Eurycoma longifolia Jack (Fam.: Simaroubaceae), known as Tongkat Ali (TA), has been known as a symbol of virility and sexual power for men. Metabolic profiling of the aqueous extract of E. longifolia (AEEL) using UPLC-MS/MS in both positive and negative modes allowed the identification of seventeen metabolites. The identified compounds were classified into four groups: quassinoids, alkaloids, triterpenes, and biphenylneolignans. AEEL is considered safe with oral LD50 cut-off >5000 mg/kg. Oral administration of 50, 100, 200, 400, or 800 mg/kg of AEEL for 10 consecutive days to Sprague-Dawley male rats caused significant reductions in mounting, intromission, and ejaculation latencies and increased penile erection index. AEEL increased total body weight and relative weights of seminal vesicles and prostate. Total and free serum testosterone and brain cortical and hippocampal dopamine content was significantly elevated in treated groups with no significant effects on serotonin or noradrenaline content. The World Health Organization defines infertility as the inability of a couple to bring a pregnancy to term after one year or more of regular, unprotected sexual intercourse or achieve conception . InfertiEurycoma longifolia Jack or “Tongkat Ali” is a herb which has been claimed to possess various medicinal properties. Traditionally, people believe that this herb can be used as remedies for sexual dysfunction, constipation, cancer, leukemia, exercise recovery, loss of libido, aging, stress, high blood pressure, malaria, osteoporosis, diabetes, fever, and glandular swelling + at m/z 351.100 which is corresponding to the molecular formula C18H19ClO5. The presence of a chlorine atom was confirmed by the appearance of an isotope peak [M+2]+ at m/z 352.000 in addition to a peak at 315.010 corresponding to [(M+H)-HCl]+. This compound was identified as Eurycolactone B [ 2 (Rt 20.05 min) showed a molecular ion [M+H]+ at m/z 365.0000 which could be correspondent to the molecular formula C19H24O7 that produced MS2 fragment at m/z 347.000 and 318.900 equivalent to [(M+H)-H2O]+ and [(M+H)-CH2O2]+, respectively. This fragmentation pattern is characteristic to the quassinoid 6α-hydroxyeurycomalactone + at m/z 409.000 (C20H24O9) and showed a daughter ion at m/z 391.000 corresponding to the loss of a water molecule [(M+H)-H2O]+ and another ion at 345.100 corresponding to [(M+H)-CO]+. This compound was identified as Eurycomanone [ 4 (Rt 23.63 min) showed a molecular ion at m/z 413.200 and a MS2 m/z 395.100 and 376.800 with the loss of 18 and 36 amu corresponding to [(M+H)-H2O] and [(M+H)-2H2O], respectively. This compound was regarded as 13β,18-dihydroeurycomanol (C20H28O9) + at m/z 383.200 with a daughter ion peak at m/z 365.100 [(M+3H)-H2O] due to the loss of a water molecule. This peak was identified as 15β-Hydroxyklaineanone [ 9 (Rt 38.36 min) also showed a molecular ion peak [M+H]+ at m/z 413.100 corresponding to the molecular formula C20H28O9; it showed MS2 at m/z 395.200 [(M+H)-H2O] that resulted from a loss of a water molecule; this quassinoid was identified as 5α, 14β, 15β-Trihydroxyklaineanone [ 11 (Rt 45.76 min) showed a molecular ion peak at m/z 455.102 (C22H30O10) and gave daughter ions at m/z at 454.100 and 436.000 corresponding to M+ and [M-H2O]+. This compound was identified as 6α-Acetoxy-14,15β-dihydroxyklaineanone − at m/z 443.300 consistent with the molecular formula C20H28O11. This peak gave daughter ions at m/z 427.100 and 413.000 due to the loss of -CH and -CH2O, respectively. It was identified as 13β,21-dihydroxyeurycomanol. Peak 8 (Rt 29.98 min) was identified as 14,15β-Dihydroxyklaineanone as it showed a molecular ion peak [M-H]− at m/z 395.100 and MS2 at m/z 377.000 due to the loss of H2O (18 amu), 359.100 with the loss of 2H2O (36 amu), and 349.300 due to the loss of –CH2O2 [ 10 (Rt 38.43 min) was with a molecular ion at m/z 315.100 corresponding to the molecular formula C18H20O5 and had fragmentation ions at m/z 271.200 and 256.001 due to the loss of CO2 [(M-H)-CO2] and [(M-H)-CO2-CH3]. This fragmentation pattern was in consistence with that of Laurycolactone B [ 12 (Rt 45.91 min) was identified as (α/β-epoxide) Ailanthone (C20H24O8) with a base peak m/z 391.100 corresponding to [M-H]− and MS2 at m/z 373.100 [(M-H)-H2O] and 363.200 [(M-H)-CO]. Peak 13 (Rt 46.18 min) was with molecular ion at m/z 573.122 corresponding to the molecular formula C26H38O14 and was identified as Iandonoside B; it showed MS2 at m/z 555.000 [(M-H)-H2O], 513.900 [(M-H)-C2H3O2] + at m/z 251.200 corresponding to the