ICCG, FAST-PETase,HotPETase, and PES-H1L92F/Q94Y). We show that FAST-PETaseand HotPETase have intrinsic limitations that may not permit theirapplication on larger reaction scales, mainly due to their relativelylow depolymerization rates. With 80% PET depolymerization, PES-H1L92F/Q94Y may be a suitable candidate for industrial reactionscales upon further rounds of enzyme evolution. LCCICCG outperforms the other enzymes, converting 98% of PET into the monomericproducts terephthalic acid (TPA) and ethylene glycol (EG) in 24 h.In addition, we optimized the reaction conditions of LCCICCG toward economic viability, reducing the required amount of enzymeby a factor of 3 and the temperature of the reaction from 72 to 68°C. We anticipate our findings to advance enzymatic PET hydrolysistoward a coherent assessment of the enzymes and materialize feasibilityat larger reaction scales.In recent years, enzymatic recycling of the widely usedpolyesterpolyethylene terephthalate (PET) has become a complementary solutionto current thermomechanical recycling for colored, opaque, and mixedPET. A large set of promising hydrolases that depolymerize PET havebeen found and enhanced by worldwide initiatives using various methodsof protein engineering. Despite the achievements made in these works,it remains difficult to compare enzymes’ performance and theirapplicability to large-scale reactions due to a lack of homogeneitybetween the experimental protocols used. Here, we pave the way fora standardized enzymatic PET hydrolysis protocol using reaction conditionsrelevant for larger scale hydrolysis and apply these parameters tofour recently reported PET hydrolases (LCC Consequently, more sustainablesolutions that are in line with a circular economy are urgently needed.PET, composed of monomers linked by ester bonds, can be enzymaticallyhydrolyzed, yielding the products terephthalic acid (TPA) and ethyleneglycol (EG), which are suitable for a resynthesis of the polymer aftertheir purification. Nearly 20 years ago, the first hydrolase, a cutinase,was shown to specifically depolymerize PET.8 Sincethen, many other hydrolases have been isolated and enhanced throughprotein engineering.11 In 2020, leaf-branchcompost cutinase (LCC12) was engineeredinto a quadruple variant called LCCICCG to meet industrialrequirements.13 This study showed thatmonomers obtained through enzymatic hydrolysis under industry-relevantconditions could be purified and reused to obtain virgin PET, pavingthe way for the industrial deployment of enzyme-based PET depolymerization.Recently, FAST-PETase14 and HotPETase,11 two engineered variants of the poorly thermostable IsPETase15 from the bacterium Ideonella sakaiensis, were reported to show betterPET hydrolyzing performances than LCCICCG. Last, PES-H1L92F/Q94Y, a double variant of a metagenome-derived cutinase,was also shown to be a promising candidate for the deployment of enzyme-basedPET recycling solutions.16 A direct comparisonof the catalytic performances and potentials for larger scale applicationsremained very limited since the experimental parameters of all ofthese studies were widely different. Moreover, to adequately translatethese enzymatic performances into a relevant large-scale industrialdeployment, a list of key parameters should also be considered.18 Such key parameters are (i) PETcrystallinity and associated pretreatment, (ii) surface of exchangeand associated pretreatment, (iii) temperature of the enzyme-basedPET depolymerization accounting for Arrhenius’ law, polymer’sglass transition temperature (Tg), thermalinduced crystallization, and enzyme thermostability, (iv) enzyme catalyticefficiency, to compete with PET crystallization kinetics, (v) PETconcentration in the reactor, (vi) yield of the enzyme-based PET depolymerization,(vii) composition of the final products, and (viii) enzyme expressability are decisive factors. Manystudies have demonstrated that PET hydrolases preferentially act onthe amorphous regions of PET,21 and to the best of our knowledge,no PET hydrolases have been reported to act efficiently on highlycrystalline forms of the polymer, typically found in consumer products.22 It thus appears crucial to perform a feedstock pretreatment to transformsemicrystalline PET to its amorphous state in order to reach the highlevel of PET conversion (>90%) necessary to meet techno-economicgoals23 and to meet process-based lifecycle assessmentof virgin PET production24 23 , rendering its recovery difficult. Additionally, consideringthe high price of PET waste and the cost of postreactional waste treatment,a minimum PET conversion of 90%  must be reached to meetthe expectations of an economically viable industrial recycling process28 (Despite the many benefits of syntheticpolymers (plastics), theirinadequate end-of-life management is a global threat to the environment,affecting ecosystems globally and posing a serious health warning.sability1. The chuction24 1. Anothe OPEX)23 1. Enzymechains34 1. Howeveon rates 1. Anotherocess28 1.14 (–1 of extracellular protein) is crucial when operatingan industrial unit, mainly for OPEX considerations, but only a fewindustrial contractors can achieve an appreciable expression yieldusing dedicated industrial hosts  that would be lost during the purification scheme ifa dedicated postdepolymerization treatment is not amended14 1. Finallal hosts 1. This sICCG,13 FAST-PETase,14 HotPETase,11 and PES-H1L92F/Q94Y.16 First, their respective PET depolymerization performanceswere evaluated using a standardized substrate (micronized amorphousGoodfellow film) at five different reaction temperatures matchingthe optimum conditions reported in the corresponding studies. Furthermore,larger scale setups using amorphized micronized postconsumer PET bottleflakes were implemented as a proof of principle for economic feasibility.We found that FAST-PETase and HotPETase performances were markedlydeteriorated once evaluated in the industrial-process-relevant–experimentalsetup used here and that LCCICCG outperformed all otherenzymes tested. We conclude that a standardization of experimentalreaction parameters of enzymatic PET degradation, as provided in thisarticle, is advisable to advance enzymatic PET hydrolysis toward industrialapplications.Taking all these industrial key parameters into consideration,we evaluated the performances of four recently reported pioneeringPET hydrolases, which are LCC36 Amorphous Goodfellow film is a commerciallyavailable substrate suitable to compare enzyme performances. Exchangesurface is a key parameter for the efficient hydrolysis of PET, andseveral PET hydrolases have shown very poor performances on amorphousPET film compared to amorphous PET powder.22 Consequently, cryo-ground Goodfellow films sieved under 500 μm(Gf-PET) appear as good candidates for further enzymatic reactions.Comparative performance assessments were performed using an initialPET concentration of 2 gPET L–1  in a low ionic strength buffer but sufficientto keep a stable pH over a theoretically full PET conversion, where10.4 mM of TPA would be released. To facilitate the evaluation ofthe four different biocatalysts studied here ,16 the enzyme/substrate ratio , the specific activity , as well as the final PET conversion obtainedafter a given time of enzyme treatment are provided as already pointedout in recent reviews.39 All biocatalysts were purified from Escherichia coli as the expression host  and subjected to differential scanningfluorimetry (DSF) for melting temperature assessments . The evaluated Tm matched the literature values (Table S1). FAST-PETase has the lowest melting temperature (63.3 °C),followed by PES-H1L92F/Q94Y (77.6 °C), HotPETase (80.5°C), and LCCICCG (91.7 °C). As mentioned above,it is very important to perform PET depolymerization at a high temperature,close to the Tg of PET which is near 70°C43 in aqueous solution. Enzymes’ performances were evaluatedat low (nonsaturating) enzyme concentration (0.2 mgenzyme gPET–1) using 2 gPET L–1 and at five temperatures to match the optimal temperaturerange reported in the literature for each enzyme . The maximal temperature of 68 °C was chosento avoid PET recrystallization, which impedes enzymatic PET hydrolysisfor all known enzymes. In the light of the literature, performancesof FAST-PETase, PES-H1L92F/Q94Y, and LCCICCG were evaluated using 0.1 M phosphate buffer at pH 8.0 while performancesof HotPETase were monitored using 0.05 M glycine–OH bufferat pH 9.2, as reported in the earlier study.16 Kinetics ofPET depolymerization were assessed using UV absorbance analysis, whereall soluble products released during PET depolymerization  can be accounted for. Theresults of these PET