood. The current literature describes how children with peanut, tree nut, or shellfish allergies report some of the most severe IgE-FA–related reaction symptoms,IgE-FA–related health care utilization was significantly higher for adults with solely childhood-onset IgE-FA compared to those with adult-onset IgE-FA. This includes higher rates of physician-confirmed IgE-FA diagnosis, EAI prescriptions, diagnostic testing, and ED visits, all of which support previous research showing that adults with adult-onset IgE-FA have lower levels of health care usage.,While our findings suggest that adults with adult-onset IgE-FA do not report high levels of allergist care, the survey data from adults with childhood-onset IgE-FA suggest that efforts to improve access to allergist care alone may be insufficient for improving IgE-FA management among the broader adult population with IgE-FA from a psychosocial perspective. Adults with solely childhood-onset IgE-FA had greater health care utilization than adults with solely adult-onset IgE-FA. Direct allergy care and hypervigilant management may be contributing to lower QoL because the group of adults with childhood-onset IgE-FA reported the greatest burden of disease. Previous literature describing the effect of EAI prescriptions and diagnostic testing  on psychosocial well-being show that there exists an association with high levels of stress and anxiety.,,,,,,,,,Our finding that an increased number of comorbid conditions resulted in increased odds of an adult having childhood-onset IgE-FA onset aligns with research that describes a strong relationship between childhood-onset IgE-FA and specific atopic comorbid conditions, such as asthma and atopic dermatitis.,,Adults with both childhood-onset and adult-onset IgE-FA reported the highest effect of IgE-FA on QoL, even after accounting for key sociodemographic and clinical allergy characteristics. These adults have likely faced certain known factors of living with IgE-FA associated with diminished QoL for the longest period of time, such as constant allergen avoidance, fear of allergic reaction, and diagnostic testing.With population-based surveys, there are some notable limitations, specifically related to likely self-reporting and recall bias relating to reported allergic reaction symptomatology and its attribution to specific perceived causative foods. Because the current reference standard of IgE-FA diagnosis is a double-blind placebo-controlled food challenge, which is not feasible for a population-based survey, stringent criteria regarding timing and type of symptoms were utilized to screen for the presence of convincing IgE-FA. As such, it is possible that some individuals identified as having “convincing IgE-FA” in the present study may in fact have distinct, non–IgE-mediated conditions—or none at all—and conversely, that some individuals with true IgE-mediated allergy may not be captured by our algorithm. However, by using the present “convincing IgE-FA” case definition, we are assessing the subpopulation of US adults who either truly do have IgE-FA or who do not actually have IgE-FA but are living their daily lives as if they do, and therefore experiencing the associated psychosocial burden.Among adults with FA, there are differences in reported reaction severity, health care utilization, comorbid conditions, and QoL according to the timing of FA onset, child or adult.Among the 3 groups of adults , there were differences in reaction severity, health care utilization, comorbid conditions, and QoL. In general, adults with childhood-onset IgE-FA reported more severe IgE-FA–related reaction symptoms and higher rates of physician-confirmed diagnoses and EAI prescriptions. The differences in reported comorbid conditions suggest that there may be differing mechanisms of IgE-FA development between childhood- and adult-onset IgE-FA. Overall, the phenotypic variances that are based on timing of IgE-FA onset reveal that tailored management strategies may be necessary in addition to the need for guideline-informed care, including physician diagnoses and EAI prescriptions. The age at which IgE-FA develops needs to be accounted for in the creation of treatment plans for adults with IgE-FA. As a result of these phenotypic differences, future research should further articulate possible mechanisms contributing to solely adult-onset IgE-FA because the current data highlight different factors than those known to be associated with solely childhood-onset IgE-FA.


