The application of the nanofibers as a reinforcing agent in the recycled linerboard considerably improved the mechanical properties , counteracting the loss of mechanical properties suffered during recycling when using chemically pretreated cellulose nanofibers from horticultural residues and vine shoots. It was concluded that this technology surpasses the mechanical reinforcement produced by conventional mechanical refining used in the industry and extends the number of recycling cycles of the products due to the non-physical modification of the fibers.The hornification processes undergone by the fibers in the paper industry recycling processes lead to the loss of properties of the final products, which exhibit poor mechanical properties. Among the most promising solutions is the reinforcement of secondary fibers with cellulose nanofibers. The present work addresses two important issues: the efficient production of cellulose nanofibers from scarcely exploited agricultural wastes such as horticultural residues and vine shoots, and their application as a reinforcement agent in recycled linerboard recycling processes. The effect of the chemical composition and the pretreatment used on the nanofibrillation efficiency of the fibers was analyzed. Chemical pretreatment allowed a significantly higher nanofibrillated fraction (45–63%) than that produced by mechanical (18–38%), as well as higher specific surface areas (>430 m The environmental problems faced by society, together with the existing economic challenges, have led industries to adopt more and more measures for the application of the circular economy in their processes as the main axis of sustainable development. The paper and linerboard industry in among these sectors that have adopted policies for the recycling of its products. The main strategy for this purpose is the use of recycled secondary fibers in the production of linerboard. Unfortunately, the products obtained have worse mechanical performance than with the original fibers due to the hornification process .Hornification refers to dry fibers that do not fully reswell in water, forming numerous hydrogen bonds between the microfibrils during drying . This prCellulose nanofibers (CNF) have been used at various stages of paper and linerboard production  as a reinforcing agent, retention system component, printing quality aid, coating binder and barrier agent that controls water vapor and oxygen permeability. It is also used as a substitute for inorganic fillers in paper sheets to maintain the mechanical resistance . There aDespite the advantages reported by the addition of CNF in paper slurries, the costs of manufacturing and applying CNF in this type of industry are not completely clear. On the one hand, chemical pretreatment (TEMPO-mediated oxidation) applied to cellulose pulps prior to their passage through the high-pressure homogenizer has been recognized as the most effective pretreatment. However, the cost of the chemical reagents involved is high, making this process difficult to scale up to industry. On the other hand, it has been shown that the presence of residual lignin and hemicelluloses in the fibers facilitates the mechanical dismantling of the cellulose fiber structure, improves the dehydration of the suspensions, and reduces their hydrophilicity, so it might not be necessary to use CNF, prior bleaching treatment of the cellulose pulp, but rather lignocellulose nanofibers (LCNF) could be used . In any To all this must be added the raw material from which the (L)CNF is obtained. Normally, wood raw materials are used but, in recent years, the use of agricultural residues for this purpose has aroused great interest. This is due to the fact that agricultural and agri-food residues are a highly available and low-cost resource . In 2021In this context, the aim of the present study was to study the viability of two different Spanish agricultural residues to produce LCNF and CNF, specifically a mixture of tomato, eggplant and bell pepper residues, and vine shoot residues, and subsequent application as reinforcement in recycled linerboard, as a solution for the paper and linerboard industry. This study evaluated two different pretreatments to produce the nanofibers as well as the effect of residual lignin for this purpose.Capsicum annuum), tomato (Solanum lycopersicum) and eggplant (Solanum melongena) horticultural residues were supplied by Cooperativas Agrícolas de Almería  as a vegetable mixture of identical proportions. The vine shoots (Vitis vinifera) were supplied by Instituto de Investigación y Formación Agraria y Pesquera (IFAPA) from an ecological crop in Cabra . Prior to all the experimental work, the biomass was stored, dried and ground to reduce the particle size to 0.25–0.40 mm according to the T264-cm-88 standard [Bell pepper , sodium hypochlorite , and sodium bromide  supplied by Honeywell FlukaTM, acetic acid (≥99.7% purity) supplied by ACS reagent, acetone and ethanol from Alcoholes del Sur S.A., 2,2,6,6-tetramethy-1-piperidinyloxy  and colloidal silica (LUDOX® HS-40) supplied by Sigma Aldrich , and cationic starch (Vector SC 20157) supplied from Roquette .Chemical reagents used in this work consisted of sodium hydroxide  from PanReac AppliChem ITW Reagents, sodium chlorite . Cellulose pulps were characterized following TAPPI standards as described for the raw materials.Cellulose pulps, both from horticultural residues (HR) and vine shoots (VS), were obtained following the same semi-chemical process. For this purpose, the raw material was pulped in a 15 L batch reactor under an environmentally-friendly soda process conditions , following the protocol reported in previous investigations . The pulwt. and passed through a GEA Niro Panda Plus 2000 high-pressure homogenizer (HPH), following optimized sequence [Both unbleached (UBP) and bleached (BP) pulps were used to produce LCNF and CNF, respectively. Before (L)CNF production, two different pretreatments were assessed: mechanical and chemical pretreatment. In the case of mechanical pretreatment, the pulp was refined in a PFI mill until a Schopper–Riegler degree (°SR) of 90 (ISO 5264-2:2002) was achieved. Then, a suspension was prepared with the pretreated fibers at 1.5% sequence . The chesequence . The reaσ) and diameter (d) of the nanofibers were estimated, following previously published methodologies [The nanofibrillated fraction for each nanofiber suspension was determined by centrifuging a 0.1% nanofiber suspension for 20 min at 10,000 rpm . Likewisdologies , accordiThe degree of polymerization (DP) was determined from the intrinsic viscosity measurements, according to UNE 57-039-92 . The DP TM instrument  by the attenuated total reflectance (ATR) technique, recording the spectra from 4000 to 400 cm−1 with a resolution of 4 cm−1 collecting a total of 20 scans. The crystallinity of the samples was determined from the X-Ray diffraction (XRD) patterns which were recorded using a Bruker D8 Discover with a monochromatic source CuKα1 over an angular range of 10–80° at a scan speed of 0.025°/s in reflection mode. The crystallinity index (CI) was calculated from the intensity of the peak corresponding to (200) plane of cellulose crystalline and the minimum intensity between the peaks associated to (200) and (110) planes following the equations described by Segal et al. [The chemical structure of the nanofibers was analyzed by Fourier Transform Infrared (FTIR) spectroscopy using a Spectrum Twol et al. .2. A batch of 10 linerboard sheets was produced for each type of nanofibers. Finally, the linerboard sheets were conditioned at 25 °C and 50% RH prior to the mechanical characterization. In order to compare the reinforcement of the nanofibers of this work with the methods applied in the industry such as the mechanical beating, a batch of linerboard sheets formed by linerboard refined fibers in a PFI beater at 2000 and 3000 rev was formed.The reinforcing effect of nanofibers addition on recycled linerboard was studied by adding them in the formulation of linerboard sheets at 3% on fiber dry weight. For this purpose, 30 g of recycled linerboard fibers were disintegrated in a pulp disintegrator for 30 min at 3000 rpm. The nanofibers were then added to the linerboard suspension and disintegrated for 1 h at 3000 rpm. Subsequently, 0.5% cationic starch and 0.8% colloidal silica were added (on fiber dry basis). All this mixture was kept under constant agitation for 30 min before linerboard sheet formation in an ENJO-F-39-71 sheet former, according to TAPPI T 205 sp-02 standard , with a The mechanical characterization of the obtained linerboard sheets was carried out by determining the breaking length and Young’s modulus, burst index, and tear index according to TAPPI standards T-494-om 01, T-403-om 97 and T-414-om98, respectively .Among the most important factors underlying the valorization of agricultural residues is their chemical composition, which will determine their suitability for the production of cellulose pulps. The chemical composition of horticultural residues and vine shoots as well unbleached and bleached cellulose pulps obtained is displayed in For both residues, the α-cellulose content was increased after the pulping process (50.45 and 51.89% for UBPs) indicating the suitability of the chosen treatment conditions. Additionally, the bleaching process employed in both UBPs was effective in removing lignin, which decreased to less than 8%, further enriching the pulps in α-cellulose content (more than 60%). The α-cellulose content determined in HR and VS was similar to other agricultural residues used successfully in the production of cellulose nanofibers such as olive prunings (41%) , and cer800), cationic demand (CD), carboxyl content (CC) and morphology  is an indirect indicator of nanofibrillated fraction resulting in higher light scattering (lower transmittance) as the fraction of nanofibers decreases. This parameter highlights once again the higher nanofibrillation produced in chemically pretreated fibers. Regarding the effect of lignin on the nanofibrillation efficiency, it was evidenced that for both pretreatments, a high lignin content results in a lower nanofibrillation of the fibers. Indeed, this is due to the secondary lignin oxidation reactions produced by the reagents during TEMPO-mediated oxidation that decreases the reagent available for cellulose oxidation, and by the higher bonding of the fiber-forming components due to the presence of lignin that hinders fibrillation during mechanical pretreatment [Generally, it was observed that the nanofibrillated fraction was much higher when chemical pretreatment was performed (45–63%) than mechanical pretreatment 18–38%). This is because the high surface charge conferred to the fiber during pretreatment due to the oxidation of the primary C6 alcohol of the cellulose into carboxyl groups facilitates its subsequent delamination during the nanofibrillation process [8–38%. Threatment . Slight reatment .A parameter directly related to the specific surface area is the CD, since it determines the interaction capacity of the anionic surface of the nanofibers with the surrounding area. The CD of the nanofibers is influenced by the CC and the specific surface area. As expected, it was observed that the nanofibers obtained by TEMPO-mediated oxidation showed a much higher CD (620–1230 µeq·g/g) compared to the mechanical ones (301–689 µeq·g/g) due to the conversion of alcohols to carboxyls. The effect of lignin on TEMPO oxidization efficiency is clearly seen in the lower carboxyl content and lower CD of LCNF compared to CNF. As mentioned above, this fact can be explained by the lower specificity of the cellulose oxidation reaction for secondary lignin oxidation reactions. As for those subjected to mechanical pretreatment, these differences are driven by the lower fibrillation performance of LCNF. The CC followed the same trend of results. The bleaching process in the pulp removes the lignin, leaving the fibers more accessible for TEMPO-mediated oxidation, hence the highest CC values occur in TCNF , thus ob−1 due to the aromatic and aliphatic O-H stretching vibrations [−1 is related to the stretching of the C-H and CH2 bonds of the aromatic structures of cellulose and hemicelluloses [−1, is assigned to carboxyl groups stretching. This band sharpened to a more intense peak in the case of chemically pretreated fibers, resulting from asymmetric stretching of the carboxyl groups present due to TEMPO-mediated oxidation [−1 corresponds to the stretching of the aromatic ring of the lignin, hence it is only observed in LCNF and TLCNF spectra, indicating the success in the bleaching process for lignin removal [−1 is attributed to the deformation of the symmetric C-H and CH2 bond of cellulose and hemicelluloses. The peaks at 1105 and 1030 cm−1 are associated with the stretching of the CH-OH and CH2-OH bonds of the secondary and primary alcohols, respectively, in the carbohydrates present. Finally, the peak at 896 cm−1 corresponds to the β-glycosidic bonds of cellulose and hemicelluloses [The chemical structure of the different nanofibers was also analyzed by FTIR-ATR spectroscopy . The spebrations . The pealluloses . The banxidation . In addi removal . The pealluloses .The X-ray diffraction patterns as well as the crystallinity index (CI) data of all samples are also shown in In general, good mechanical performance of linerboard sheets is affected by fiber strength, specific bond area, forming process and residual stress distribution. Any treatment that improves one of these characteristics will favorably affect the mechanical properties of the final products . TherefoThe breaking length of the industrial paper in the machine direction has a value of 5656 m. Considering the anisotropy/isotropy ratio of 1.65, the breaking length of sheets made in isotropic former should be 3428 m. The Ref. sample shows a breaking length of 1895 m, so a significant decrease in mechanical properties due to the hornification process was observed. In general, it was observed that the application of the nanofibers obtained in this work as a reinforcing agent in recycled linerboard considerably improved the mechanical properties of the sheets obtained. Among the samples obtained, only the incorporation of HR-TCNF and VS-TCNF managed to counteract the loss of mechanical properties suffered, giving rise to 3429 and 3923 m, respectively. So, in this sense, recycled linerboard fibers reinforced with HR-TCNF and VS-TCNF would make it possible to obtain recycled linerboard with the characteristics of industrial linerboard. A greater specific surface area of the nanofibers, as in the case of HR-TCNF and VS-TCNF, leads to greater interaction between adjacent fibers resulting in a reinforcing effect. The nanofibers act as promoters of fiber–fiber adhesion, generating an embedded network between the matrix that contributes to increase the load-bearing capacity of the linerboard . In bothIn the case of Young’s modulus, the improvement of the linerboard sheets by adding the nanofibers was evident, following the same trend as that for breaking length. In the case of HR nanofibers, the best results for Young’s modulus were obtained with HR-LCNF and HR-TCNF. In both cases, this improvement was more than 220%. When VS nanofibers were used, the best results for Young’s modulus were achieved when using VS-CNF and VS-TCNF. It seems that, in this case, the pretreatment applied to obtain nanofibers had no influence, but the absence of lignin was decisive in obtaining optimum results. For this raw material, the improvement achieved was also almost 240%. This improvement in mechanical properties is due to the high specific surface area of the fibers introduced into the recycled linerboard, which will also produce a higher swelling capacity. These facts result in a higher number of inter-fiber bonds per unit, increasing the relative bond area. Mechanical beating resulted in 1.15 and 1.43 GPa for 2000 and 3000 rev, respectively. The use of HR and VS nanofibers obtained by any pretreatment and regardless of lignin content, outperformed the mechanical beating values at 2000 rev. The beating at 3000 rev was only surpassed in the case of the best reinforcements, as explained above. These data again corroborate the suitability of agricultural residue-derived nanofibers for use in this type of process as a substitute for mechanical beating. Several authors have demonstrated the suitability of various agricultural residues such as bamboo or oil palm tree for the preparation of nanofibers and their subsequent incorporation into composite materials by improving Young’s moduli ,37.Tear index showed no major improvements with respect to recycled linerboard or the ones with the fibers subjected to mechanical beating, except for TCNF. As occurred for the breaking length, HR-TCNF and VS-TCNF improved the tear index by 27% over the mechanical beating. Regarding the burst index, only in the case of VS-TCNF were the values of mechanical beating slightly exceeded. This index describes the intensity of the material when resisting a disc-shaped diaphragm that attempts to burst it. Properties such as fiber length, fiber quality, internal sizing and surface treatment also influence the burst index of the final material . The datAnother parameter to be considered when applying reinforcement treatments in the recycling processes of paper production is the drainability of the slurries. This parameter is indicative of the capacity of a given pulp to retain water and, therefore, its value affects the production rate of a given paper or linerboard machine. The results obtained show the suitability of unexplored agricultural residues such as horticultural residues and vine shoots to produce nanofibers with suitable characteristics for their application as a reinforcing agent in linerboard recycling process. This production of nanofibers can be carried out by mechanical or chemical pretreatment, depending on the costs to be assumed. Moreover, there will be no need for the removal of the lignin present, thus reducing process costs due to the elimination of bleaching steps and reducing the environmental impact. As discussed in previous works, the high mechanical strength obtained in this work allows the adoption of several market strategies. The first is to reduce the basis weight or increase the mineral filler content, thus reducing the consumption of fibrous raw materials and, therefore, the costs of the process. The next would be the introduction of these materials in market niches with higher technical requirements . Prior tBoth HR and VS resulted in nanofibers with good properties for application as reinforcement in linerboard sheets.2/g, respectively, in addition to nanofibrillated fractions of more than 60%.The most remarkable nanofibers were HR-TCNF and VS-TCNF, which presented a cationic demand of 1043.54 and 1227.91 µeq·g/g, a carboxyl content of 148.12 and 168.93 µeq·g/g, and a specific surface area of 436 and 516 mThe use of the nanofibers obtained as reinforcement in the linerboard resulted in an improvement of the mechanical properties of the recycled linerboard. The most significant increase was obtained when using HR-TCNF and VS-TCNF, which offset the loss of mechanical properties suffered during recycling. However, these samples were the ones that greatly reduced the drainability of the suspensions.It was concluded that the use of cellulose nanofibers as a reinforcing agent outperforms the mechanical reinforcement produced by conventional mechanical refining used in the industry and extends the number of recycling cycles of the products due to the non-physical modification of the fibers.Tomato, eggplant and bell pepper horticultural residues and vine shoots have been used to produce cellulose nanofibers by different pretreatments and from cellulose pulps with different compositions. The following conclusions are drawn from this work:


A distinct population of dopamine neurons in the substantia nigra pars lateralis (SNL) has a unique projection to the most caudolateral (tail) region of the striatum. Here, using two electrochemical techniques to measure basal dopamine and electrically evoked dopamine release in anesthetized rats, we characterized this pathway, and compared it with the ‘classic’ nigrostriatal pathway from neighboring substantia nigra pars compacta (SNc) dopamine neurons to the dorsolateral striatum. We found that the tail striatum constitutes a distinct dopamine domain compared with the dorsolateral striatum, with consistently lower basal and evoked dopamine, and diverse dopamine release kinetics. Importantly, electrical stimulation of the SNL and SNc evoked dopamine release in entirely separate striatal regions; the tail and dorsolateral striatum, respectively. Furthermore, we showed that stimulation of the subthalamic nucleus (STN) evoked dopamine release exclusively in the tail striatum, likely via the SNL, consistent with previous anatomical evidence of STN afferents to SNL dopamine neurons. Our work identifies the STN as an important modulator of dopamine release in a novel dopamine pathway to the tail striatum, largely independent of the classic nigrostriatal pathway, which necessitates a revision of the basal ganglia circuitry with the STN positioned as a central integrator of striatal information. We showed that neurons in the substantia nigra pars lateralis (SNL) constitute a dopamine pathway projecting to the tail of the striatum, a dopamine domain distinct to the dorsolateral striatum which receives input from the substantia nigra pars compacta (SNc). Furthermore, the subthalamic nucleus (STN) exclusively modulates dopamine release in the tail striatum, likely via the SNL. Our work necessitates a revision of the basal ganglia circuitry with the STN positioned as a central integrator of information from both the dorsolateral (motor) and tail (sensory) striatum. DATdopamine