depolymerizations are shown in Numerous strategies have successfullybeen developed to improve PET hydrolase performances in heterogeneoussystems. Nevertheless, there is still a lack of a standardized approachto rationalize the kinetics of these interfacial enzymes, thereforehampering fundamental and comparative descriptions of PET-hydrolases.ion host S1 and suion host S1 and suTPAeq h–1 mgenzyme–1 whenPET depolymerizationis performed at 45 °C and exhibited its highest SA at 50 °C,(348 μmolTPAeq h–1 mgenzyme–1) (Table S2) where the reactionreached a PET conversion of 10% of hydrolysis after 24 h before stoppingthe reaction (Table S2). When PET depolymerizationwas performed at 60 °C and above, FAST-PETase suffered from itslow thermostability and appeared to be destabilized very rapidly,with no significant activity detected. As expected for a more thermostableenzyme, the SA of HotPETase was improved from 211 μmolTPAeq h–1 mgenzyme–1 at45 °C up to 1351 μmolTPAeq h–1 mgenzyme–1 at 65 °C (Table S2). However, while 25% substrate conversionwas achieved at 60 °C, only 12% was reached at 65 °C, reflectingHotPETases’ low stability at temperatures higher than 60 °C(Table S2). Likewise, PES-H1L92F/Q94Y showed its highest SA at 65 °C (481 μmolTPAeq h–1 mgenzyme–1) (Table S2), and depolymerization stopped rapidlyat temperatures higher than 60 °C (ICCG showed its highest SA at 68 °C (963 μmolTPAeq h–1 mgenzyme–1) (Table S2), and its overall stability allowedthe reaction to proceed over 48 h at 68 °C (Table S2). From this first performanceevaluation, we decided to use each enzyme under its best temperaturecondition  to reassess the enzyme performances under an upscaled PET depolymerizationassay, including the new constraints previously described.FAST-PETase has a SA of 246 μmolan 60 °C 1c. Whileat 68 °C 1d, where5Gf-PET appears to be an appropriate uniform PETsubstrate accessible to everyone to perform a comparative evaluationof PET hydrolases through SA and stability studies. Nevertheless,it is of great interest to further corroborate these initial performancesby conducting enzyme-based depolymerization on a larger scale  using a significant amount of postconsumerwaste polymer (PcW-PET). Even though postconsumer bottle flakes arereadily available globally from various suppliers, these PET samplescan be produced by crushing a mix of water, soda, milk, or cosmeticsbottles. Still, this crushed material is highly crystalline , which makes it recalcitrant to enzymatichydrolysis. A suitable amorphous material was obtained by rapidlycooling previously melted PET pellets in an extruder at 265 °Cand cryogrinding them into sieved PET powder with a particle sizeof less than 500 μm.24 To maximize batch productivity, we aimed fora maximum PET loading in the reactor while ensuring that the disodiumterephthalate generated would remain soluble. Considering the disodiumterephthalate solubility (13% w/w),35 theaddition of a 20% (w/w) NaOH solution for pH regulation and the transformationof PET solid polymer into two water-soluble products, the initialPET waste concentration can be set at 15.5% (w/w). However, withinan industrial facility, an additional water volume must be consideredto clean the reactor, to flush circuits from the reactor to the filter,and to wash the postreaction filtrate. Two loadings of PET were thentested: 16.5% (w/w) and 20% (w/w), considering a post reaction additionof water of 3.5 and 20%, respectively.The investmentexpenditure (CAPEX) dedicated to the depolymerization section mainlyrelies on the productivity of the PET depolymerization, expressedin grams of products released per liter and per hour. This productivityis a function of three parameters: (i) the concentration of PET waste,(ii) the kinetics of the reaction, and (iii) the final conversionof PET and hence the yield of TPA and EG recovered, as underlinedby a life cycle assessment study of enzymatic PET recycling.L92F/Q94Y, and 68 °C for LCCICCG (Table S3). PETsubstrate conversion reached 15% after 24 h using FAST-PETase at 50°C (Table S3), but the PET depolymerizationrate slowed down considerably very early on (2 h after the beginningof the reaction), indicating insufficient stability of this enzymeeven at 50 °C .A maximum productivity of 10.8 gTPAeq L–1 h–1 can be estimated, but it decreases to an averageproductivity of 1.57 gTPAeq L–1 h–1 over 24 h stability, and the catalytic propertiesof HotPETase, including product inhibition, are still not alignedwith the requirements for its implementation in an enzyme-based PETdepolymerization process on the industrial scale. In contrast, the80% PET conversion obtained after 24 h (96% after 48 h) when usingPES-H1L92F/Q94Y at 60 °C (S3 and Table S3) emphasizes continuous enzyme performancewhen increasing the scale of the PET depolymerization assay. A maximumproductivity of 15.5 gTPAeq L–1 h–1 can be estimated as well as an average productivityof 4.75 gTPAeq L–1 h–1 over 24 h (ICCG converting 97% of the PET introduced into TPAand EG in 24 h (Table S3) with an average productivityof 5.8 gTPAeq L–1 h–1 and a maximum productivity of 28.6 gTPAeq L–1 h–1  PcW-PET depolymerizations were performed at 50 °Cwhen using FAST-PETase, 60 °C for HotPETase and PES-H1at 50 °C 2. Conseq LCCICCG S3. PETs LCCICCG S3. PETs LCCICCG S3. PETs LCCICCG S3. PETsTable S3). After around 3 h of reaction time using FAST-PETase, HotPETase,PES-H1L92F/Q94Y, and LCCICCG, MHET accountsfor 58, 21, 57, and 43% of the acidic products released , respectively. Conversely, after24 h, TPA becomes predominant and represents 64, 100, 78, and 100%of the soluble acidic products released, respectively. At 48 h, MHETrepresents only 1% of the total products released by PES-H1L92F/Q94Y. Using these values, and considering that 2 mol NaOH is used toneutralize 1 mol TPA while 1 mol NaOH is used to neutralize 1 molMHET, 18.1, 26.4, 90.1, and 97.3% PET conversion were effectivelyreached after 24 h for FAST-PETase, HotPETase, PES-H1L92F/Q94Y, and LCCICCG, respectively (S3 and Table S3), and 96.2% was reached after 48 h forPES-H1L92F/Q94Y. These values agree with PET conversioncalculated from the residual dry weight assessment performed at theend of the reaction (Table S3) . Interestingly, when consideringthe whole reaction using LCCICCG at 68 °C, we noticedthat 58 and 79% PET conversion can be achieved in only 3 and 6 h,respectively, illustrating that the last 18 h of the PET depolymerizationis mostly dedicated to MHET hydrolysis. This MHET hydrolysis can becatalyzed by the enzyme, but it was also demonstrated to be due toits spontaneous hydrolysis.44 Such instabilityof the MHET might also be accelerated when PET depolymerization isperformed at a higher pH, as it is the general rule for ester hydrolysis45 and could explain the lack of MHET accumulationobserved during the PET depolymerization at pH 9.2 using HotPETase.Notably, during the first hours of the reaction, MHETrepresentsa significant amount of the degradation products . With theexception of PES-H1L92F/Q94Y, the increase in productivityis linear between the two PET loadings. For PES-H1L92F/Q94Y, the productivity is slightly decreasing between 16.5 and 20% loading.At 20% PcW-PET loading, the overall behavior is like the one observedat 16.5% for the four enzymes  PET depolymerization fromthe perspective of a viable large-scale biotechnological process development.We postulated that studies solely relying on enzyme specific activityas readouts were unable to assess the true applicability of the enzymesfor industrial deployment. One of the reasons is very likely the numberof new constraints to be considered on a larger scale. As a firststep, we have therefore performed a standardized small-scale PET depolymerizationstudy of four engineered PET hydrolases  to evaluate their optimal temperaturecondition. Such standardization comprised the use of cryo-ground amorphouscommercial Goodfellow film with known particle size distribution,a fixed enzyme/substrate ratio (mgenzyme gPET–1), and a fixed polymer concentration (gPET L–1). Enzymes’ performances were evaluatedat such small scale by determining standardized parameters such asfinal PET conversion/terephthalic acid formed after a given time andenzyme-specific activity values (μmolTPAeq h–1 mgenzyme–1 or gTPAeq h–1 mgenzyme–1) as recommended elsewhere.37 Then, athorough comparison of the four PET hydrolases was performed in bioreactorsusing high concentrations of pretreated postconsumer PET waste material(165 and 200 gPET kg–1) to mimic theconditions of an industrial process. Parameters such as the averageand maximum productivity (expressed in gTPAeq L–1 h–1), the PET conversion/terephthalic acid formedafter a given time, and the purity