molecular formula C15H10O2N2. It showed also daughter ions at m/z 236.100 and 233.000 due to the peaks [(M+H)-CH3] and [(M+H)-H2O], respectively. This compound was identified as 9-methoxycanthin-6-one [ 15 (Rt 46.69 min) was with a molecular ion peak [M-H]− at m/z 279.000 corresponding to the molecular formula C16H12O3N2. MS2 appeared at m/z 264.000 [(M-H)-CH3], 251.100 [(M-H)-CO], and 237.000 [(M-H)-C2H2O]. This compound could be identified as 5,9-dimethoxycanthin-6-one [ 16 (Rt 47.45 min) had a molecular ion peak [M-H]− at 265.100 corresponding to C15H10O3N2. Its daughter ions MS2 were detected at m/z 250.200 [(M-H)-CH3], 247.300 [(M-H)-H2O], and 222.200 [(M-H)-CHNO]; thus this was identified as 11-hydroxy-10-methoxycanthin-6-one [Three alkaloids of cathin-6-one type were also detected: one was detected in the positive ionization mode as peakin-6-one . Peak 16 17 (Rt 47.91 min) that had a molecular ion peak [M+H]+ at 389.000 (C21H24O7) with MS2 at 371.110 [(M+H)-H2O], 357.001 [(M-H)-CH4O], and 343.100 [(M-H)-C2H6O] which was consistent with the biphenylneolignan;2-hydroxy-3,2′,6′-trimethoxy-4′--5-(3-hydroxy-1-propenyl)-biphenyl [Furthermore, a biphenylneolignan was observed in the positive ionization mode detected as Peakbiphenyl . The ide389.000 C1H24O7 wi E. longifolia extract is considered unclassified according to the OECD with oral LD50 cut-off >5000 mg/kg.A repeated single oral dose of AEEL (2000 mg/kg) did not show any mortality at 24 h after administration. Therefore,Oral administration of AEEL  caused dose-related enhancement of male rat sexual behavior . This waThe oral administration of AEEL resulted in significant  increases in total body weight (TBW) of male rats at 200, 400, and 800 mg/kg, respectively . Also, rOur data indicated that AEEL oral administration to male rats leads to significant elevation of total testosterone serum level by 67.1%, 127.8%, and 288.9% of control level at 200, 400, and 800 mg/kg, respectively . InteresA reverse pattern was observed with FSH and LH levels. There were significant decreases in both FSH and LH levels at higher doses , as compared to the corresponding control values. Serum levels of FSH were decreased by 21.4%, 18.9%, and 29.5% of the control level at 200, 400, and 800 mg/kg, respectively . The decOur evaluation indicated that AEEL oral administration to male rats resulted in significant elevation of cortical dopamine level at 200, 400, and 800 mg/kg . However E. longifolia has been reputed by Malays as a traditional remedy used as an adaptogen for energy and vitality and is well known for its aphrodisiac activities [ E. longifolia as an aphrodisiac herb is well-recognized, there is a paucity of information on the possible underlying mechanisms. Therefore, the present study was conducted to substantiate the aphrodisiac activity of E. longifolia.In Malaysia,tivities . Althoug E. longifolia to discriminate between two samples cultivated in two different locations in Malaysia using the positive ionization mode [Initially, metabolic profiling of AEEL was performed using UPLC-MS/MS. A previous study was conducted which involved the LC-MS/MS analysis of the aqueous extracts ofion mode . Here we50 of AEEL was determined in Sprague-Dawley rats according to OECD Guideline #423(OECD)  to male rats resulted in obvious improvement of sexual behavior. This is evidenced by significant reductions in mounting, intromission, and ejaculation latencies and significant increase in penile erection index, at high dose levels. These findings gain support by several studies highlighting the aphrodisiac activities of E. longifolia which indicated an improvement in all parameters of sexual behavior towards receptive females [ E. longifolia promoted the growth of both rat ventral prostate and seminal vesicles [LDary File ). E. lon females –28. In aAssessment of the impact of AEEL on the pituitary-gonadal axis indicated significant elevation of the serum levels of total and free testosterone. This was confirmed by other studies which showed an increase in testosterone concentration after treatment of male rats with AEEL . RationaDue to their key roles in behavioral functions including sexual behavior, the brain cortical and hippocampal contents of dopamine, serotonin, and noradrenaline were evaluated. Brain cortex and hippocampus are important structures in the sexual reward system , 31. Dop E. longifolia was reflected on the possible negative feedback actions on LH and FSH levels as well as modulation of related brain neurotransmitters. Androgens modulate male sexual behavior