transporterDLSdorsolateral striatumFSCVfast‐scan cyclic voltammetryFSCAVfast‐scan controlled‐adsorption voltammetryMFBmedial forebrain bundleSNcsubstantia nigra pars compactaSNLsubstantia nigra pars lateralisSNrsubstantia nigra pars reticulataSTNsubthalamic nucleusVGlut2vesicular glutamate transporter 2VTAventral tegmental area1Dopamine release in the striatum of the basal ganglia is a fundamental process that underlies many core brain functions including motor control, motivation, and reinforcement learning  regions. Interestingly, recent findings have highlighted the tail striatum as an additional functional domain of the striatum  and fast‐scan controlled‐adsorption voltammetry (FSCAV) to make a robust comparison of evoked and basal dopamine between the tail and dorsolateral striatum and determine what role the STN has in modulating dopamine release in these two striatal regions.22.1In vivo experiments were conducted on anesthetized male Wistar rats . Prior to experiments, rats were housed in cages in a humidity and temperature‐controlled environment with a 12‐h dark/12‐h light cycle and unrestricted access to food and water. All experimental procedures were conducted with approval from the Animal Ethics Committee of the University of Auckland (AEC 001748), in accordance with the New Zealand Government Animal Welfare Act.2.22 saturation (~99%) were continuously monitored . Regular subcutaneous injections of sterile 0.9% NaCl were administered every 1–2 hrs (200 μl). Experiments were performed from about 8 a.m. to 9 p.m., with animals anesthetized at 8–10 a.m. At the end of experiments, rats were sacrificed by decapitation and the brain was removed. A small block of tissue containing the relevant brain structures was cut and placed in fixative  for 48 hrs prior to washing and storage in phosphate‐buffered saline. The experimental procedure is outlined in Figure Rats were anesthetized with urethane  and, after reaching the surgical plane of anesthesia, were secured in a stereotaxic frame . Urethane was chosen as the appropriate anesthetic due to its use in other studies involving the electrochemical detection of dopamine . Calibration curves were obtained by adding aliquots of concentrated dopamine hydrochloride solution  in 100–250 nM steps.Carbon‐fiber microelectrodes were manufactured as described previously  or tail striatum . Electrode potential was controlled with customized WCCV software  interfaced with a potentiostat  and a PCI Data Acquisition card . For FSCV, the electrode potential was scanned as a triangular waveform  at 10 Hz. The resultant current was filtered (10 kHz) and recorded using the potentiostat and WCCV software. After insertion of the carbon‐fiber microelectrode into the brain, recordings started following a short period (5–10 min) of scanning to allow electrode stabilization.2.3.2FSCAV was performed to measure absolute  levels of dopamine concentration. Voltage commands applied to the microelectrode alternated between a triangular waveform  repeated at 100 Hz (10 s) and a constant DC potential . This pause in scanning allowed for maximal adsorption of dopamine to the carbon‐fiber surface  was lowered into the brain (1 mm/min) to above either the MFB , SNc , SNL  or STN . The electrode was then slowly advanced while stimulating every 50–200 μm until a maximal evoked response was observed. Stimulation sites were confirmed following histological analysis post‐experiment. Bi‐phasic constant current stimuli  were generated using a stimulus isolator  with timing parameters controlled by WCCV software. The electrical stimuli were applied so that there was no overlap with the FSCV scans to avoid noise caused by the stimulus artifact.2.5Electrochemical data were analyzed using WCCV software and Excel (Microsoft). For analysis of FSCV recordings, the current at the dopamine oxidation peak was converted to dopamine concentration using calibration data . An increase in dopamine concentration was only considered significant if the cyclic voltammogram had an oxidation peak between 0.5 and 0.6 V at least 1.5 times the noise level, otherwise they were excluded from the FSCV data. The reproducibility of evoked responses per stimulation site  is reported in the figures. Values of interest including amplitude and time to peak were calculated using Excel. Differentiation (first derivative) of the dopamine response was performed in OriginPro 2021 (OriginLab Corporation) to determine profiles of velocity of dopamine release. For FSCAV recordings, a segment (between +0.4 V and + 0.9 V) of the 10th scan after the pause was integrated using WCCV software. This scan was chosen because of its high selectivity for dopamine over its metabolites and other electrochemically active species . Statistical tests were performed in SPSS (IBM) and graphical presentation was performed using OriginPro 2021. Statistical tests included two‐tailed paired and independent samples t‐tests. Normality was tested using Shapiro–Wilk's test and homogeneity of variance was tested using Levene's test. If these assumptions were violated, an equivalent non‐parametric test (Mann–Whitney U test) was performed. Differences were considered statistically significant if p < 0.05. No test for outliers was conducted and no outliers were removed. Data are presented as mean ± SEM in the text, and in figures as either box and whisker plots depicting the minimum, first quartile, mean, third quartile and maximum or as bars representing the mean ± SEM. This study was not pre‐registered.A total of 46 Wistar rats were used in this study. No randomization or blinding was performed in this study, and brain regions (recording and stimulation sites) were assessed in an arbitrary order. Sample size numbers represent the number of brain hemispheres. No sample size calculation was performed prior to the study and this study was exploratory. Post‐hoc power analysis calculated a minimum sample size of 3 , validating the sample sizes used in this study  and rostral tracts . Going deeper, two distinct peaks of dopamine concentration were seen in the caudal tract  within the same tract at caudal and rostral locations Figure . In the t Figure  correspot Figure . In the t[34] = 13.1, p < 0.0001, paired t‐test; n = 35; Figure Further recordings from the depth of the first peak in each tract revealed that basal dopamine was consistently lower in the tail striatum compared with the dorsolateral striatum  following electrical stimulation of the MFB . Repeated stimulation  evoked consistent dopamine release with minimal variation in amplitude .3.3To further evaluate the novel dopamine pathway, dopamine release was recorded in both the tail and dorsolateral striatum sequentially (randomized order) in response to electrical stimulation of the SNL and STN and compared with SNc‐evoked dopamine release.n = 8; Figure As expected, SNc stimulation (DV 8.4 ± 0.1) evoked dopamine release in the dorsolateral striatum with an amplitude of 57 ± 13 nM (n = 4), but no dopamine release in the dorsolateral striatum  evoked small dopamine release in the tail striatum , consistent with anatomical evidence of a significant STN to SNL projection  evoked small dopamine release in the tail striatum , which was then sustained at a slower rate  for the duration of the stimulation  to near‐zero  in the second phase despite ongoing stimulation  compared with the dorsolateral striatum , plateau upward velocity (R2 = 0.86), and peak downward velocity (R2 = 0.88).Investigation of dopamine release and clearance kinetics can reveal information about the modulation of dopamine transmission, and how it differs between the two striatal domains. The velocity of dopamine release in the tail and dorsolateral striatum was determined by differentiating , based on FSCV, to describe high resolution depth profiles of basal dopamine in rostral and caudal striatal regions with sub‐millimeter resolution. These profiles closely matched the underlying anatomically and functionally defined regions of the striatum  and matrix (Desban et al., 2 autoreceptor regulation (Davidson & Stamford, Both basal dopamine and evoked release were consistently lower in the tail striatum compared with the dorsolateral striatum. Furthermore, measures of dopamine release kinetics including peak upward velocity, plateau upward velocity, and peak downward velocity were significantly slower in the tail striatum compared with the dorsolateral striatum. Dopamine release kinetics were remarkably similar within each dopamine pathway irrespective of stimulation site, and were instead determined by the striatal region recordings were made in. Several possible reasons could explain all these differences between the tail and dorsolateral striatum. Firstly, expression of tyrosine hydroxylase is lower in the tail striatum (Miyamoto et al., 4.2Anatomical studies have recently described a midbrain projection to the tail striatum from a distinct population of dopamine neurons residing in the SNL (Jiang & Kim, The key finding here was that SNL stimulation evoked dopamine release exclusively in the tail striatum, which is consistent with studies describing this projection (Jiang & Kim, 2 autoreceptors, these are not expressed in the SNL (Fu et al., 2 autoregulation on tail striatum dopamine release are required.SNL‐evoked dopamine release in the tail striatum was not only smaller in amplitude but was also less reproducible than SNc‐evoked dopamine release in the dorsolateral striatum. There are several possible reasons for why this occurred. As previously discussed, dopamine neurons in the SNL are sparsely organized (Fu et al., As described previously, stimulation of the MFB evoked dopamine release in the dorsolateral striatum consistent with other studies (Covey & Garris, 4.3dorsolateral striatum (Covey & Garris, We showed that the STN has a unique role in the basal ganglia circuitry by exclusively modulating dopamine neurons of the SNL and evoking dopamine release in the tail striatum. This observation is consistent with a viral‐vector tracing study showing that tail striatum‐projecting dopamine neurons receive STN input (Menegas et al., Furthermore, the anatomical and electrophysiological evidence shows that there is only a minor glutamatergic afferent projection from the STN to dopamine neurons in the SNc that would support STN‐evoked dopamine release in the dorsolateral striatum (Chang et al., In light of our current finding that the STN exclusively modulates tail striatum‐projecting SNL dopamine neurons, and that the STN has a unique hyperdirect input from the motor cortex (Nambu et al., It is important to consider this revised basal ganglia circuitry in the context of Parkinson's Disease, especially given that in Parkinson's patients there is a greater reduction of dopamine in caudal portions of the putamen (Frost et al., P.S.F and J.L designed the research; K.L.T performed the research and analyzed the data; K.L.T and P.S.F wrote the paper; P.S.F and J.L reviewed and edited the paper.The authors declare no competing interests.https://cos.io/our‐services/open‐science‐badges/This article has been awarded an Open Materials Badge because it provided all relevant information about the components of the research methodology needed to reproduce the reported procedure and analysis. More information about the Open Science Badges can be found at