Montagnia macrospora is a freshwater red alga introduced from South America to East Asia via the global aquarium trade. The earliest occurrence record of this alga in Taiwan is dated 2005. To determine whether M. macrospora has become invasive in Taiwan and to understand the traits that facilitated its invasion, we took a multifaceted approach that combines examination of ecological background and population genetic analysis. Our island‐wide survey showed that M. macrospora is widespread in the field across Taiwan, where the climate greatly differs from that of South America, and can self‐sustain for nearly a decade. Our population genetic analysis revealed a lack of genetic diversity of M. macrospora in Taiwan, consistent with the hypothesis that the alga expanded through asexual reproduction. Moreover, during our long‐term ecological assessments and field surveys, we observed that M. macrospora is an ecological generalist that can survive in a wide range of temperature, pH, illumination, and nutrient enrichment. Taken together, our data suggest that M. macrospora has successfully invaded the freshwater ecosystems of Taiwan, likely due to its ability to disperse asexually and to grow under broad environmental conditions. We hope that our study brings attention to invasive freshwater algae, which have been overlooked in conservation planning and management.Invasive freshwater macroalgae are rarely described.  Studies of freshwater red algae that combine several aspects of freshwater macroalgae biology and invasion biology, such as ours, are rare. Our study fills an important knowledge gap that will aid in the conservation planning and management of invasive freshwater red macroalgae that are overlooked or are yet to be discovered. Introduced alien species might cause negative ecological and socioeconomic impacts on the non‐native ranges when they become widespread invasive species  is a recently reported introduced case , Hong Kong, Taiwan, and Okinawa (Japan). A genetic analysis based on the plastid marker rbcL has suggested that there were at least two independent introductions of M. macrospora into East Asia , which are traded between South America and Taiwan , which is a widely used marker to evaluate interpopulation genetic variation in freshwater red algae . For nutrient measurements, water samples of 1000 ml were collected and stored in an ice box during transportation. Then, measurements of four nutrients  were taken in our laboratory using a SMARTSpectro spectrophotometer (LaMotte).To assess the distribution of s Figure . We meas2.2M. macrospora between its native range in South America and its non‐native ranges in Taiwan, we compared the climate of the 16 sampling locations of M. macrospora in South America surveyed in previous studies . To exclude highly correlated and redundant variables, we conducted a multicollinearity test using the variance inflation factor (VIF) using the R package usdm version 1.1‐18 .To examine the climatic difference of 2.3M. macrospora were collected from aquaria and field locations  in Taiwan  and COX3R (5′‐GGATCTACWAGATGRAAWGGATGT C‐3′) under the PCR conditions described in Vis et al. .Thirty specimens of an Table . Two adds et al. . Sanger cox2‐3 sequences of M. macrospora specimens found in the native range of the alga in South America from GenBank . We conducted the surveys once a month except in December 2012 due to logistic issues and weather conditions. The four sites were 10 m apart from each other, going from downstream (site 1) to upstream (site 4) Figure . The strM. macrospora at each site, a transect was placed underwater across the width of the stream bed  (the width ranged between 50 to 380 cm), then five 10 × 10 cm quadrats were positioned along the transect at equal distance. The distance between quadrats varied depending on the total length of the transect. The percent cover of the algae was calculated as the average of the percent cover of the five quadrats of the site. M. macrospora was identified in situ based on the color (blue‐green) and the overall external morphology of the thallus. To further confirm the species identity of the algae, five specimens per site were randomly collected from the quadrats and brought back to the laboratory. This was followed by cox2‐3 molecular verification for random samples as a training process for confirming our morphological identification. Eleven of these sequences were utilized for the population genetic analysis (see Section −2 s−1), water temperature (ºC), pH, conductivity (µS cm−1), turbidity (mg L−1), water velocity (m s−1), and