of the products were carefully assessed. We find that a typical weakness of the PETdegrading enzymes is the deterioration of the activity over a long–timereaction that is in turn required to achieve 100% substrate conversion.FAST-PETase showed convincing performances in terms of specific activitiesat both reactor scales, but the kinetics of PET conversion in thereactor dramatically slowed down to reach less than 20% at 24 h. Thisobservation appears to be in-line with the previous study where FAST-PETaseenabled a 90% PET depolymerization from a 45 gPET L–1 solution  after14 days at 50 °C but necessitating a daily replenishing of freshenzyme solution  tocompensate for the low stability of the enzyme.14 HotPETase, a more thermostable variant of the IsPETase, can achieve a higher conversion of 26% at 24 h. Likewise,Bell et al. have shown a substantial decrease in PET conversion after5 h of reaction using HotPETase correlated to enzyme deactivationbut not to product inhibition or PET.11 Comparatively, PES-H1L92F/Q94Y and LCCICCG appeared more stable over time in reactor conditions, and near-completePET conversions (∼98%) were attained within 48 and 24 h, respectively,at 16.5% PET loading . LCCICCG and PES-H1L92F/Q94Y thusappear as good candidates for the deployment of an enzyme-based-PET-depolymerizationprocess.In the past few years, numerouspublications dealing with the discoveryand engineering of PET hydrolases have been released. To improve theactivity and thermostability of such PETases, various concepts ofprotein engineering were implemented, such as rational design,L92F/Q94Y in the reactoris intriguing.Its Tm is lower than that of HotPETase and in small reactors, at low PET concentration,and in nonsaturating enzyme conditions, the reaction stops rapidlyat less than 12% conversion. This contradiction cannot be simply explainedby low stability or inhibition by the products. Further investigationsare necessary to solve this mystery.The behavior of PES-H1ICCG performance,13 reactionconditions have been largely improvedin this work. While 95% PET conversion was previously obtained after24 h using 3 mgenzyme gPET–1 at 72 °C,13 98% PET conversion wasobtained here by introducing 1 mgenzyme gPET–1 at 68 °C. Reducing the temperature of thereaction by 4 °C enabled the PET recrystallization kinetics tobe reduced, which favors obtaining high conversion. The positive outcomewas a reduction of the amount of enzyme by a factor of 3 while increasingthe final PET conversion in the same time frame and consequently decreasingthe cost of waste treatment. Such enzyme performance is meeting oneof the bottlenecks described in a comprehensive LCA study of enzymaticrecycling of PET, highlighting the need to achieve conversions greaterthan 90% at high PET concentrations to minimize postreaction waste.28 Finally, TPA and EG monomers are exclusively produced without theaccumulation of MHET in the solution, simplifying the purificationscheme of the products.Since the last study describingLCC28 Ultimately, new acid-tolerant PET hydrolases able to perform efficientPET depolymerization with no  need of soda for pH regulationwould also be of great interest to further strengthen the conceptof enzymatic PET hydrolysis in the frame of a circular economy.We believe that our study of PET hydrolaseswill contribute tofinding a consensus on the methods used and key parameters to considerwhen performing a large-scale PET depolymerization. Such a consensusis necessary for the deployment of the first generation of enzymaticPET recycling processes at an industrial scale. We also believe thatthis field will further benefit from new technologies to emerge aswell as new superior enzymes being able, for instance, to performefficient degradation of semicrystalline PET to minimize the PET pretreatmentsteps as underlined in a previous study.13 Gf-PET materialhas a Tg of 76.5 °C, a percentageof crystallinity of 7.7%, and is constituted of particles with sizeslower than 500 μm. PcW-PET material has a Tg of 78.4 °C, a percentage of crystallinity of 14.6%,and is constituted of particles with size lower than 500 μm(D90 < 400 μm and D50 between 200 and 250 μm).Amorphous commercial PET wasprovided by Goodfellow Cambridge Ltd.  and postconsumer colored-flake PET waste were provided bySorepla Technologie SA, a recycling company .Amorphous commercial PET powder (Gf-PET) and postconsumer colored-flakePET waste powder (PcW-PET), with 98% PET content, were prepared aspreviously described.L92F/Q94Y,16 FAST-PETase,14 and HotPETase11 were synthesized withcodon optimization for expression in E. coli cells  into the pET-26b(+)  vector between NdeI and XhoI restriction sites. The geneencoding for the leaf-branch compost cutinase (LCC) variant ICCG wascloned as described previously.13 A listof all nucleotide and amino acid sequences of the genes used in thestudy is provided in the Supporting Information.The genes encoding PES-H1E. coli BL21 (DE3) competent cells by cultivation in ZYM auto-induciblemedium46 for 23 h at 21 °C. The E. coli cells were harvested by centrifugation  and suspended in lysis buffer . Cells were disrupted bysonication on ice, and the lysate was clarified by centrifugation. The soluble fractionwas applied to TALON metal affinity resin . After unboundproteins were washed with the lysis buffer supplemented by 10 mM imidazole,bound proteins were eluted with elution buffer . The buffer was finally exchangedfor storage buffer  using Hiprep 26/10desalting column . Purified protein concentrationwas determined based on the calculated molar extinction coefficientat 280 nm. Protein purity was evaluated by SDS–PAGE analysis.Genes were expressedin ICCG,FAST-PETase, HotPETase, and PES-H1L92F/Q94Y by determiningtheir melting temperature (Tm). Proteinsamples were prepared at a concentration of 6.25 μM and storedin buffer consisting of potassium phosphate  for LCCICCG, FAST-PETase, and PES-H1L92F/Q94Y, or in glycine–OHbuffer 50 mM, pH 9.2 for HotPETase. The SYPRO Orange dye 5000×stock solution in DMSO was first diluted to 250× in water. Proteinsamples were loaded onto a white clear 96-well PCR plate  with each well containing afinal volume of 25 μL. The final concentration of protein andSYPRO Orange dye in each well was 6 μM and 10×, respectively.Loaded volumes per well were as follows: 24 μL of the 6.25 μMprotein solution and 1 μL of the 250× SYPRO Orange dilutedsolution. The PCR plates were then sealed with optical-quality sealingtape and spun at 2000 rpm for 1 min at room temperature. DSF experimentswere then carried out using a Bio-Rad CFX96 real-time PCR system seton the FRET channel to use the 450/490 excitation and 560/580 emissionfilters. The samples were heated from 25 to 100 °C at the rateof 0.3 °C s–1. A single fluorescence measurementwas taken every 0.03 s. The Tm was determinedfrom the peak of the first derivatives of the melting curve usingthe Bio-Rad CFX Manager software. Tm valuescorrespond to the average of three measurements.DSF was used to assess the thermostability of LCCenzyme mL–1 (0.7 μM of enzyme) solution of purified protein  in100 mM potassium phosphate buffer, pH 8.0 for LCCICCG,FAST-PETase, and PES-H1L92F/Q94Y, or in glycine–OHbuffer 50 mM, pH 9.2 for HotPETase. Samples were harvested at 2, 4,6, 9, 24, 48, 72, and 96 h of reaction time and analyzed by ultravioletlight (UV) absorbance measurements at 242 nm for the determinationof PET depolymerization kinetics (see below). Reactions were performedin triplicate.A 49 mL aliquot of potassium phosphate buffer  or glycine–OH buffer  was combinedwith 100 mg of Gf-PET in a 100 mL glass bottle and incubated at 45,50, 60, 65, or 68 °C in a stirring dry bath 15–100  under agitation at 200 rpm until the solutionreached the desired temperature. The depolymerization was initiatedby adding 1 mL of a 0.02 mgTPAeq L–1 h–1 were estimated from a lineartime frame of the NaOH consumption kinetics generated using FAST-PETase,HotPETase, PES-H1L92F/Q94Y, or LCCICCG. Averageproductivities in gTPAeq L–1 h–1 were estimated after 24 h of reaction. An additional average productivityin gTPAeq L–1 h–1 wasspecifically estimated after 48 h of reaction when using PES-H1L92F/Q94Y.PET depolymerizationreactions at 20% PET (w/w) were performed using 49 mg of purifiedprotein (1.7 μmol) prepared in 195 mL of potassium phosphatebuffer  or glycine–OH buffer that was combined with 50 g of PcW-PET (98% purity). PET depolymerizationreactions at 16.5% PET (w/w) were performed using 40.43 mg of purifiedprotein (1.4 μmol) prepared in 203.75 mL of potassium phosphatebuffer  or glycine–OH buffer that were combined with 41.25 g of PcW-PET (98% purity). Reactionswere performed in a 500 mL Benchtop