and act at both the central and peripheral nervous system levels [Collectively, the observed enhancement of free testosterone levels bym levels . The tesm levels . E. longifolia in the improvement of sexual behavior may be attributed to its active constituents such as quassinoids and in particular the major one, eurycomanone which was detected as peak 3 in UPLC-MS/MS analysis of AEEL. Eurycomanone was reported to induce testosterone production [The positive effect ofoduction  and was oduction . Moreoveoduction . E. longifolia roots aqueous extract in male Sprague-Dawley rats. This can be attributed, at least partly, to elevation of blood testosterone level as well as enhancement of brain cortical and hippocampal dopamine content.The current data confirm the aphrodisiac and anabolic activities of


Consequently, the Phe/Tyr ratio decreased significantly (p < 0.05) in the majority of patients treated. In conclusion, LNAA treatment increases Tyr levels in sub-optimally controlled adult PKU patients, while offering the potential to improve their adherence to treatment.The standard treatment for phenylketonuria (PKU) is a lifelong low-phenylalanine (Phe) diet, supplemented with Phe-free protein substitutes; however, adult patients often show poor adherence to therapy. Alternative treatment options include the use of large neutral amino acids (LNAA). The aim of this study was to determine the Phe, tyrosine (Tyr), and Phe/Tyr ratio in a cohort of sub-optimally controlled adult patients with classical PKU treated with a new LNAA formulation. Twelve patients received a Phe-restricted diet plus a slow-release LNAA product taken three times per day, at a dose of 1 g/kg body weight (mean 0.8 ± 0.24 g/kg/day), over a 12-month period. The product is in a microgranulated formulation, which incorporates all amino acids and uses sodium alginate as a hydrophilic carrier to prolong its release. This LNAA formulation provides up to 80% of the total protein requirement, with the rest of the protein supplied by natural food. Patients had fortnightly measurements of Phe and Tyr levels over a 12-month period after the introduction of LNAA. All patients completed the 12-month treatment period. Overall, adherence to the new LNAA tablets was very good compared with a previous amino acid mixture, for which taste was a major complaint by patients. Phe levels remained unchanged ( Phenylketonuria  is an autosomal recessive genetic disorder caused by mutations of the gene encoding the phenylalanine hydroxylase  enzyme ,4. The PIn our recent findings, which are representative of the whole Italian PKU population, habitual consumption of low-protein foods was reported by 87% of patients, while the mean use of amino acid mixtures was low: only 18.5% of patients reported an optimal consumption . The mosIn recent years, alternative or complementary treatments were proposed. New protein substitutes, including large neutral amino acids (LNAA) and glycomacropeptide, and pharmacological treatments, such as sapropterin dihydrochloride (BH4) and phenylalanine ammonia lyase enzyme, were successful in the treatment of adult PKU patients ,13,14.In the pathophysiology of PKU, modification of amino acid transport across the blood–brain barrier (BBB) plays an important role with the increase of brain Phe influx competing with other LNAAs at the same transport level . TherefoThe LNAA transporter facilitates the transport of LNAA across the BBB. Elevated blood levels of Phe is predicted to increase the uptake of Phe at the expense of other LNAAs, resulting in the disruption of brain amino acid homeostasis. In PKU patients, administering large amounts of Phe-free LNAAs competes with Phe and may reduce the influx of Phe into the brain .Nowadays, two different LNAA treatment strategies are reported in the literature: LNAA alone or in combination with a low-Phe diet . All stuThe aim of our study was to evaluate plasma Phe, Tyr, and their ratio in a cohort of sub-optimally controlled classical adult PKU patients treated with a new LNAA formulation combined with a low-Phe diet.In total, 12 adult PKU patients from the Division of Inherited Metabolic Diseases, University Hospital of Padova, Italy, were selected to participate in the study because of their persistently low diet adherence due to their refusal to take supplementation with an amino acid mixture. These patients were already on a Phe-restricted diet and amino acid supplementation covering 80% of their total protein intake.Diagnosis of PKU was performed by neonatal screening, and all patients were treated with a Phe-restricted diet from birth. All 12 patients had a PAH genotype and BH4 loading test showing a lack of response to the PAH cofactor sapropterin dihydrochloride.Dietary intake was not recorded during the study due to its long treatment period ; therefore, detailed information on dietary habits is not available.The principles of Good Clinical Practice were adhered to throughout the study, in accordance with the Declaration of Helsinki and ICH/GCP. The study was approved by the Ethical Committee of Padua University Hospital, Italy. Written informed consent was obtained from all participants.