water depth (cm) were measured at each of the four sites by using a portable digital photometer , a thermometer, a pH meter , a meter for both conductivity and turbidity , a flow probe , and a ruler, respectively. The levels of four nutrients  were measured as described above. Precipitation data at Zhongli automatic weather station, located in Zhongli District, Taoyuan City was retrieved from the Central Weather Bureau, Taiwan (https://www.cwb.gov.tw), and hours of daylight were retrieved from the National Oceanic and Atmospheric Administration (NOAA) .The four sites showed variation in vegetation and surrounding structures, which caused differences in illumination. Site 3 was most exposed to sunlight, and the other three sites showed different degrees of tree canopy shading. Site 1 was muddier than the other sites, sites 2 and 3 had cemented stream beds, and site 4 had silt in its bed. To calculate the percent cover of M. macrospora and environmental variables among the four sites and determined which environmental variable was significantly correlated with the percent cover for each site. First, to determine whether there was any variation in the percent cover of M. macrospora and environmental variables among the four survey sites, we applied a one‐way Analysis of Variance (ANOVA), followed by multiple comparisons among each pair using a post hoc Tukey's test if significant effects were detected. Prior to ANOVA, the light intensity of each month was normalized among the four sites based on the relative light percent difference . Second, to examine the effects of the environmental variables on the percent cover of M. macrospora at each site, we applied multiple regression analyses. Ten environmental variables were included in the analysis: water temperature, pH, conductivity, turbidity, ammonia, phosphate, nitrite, nitrate, accumulated precipitation one week before collection, and day length. Prior to the regression analysis, the ten variables were standardized as z‐scores. We did not include light intensity, water velocity, and water depth, because they were affected by rain clouds and rainfall during or before our field work. If significant effects were detected, we additionally calculate the Pearson correlation coefficients among the ten selected environmental variables, as some of them had the potential to be highly correlated to each other . All the statistical analyses were performed in R  locations in Taiwan . Similarly, the nutrient levels varied widely across the sites in Taiwan. The levels of ammonia ranged from 0.18 to 0.76 mg L−1; Taichung Industrial Park Outlet was an outlier, with a level of ammonia of 146 mg L−1 in 2014 (at least 150 times higher than the other locations) that decreased to 1.21 mg L−1 in 2021. The nitrite levels ranged from 0.02 mg L−1 to 1.4 mg L−1. The nitrate levels ranged from 1 mg L−1 to 6 mg L−1; Taichung Industrial Park Outlet and Nanshi river were outliers, with 101 mg L−1 and 260 mg L−1 in 2014 (at least 20 and 50 times higher than the other locations), respectively. The phosphate levels were low, ranging from 0.01 to 0.27 mg L−1; again, Taichung Industrial Park Outlet was an outlier with 109 mg L−1 in 2014 and 210 mg L−1 in 2021, at least 400 times higher than the other locations  compared to most of the sites in Taiwan, while the level of nitrate (1.1 mg L−1), nitrite (0.22 mg L−1), and phosphate (0.79 mg L−1) were lower  , and a less shaded site (site 1) had higher light intensity (3 to 8000 µmol photons m−2 s−1). In contrast, site 3 was a well‐lit, open site that had the highest light intensity .Over our 14‐month field surveys in the Taoyuan stream, of all the parameters measured, the percent cover of ) Figure . Over thM. macrospora at site 3 followed a seasonal pattern. It was significantly correlated with day length and went as high as 36.4% during the winter when the days were shorter (10 h 49′) and the water temperature was lower , and it went as low as 1.2% during the summer when the days were ~3 h longer and the water temperature was higher (up to 27.3°C)  Figure .M. macrospora . We observed that M. macrospora bloomed in 2021, its percent cover exceeded 50% of all available substrates, such as bamboo shoots, grass shoots, and fishing nets . Besides V. dubyana, these species are not native to Taiwan or Asia. Also, we found three freshwater red algae that are commonly present in aquaria: Compsopogon caeruleus, Nemalionopsis shawii, and Sheathia dispersa . In our monthly survey of the Taoyuan stream, M. macrospora was found with other aquarium‐associated macrophytes, indicating