F1 0.5 MB Bioreactor . Temperature regulation was performed in the water-jacketedbioreactor, and a double Rushton impeller was used to maintain constantagitation at 800 rpm. The pH value was regulated to pH 8.0 or 9.2by the addition of a 20% NaOH (w/w) solution using the ROSITA 2.0software . The kinetics of the PET depolymerizationwas followed based on NaOH consumption, considering the exclusiveproduction of TPA and EG. Terephthalic acid has two carboxylic acidgroups; therefore, 1 mol of NaOH titrates 0.5 mol of terephthalicacid. Thus, the conversion of PET to terephthalic acid can be easilycalculated from the amount of NaOH consumed. In addition, sampleswere harvested at different time points and analyzed by UHPLC (seebelow) to adjust PET conversion overtime by considering the TPA/MHETratio (1 mol of NaOH is consumed to titrate 1 mol of the monoacidMHET). The final yield of the PET depolymerization assay was determinedeither by NaOH consumption or by dry weight determination of residualPET. To determine dry weight of residual PET, the entire reactionsolution, including solid particles, was filtered through a 12 to15 μm grade 11 ashless paper filter  and dried. Maximum productivities in g47 Briefly, the absorbance of the reaction mixturesin the ultraviolet region of the light spectrum (at 242 nm) indicatesthe release of soluble TPA or its esters from the insoluble PET substrate. Standard curves of TPA, MHET, andBHET were performed at 242 nm using an Eon Microplate Spectrophotometer. An average coefficient of 16,400 M–1 cm–1 corresponding to a combination of these productswas used. Samplings performed at different times  during the hydrolysis of Gf-PET wereanalyzed by absorbance reading at 242 nm. If necessary, samples werediluted in potassium phosphate buffer . The absorbancevalue is used to calculate the overall sum of soluble PET hydrolysisproducts according to the Lambert–Beer law. The SA of PET hydrolysisin μmolTPAeq h–1 mgenzyme–1 was determined in the linear part  of the hydrolysis curve of the reaction.Alternatively, when enzymes suffered from poor thermostability, specificactivity was determined between 0 and 2 h of reaction time. The termTPAeq corresponds to the sum of soluble products releasedfrom the hydrolysis the PET polymer .Kinetics of Gf-PET enzymatic depolymerization were followed byUV light absorbance using a method adapted from Zhong-Johnson et al.2S04 at 1 mL min–1 through a Discovery HS C18 HPLC column  equipped with a precolumn . TPA,MHET, and BHET were quantified according to standard curves, preparedfrom commercial TPA and BHET  and in-housesynthesized MHET,13 under the same conditionsas for the samples.The concentrations of TPA, MHET, and BHET were monitored by UHPLC.When required, samples were diluted in potassium phosphate buffer. Then, 150 μL of methanol and 6.5 μLof HCl 6 N were added to 150 μL of a (diluted) sample. Afterhomogenization and filtering through a 0.45 μm syringe filter,20 μL of the sample was injected into a UHPLC column. The chromatographysystem used was a Vanquish UHPLC system  equipped with a pump module, an autosampler, a columnoven thermostated at 25 °C, and a UV detector at 240 nm. TPA,MHET, and BHET were separated using a gradient of methanol (30 to90%) in 1 mM H


Quinoa is an important economic crop, drought is one of the key factors affecting quinoa yield. Clarifying the adaptation strategy of quinoa to drought is conducive to cultivating drought-tolerant varieties. At present, the study of quinoa on drought stress-related metabolism and the identification of related metabolites are still unknown. As a direct feature of biochemical functions, metabolites can reveal the biochemical pathways involved in drought response.Here, we studied the physiological and metabolic responses of drought-tolerant genotype L1 and sensitive genotype HZ1. Under drought conditions, L1 had higher osmotic adjustment ability and stronger root activity than HZ1, and the relative water content of L1 was also higher than that of HZ1. In addition, the barrier-to- sea ratio of L1 is significantly higher than that of HZ1. Using untargeted metabolic analysis, a total of 523, 406, 301 and 272 differential metabolites were identified in L1 and HZ1 on day 3 and day 9 of drought stress. The key metabolites  accumulated differently in quinoa leaves. and HZ1 had the most DEMs in Glycerophospholipid metabolism (ko00564) and ABC transporters (ko02010) pathways.These results provide a reference for characterizing the response mechanism of quinoa to drought and improving the drought tolerance of quinoa.The online version contains supplementary material available at 10.1186/s12870-023-04467-6. Eucommia ulmoides leaves decreased significantly, and the stomatal area, stomatal size and stomatal aperture decreased [Drought is extremely unfavorable to agriculture and livestock, especially drought is one of the key environmental stress factors affecting crop growth and production . Droughtecreased . Li ‘s recreased . Thus, pAt present, there are about 200,000-1000000 metabolites in plants . Under sChenopodium quinoa Willd) is an annual dicotyledonous herb. Its seeds contain all the nutrients required by the human body, and the proportion of amino acids is balanced. It has extremely high edible value and medical and health care value such as anti-cancer [Quinoa  were selected for research, both from Gansu Academy of Agricultural Sciences, they were identified by Gansu Academy of Agricultural Sciences. Pest-free, plump and size - consistent quinoa seeds were selected for this study. Quinoa seeds were washed three times with distilled water, soaked in 95% ethanol for 5–6 min, and finally disinfected with 1% sodium hypochlorite for 5 min. Seeds were sown in pots containing 2 kg sandy loam soil, 10 seeds per pot, covered with 0.1 cm thick vermiculite, placed in a greenhouse, and watered normally. During the experiment, the greenhouse environment was maintained at 25 °C / 18 ± 1 °C (day/night), and the relative humidity was 75 ± 5%. When quinoa seedlings grew to 2 months(Four leaves one heart period), the experimental group was treated with 20% polyethylene glycol-6000 (PEG-6000) to simulate osmotic stress (ψs = -0.49 MPA), and the control group was well watered. All quinoa leaves were taken on days 0,3,6,9 and 12 after treatment. Each biological replicate contained six individual plants under drought stress. The sample leaves were taken from top to bottom from the third to fourth leaves.Leaves were collected from control and drought plants at noon on 3 and 9 days of drought stress. Immediately after collection, freeze in liquid nitrogen and store at -80 ° C. Samples were lyophilized for 72 h and ground using TissueLyser, and lyophilized powders (50 mg) were used for untargeted global metabolite analysis based on UPLC-HRMS. 50 mg samples were weighed, and 1000µL of extract containing internal standard ( 1000:2 )  was added, and vortexed for 30 s ; add ceramic beads, 45 Hz grinder processing 10 min, ultrasonic 10 min ( ice water bath ) ; stand at -20 °C for one hour ; the sample was centrifuged at 4 °C, 12,000 rpm for 15 min; carefully remove 500 µL supernatant in EP tube ; drying the extract in a vacuum concentrator ; the dried metabolites were redissolved with 160µL extract ( acetonitrile-water volume ratio:1: 1). Vortex 30 s, ice bath ultrasound 10 min; the sample was centrifuged at 4 °C, 12,000 rpm for 15 min; carefully take out 120µL supernatant in 2mL injection bottle, each sample take 10µL mixed into QC samples for testing.Undetermined metabolomics analysis was performed on UPLC-HRMS (model: Acquity I-Class PLUS and Xevo G2-XS Q Tof mass spectrometer). Chromatographic separation was obtained on an Acquity UPLC HSS T3 column (1.8 μm 2.1*100mm). Both positive and negative ion modes were: mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile, injection volume was 1uL, the total running time of each sample was 51.35 min. Waters Xevo G2-XS QTOF high resolution mass spectrometer can collect primary and secondary mass spectrometry data in MSe mode under the control of the acquisition software . In each data acquisition cycle, dual-channel data acquisition can be performed on both low collision energy and high collision energy at the same time. The low collision energy is 2 V, the high collision energy range is 10 ~ 40 V, and the scanning frequency is 0.2 s for a mass spectrum. The parameters of the ESI ion source are as follows: capillary voltage: 2000 V (positive ion mode) or-1500 V (negative ion mode); cone hole voltage: 30 V; ion source temperature: 150 °C; desolvation gas temperature 500 °C; reverse blowing gas flow rate: 50 L / h; desolvation gas flow