POLE mutations or p53 abnormalities, respectively. However, there are still several issues to be resolved, such as the prognostic value of the TCGA classifier in each FIGO stage, or the type of adjuvant treatment most suitable for each molecular group. Other issues regard the prognostic stratification of the mismatch repair-deficient and “no specific molecular profile” groups, which currently follows the same criteria; however, the former seems to be prognostically consistent regardless of FIGO grade and histotype, whereas the latter appears highly heterogeneous. Numerous clinical, histological, immunohistochemical and molecular markers have been proposed to refine the TCGA classification, but their prognostic value is still undefined. Hopefully, prospective data collected in the next years will help resolving these issues.In the last years, the TCGA-based molecular classifier have been progressively integrated in the management of endometrial carcinoma. While molecular assays are increasingly available across pathology laboratories, the additional costs will expectedly be compensated by a reduction in overtreatments and a prevention of recurrences. The additional time might be shortened by assessing molecular markers on biopsy specimens. Retrospective data suggest that the molecular classifier will have a major impact of on the risk stratification, with many patients having their risk class down- or upstaged based on  The last years have seen an unprecedented revolution in the approach to endometrial carcinoma (EC). Such revolution began with the publication of the study by The Cancer Genome Atlas (TCGA) in 2013, which showed that EC could be subdivided into 4 molecular prognostic groups based on mutational burden and somatic copy number variations. Since then, numerous studies have contributed to the translation of the TCGA findings into routinary usable protocols for the management of EC [POLE-mutant” group (surrogate of the “ultramutated” group), the “p53-abnormal” group (surrogate of the “copy number-high” group), and the “no specific molecular profile group” . These surrogates have allowed several research groups to test the TCGA classification on their EC series and confirm its prognostic value [A crucial step in this process has been the validation of cheaper and easier surrogates of the complex and expensive molecular analyses performed by TCGA. Therefore, the use of immunohistochemistry for mismatch repair (MMR) proteins and p53 protein and of polymerase-ε sequencing has been found able to replace the molecular assessment of mutational burden and copy number variations. On this account, EC can now be subdivided into the “MMR-deficient” group (surrogate of the “hypermutated” group), the “ic value ,3,4,5.POLE-mutant cases and poor prognosis for the p53-abnormal cases [Given that the TCGA study only included endometrioid and serous EC, several studies have assessed the molecular classification on other histotypes, including clear cell, carcinosarcoma, and undifferentiated/dedifferentiated carcinoma, consistently showing a good prognosis for al cases ,5. The Tal cases ,6. Numeral cases ,10,11,12POLE-mutant ECs, which are categorized as “low-risk” when confined to the uterus, regardless of any other clinicopathological or molecular factors, and (ii) p53-abnormal ECs, which are categorized as “high-risk” in the presence of myoinvasion, regardless of any other factor [In 2020 (7 years after the publication of the TCGA study), the updated ESGO-ESTRO-ESP guidelines adopted the molecular prognostic classification in the risk stratification of EC. Two crucial updates regard (i) r factor .As observed by Oberndorfer et al. in their recently published paper , a high POLE analysis to a subset of patients [POLE at all [Possible limiting factors of the TCGA classifier discussed in the Literature are the additional costs and the limited availability of molecular analyses. In this regard, limiting patients , or evenE at all , have beE at all , the addPOLE on diagnostic biopsy specimens. In fact, it has been shown that there is high concordance in the assignment of molecular groups between biopsy and hysterectomy specimens [POLE analysis. Regarding immunohistochemistry, it appears advisable to perform it on the hysterectomy specimen, as it might reveal, for example, a subclonal p53-abnormal component [Another issue raised by Oberndorfer et al. is the additional time required for molecular analyses (18.5 days on average in their series) . This lepecimens . In ordeApart from the problems highlighted by Oberndorfer et al., there are further issues that have been raised with the use of the molecular classifier. Amant et al. highlighted that it is still unclear how the TCGA classifier impacts the prognosis in each FIGO stage, and that prospective studies are needed to resolve this point. Furthermore, they underlined that the type of adjuvant treatment most suitable for each group is not defined .Merlotti et al. raised the question whether it is reasonable to treat p53-abnormal low-grade ECs with superficial myoinvasion as high-risk tumors . These cAnother issue regards whether it is meaningful to substratify MMR-deficient ECs according to FIGO grade and histotype ,5. In faThe prognostic heterogeneity of NSMP ECs appears as another unresolved issue. Oberndorfer et al. adopted the classification proposed by Stelloo et al. for ESMO high-intermediate risk ECs, which involves CTNNB1 exon 3 mutation and L1CAM overexpression ,14; thesHopefully, the prospective and large-scale assessment of the TCGA classification will allow resolving these issues and obtaining the necessary data for an optimal management of EC.


Below these values, it is likely that crosslinking has not been maximized and that the oxidizing environment is weakening the gelatin. Above these values, the loss in adhesiveness may result from the disruption of the alpha helixes due to the large number of phenolic compounds as well as the maintenance of an oxidizing environment. Overall, this investigation shows the possibility to design strongly adhesive hydrogels to metal surfaces by blending gelatin with polyphenols in oxidative conditions.The synthesis of surgical adhesives is based on the need to design glues that give rise to strong and fast bonds without cytotoxic side effects. A recent trend in surgical adhesives is to use gel-forming polymers modified with catechol groups, which can undergo oxidative crosslinking reactions and are strongly adhesive to all kinds on surfaces in wet conditions. We previously showed that blending gelatin with catechol can yield strong adhesion when the catechol is oxidized by a strong oxidant. Our previous work was limited to the study of the variation in the sodium periodate concentration. In this article, for an in-depth approach to the interactions between the components of the gels, the influence of the gelatin, the sodium periodate and dopamine/(pyro)catechol concentration on the storage (G′) and loss (G″) moduli of the gels, as well as their adhesion on steel, have been studied by shear rheometry. The hydrogels were characterized by infrared and UV-Vis spectroscopy and the size of their pores visualized by digital microscopy and SEM after freeze drying but without further additives. In terms of adhesion between two stainless steel plates, the optimum was obtained for a concentration of 10%  In this investigation, TA was used as a hydrogen bond donor, whereas the 4-arm modified poly(ethylene glycols) (PEG) worked as a hydrogen bond acceptor to produce a strong glue, enabling a 2.5 fold enhancement of the adhesion strength between the epidermic side porcine skin with respect to fibrin glue [w/v) was blended with pyrocatechol or pyrogallol (but a with weak contribution to adhesion) at 10 mM, with increasing concentrations in sodium periodate. In the case of catechol , we demonstrated a 2.7-fold increase in adhesive strength and a 21-fold increase in debonding energy for 10% w/v gelatin + 10 mM catechol oxidized with 30 mM NaIO4  [4 gels were evaluated using a rheometer. The obtained results were discussed on the basis on the possible reactions of oxidized catechol or dopamine with gelatin chains and on a morphological basis after quantitative evaluation of the pore size of the gels containing variable amounts of catechol or dopamine. For the sake of simplicity, we will adopt the following nomenclature for the investigated hydrogels: GxCyNz or GxDyNz, where G, N, C, and D represent gelatin, sodium periodate (NaIO4), catechol, and dopamine, respectively. In this nomenclature, x, y, and z are the weight fraction of gelatin (% w/v), the molar concentration of catechol or dopamine, and the molar concentration of NaIO4, respectively. The rationale between comparing catechol with dopamine was the presence of an additional ethylamine group on dopamine  . The predopamine , which m4. The gelation kinetics are displayed in w/v in gelatin. Below 10% w/v, G′ scales as C4.6±0.3  and in the high (above 10% w/v) concentration range. This is a strong indication of the fractal nature of the gelatin gels investigated in the presence of 10 mM NaIO4 [w/v was used in all the forthcoming experiments.The gelation kinetics of gelatin were first investigated as a function of the gelatin concentration in the presence of 10 mM NaIOC4.6±0.3 , whereastin gels ,40. It mmM NaIO4 . Hence, 4 concentration, the oxidant that will be used to oxidize the introduced catechol or dopamine, in order to investigate its effect on the rheological properties of gelatin itself. Indeed, it is well known that such a strong oxidant  is able to oxidize vicinal diols or amino alcohols  to produce formaldehyde [4, the gel loses only about 20% of its elasticity compared to the pristine gelatin gel. This moderate decrease in storage modulus is acceptable from a mechanical point of view. For this reason, we will restrict our investigation to 10% (w/v) gelatin gels and a NaIO4 concentration of 10 mM, and the investigated variable isthe catechol or dopamine concentration.We then increased the NaIOaldehyde , and sucG10YNC10 and of the G10YND10 gels displays a maximum close to y = 20 mM in either catechol or dopamine  than the dopamine-containing ones. This trend is conserved over the whole investigated concentration range in either catechol or dopamine x-co-(lactic acid)1−x) used to glue aluminium or steel plates displays adhesion strengths higher by one order of magnitude [The values of the optimal adhesion strength obtained in this investigation, close to 100 kPa, must be compared with data from the literature. Most of these data, with