® Micro R (PIAM). This product is registered in Italy for the treatment of PKU under medical prescription, and it is under registration in Turkey and Spain. Because the product does not contain vitamins or minerals, all patients in our study were regularly supplemented with multivitamins and minerals.The LNAA formulation used in this study was NeutrAfenilThe LNAA formulation is a granulation that can be easily swallowed with water or other liquids. The amino acids are incorporated in microgranules that are coated with a methylcellulose film to prevent any unpleasant taste related to the amino acid mixture. The cap of the bottle can be used to measure 17 grams of amino acids.The most significant characteristics of the LNAA formulation are improved palatability (unflavored taste), its prolonged-release formulation due to sodium alginate as a hydrophilic carrier that provides a physiological absorption similar to that of natural protein, and the high concentration of Tyr in comparison with other available products.The composition of the LNAA formulation is shown in Table 1The demographic characteristics of the patient population are shown in The LNAA supplement was taken three times per day over a 12-month period , at a dose of 1 g/kg body weight. No other amino acid mixture was given to the patients and no changes were made to their low-Phe diet.Patients collected fasting blood spots at home every two weeks in the morning before taking LNAAs. Measurements of Phe and Tyr levels were done on these dried blood spots (DBS) using liquid chromatography/tandem mass spectrometry, and the Phe/Tyr ratio was calculated. The consumption of LNAA was calculated by the dietitian every six months. All patients stated that, due to their commitments, they generally had lunch or dinner out of the home, with a high intake of Phe .p-value of <0.05 was considered significant. Categorical variables are expressed as N (%), and quantitative variables are expressed as mean ± SD.The Wilcoxon paired sample T-test was adopted to compare data sets acquired from the patients before and after the start of LNAA therapy. A p = 0.008 and p = 0.0039, respectively); no significant differences were identified for the other components.Patient nutritional data prior to and after LNAA were introduced, including the total protein intake, medical foods intake, and Phe and Tyr intake, are summarized in In the 12-month period prior to the introduction of LNAA, the mean ± SD Phe and Tyr values ranged from 628 ± 148 to 1033 ± 198 μmol/L and from 32 ± 7 to 87 ± 35 μmol/L, respectively, while the mean ± SD Phe/Tyr ratio ranged from 9.7 ± 2.2 to 19.9 ± 2.8 μmol/L, and the DBS frequency ranged from 5 to 38 (median = 19): 3 patients (25%) sent more than 24 DBS samples. Phe and Tyr values, Phe/Tyr ratio, and DBS frequency are shown for each patient in p = 0.57 and p = 0.95, respectively).All 12 patients completed the 12-month trial. At baseline and at the end of the study, patients had a mean ± SD weight of 70.0 ± 18 kg and 70.4 ± 18 kg, respectively, and a mean ± SD BMI index of 24.80 ± 4.83 and 24.92 ± 5.11, respectively. Neither weight nor BMI were significantly different when compared prior to and after the introduction of LNAA (Adherence to LNAA supplementation was very good.p = 0.0088) ( 0.0088) .p = 0.0195) (p < 0.05) after 12-months of treatment with LNAA; in 2 patients, the Phe/Tyr ratio showed a small increase   . Compare < 0.16) .We also report data which illustrates the level of Phe and Tyr at baseline, after 6 months (short term), and after 12 months (long term) of LNAA treatment for each patient .p = 0.00458), but then increased significantly (p = 0.0522) in the last six-month treatment period with LNAA (p = 0.324) or from 6 to 12 months (p = 0.1400)  (ith LNAA . Althoug 0.1400) , suggestIn this study, blood Tyr levels were significantly elevated in the patient cohort for the entire duration of the study. The mean percentage variation