rate: 800 L/h. mass nucleus ratio (m/z) Collection range: 50-1200.The raw data collected using MassLynx V4.2 is processed by Progenesis QI software for peak extraction, peak alignment and other data processing operations, based on the Progenesis QI software online METLIN database and Biomark’s self-built library for identification, and at the same time, theoretical fragment identification and mass deviation All are within 100ppm.Principal Component Analysis (PCA) was performed with R language (R-3.1.1) and software package: scales, GGPLOT2, GGREPEL, scatterplot3d. It was used for preliminary understanding of the overall metabolic differences between groups and the magnitude of variability between groups. R language (R-3.1.1) was used for correlation analysis and software package: pheatmap. The difference multiple was analyzed by R language (R-3.1.1), software package: GGPLOT2. Orthogonal projections tolatent structures- discriminant analysis (OPLS-DA) was used to maximize differences in metabolic characteristics between control and drought groups, enabling detection of metabolites present in biological samples, using r language (R-3.1.1), software Package: ropls , VIP thrSoluble protein was determined by Coomassie brilliant blue method . SolubleOne leaf of quinoa was selected from each replicate and repeated for 3 times. A 1.5 cm × 1.5 cm cube was cut from the middle of the leaf connected to the midrib, fixed in FAA fixative , and stored in a refrigerator at 4 °C for routine paraffin sectioning. The materials were fixed by FAA for more than 24 h, dehydrated and transparentized by ethanol and xylene series, immersed in wax, and embedded. Slices (slice thickness 8 μm) were stained with safranine-fast green, and then sealed with neutral gum to make permanent sections. The sections were observed and photographed under a microscope. Each treatment was observed in 6 fields of vision. The structural parameters of leaf palisade tissue, sponge tissue, upper epidermis, lower epidermis and veins were observed and analyzed by ImageJ software.One quinoa leaf was selected from each replicate and repeated three times. A 1 cm × 1 cm square was cut from the side of the main vein in the middle of the leaf, fixed in an electron microscope fixative , and stored in a refrigerator at 4 °C for electron microscope scanning. Each treatment was observed under a scanning electron microscope and photographed in 15 fields. The number of stomata and the number of closed stomata were counted from the field of view of a 100 × mirror, and the stomatal density (number/mm2) and stomatal closure percentage were calculated. The area, length and width of each stoma were measured by ImageJ software in two 400 × visual fields. The length of stoma was the longest value parallel to stoma and the width of stoma was the widest value perpendicular to stoma. The average values of area, length and width of stoma in two visual fields were calculated. Each treatment was repeated 3 times and the results were averaged.Stomatal density (number·mm-2) = stomatal number / visual field area.Percentage of closed stomata (%) = Number of closed stomata per visual field / Number of stomata per visual field × 100.SPSS 13.0 was used to analyze the physiological parameters. All treatments were repeated 3 times. Mean values and calculated standard errors are reported.As shown in Fig. Combined with early physiological indicators, the 9th day quinoa leaves were selected to observe the effects of drought stress on leaf anatomical structure Table . It can Figure PLS-DA analysis was performed on drought and control conditions of drought-tolerant and drought-sensitive genotype materials at two different time points Fig. . The firThe KEGG annotation analysis showed that 846 metabolites Figure  were invAs shown in Fig. Based on the OPLS-DA model, the differential metabolites (DEMS) were screened by VIP value (VIP > 1) and P < 0.05. In L1,523 DEMs were identified on the 3rd day under drought stress, of which 102 DEMs were up-regulated and 421 DEMs were down-regulated, and 406 DEMs were identified on the 9th day under drought stress Table -Table S2In 4 group comparisons , we found 59 differentially expressed metabolites that were co-expressed; Among them, 47 differential metabolites were all down-regulated in the comparison of 4 groups after drought stress, and 5 differential metabolites were all up-regulated in the comparison of 4 groups after drought stress, 5(S)-hpete, Theasapogenol a, 8(R)-HETE, planagonine and His Gly Val; 3-phenyl-1-propanol were up-regulated in HZ1 and down-regulated in L1. Among them, 18 metabolites showed significant differences between treatment and control  and 42(44 DEMs) metabolic pathways were enriched in L 1 on Days 3 and 9 under drought stress, respectively, the top 20 metabolic pathways were showed in Fig. On Day 3 of drought stress, 37 DEMs in HZ1 were assigned to 42 metabolic pathways, with the most DEMs  in the Glycerophospholipid metabolism (KO00564) versus ABC transporters (KO02010) pathway; The next were Arachidonic acid metabolism (KO00590) pathway , Tryptophan metabolism (KO00380) pathway , Vitamin B 6metabolism (KO00750) pathway (5.5.56%), Isoquinoline alkaloid biosynthesis (KO00950) pathway (5.5.56%), Aminoacyl-tRNA biosynthesis (KO00970) pathway (5.5.56%), Nicotinate and nicotinamide metabolism (KO00760) pathway (5.5.56%), Glucosinolate biosynthesis (KO00966) pathway (5.5.56%), Glycine, Glycine, Glycine, Glycine and nicotinamide, serine and threonine metabolism (Ko00260) pathway (5.5.56%), 2-Oxocarboxylic acid metabolism (KO01210) pathway (5.5.56%), Phenylpropanoid Biosynthesis (KO00940) pathway (5.5.56%), Biosynthesis of amino acids (KO01230) pathway (5.5.56%), alpha-Linolenic acid metabolism (KO00592) pathway (5.5.56%) and Tropane, piperidine and pyridine alkaloid biosynthesis (KO00960) pathway (5.5.56%). In addition, 27 metabolic pathways each contained 1 DEMs, and the significantly enriched metabolic pathway was Glycerophospholipid metabolism (KO00564). On the 9th day of drought stress, 34 DEMs in HZ1 were assigned to 37 metabolic pathways. Arachidonic acid metabolism (KO00590) pathway , Phenylpropanoid biosynthesis (KO00940) pathway , and ABC transporters (KO02010) pathway had the most DEMs  Glucosinolate Biosynthesis (KO00966) pathway , Vitamin B 6metabolism (KO00750) pathway , Zeatin Biosynthesis (KO00908) pathway , Linoleic acid metabolism (KO00591) pathway , Glycine, serine and threonine metabolism (Ko00260) pathway , Biosynthesis of amino acids (KO01230) pathway , Tropane, piperidine and pyridine alkaloid biosynthesis (KO00960) pathway . In addition, there were 27 metabolic pathways with 1 dems, and the Vitamin B 6 metabolism (KO00750) pathway and Zeatin biosynthesis (KO00908) pathway were significantly enriched.Based on the KEGG metabolic pathway, we found that the α-Linolenic acid metabolism pathway was enriched in many different metabolites Fig. . We founThe ability of plants to maintain high water status under drought stress is an important strategy for them to cope with drought stress, and changes in relative water content are the best method for assessing water scarcity and physiological water status under water stress , higher The accumulation of amino acids in plants under drought stress, such as aromatic amino acid, is beneficial for plants to cope with drought stress. Increases in amino acid levels are thought to enhance plant resilience by influencing various physiological mechanisms, such as the regulation of osmolarity changes, ROS detoxification, and the regulation of intracellular pH levels . DroughtIn this study, we observed that the contents of tyrosine, tryptophan and L-Phenylalanine aromatic amino acid increased under drought stress Table , and a sNucleotide metabolism is one of the important components of metabolic pathways, in which nucleotide is an important component of nucleic acid. For the synthesis of carbohydrates, lipids, peptides, and secondary metabolites, nucleic acids provide nucleic acids as the ultimate energy source [Plant lipids are diverse and essential to cells. As hydrophobic barriers of membrane, they are essential for the integrity of cells and organelles. Furthermore, lipids are stored in seeds in the form of chemical energy, and they act as signaling molecules to regulate cellular metabolism . The majThe results showed that the leaves treated with 20% PEG6000 in quinoa genotypes with different sensitivity to drought had different mechanisms of metabolite accumulation and regulation, it is of great significance to better understand the mechanisms of abiotic stress tolerance. In a word, polypeptides, organic acids and amino acids are the main metabolites changed by drought stress. Tyr His Leu Cys, Gln Lys Cys Phe, Tyr Phe Tyr Phe, pentadecanoic acid, 5(S)-hpete, 8(R)-HETE, proline, L-Phenylalanine were shown after 20% PEG 6000 treatment, they can be considered as major drought stress-specific markers and osmoprotectants. Metabolomics approaches will improve our understanding of the mechanisms underlying quinoa dehydration and will become increasingly important in the future.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6