the exception of the data of TA blended with 4-arm PEGs , poly, the pH, the molecular mass of the gelatin, and other physicochemical conditions. Interestingly, the G10C80N10 hydrogels remain stable up to the highest investigated temperature whereas the G10D80N10 gels undergo the gel–sol transition at a temperature which lies even below the gel–sol transition temperature of G10N10 hydrogels. This highlights, as already suspected from the data in (1)G10zNC10 or G10zND10 hydrogels are optimal for a molar concentration corresponding to a 2-fold molar excess of catechol or dopamine with respect to NaIO4, and correspond to an optimal crosslinking density with gelatin. A priori, such a ratio is unexpected because the oxidation of catechol or dopamine produces a quinone with the loss of two protons and two electrons, whereas the IO4− anion is reduced in the IO3− anion in a two electron process. One would hence expect an optimal catechol or dopamine concentration close to 10 mM, and hence a catechol/dopamine ratio equal to 1. The finding that it occurs close to 20 mM might be due to the fact that the oxidation of catechol or dopamine by NaIO4 is a fast reaction , and it(2)The difference between catechol and dopamine at high concentrations  C is probWe will now explore explanations as to why an optimal catechol or dopamine over NaIOatin gel . To begidopamine . These gterature , gelatin4 and the addition of either catechol or dopamine  and the others [−1/I1235 cm−1) of 0.9701. The latter was the basis for evaluating the percentage of disruption given by the addition of phenolic compounds and periodate (−1/I1235 cm−1) ratio from 1.2% to 2.8% for all species combined except for the G10D801N0 hydrogel, which shows a notable variation of 4.6%. It then becomes possible to assume that the addition of phenolic species somewhat disrupts the To gain additional information about the formation of Michael addition or Schiff base formation between the oxidized catechol or dopamine species, we also performed some characterization by means of infra-red spectroscopy. Some typical spectra are displayed in e others . Howevere others . In the eriodate . It was  gelatin .To infer such a possibility and more generally to produce the influence of the added catechol or dopamine on the morphology of hydrogels, we performed a morphological characterization, combining optical reflection microscopy and scanning electron microscopy (SEM). Indeed, a change in the pore size and in the pore volume of porous materials has a strong influence on their mechanical properties . One wouG10zNC10 and G10zND10 investigated hydrogels. Some representative pictures are given in G10zNC10 hydrogels and in G10zND10 hydrogels.The preparation of the gels for morphological characterization will be described in the reach a plateau value close to 250 and 175 µm, respectively, for a catechol or dopamine concentration close to 5 mM. As a consequence, the anisotropy factor also plateaus at around 75 µm for the same catechol or dopamine concentration. A more detailed representation of the pore size distribution is given in 4 or dopamine/NaIO4 ratio allowing for an optimal chemical post-oxidation reaction with nucleophiles present on the gelatin, and that the pore diameter is not an important parameter to explain the data shown in The largest pore size as well as the smallest pore size distribution were determined by analysis of the optical micrographs. It appears, for both kinds of gels, that the pore size increases slightly when the catechol or dopamine concentration increases from 0 to 5 mM. However, qualitatively , the porAs an additional remark: in none of the acquired images, or at the resolution of those images, did we observe solid particles which could originate from the oxidation process, in particular of dopamine. However, such very small particles or polymers, in the nanometer size range, may form in the presence of gelatin, as found in the presence of other, but non-gelling, proteins .w/v gelatin and 10 mM NaIO4 represent the ideal concentration of gelatin and oxidant for the purpose of studying the influence in phenolic compounds concentration on the gels’ mechanical and adhesive properties. The optimum was obtained at 20 mM of phenolic compounds of either catechol or dopamine. Below these values, it is likely that crosslinking has not been maximized and that the oxidizing environment has weakened the gelatin. Above these values, the loss in mechanical and adhesive properties may result from the disruption of the alpha helical contacts responsible for the gelation of gelatin due to the large number of phenolic compounds as well as the maintenance of an oxidizing environment. Our results show the adhesive strength of the G10C20N10 and of the G10D20N10 gels, specifically at 100 kPa, can compete with adhesives where the catechol moiety has been grafted to the polymer chains. Because their preparation is the result of a rapid (less than 5 min) one-pot process, the hydrogels investigated are ideal candidates for use as a biological glue. In the future, the swelling properties and the cytotoxicity of the catechol- or dopamine-containing hydrogels will be investigated, as well as their adhesiveness to tissues such as skin or muscles. In the present investigation, we focused on the adhesion on steel as a prototypal alloy, but the cytotoxicity aspect has not been considered yet. Indeed, even if the preparation of the adhesive gels is much easier than for those in which the catechol is grafted to the polymer, we may not exclude, particularly at high catechol or dopamine concentrations, that the gels release some none-covalently bound and cytotoxic species. As an additional perspective, the influence of condensed polyphenols such as resveratrol or epigallocatechin gallate on the mechanical and adhesive properties of the gelatin-based hydrogels is worth investigating.We showed that 10% All solutions were made from double distilled and deionized water . Pyrocatechol (ref. P0381), sodium periodate (ref. 311448), dopamine hydrochloride (ref. H8502), and gelatin (ref. G9382) were purchased from Sigma-Aldrich . All products were used without further purification. The pH of the 50 mM sodium acetate  buffer was adjusted to 5.0 using concentrated hydrochloric acid. The mother solutions of pyrocatechol and dopamine were prepared during the heating process of the buffer solution, before the beginning of each gelation experiment.4), catechol, and dopamine, respectively. In this nomenclature, x, y, and z are the weight fraction of gelatin (% w/v), the molar concentration of catechol or dopamine, and the molar concentration of NaIO4, respectively. The volume of buffer solution has been adjusted  in order to reach a final and constant volume of 10 mL. The flask was closed and placed on a hot plate set at 75 °C. Gelatin powder was slowly added (to reach 10% or 20% w/v) and eventually a pyrocatechol or a dopamine solution was added. To start the oxidation process of catechol or dopamine, the concentrated NaIO4 solution was finally added. This concentrated NaIO4 solution was prepared at a 10-fold higher concentration than the expected final concentration. After homogenization with a magnetic stirrer (200 rpm), 0.6 mL of the hot mixture was used for rheological characterization in a cone plate geometry. However, 1.6 mL of the whole and hot sol was deposited on the cleaned stainless steel plate to evaluate the adhesion strength of the gels. The temperature of the horizontal plate was fixed at 25.0 ± 0.1 °C and controlled with a Peltier element. The deposition of the pre-gel mixture on the lower stainless steel plate corresponds to the beginning of the gelation process in all cases.For the sake of simplicity, we will adopt the following nomenclature for the investigated hydrogels: GxCyNz or GxDyNz, where G, N, C, and D represent gelatin, sodium periodate . The force was measured every 10 ms up to separation, at which the force was equal to zero, corresponding to a complete rupture. A photograph of both plates was then taken in order to estimate if the rupture was of an adhesive (the gel being removed from one of the plates) or cohesive (gel parts being adherent on both plates) nature. The adhesive strength was obtained by dividing the maximal force obtained in such experiments by the area of the upper plate of the rheometer, specifically 3.14 cm2, neglecting its roughness. This yielded the adhesive strength of the corresponding gel. The specific energy of the rupture of the adhesive contact was calculated by integrating the area under the peeling curves after transformation of the time into a plate-to-plate distance and normalizing by the area of the upper plate, which was also 3.14 cm2.All rheological experiments were performed with a Kinexus Ultra rheometer  fitted with a dynamometer limited to 50 N. The gelation kinetics and the temperature measurements were performed in the cone–plate geometry with an upper cone 4 cm in diameter and an angle of 174°. The distance between the apex of the cone and the lower plate was equal to 150 µm. However, the adhesion strength experiments were performed in a plate–plate geometry with an upper plate disk 2.0 cm in diameter. All the plates, cones, and disks were stainless steel. Before each experiment, they were cleaned with soap, hot distilled water, and ethanol. All the gelation kinetics were followed at a constant frequency of 1 Hz, at a constant strain of 1%, and at a constant temperature of 25 °C. The gelation kinetics were followed over the course of 3 h, and a new data point was performed every 30 s. After completion of some of the gelation kinetics, a temperature sweep experiment was performed from an initial temperature of 25 °C to a final temperature of 50 °C at a heating rate of 1.0 °C/min2 spectrophotometer  in quartz cuvettes. The reference cuvette contained sodium acetate buffer (50 mM).UV-Vis spectra of freshly prepared solutions were measured with a double beam mc−1, with a resolution equal to 4 cm−1. A total of thirty-two scans were performed for each of the samples. The results were processed using the OMNIC program (Version 9.2.46). The samples come from the powdering of hydrogels previously dried in the open air. The spectra were processed using Origin Pro 2022 software.The spectra of samples were recorded using a Thermo Fisher Scientific Nicolet 380  FTIR spectrophotometer. The spectra of all samples were recorded in the wavenumber range 4000–400 cmA VHX-5000 microscope  fitted with a VH-Z500R objective (Zoom Recognition Lens × 500 to 5000) and a white LED light source was used to image the same gels as those prepared for SEM characterization. Statistical analysis was performed with the Sigma-Plot software (Version 11.0). Pore size measurements were made on at least three different samples, with a minimum of 400 measurements per type of hydrogel.First, we tried to freeze-dry the hydrogels after 5 min of freezing in liquid nitrogen, but this treatment induced extensive cracks in the obtained material, as shown in