increased after 12 months of treatment, and the difference in Tyr levels compared with baseline was statistically significant. Even in the short-term, Tyr values showed an increase, which was also confirmed in the long-term (12 month), with an increase in 67% of patients. This increase also reflects the high amount of Tyr present in the LNAA formulation in comparison with other available products. Changes of Tyr levels were also confirmed by the decrease in Phe/Tyr ratio during all periods of the study.In our study, mean blood Phe levels did not change significantly in the 12 months before starting LNAA therapy compared with the 12-month period after the initiation of LNAA therapy. Indeed, patients still showed poor control over Phe levels. These data support previous studies  that didIn our study, we confirmed these results over a short-term period, but a decrease of Phe values was not confirmed over a long-term period (12 months). Indeed, at the end of this study, Phe levels returned to high levels in 75% of patients. Blood Phe concentration remained unchanged after one and six months of LNAA supplementation in a small cohort of six patients with classical PKU . NonetheIn a previous study, which focused on adult Italian patients, we showed that dietary adherence was poor, with reduced daily use of amino acid supplements (<4–5 times/day in 82% patients) . The surThe use of LNAAs in the treatment of patients with PKU was suggested as early as 1948 and offers an alternative complementary treatment option; however, the optimal composition of LNAA is still unknown .Studies that have evaluated LNAA in PKU patients ,27 are rIn 2006, Matalon and colleagues evaluated a new formulation of LNAA (with administration of 0.5 to 1 g/kg/day) and reported that blood Phe concentrations decreased after one week without any change in dietary practice . A folloThe most recent report on the use of LNAA demonstrated a positive correlation between blood Phe reduction and LNAA intake in 12 PKU children after four weeks of treatment . NotablyData on brain Phe levels in patients treated with LNAA are scarce. Pietz et al. showed that LNAA had the ability to block Phe transport into the brain . In thisIn animal experiments using PKU mice on a normal diet supplemented with LNAA, van Vliet and colleagues showed significantly reduced brain Phe concentration and increased brain serotonin and norepinephrine, but no significant changes in dopamine concentration . The samThe change in LNAA levels detected in plasma and the brain reflects the different distribution of LNAA in plasma, in the neurovascular unit, and the actions of the specific transporter systems across the BBB. The PAH, which converts Phe into Tyr, is not expressed in the brain. Experimental models showed that Tyr is the LNAA with the most consistent correlation between serum ratio and brain tissue levels . Indeed,The complexity of the neurovascular unit, which comprises the BBB endothelium, the astrocytes, and neurons, also makes any direct comparison between LNAA data achieved from experimental models with those of the human brain difficult .Most patients in our cohort showed nonsignificant biochemical changes (blood Phe levels) at the end of the LNAA treatment period, which is contradictory to some of the previously published studies. However, previous studies were carried out over very short time periods (less than 12 months), whereas patients in the current study were treated with LNAA for a 12-month period. Indeed, in the short term (less than 6 months), a decrease in Phe levels was detected, but Phe levels returned to baseline levels for the majority of patients by the end of the 12-month treatment period .Another possible contributing factor to the nonsignificant reduction of Phe levels is the age of the patients. In our cohort, the patients were all adults, whereas, in the Concolino study, they were pediatric patients, with a better therapy adherence .Limited data are available regarding Tyr values and Phe/Tyr ratio in patients on LNAA supplementation. The LNAA product used in the current study is very rich in Tyr and Trp in comparison with other products on the market and offers a new extended-release formulation. Both these characteristics can help avoid fluctuation of blood Phe in adults. Phe fluctuations were already suggested as a major source of neurotransmitter perturbation, with subsequent behavioral complications for PKU patients . More reMoreover, high levels of Tyr may promote an increased brain monoaminergic neurotransmitter concentration . Three