These results were more evident in females. Our study corroborates glial involvement in AD pathology, in which cannabinoid signaling plays an important role. 3×Tg-AD mice born with hippocampal astrocytes with differential gene expression of the ECS associated with an innate attenuation of their activity. In addition, we show that there are sex differences from birth in this AD animal, which should be considered when investigating the pathogenesis of the disease.Alzheimer’s disease (AD) is a common neurodegenerative disease. In AD-associated neuroinflammation, astrocytes play a key role, finding glial activation both in patients and in animal models. The endocannabinoid system (ECS) is a neurolipid signaling system with anti-inflammatory and neuroprotective properties implicated in AD. Astrocytes respond to external cannabinoid signals and also have their own cannabinoid signaling. Our main objective is to describe the cannabinoid signaling machinery present in hippocampal astrocytes from 3×Tg-AD mice to determine if they are actively involved in the neurodegenerative process. Primary cultures of astrocytes from the hippocampus of 3×Tg-AD and non-Tg offspring were carried out. We analyzed the gene expression of astrogliosis markers, the main components of the ECS and Ca Alzheimer’s disease (AD) is a progressive disorder that is characterized clinically by memory loss and pathologically by extracellular accumulation of beta-amyloid peptide (Aβ), neurofibrillary tangle formation, extensive neuroinflammation, synaptic dysfunction, neurodegeneration, and brain dysfunction ,2,3. TheNeuroinflammation, in which astrocytes and microglia play a key role, has recently been associated with AD pathogenesis. It was shown that this is not simply a consequence of the deposition of beta-amyloid plaques and neurofibrillary tangles ,5.Thus t2+ concentrations , with decreased Tnfa mRNA levels in hippocampal astrocytes of 3×Tg-AD female offspring compared to 3×Tg-AD male astrocytes  > 4.677; ons test D.Il6 mRNA expression was observed , with an overall decrease in Il6 expression in 3×Tg-AD female astrocytes compared to non-Tg (WT) female and 3×Tg-AD male groups  = 13.65 3; Cnr2 mRNA expression was found . An interaction between the two factors analyzed was also found in the Cnr2 expression . Hippocampal astrocytes from 3×Tg-AD female offspring had a lower level of Cnr2 than WT female astrocytes  astrocytes compared to the WT male group  = 6.881; p < 0.05], with an overall decrease in Daglb mRNA levels in 3×Tg-AD female astrocytes compared to 3×Tg-AD male group  = 4.167; p < 0.05].Two-way ANOVA also showed an interaction between two factors analyzed on Dagla, Mgll, and Faah were found.No effects of the factors analyzed on the expression of p < 0.05] and an interaction between sex and genotype in MAGL protein expression . Tukey post hoc analysis indicated a decreased of MAGL expression in hippocampal astrocytes from female offspring born to 3×Tg-AD mothers compared to WT female astrocytes  = 5.558; p < 0.05]. The uncorrected Fisher’s LSD post hoc test showed lower FAAH protein level in 3×Tg-AD male astrocytes compared to WT male group  = 6.416; To analyze the balance between endocannabinoid production and degradation, we calculated the ratio between the enzymes of synthesis and degradation of acylethanolamides and acylglycerols.p < 0.05]. Tukey post hoc analysis indicated an increased DAGLα/MAGL ratio in 3×Tg-AD female astrocytes compared to 3×Tg-AD male and WT female groups  = 7.081; p < 0.05] and an interaction between sex and genotype  in the NAPE-PLD/FAAH ratio. Tukey post hoc analysis indicated that hippocampal astrocytes from 3×Tg-AD male offspring had a higher NAPE-PLD/FAAH ratio than WT male astrocytes  = 7.187; No effects of the factors analyzed on the expression of DAGLα, MAGL, and DAGLβ/MAGL were found.Regarding the endocannabinoid levels measured in the culture medium: 2-Arachidonoylglycerol (2AG), 2-Linoleoylglycerol (2LG), palmitoleoylethanolamide (POEA), linoylethanolamide (LEA), palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and N-stearoylethanolamine (SEA).p < 0.05]. Although the uncorrected Fisher’s LSD test did not show significant differences between groups, we observed an overall increase in LEA levels in 3×Tg-AD astrocytes compared to WT (non-Tg)  = 4.638; p < 0.05], with an overall decrease in POEA and OEA levels in 3×Tg-AD astrocytes compared to WT, although these differences are not significant applying the uncorrected Fisher’s LSD post hoc test  > 4.963; p = 0.3255; female 3×Tg-AD vs. female WT p = 0.4667; No effects of the factors analyzed on 2AG, 2LG, PEA, and SEA levels were found. Despite the non-statistical significance of the results, we also observed evident differences between the groups analyzed in the levels of 2AG  = 5.152; p < 0.05] and an interaction between sex and genotype  were also found. Post hoc analysis (Tukey’s multiple comparisons test) showed higher levels of P2x5 in WT female astrocytes than males but decreased P2x5 mRNA expression in 3×Tg-AD female astrocytes than 3×Tg-AD male group  = 14.18; Gls mRNA expression , with 3×Tg-AD female astrocytes showing a lower level of Gls than WT female and 3×Tg-AD male groups  and subcellular level  in the hippocampal astrocytes of 3×Tg-AD female animals at PND 2-3.We found a profile similar to that of cannabinoid receptors, with an overall decrease in the main Ca2+ levels of astrocytes. In addition, CB1 receptors in astrocytes are also found in the mitochondrial membrane (mtCB1) [2+ flux between the endoplasmic reticulum and mitochondria; moreover, MCU inhibition affects subcellular spread of Ca2+ [2+ transit regulated by mtCB1, presenting a low profile of all the calcium receptors  , and its of Ca2+ . All the2+, and thus, producing the release of astrocytic glutamate [Gls; Nsmf; Glutamate is the main excitatory neurotransmitter; however, it can induce neuronal damage through excitotoxicity, which results from overactivation of glutamatergic receptors. This glutamate toxicity is implicated in the pathogenesis of AD and astrocytes may play a key role in it. Glutamate release from astrocytes contributes to NMDA-related long-term depression. This mechanism is controlled by CB1 activation of astrocytes, increasing the concentration of Calutamate . The hipFinally, it should be noted that most of the results presented show a clear sexual dimorphism, with changes in the markers analyzed being more present in females than in males. We found an interaction of sex and genotype in many of our results, i.e., the changes induced by genotype are sex-dependent.In humans, a sex difference in AD prevalence is described, but less so in brain pathology and underlying molecular mechanisms. In the 3×Tg-AD animal model, female mice show more amyloid plaques, neurofibrillary tangles, neuroinflammation, and cognitive deficits than males at 12 months of age . Our stuThe present study has several limitations. First, we were unable of measuring anandamide because of technical reasons. Anandamide is a relevant modulator of astrocytic networks through its role in the modulation of gap junctions-mediated calcium signals . Second,An important aspect not studied in the present work is the role of the alternative pathway for enocannabinoid synthesis and metabolism, involving the ABHD family of enzymes. The availability of free arachidonic or other poly-unsaturated fatty acids potentially engaging with the formation of prostaglandins and endoperoxides is very relevant and deserves further research.In any case, the data are sufficient to support the existence of sex and genotype dimorphic alterations in astrocytes, demanding a carefully differential experimental approach.All procedures were conducted in strict adherence to the principles of laboratory animal care  following the European Community Council Directive (86/609/EEC) and were approved by the Ethical Committee of the University of Málaga. Special care was taken to minimize the suffering and the number of animals needed to perform the procedures.The 3×Tg-AD-AD (Jackson Laboratory) mouse model combines mutant hAPP (Swedish), PSEN1 (MM146V), and tau (P301L) transgenes, resulting