Poly(methyl methacrylate) (PMMA) is widely used in aviation, architecture, medical treatment, optical instruments and other fields because of its good transparency, chemical stability and electrical insulation. However, the application of PMMA largely depends on its physical properties. Mechanical properties such as tensile strength, fracture surface energy, shear modulus and Young’s modulus are increased with the increase in molecular weight. Consequently, it is of great significance to synthesize high molecular weight PMMA. In this article, we review the application of conventional free radical polymerization, atom transfer radical polymerization (ATRP) and coordination polymerization for preparing high molecular weight PMMA. The mechanisms of these polymerizations are discussed. In addition, applications of PMMA are also summarized. Poly(methyl methacrylate) (PMMA), commonly known as plexiglass, is a kind of polymer synthesized by free radical polymerization, ionic polymerization and coordination polymerization ,2,3. Com6 Da can be defined as ultrahigh molecular weight PMMA and the PMMA with a number-average molecular weight exceeding 105 Da can be defined as high molecular weight PMMA [I]0.5(fkd/kt)0.5  and the kinetic chain length (υ) should follow the equation of υ = kp[M]/2(fkdkt[I])1/2 )1/2 . It indintration . TherefoAzodiisobutyronitrile (AIBN) and benzoyl peroxide (BPO) are the most commonly used initiators in conventional radical polymerization. In a conventional free radical polymerization of MMA, PMMA can be synthesized by bulk polymerization, emulsion polymerization and suspension polymerization. High molecular weight PMMA can be prepared by bulk polymerization. However, it is difficult to remove the generated heat in the polymerization, resulting in a generation of gel effect and side reactions. Therefore, there is still obstacles to preparing high molecular weight PMMA with high yield through bulk polymerization. Compared with ionic polymerization, conventional free radical polymerization is more resistant to water. Consequently, dispersion polymerization, suspension polymerization and emulsion polymerization using water or other solvents as medium are proposed and used to synthesize high molecular weight PMMA.2), generating stable and inactive peroxyl radicals. Therefore, it is important to solve the problem of oxygen inhibition in a radical polymerization. Degassing of the polymerization environment with an inert inactive gas, such as argon or nitrogen, is an effective method to prevent oxygen inhibition.Commonly, in a free radical polymerization, the initiating and chain growing free radicals are easily quenched by oxygen  of 3.16 × 105 Da has been synthesized. Subsequently, Hsiao et al.  as a stabilizer, AIBN as an initiator and supercritical CO2 as a reaction medium. The effect of P(MMA-co-HEMA)-g-PFPO hyperbranched polymers with different molecular weights on molecular weight of the synthesized PMMA is investigated, and the PMMA with a high molecular weight of 3.55 × 105 Da has been synthesized. Actually, the solubility of P(MMA-co-HEMA)-g-PFPO hyperbranched polymers has a significant effect on the molecular weight distribution of PMMA, and P(MMA-co-HEMA)-g-PFPO with good solubility is beneficial for synthesizing narrow molecular weight distribution PMMA.Lepilleur et al.  report a2 as a reaction medium in the absence of stabilizers can realize the synthesis of high molecular weight PMMA :[SDS] = 130:1 (weight ratio). As an oil-soluble initiator is used in this reaction, the particle nucleation is believed to take place in micelles and follow the heterogeneous nucleation mechanism.Jiang et al. . proposen et al.  report a2 in the initiation mechanism has also been investigated in photopolymerization [P*] = kpKeq[M][I0] × [CuI]/[CuII] and the degree of polymerization (DP) follows the equation of DP = ∆M/[I0] :[BMPE]:[CuCl] = 6400:1:6.Agarwal et al.  report a2) as an initiator, CuCl as a catalyst, and dnNbpy as a ligand. The high molecular weight six-arm star PMMA with a Mw of 2.35 × 106 Da is synthesized using TP6Br as initiator. In addition, a six-arm star PMMA with a Mw of 2.10 × 106 Da is successfully synthesized using EG(Bz3Br)2 as initiator.The star polymer attracts a lot of interests because of its branched structure. Similarly to the preparation of linear polymers, the preparation of high molecular weight star polymers also requires a low extent of chain transfer and chain termination, high initiation efficiency, and moderate polymerization rate. Agarwal and coworkers  report aA high concentration of catalyst is often required to ensure the controllability of normal ATRP, which will cause an increment in side reaction and the decrement in molecular weight. The concentration of the catalyst can be significantly decreased in an electron transfer activation regeneration catalyst atomic transfer radical polymerization (ARGET ATRP), resulting in a decrease in side reactions. Therefore, the ARGET ATRP may be applied to prepare higher molecular weight polymers. The mechanism of copper mediated ARGET ATRP of MMA is shown in I) are constantly regenerated from the transition metal complex in its higher oxidation state (CuII) by the reduction in reducing agent. Therefore, the amount of Cu(II) species is much lower than the amount of reducing agent. Actually, the use of Cu(II) species can be reduced to around 10 ppm. The extent of the side reaction is reduced owing to the drastically smaller amounts of required Cu(II) species and thereby high molecular weight polymer can be prepared via ARGET ATRP  as a reducing agent. The effects of initiator concentration, catalyst concentration and reducing agent concentration on the molecular weight of PMMA have been investigated, and the high molecular weight PMMA  is successfully prepared under a molar ratio of [CuBr2]:[PMDETA]:[Sn(EH)2] = 0.1:1.5:1.5.Youk et al.  report tkp2/kt, and the propagation rate constant (kp) can be improved by increasing the pressure  = 14 mM), Fukuda et al.  systematCoordination polymerization is also an effective method for preparing high molecular weight PMMA . The mec4-AlR3, if the halogen has been substituted by cyclopentadienyl (Cp), the obtained catalyst (Cp2TiCl2-AlR3) will be soluble in the organic solvent. The polymerization rate can be increased by increasing the temperature. However, this will cause a decrement in the molecular weight. According to the literature  complexed with modified methylaluminoxane (MMAO) is used as a catalyst and toluene is used as a solvent in the polymerization of MMA. The PMMA with a Mw of 9.62 × 105 Da is prepared at 60 °C with a polymerization time of 2 h.Compared to the early transition metal, the later transition metal has a lower oxygen affinity and higher functional group tolerance. Therefore, the later transition metal can also be applied to the polymerization of polar monomers. For instance, late transition metals such as Cu(II), Pd(II), Zn(II), Cd(II) and Co(II) complexed with n of MMA ,61. Lee n of MMA . The bid2 or Cu(OAc)2 combined with N-tripod ligand is an effective catalyst for the polymerization of MMA in the presence of methylaluminoxane (MAO). The commonly used N-tripod ligands are shown in Lavastre et al.  report t2 or Cu(OAc)2 alone does not induce the polymerization, indicating that the N-tripod ligand is also a key factor for the polymerization. In Cu(OAc)2 catalyst systems, the PMMA is prepared with a yield of 23% to 40%. CuCl2 combined with ligands 1b or 1g gave a PMMA with a yield of 60%. The molecular weight of PMMA prepared in Cu(OAc)2 system is higher than that produced in CuCl2 system. In the presence of toluene, the PMMA with a Mn of 3.39 × 105 Da is synthesized in Cu(OAc)2/1b system with a polymerization time of 4 h and temperature of 30 °C.CuClN,N-bis{(1-pyrazolyl)methyl}aniline (bpmaL1) ligand as a catalyst in the presence of MMAO. A series of bidentate -cobalt (II) dichloride complexes are synthesized and presented in Lee and coworkers  report a6 g/mol-Co·h) shows the highest activity, 2w/MMAO (8.00 × 105 g/mol-Co·h) and 3w/MMAO (8.73 × 105 g/mol-Co·h) have a moderate activity, and 1w/MMAO (6.17 × 104 g/mol-Co·h) presents the lowest activity. However, the 1w/MMAO system is more suitable for preparing high molecular weight PMMA. With a polymerization temperature of 60 °C, the PMMA with a Mn of 1.13 × 106 Da and PDI of 1.75 has been synthesized in 1w/MMAO system. Later, Lee et al.  (NHC = N-heterocyclic carbenes) are an efficient catalyst for the polymerization of styrene and phenylacetylene [2 can be used as a catalyst for the polymerization of MMA in the presence of MAO [5-C5H4R)(X)(NHC) ] complex can also be used as a catalyst for the polymerization of MMA :[RAFT agent]:[AIBN] = 1.20 × 104:1:0.2.Rzayev et al. report a RAFT of MMA by using AIBN as an initiator and cyanoisopropyl dithiobenzoate as a RAFT . The eff3SiOLi) [Mn = 8.25 × 105 Da) and low polydispersity index (PDI = 1.16) has been prepared under a molar ratio of [MMA]:[initiator] = 3000:1.Kitayama and coworkers report an anionic polymerization of MMA using α-lithioisobutyrate (Li-iPrIB) as initiator in the presence of lithium trimethylsilanolate (Me3SiOLi) . The molMn upped to 107 Da through plasma-initiated polymerization [6 Da.Since it was discovered by Osada and coworkers , the plarization ,72. The rization . The 2-cMn of 4.65 × 106 Da and a Mw of 8.08 × 106 Da is synthesized at 70 °C using 2-bromoisobutyric acid ethyl ester (EBiB) as an initiator in the presence of catalytical amount (10.1 ppm) of Pd NPs [Nanoparticles have high catalytic activity due to their large surface energy. In our group, the polymerization of MMA with organic halide as initiator in the presence of palladium nanoparticles has been investigated. The ultrahigh molecular weight PMMA with a f Pd NPs .As the development of research, much more polymerization methods are reported and used for the preparation of high molecular weight PMMA ,76. SomeMυ) up to 106 Da. Therefore, the synthesized high molecular weight PMMA will further expand the application value of PMMA.High molecular weight PMMA and its copolymers are important precursors for the preparation of polymer materials with high light transmittance, excellent electrochemical performance and mechanical properties. PMMA and its copolymers or composites are widely used in electronic equipment, medical technology, and polymer membranes areas. The relationship between molecular weight and physical properties is deeply investigated ,78. For Mυ) range from 1.7 × 105 Da to 7.5 × 105 Da are successfully used for the preparation of PMMA bone cements [Osteoporotic vertebral compression fracture (OVCF) is one of the most common complications of osteoporosis. At present, percutaneous vertebroplasty (PVP) is widely used for the treatment of OVCF. PMMA bone cement shows an important function in PVP. In a PVP surgery, PMMA bone cement is