mResults from our study suggest that the period of biochemical and clinical follow-up should be increased in comparison with what was known from previous studies. It is possible that a follow-up of 12 months is still not long enough for a chronic condition like PKU. At present, we are continuing to monitor the patients receiving LNAA treatment.There are limitations within the current study. Firstly, the results of our study are related to a relatively small group of noncompliant adult PKU patients over a long period of time. It is difficult to compare our observational study with other published studies in patients (children and adults combined) conducted over a short period of time. Although some findings may be a consequence of bias and lack of an optimal composition of LNAA, a follow-up study with an increased number of noncompliant patients should be performed before concrete conclusions can be drawn.Secondly, food intake and compliance were not regularly recorded and obtained through self-reporting. The authors believe that this closely reflects the real life of adult PKU patients; however, patient-centered data collection presents opportunities for misreporting. The ability to conduct a research study is particularly difficult when the patients are predominantly noncompliant PKU and are disengaged from active metabolic follow-up, highlighting the challenges in their inclusion in relatively complicated research procedures. This has done recruitment particularly difficult and the design of a randomized control trial study complex, as recently reported by Green et al. .Thirdly, the plasma Phe, Tyr, and their relative ratio are clinically effective biomarkers for the follow up of the disease, but due to the lack of standardization of blood sampling obtained through self-completed blood samples, caution should be taken when extrapolating the results of this study.In conclusion, LNAA supplementation to a low-protein diet should be considered in adult PKU patients who refuse amino acid mixtures. The use of a different therapeutic option, such as LNAA with its tasteless formulation, showed two major results: the increase of blood Tyr levels and the improvement of patient adherence to medication. Further studies are needed to assess the nutritional intake and metabolic status of PKU patients using LNAA for longer periods of time .


POLE ultramutated (POLEmut), mismatch repair‐deficient (MMRd), p53 mutant (p53abn) and those EC lacking any of these alterations, referred to as NSMP (non‐specific molecular profile). Several large studies confirm the prognostic relevance of these molecular subgroups. However, this ‘histomolecular’ approach has so far not been implemented in clinical routine. The ongoing PORTEC4a trial is the first clinical setting in which the added value of integrating molecular parameters in adjuvant treatment decisions will be determined. For diagnostics, the incorporation of the molecular parameters in EC classification will add a level of objectivity which will yield biologically more homogeneous subclasses. Here we illustrate how the management of individual EC patients may be impacted when applying the molecular EC classification. We describe our current approach to the integrated diagnoses of EC with a focus on scenarios with conflicting morphological and molecular findings. We also address several pitfalls accompanying the diagnostic implementation of molecular EC classification and give practical suggestions for diagnostic scenarios.Histopathological evaluation including subtyping and grading is the current cornerstone for endometrial cancer (EC) classification. This provides clinicians with prognostic information and input for further treatment recommendations. Nonetheless, patients with histologically similar ECs may have very different outcomes, notably in patients with high‐grade endometrial carcinomas. For endometrial cancer, four molecular subgroups have undergone extensive studies in recent years:  World Health Organisation (WHO) classification of tumours of female reproductive organs,In the 4th edition of the POLE); (ii) hyper‐mutated ECs characterised by microsatellite instability (MSI); (iii) a copy‐number low subgroup with a low mutational burden; and (iv) a copy‐number high group with frequent TP53 mutations.POLE ultramutated (POLEmut) EC, MMRd EC, NSMP EC and p53abn EC  project were published.C Figure . These nPOLEmut EC) and intensified treatments are considered in the presence of unfavourable factors (e.g. p53mut EC). The ongoing PORTEC4a trial  is the first clinical