in Aβ and tau pathologies.Non-transgenic  and transgenic (3×Tg-AD) female adolescent mice were individually housed in standard cages and maintained under controlled room conditions: 21 ± 1 °C room temperature, 40 ± 5% relative humidity, and a 12-h light-dark cycle (lights off at 8:00 p.m.).Females were allowed to mate with males of the same strain in their home cage for 72 h. The presence of a spermatozoa plug in the vaginal smear confirmed successful mating, which was designated as gestational day 0 (GD0). At postnatal day 2–3, 3×Tg-AD and WT offspring  were sacrificed by decapitation.n = 8; 3×Tg-AD male: n = 8; WT female: n = 8; 3×Tg-AD female: n = 8), as described previously [2 and cultured for 24 h. They were then grown in serum free media for 24 h before freezing.Primary astrocytes were derived from male and female pups at PND2 separately  levels, which did not vary significantly between groups.Real-time PCR was performed using specific sets of primer probes from TaqMan Gene Expression Assays  were homogenized in 500 µL of ice-cold lysis buffer containing Triton X-100, 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1 M ethylenediaminetetraacetic acid (EDTA), sodium pyrophosphate, sodium fluoride (NaF), sodium orthovanadate (NaOV), and protease inhibitors using a tissue-lyser system . After centrifuging at 26,000× g for 30 min at 4 °C, the supernatant was transferred to a new tube. The Bradford method was used to measure the protein concentration of the samples. A quantity of 30 µg of each total protein sample was separated on 4–12% polyacrylamide gradient gels. The gels were then transferred onto nitrocellulose membranes  and stained with Ponceau red, Membranes were blocked in TBS-T  with 2% albumin fraction V from bovine serum  for 1 h at room temperature. The primary antibodies to the proteins of interest  formic acid in water and B, 0.1% (v/v) formic acid and 0.05 mM of NaCl in acetonitrile. The initial conditions were 60% A and 40% B. The gradient was increased linearly to 100% B over 5 min, maintained at 100% B for 3 min, and returned to the initial conditions for a further 4 min with a total run time of 20 min. The mass spectrometer was a Q Exactive from Thermo Scientific with an Orbritap detector. The spectral range was recorded between 270–410 m/z in positive mode for: DHEA, POEA, AEA, LEA, 2AG, 2LG, DEA, PEA, OEA, 2OG, and SEA. The desolvation gas temperature was 230 °C, a gas flow rate of 40 mL/min, auxiliary gas 15 mL/min, and sweep gas 2 mL/min. The spray voltage was set at 3.0 kV. The compounds were identified by their exact mass with an exactitude inferior to 2 ppm. A six-point external calibration curve was prepared with concentration between 0.5–15 ng/L for SEA, DEA, DHEA, AEA, LEA, and OEA and between 10–300 ng/L for 2AG, 2LG, 2OG, PEA, and POEA and the mix of internal standards .The chromatographic separation was carried out on a HPLC (high performance liquid chromatography tandem mass spectrometry) model ultimate 3000 from Thermo Scientific. The column was ACE Excel 2 C18  maintained at 40 °C with a mobile phase flow rate of 0.3 mL/min. The composition of the mobile phase was: A, 0.1% . IBM SPSS Statistics 23 and GraphPad Prism 6 software programs were used. Subsequent multiple comparisons between groups were carried out using Tukey adjustments or simple effect analysis (Fisher’s test) in cases of factor effect but no interaction. 2+ flux at birth in hippocampal astrocytes compared to non-transgenic animals, which appears to be associated with decreased endocannabinoid signaling. Therefore, we can ensure that astrocytes not only responded to the damage that will be generated throughout the development of the disease, but that they are actively involved in the neurodegenerative process, innately having differential activity compared to wild-type ones.In this study, we characterized the primary astrocytes of the 3×Tg-AD model in terms of cannabinoid signaling related to the phenotype resulting from the expression of human transgenes for AD. Here, we demonstrate for the first time that 3×Tg-AD mice display inflammatory activity and attenuated CaSex differences in humans and in this animal model of AD have already been established, but always using older individuals. Most of the results presented now show a clear sexual dimorphism, with changes of the markers analyzed being more present in females; thus, the present study shows that sex differences in this animal model of AD are evident from birth and, therefore, sex differences should be taken into account when investigating the pathogenesis of the disease.


To update and assess the efficacy and tolerability of once weekly subcutaneous semaglutide in patients with type 2 diabetes (T2D).PubMed, Science Direct, Cochrane Library, Clinical trial, Springer, OVID, China National Knowledge Infrastructure (CNKI), WanFang Data and China Science and Technology Journal Database (VIP) were searched from inception to January 18, 2023. Randomized controlled trials (RCTs) comparing subcutaneous semaglutide with placebo or any other antidiabetic agent in adults with T2D were eligible. The risk ratio (RR) and mean difference (MD) with 95% confidence intervals (CIs) were determined to synthesize the results.I2 = 91%; MD -1.36%, 95% CI , I2 = 84%, semaglutide 0.5 and 1.0 mg, respectively], body weight reduction, blood pressure control. At the same time, subcutaneous semaglutide 0.5 and 1 mg reduced HbA1c by 0.56% (95% CI 0.32 to 0.80) and 0.63% (95% CI 0.35 to 0.91) compared to other glucose-lowering agents. For tolerability, semaglutide did not increase the incidence of adverse events (AEs) and serious adverse events (SAEs), severe or blood glucose (BG) confirmed hypoglycaemia, acute pancreatitis and diabetic retinopathy compared to placebo or active comparators, but did increase the risk of nausea, diarrhea and vomiting.A total of 17 trials enrolling 14,940 T2D patients were included. For efficacy, compared with placebo, semaglutide exhibited beneficial effects on glycosylated hemoglobin A1c (HbA1c) control [MD -0.97%, 95% CI , Semaglutide has a better effect on glycaemic control and weight loss than other therapies. Nevertheless, semaglutide was associated with increased incidence of gastrointestinal-related disorders. Further large, multicenter randomized controlled clinical trials are still needed to obtain more robust evidence to better guide clinical treatment decisions.The online version contains supplementary material available at 10.1186/s13098-023-01195-7. Type 2 diabetes (T2D) is a metabolic syndrome characterized by long-term hyperglycemia, which is caused by insulin resistance and/or impaired pancreatic β-cell function . AccordiSemaglutide is a newly approved GLP-1 RA that reduces glucose levels by improving β-cell response, inhibiting glucagon secretion, and delaying gastric emptying , 5. In aTo date, in addition to the network meta-analysis, four meta-analyses have evaluated the efficacy and tolerability of subcutaneous semaglutide in T2D patients –11. In tThere are two main aims to update the meta-analysis of subcutaneous semaglutide. Firstly, four meta-analyses have been evaluated the efficacy and tolerability of subcutaneous semaglutide in 2018 . Since tThis research was operated according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement , 19, whiThe following electronic databases were searched: PubMed, Science Direct, Cochrane Library, Clinical trials, Springer, OVID, China National Knowledge Infrastructure (CNKI), WanFang Data, and the China Science and Technology Journal Database (VIP) from inception to January 18, 2023. The selected terms and search combinations were: “semaglutide” or “NN9535” in combination with “Diabetes Mellitus, Type II” or “Diabetes Mellitus, Noninsulin-Dependent” or “T2D”. The detailed search strategy is provided in Table Two reviewers (Hu and Su) independently identified studies and did screening and data extraction, while any disagreements were resolved by the third reviewer (Fan). RCTs that compared once-weekly subcutaneous semaglutide with placebo or any other antidiabetic agent in adults with T2D were included. Search results were imported into Endnote, Clarivate Analytics, a reference management software, for deduplication. After removing duplication, two reviewers independently screened all records by title and abstract in duplicate. Subsequently, potentially eligible records were assessed in full text.1c ≥ 7%; (2) treatment duration ≥ 12 weeks; (3) the primary outcome for this meta-analysis must be reported in the trial: reduction in HbA1c. The main exclusion criteria were: (1) RCTs not for T2D, but obesity, impaired glucose