injected into the injured vertebra of the patient, which can quickly relieve pain, stabilize and strengthen the injured vertebra, and restore the height and angle of the injured vertebra ,80,81. H cements , indicatIC) of bone-PMMA with nano MgO particles or micro MgO particles, finding that the KIC of bone-PMMA with nano MgO particles and bone-PMMA with micro MgO particles are much higher than the KIC of bone-PMMA [Inadequate strength at the cement/bone interface is one of the main drawbacks of PMMA bone cement in the current orthopedic surgeries. PMMA cement strength, surface roughness properties and osteo-blast cell growth can be improved by incorporating additives such as MgO, chitosan and hydroxyapatite to PMMA ,85,86. Kone-PMMA .Lin and coworkers report that the bone/cement interfacial strength can be enhanced by partially degradable PMMA/Mg composite bone cement (PMC) . This rew/w are added to PMMA cement, finding that the mechanical behavior of PMMA cement can be modified by the addition of CS and CSMCC. Bioactive PMMA surfaces at the site of implantation are obtained via the addition of amphiphilic molecule phosphorylated 2-hydroxyethylmethacrylate (HEMA-P) in PMMA bone cement [Roldo and coworkers report a preparation and characterization of antibacterial PMMA composite cement . The chie cement . The add5 make no contribution to the plastic toughening of the material [5 display optimum fracture strength properties [Mn > 105 Da) should be a suitable candidate for dental material.PMMA resin is commonly used dental material because of the low cost and lightweight performances. However, the properties such as tenacity, fracture strength of PMMA used for dental material should meet a suitable value. Kusy and coworkers find that the fractions of viscosity-average molecular weight less than 10material . Huggettoperties . TherefoBoth heat- and cold-cured PMMA materials are used for the relining of dentures . Heat-cuAcrylic (PMMA) teeth are a novel dental material which is manufactured by compression or injection molding techniques. Compared to heat-cured PMMA, acrylic teeth are less brittle owing to the high resilience and flexibility. Distinct from porcelain teeth, acrylic teeth are lightweight and do not cause clicking sounds . Howeverw/w) on the property of the material have been studied. The tensile properties and fracture toughness are enhanced by the addition of 7% w/w of eggshell powder, while the elongation percentage at break and impact strength are decreased compared with other specimens. Eggshell has a poor dispersion ability in PMMA and this may cause a formation of agglomerates in the PMMA matrix. This might be a main reason for the decrement in percentage at break and impact strength. A similar result is obtained when using some other nanofillers as additives [Alaa Mohammed and coworkers prepare a new composite material via the mixing of eggshell powder and PMMA resin . The effdditives .The addition of silica to PMMA has a negative impact on flexural strength. However, the flexural strength can be slightly improved by using silanized feldspar as an additive. Raszewski and coworkers find that properties such as Brinell hardness, elastic modulus, maximal displacement, and flexural strength of PMMA modified with silanized feldspar are obviously improved . In addiVarious types of PMMA materials such as heat-cured PMMA, cold-cured PMMA and light-cured PMMA are used for denture repair . The heaThe PMMA-based polymer nanocomposites have attracted the considerable interest of chemists due to their excellent optical, mechanical and electrical properties ,103,104.Polymer field-effect transistors (PFETs) have attracted the significant attention of scientists owing to their potential applications in smart card, displays and sensor ,106. TheMao and coworkers reported that the electron and hole mobilities in polymer field-effect transistors can be enhanced by tailoring the molecular weight of polymeric dielectric . PMMA wiMw = 1.2 × 105 Da) exhibit a large electron mobility of 0.30 cm2 V−1 s−1 but show a low hole mobility of 0.01 cm2 V−1 s−1  for electron traps decrease from 5.46 to 1.38 × 1011 cm−2 and the trap density (Ntrap) for hole traps decrease from 3.64 to 1.10 × 1011 cm−2. Therefore, using high molecular weight PMMA as dielectric is beneficial for simultaneously enhancing electron and hole mobilities. Consequently, high molecular weight PMMA is an excellent candidate for electrical applications.It shows that the PFETs based on PMMA  have also attracted significant attention owing to their potential applications in electronics . DinaphtMw = 3.50 × 105 Da) is an excellent material for the preparation of Sm/PMMA luminescent composite fiber [3+ ion.Recently, the rare earth luminescent materials have attracted considerable attention owing to their potential applications in optoelectronic devices . Howeverte fiber . CompareMw = 3.50 × 105 Da) and poly (ethyl methacrylate) (PEMA) nanocomposite via casting method [The Co doped ZnO nanoparticle (NP) is used to prepare PMMA (g method . The conMn = 1.00 × 106 Da), PEG and stearic acid can be used as a binder for the MIM of 316L stainless steel powder [Metal injection molding (MIM) is an important method for the production of small parts with complex shape. The key factor in MIM is the selection of the binder, which should permit the mixing and injection molding of feedstocks with high powder loading. Ideally, the debinding should occur in a short time without causing defects. Bakan et al. report that the complexes consisted of PMMA  with different free volumes (FFV) are synthesized in different solvents  [2, N2, CO2) in the PMMA membrane are studied, finding that the gas permeability is increased with the increase in FFV. Moreover, they have found that the high molecular weight PMMA membranes possess higher gas permeability than the low molecular weight membranes.Membrane gas separation technology is widely used in nitrogen purification, oxygen enrichment, hydrogen recovery from reactor purge gas, and stripping of carbon dioxide from natural gas . Comparettleness . Fu and  ketone) . This me5–107 Da have been prepared and reported by chemists. The mechanisms of some polymerization systems have also been summarized. Finally, this work also proides a brief description of the application of high molecular weight PMMA in medical, optical and electricity, and other areas. We hope that this work can bring more research insights into this field.Compared to low molecular weight PMMA, the high molecular weight PMMA has a better mechanical strength. The application value of PMMA is further expanded due to the synthesis of high molecular weight PMMA. This review summarized the application of conventional free radical polymerization, ATRP, RAFT, plasma-initiated polymerization and ionic polymerization on the preparation of high molecular weight PMMA. A series of linear PMMA, star-shaped PMMA and block polymers with molecular weights between 10


M. micrantha has caused huge ecological damage and economic losses worldwide due to its rapid growth and serious invasion. However, the underlying molecular mechanisms of its rapid growth and environmental adaption remain unclear. Here, we performed transcriptome and small RNA sequencing with five tissues of M. micrantha to dissect miRNA-mediated regulation in M. micrantha. WGCNA and GO enrichment analysis of transcriptome identified the gene association patterns and potential key regulatory genes for plant growth in each tissue. The genes highly correlated with leaf and stem tissues were mainly involved in the chlorophyll synthesis, response to auxin, the CAM pathway and other photosynthesis-related processes, which promoted the fast growth of M. micrantha. Importantly, we identified 350 conserved and 192 novel miRNAs, many of which displayed differential expression patterns among tissues. PsRNA target prediction analysis uncovered target genes of both conserved and novel miRNAs, including GRFs and TCPs, which were essential for plant growth and development. Further analysis revealed that miRNAs contributed to the regulation of tissue-specific gene expression in M. micrantha, such as mmi-miR396 and mmi-miR319. Taken together, our study uncovered the miRNA-mRNA regulatory networks and the potential vital roles of miRNAs in modulating the rapid growth of M. micrantha. Mikania micrantha  is native to tropical America and is listed as one of the 10 most invasive weeds in the world [M. micrantha is also known as the “plant killer”. The rapid elongation of the cane makes it climb to the top of other plants, prevent other plants from receiving enough sunlight, and hinder their growth and reproduction, which eventually leads to the death of other plants and the destruction of genetic and species diversity [he world . Due to he world , this plhe world ,5,6. In iversity ,7.M. micrantha, scientists analyze the population genetics of this species to trace its origin and the invasion history by using several molecular markers [M. micrantha grown in the southeastern region of Guangdong province and use ISSR (inter-simple sequence repeats) [M. micrantha. In Mexico, Bravo-Monzon et al. analyzed the genetic structure of native Mikania populations with six SSR markers [M. micrantha genome may have significant effects on its growth, metabolism, and defense responses.To figure out the factors that contribute to the rapid growth and notorious invasion of  markers ,9,10. Strepeats) ,12, SSR repeats) ,9,13, AFrepeats) , and SNP markers . These aM. micrantha genes. Zhou et al. showed that ABA treatment, dehydration and high-salt conditions could induce the expression of a two-fingered C2H2-type ZFP gene (MmZFP1), which plays a key role in drought resistance [M. micrantha have homologous sequences from existing proteins in the NCBI database [M. micrantha [M. micrantha, Liu et al. identified a genome-wide duplication event and recent segmental duplications, which may contribute to its quick adaption to the environment. They also found that the metabolites of M. micrantha could improve the nitrogen utilization rate by enriching the microbes that participate in nitrogen cycling pathways. This plant species could absorb CO2 at night to further supplement the carbon fixation, making it obtain higher photosynthetic capacity and improve the photosynthetic efficiency of the stem [A few studies have begun to uncover the function of sistance . By trandatabase . Based oicrantha . In 2020the stem .Arabidopsis taproots, miR163 targets Paraxanthine Methyltransferase 1 (PXMT1) in a light-dependent manner at a suitable temperature to affect the root length [growth-regulating factors (GRFs) to affect the development of leaves, but also affects the length and cell cycle of cells in the root elongation zone by regulating the target gene basic helix–loop–helix transcription factor 74 (bHLH74) [Arabidopsis and overexpression of miR3954 in kumquat result in the early flowering of transgenic plants [Epigenetic regulation is another vital aspect in modulating plant growth and development, as well as adaption to environment. MicroRNAs (miRNAs) are a class of endogenous small RNAs (sRNAs) of about 20–24 nucleotides (nt) in length and regulate their target genes mainly at the post-transcriptional level [t length . The miR(bHLH74) ,23. Bothc plants .M. micrantha are rarely reported. In this study, we performed small RNA-seq and RNA-seq with five tissues of M. micrantha, including root, stem, leaf, shoot apex, and flower. Hundreds of conserved and novel miRNAs were identified, many of which were also differentially expressed among the five tissues. Integrated analysis uncovered the miRNA-mRNA regulatory networks in M. micrantha and revealed the potential roles of miRNAs in modulating the rapid growth and development of M. micrantha, which will facilitate further research on functional genomics and effective control of the rapid growth of this species.In plants, the sequences of miRNAs are completely or almost completely complementary to those of their target gene transcripts, and miRNAs repress the expression of target genes by cleaving the target transcripts or inhibiting the translation. The continuous development of high-throughput sequencing platforms and integrated analysis of small RNA and transcriptome enabled the large-scale identification of miRNAs and their target genes in diverse plant species, including non-model plants . So far,M. micrantha. Three biological replicates were prepared for each tissue, and the results were highly producible , 6 Dicer-like (DCLlike), 4 double-stranded RNA binding proteins (DRBlike), 1 HUA Enhancer 1 (HENlike), 10 RNA-dependent RNA polymerases (RDRlike), and 1 Serrate (SElike) , and 11 Nucleotidyl Transferases (3 HESOlike and 8 NTPlike). In addition, 3 DNA methyltransferases (CMTlike) were predicted, which could regulate RNA-directed DNA methylation (RdDM), suggesting that the RdDM pathway may present in M. micrantha. A total of 10 putative RNA polymerases (POLlike) were found as well, among which plant-specific Pol IV and Pol V should be included. Most of these predicted genes were highly expressed in the five tissues, especially in flower and shoot apex tissues, indicating that sRNA regulatory pathways exist in M. micrantha and potentially modulate its growth and environment adaption.Besides coding genes, many long non-coding RNAs and small RNAs, especially miRNAs, were reported to play essential roles in plant growth and development. To determine whether small RNAs are produced in escribed ,33, and (SElike) . Some geM. micrantha, we constructed sRNA libraries with RNAs extracted from the five tissues. Three biological replicates were prepared for each tissue. Around 414.38 M raw reads were obtained in the flower, leaf, root, shoot apex, and stem in total. After filtering low-quality reads and trimming adapters, approximately 320.3 M reads were obtained, and the reads aligned to the genome were 13.19, 17.95, 4.51, 22.68 and 22.78 M in the flower, leaf, root, shoot apex, and stem, respectively  was the most abundant one, with around 33,995 reads per million (RPM)  . When loon (RPM) a. Sequen members b, and thIn addition to conserved miRNAs, we predicted novel miRNAs in five tissues via integrated analysis of sRNA-seq and RNA-seq according to previous described criteria . A totalArabidopsis miR172 is predominantly highly expressed in the floral organ to repress the translation of its target APELA 2 (AP2), which is important for flowering [Arabidopsis miR163 is only abundant in the root and regulates the root length by targeting PXMT1 [In plants, many miRNAs exhibit specific temporal and/or spatial expression patterns, and their abundance is precisely and dynamically controlled via the balance between miRNA biogenesis and degradation pathways. For example, lowering . In contng PXMT1 .M. micrantha miRNAs are also differentially expressed among different tissues, all identified conserved and novel miRNAs in the five tissues were compared with each other with the cutoff |log2(fold-change)| ≥ 1 and FDR ≤ 0.05. The analysis result showed that many mmi-miRNAs were differentially expressed in the five tissues  transcription factors [SlHB15A (belong to HD ZIP-III family) regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures [TEOSINTE BRANCHED 1 (TB1)/CYCLOIDEA (CYC)/PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR 1 (PCF1) (TCP) genes [TCP genes or downregulation of miR319 results in delayed leaf senescence in Arabidopsis [Camellia sinensis, CsTCPs, targeted by CsmiR319b, regulate shoot tip development and catechin biosynthesis [M. micrantha.In order to verify whether  tissues . Among t tissues a. Other  tissues . As for  factors ,38, whiceratures . In addiP) genes ,41,42. Tbidopsis . In tea ynthesis . The difM. micrantha.Novel miRNAs exhibited different spatial expression patterns as well b. For inM. micrantha, we predicted their target genes using psRNA Target. With the standard of expectation set as 3, a total of 539 mmi-miRNAs corresponding to 4541 potential target genes were obtained, forming more than 12,000 miRNA-target pairs  gene family, which were conserved among plants and regulated the vegetative-productive phase transition. The mmi-miR858 family was predicted to target many MYB transcription factors which may participate in GA responses. Target genes for novel miRNAs were also predicted. In total, 192 novel miRNAs could target 1553 putative genes, but further validation of these miRNAs and their targets is required.To explore the regulatory functions of miRNAs identified in et pairs . In thiselopment ,45,46. NArabidopsis, miR845 is reported to target the long terminal repeat (LTR) retrotransposons in pollen to trigger the production of 21–22-nucleotide (nt) epigenetically activated small-interfering RNAs (easiRNAs) via Pol IV, and these easiRNAs are required for the triploid block [M. micrantha, the potential targets of mmi-miR845 included genes encoding ribosomal proteins, protein kinases, zinc finger proteins, histone methyltransferases, proteasome subunits and so on; many of these targets were related to gene expression regulation. Intriguingly, the gene encoding retrotransposon gag protein was also the putative target of mmi-miR845, suggesting that easiRNAs may be produced in M. micrantha pollen and required for post-fertilization genome stability and seed viability.Surprisingly, we found that the mmi-miR845 family, consisting of mmi-miR845a/b/c, may target 585 genes. In id block . In M. mM. micrantha  [GRFs in the shoot apex tissue will facilitate the growth of SAM, thereby promoting the growth of leaves and the rapid growth of M. micrantha. Similarly, the abundance of the mmi-miR319 family was lower in M. micrantha leaf than in other tissues, implying that its target gene TCPs were highly expressed in leaf tissue. A previous study reported that miR319 was involved in the regulation of plant leaf morphology by targeting the TCP2, TCP3, TCP4, TCP10, and TCP24 genes [Arabidopsis, the silence of miR319 or overexpression of TCPs caused delayed leaf senescence [M. micrantha leaf tissue will be beneficial to leaf morphology maintenance and senescence delay, enabling leaves to maintain a high photosynthetic efficiency, thereby promoting the rapid growth of M. micrantha. Taken together, these results indicate that miR396 and miR319 play important roles in regulating the growth and development of M. micrantha.Similar to other plants, the mmi-miRNAs exhibit differential expression patterns among different tissues, which could facilitate the regulation of tissue-specific gene expression and the exuberant vitality of cked SAM ,45,46, i(leaves) ,52. Ther24 genes ,42. In anescence . These rM. micrantha vines were collected from Shenzhen University (Lihu Campus) and cut into several branches with two stem nodes and a leaf on the apical side. Then the morphological base end was cultured in water under low light conditions for approximately 1 week. Plants with root lengths of approximately 4–5 cm were transferred to Hoagland liquid medium and grown in the growth room with daily cycles of 16 h/light and 8 h/dark at 24 °C for 60 days. The flower, leaf, shoot apex, stem, and root tissues were collected for RNA extraction; three biological repeats were prepared for each tissue.Wild-type M. micrantha flower, leaf, shoot apex, stem, and root tissues using TRIzol  according to the manufacturer’s instructions. The RNA integrity and concentration were determined with NanoDrop . The small RNA libraries were constructed as described before [Total RNA was extracted from d before  and sequArabidopsis was constructed, dividing the 20,000 genes into 30 modules. To specifically quantify the unique correlations between individual gene modules and each sample, the correlation coefficients of gene clustering modules and sample features were analyzed. Use plot Eigengene Networks to create modular cluster plots and modular heatmaps. Extract the genes in each module and use pheatmap to draw the gene expression heat map of the module and add tissue type grouping information. Finally, the correlation coefficient matrix between genes was imported into cytoscape as interaction information to create a gene co-expression network.WGCNA analysis was performed as described before ,55. FirsM. micrantha reference genome (ASM936387v1) by AUSPP with the mode  [http://www.miRBase.org/, accessed on 7 December 2021), and the matched reads were defined as conserved miRNAs. The potential novel miRNAs were predicted using miRDeep-P2 software  [The raw data were processed and mapped to the mallRNA) ,56. All mallRNA)   . The nor, China)  with |lo, China) . The seqhttps://www.zhaolab.org/psRNATarget/, accessed on 7 December 2021) with a maximum expectation of 3.miRNA target genes were predicted using the psRNATarget program  (https:/M. micrantha. We also identified numerous conserved and novel miRNAs in M. micrantha and predicted the targets of mmi-miRNAs. According to the abundance and family size, mmi-miR396, mmi-miR165/166, and mmi-miR319 families may play the most important roles in regulating the growth and development of M. micrantha by targeting GRF genes, HD ZIPIII genes, and TCP genes, respectively. In addition, the differential repression of miRNAs among the five tissues may contribute to the regulation of tissue-specific gene expression and facilitate the fast growth of M. micrantha. Our results not only revealed a miRNA-mRNA regulatory network in M. micrantha, but also indicated the potential roles of miRNAs and their target genes in the fast growth and development of M. micrantha. These findings will provide new insights into dissecting the molecular mechanism of the rapid growth of M. micrantha and controlling the serious invasion of these invasive plants.We performed co-expression network analysis of RNA-seq from five tissues to explore many candidate genes responsible for the rapid growth and development of