that the alga is likely released into the environment via the disposal of aquarium water and/or unwanted aquatic plants. This observation is consistent with the findings by Zhan et al. . This result is consistent with the hypothesis that a founder population of M. macrospora from the native range was introduced in a single event. Then, its spread was probably followed by rapid clonal expansion via asexual reproduction among the local aquarium shops or farms, or by secondary dispersal via waterfowl or rats as viable algae can be found on their feet or in their gut ; in the Senbaru‐ike Pond, Okinawa from 2006 to 2013 (~eight years); and in the Taichung Industrial Park Outlet from 2014 to 2021 (~eight years)  in cool to warm waters (~17 to ~30°C); (2) in acidic to basic waters ; (3) under shaded (<65 µmol photons m−2 s−1) and exposed habitats (>500 µmol photons m−2 s−1); (4) in oligotrophic/mesotrophic waters and highly eutrophic waters ; and (5) on abiotic  and biotic substrates . Our comparison between the climate of the M. macrospora locations in South America and Taiwan showed that the two environments mainly differ in diurnal and annual temperature oscillations, and precipitation, with overall greater temperature oscillations but less precipitations in Taiwan. This observation further supports that M. macrospora is an ecologically generalist species. M. macrospora was reported in Taiwan in the Taoyuan stream from 2005 to 2013 and in the Taichung Industrial Park Outlet from 2013 to 2021, suggesting that its population can self‐sustain and even bloom in new environments for a long period of time.Studies have shown that species of freshwater red algae usually are not ecologically generalists. Most species of freshwater red algae exhibit a narrow range of environmental preferences, for example, dim light conditions, moderate temperature fluctuations, certain acidity, and clean waters . It will be crucial to further examine the relative contribution of these factors on the growth of M. macrospora in future controlled experiments. Nevertheless, these observations suggest that the areas in East Asia with such environmental properties may have a higher risk of invasion by M. macrospora from the global aquarium trade. For instance, the open and less‐shaded aquatic ecosystems in higher latitudes  that have strong seasonal patterns are likely most vulnerable to the invasion by M. macrospora.Our monthly temporal surveys in the Taoyuan stream indicate that constant illumination and strong seasonality  favor the growth  of M. macrospora was the dominant species in the periphyton community in the Taoyuan stream for over a year and that (2) it was extensively growing on other substrates in the Taichung Industrial Park Outlet, we speculate that M. macrospora, owing to its ecophysiological flexibility, potentially exerts an ecological impact by outcompeting native macroalgae. Recognizing M. macrospora as an invasive species is the very first step to understand its impact on the invaded regions, such as Taiwan and probably other regions of East Asia.Identification of invasive species is important to conservation planning and management, especially in islands. Islands are key hubs of the global aquarium trade , suggesting that there may be more species of invasive freshwater macroalgae introduced via the global aquarium trade that have gone undetected and unrecognized. We encourage researchers who specialize in conservation biology and management with an interest in algae ecology to collaborate with phycologists in the future to improve our understanding of the impacts that freshwater algae make on introduced habitats.New species of invasive freshwater macroalgae are rarely reported ; Data curation ; Formal analysis ; Methodology ; Software ; Visualization ; Writing – original draft ; Writing – review & editing . Lanwai Yeh: Data curation ; Formal analysis ; Methodology ; Software ; Visualization ; Writing – original draft ; Writing – review & editing . Shing Hei Zhan: Conceptualization ; Validation ; Visualization ; Writing – review & editing . Shao‐Lun Liu: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Supervision ; Validation ; Visualization ; Writing – review & editing .https://datadryad.org/stash/share/fYc_wy2AVGrDDnfesD8XCX0itVgY6ZDoNIIbDRx_Ig.This article has been awarded Open Data, Open Materials, Preregistered Research Designs Badges. All materials and data are publicly accessible via the Open Science Framework at Supplementary MaterialClick here for additional data file.


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