tolerance, gestational diabetes or type1 diabetes; (2) papers published in form of abstracts, review articles, hoc-analysis, pharmacoeconomics research or letter and comments; (3) duplicate studies.The detailed inclusion and exclusion criteria were listed in Table The extracted information included: first author, publication year, National Clinical Trial (NCT) number, duration of treatment, intervention in each trial arm, sample size, average age, diabetes duration, baseline HbA1c, body weight. The primary outcome for this meta-analysis was the change in HbA1c from baseline. Secondary efficacy outcomes included the change in body weight, fasting plasma glucose (FPG), self-measured plasma glucose (SMPG), systolic blood pressure (SBP), diastolic blood pressure (DBP), the number of participants achieving HbA1c<7%, the proportion of patients achieving weight loss ≥ 5% and ≥ 10%, the number of participants achieving HbA1c<7.0% without severe or BG-confirmed hypoglycaemia and without weight gain. Tolerability outcomes consisted of the incidence of adverse events (AEs), serious AEs (SAEs), hypoglycaemic events (severe or BG-confirmed symptomatic). With additional tolerability outcomes, the incidence of gastrointestinal adverse events , acute pancreatitis, and diabetic retinopathy were summarized.The quality of the included studies was assessed using the Cochrane Collaboration’s Risk of Bias Tool . Two revP < 0.05 (two-tailed) was considered significant. Heterogeneity across studies was evaluated by Cochrane’s Q and I2 statistics, considering the P value less than 0.10 or the inconsistency index (I2) statistic greater than 50% indicative of significant heterogeneity  and by 1.36% , respectively ; I2 = 91%, 8 studies; MD -0.63%, 95% CI ; I2 = 97%, 12 studies for semaglutide 0.5 and 1.0 mg, respectively] ; I2 = 97%, 10 studies for semaglutide 0.5 and 1.0 mg, respectively] and in a sensitivity analysis including only trials at low risk of bias.Compared to other active comparator, semaglutide was linked to a significant reduction in HbA1c  compared with semaglutide Table .I2 = 81%, 5 studies; MD -3.98 kg, 95% CI ; I2 = 68%, 7 studies for semaglutide 0.5 and 1 mg, respectively] ; I2 = 91%, 8 studies], and by 2.87 kg , respectively , In the subgroup analysis, compared with GLP-1 RAs, body weight significantly decreased with 1.0 mg semaglutide, whereas no significant difference was detected between semaglutide 0.5 mg and GLP-1 RAs. At the same time, compared with sitagliptin, the reduction in body weight was notably greater in both doses of semaglutide. Of note, compared with 1.0 mg semaglutide, tirzepatide was associated with a significantly stronger reduction in body weight Table .I2 = 76%, 5 studies]; this effect was enhanced with semaglutide 1 mg  compared to placebo , ebo Fig. .I2 = 91%, 8 studies], and by 0.92 mmol/L  , es] Fig. .Subgroup analyses revealed that semaglutide lowered FPG significantly compared with GLP-1 RAs and sitagliptin. It’s worth noting that compared to semaglutide, tirzepatide led to significant FPG reductions Table .Compared with placebo or active comparators, treatment with semaglutide was associated with a reduction in SBP Figs.  and S6.The results of subgroup analysis showed that, compared with other GLP-1 RAs, semaglutide 1 mg appeared to have significantly reduced blood pressure compared to the other GLP-1 RAs. In addition, semaglutide was related to a significantly stronger reduction in SBP than sitagliptin Table .In our analysis of changes in diastolic blood pressure, there was no difference between semaglutide and the control group Figs , S8.Against placebo and the active comparator, semaglutide showed greater SMPG reduction. Figs , S10.In the subgroup analysis, when compared with GLP-1 RAs, the change in SMPG was notably greater in the semaglutide group. At the same time, two trials , 22 compA greater percentage of patients achieved HbA1c<7%, HbA1c ≤ 6.5%, HbA1c < 7.0% without hypoglycaemia or weight gain with semaglutide 0.5 and 1.0 mg than with placebo or the active comparator Figs -S16.Comparing semaglutide 0.5 mg and GLP-1 RAs, the RR (95% CI) were 1.13  and 0.91  in the number of patients achieving HbA1c < 7.0% and ≤ 6.5% respectively Table .In comparison with the placebo, treatment with semaglutide did not increase the incidence of any adverse events, serious adverse events Figs -S24. ResThere was no statistically significant difference in the incidence of severe or blood glucose-confirmed hypoglycaemia between semaglutide and placebo.I2 = 0%, 7 studies] ; es] Figs -S26.Subgroup analyses showed no significant difference in the occurrence of hypoglycemia between semaglutide and GLP-1 RAs or sitagliptin Table .I2 = 0%, 3 studies; RR 1.65, 95% CI ; I2 = 72%, 5 studies for semaglutide 0.5 and 1 mg, respectively] and sitagliptin , except for the incidence of diarrhea and vomiting, which did not differ between semaglutide 1.0 mg and GLP-1 RAs ; As Table .In this meta-analysis, no difference was found between the semaglutide and control groups for the incidence of acute pancreatitis (AP). For subgroup analysis, semaglutide had a lower incidence of acute pancreatitis compared with GLP-1 RAs, whereas sitagliptin had a higher incidence. But the comparison of the results of the subgroup analysis was not statistically significant Table .Regardless of the type of control arms  or the treatment dose, semaglutide was not associated with an increase in the incidence of diaabetic retinopathy (DR) Table .This updated meta-analysis, including 17 RCTs that identified a further 5 RCTs, was conducted to comprehensively evaluate the efficacy and tolerability of subcutaneous semaglutide compared with placebo or other antidiabetic medications. In general, the results of this meta-analysis suggested that semaglutide showed a superior ability for glycemic lowering, body weight reduction, and blood pressure control compared with placebo or other hypoglycemic agents. Similar results were observed in the subgroup analysis comparing sitagliptin and GLP-1 RAs, except for body weight and SBP, which did not differ between the semaglutide 0.5 mg and GLP-1 RAs. Regarding tolerability outcomes, semaglutide did not increase the incidence of AEs, SAEs, or severe or BG-confirmed hypoglycaemia compared to placebo, which is consistent with the subgroup analysis. While semaglutide 0.5 mg can slightly decrease the risk of hypoglycaemia compared to an active comparator. However, treatment with semaglutide was associated with an increased risk of nausea, diarrhea and vomiting, but did not increase the risk of acute pancreatitis and diabetic retinopathy.The long-term goal of diabetes management is to prevent chronic complications, improve the quality of life, and prolong life through great metabolic control . MeanwhiData derived from the Framingham Heart Study suggested that T2D patients have a 2.5-fold increase in the cardiovascular disease (CVD) . AccordiIn addition, previous results reported that semaglutide can significantly reduce SBP level compared with counterparts in the SUSTAIN series of clinical trials, except for SUSTAIN 8 trial . In summGastrointestinal disorders were the most reported treatment-related AEs with semaglutide, which is consistent with other GLP-1 RAs . In geneIn this research, there were no statistical differences between semaglutide and other antidiabetic drugs in the incidence of AP and DR. Since the first case of pancreatitis in patients treated with exenatide in 2006, the tolerability of the pancreas for GLP-1 RAs has been a highly controversial topic in the past decade –54. HoweThere are some limitations. Firstly, there was significant heterogeneity between the included trials that was not eliminated with sensitivity and subgroup analysis. These heterogeneities in findings could be attributed to the differences in the baseline characteristics of included trials, including race, treatment duration, background medication, control arms, and the actual dosage of semaglutide. Different antidiabetic agents used as controls could be the main reason for heterogeneity. It is worth mentioning that the dosage of semaglutide extracted from two dose-finding trials was less than the FDA-approved dose, which could underestimate the effectiveness of semaglutide and increase the heterogeneity , 28. SecIn conclusion, subcutaneous semaglutide appears to exhibite beneficial effects regarding the reduction of HbA1c, weight loss, and SBP. Treatment with subcutaneous semaglutide did not increase the risk of hypoglycemia but was associated with increased incidence of nausea, vomiting, and diarrhea.Below is the link to the electronic supplementary material.Supplementary Material 1