trial to prospectively investigate the incorporation of molecular EC characteristics in the context of patients who would currently be classified as high–intermediate risk based on clinicopathological factors alone. Standard postoperative vaginal brachytherapy (VBT) is being compared to adjuvant treatment based on an individual's molecular‐integrated risk profile, including no adjuvant treatment, VBT or external beam radiotherapy (EBRT). This randomised trial will provide essential information on how the ‘histomolecular’ entities may be used to personalise adjuvant treatment by decreasing both over‐ and undertreatment.Adjuvant treatment is currently recommended based on a patient's individual risk  comprised of a combination of clinical (age) and pathological  factors.In this review, we illustrate clinical scenarios in which integration of the molecular characteristics into the current clinicopathological classification may shift the adjuvant treatment recommendations. We also discuss how the integrated molecular entities may provide clues to specific targeted treatment modalities that can be exploited in advanced‐stage or recurrent disease. For a more detailed background on the molecular EC classification, as well as assays that may be used for the diagnosis, the reader is referred to other reviews.POLE variants in the exonuclease domain of the POLE gene comprise approximately 10% 6of all endometrioid EC (EEC),POLEultramutated’ or ‘POLEmut EC’. Typical POLEmut EC features include: presentation at relatively young age and early stage, high tumour grade with scattered tumour giant cells and a prominent lymphocytic infiltrate.POLEmut EC are mainly microsatellite‐stable (MSS), unstable cases with MMR‐protein loss have been described.TP53 mutations have been reported in up to 42% of POLEmut EC, sometimes resulting in subclonal mutant‐like p53 immunohistochemistry.POLEmut EC are illustrated in Figure Pathogenic POLEmut EC patients have an exceptionally good prognosis.POLEmut ECs in these series. Sub‐analysis of POLEmut and POLEwild‐type EC in the observation arm of the PORTEC‐1 trial (n = 245 patients with stage I intermediate risk EC), however, also showed a 10‐year recurrence‐free survival (RFS) of 100% versus 80.1%, respectively .POLEmut EC possess intrinsic characteristics that are beneficial for survival, independent of sensitivity to adjuvant treatment. This is supported by the lack of an increased radiation sensitivity in POLEmut embryonic stem cells.POLEmut EC are immunogenic due to their high mutational load, which may be a more plausible explanation for the unusually low chance of recurrences observed in these patients.POLEmut EC, similar survival rates have now been reported for patients with high‐risk POLEmut EC.POLEmut ECs raises the question of whether these patients may be unnecessarily exposed to unwanted side‐effects of radio‐ and/or chemotherapy.POLEmut EC has therefore been proposed.POLEmut EC. For high‐risk POLEmut EC, the recently presented molecular characterisation of the PORTEC‐3 trial has been highly informative.POLEmut EC showed an excellent prognosis independent of the treatment arm (EBRT versus CTRT), supporting omitting the addition of chemotherapy for patients with high‐risk POLEmut EC.Despite their association with high‐grade histology, POLEmut‐MMRd EC, POLEmut‐p53abn EC, MMRd‐p53abn EC or POLEmut‐MMRd‐p53abn EC), also referred to as ‘multiple classifier’ EC.POLEmut‐p53abn EC, in which the tumour exhibits both a favourable pathogenic mutation in the POLE exonuclease domain as well as unfavourable aberrant p53 IHC expression, such as illustrated in our case in Figure The majority of EC can be classified into one of the four molecular subgroups. However, in a small subset (3–5%) of patients molecular analysis will show more than one classifying alteration , unrelated to Lynch syndrome.POLEmut EC in that these are also associated with higher‐grade, endometrioid‐type histology and abundance of tumour‐infiltrating lymphocytes (TILs).POLEmut EC.MMR deficiency is frequent in EC 25–30%) and defined by the loss of nuclear expression of one or more MMR proteins  by the tumour cells.–30% and P = 0.002).Histologically, LVSI is defined as the presence of tumour cells within an endothelial‐lined space that lies outside the invasive border. Interestingly, identifying one focus of LVSI  has little impact on prognosis, whereas substantial LVSI  is associated with a significant increase in the risk of recurrence.Multiple studies investigating the prognostic impact of  LVSI in EC patients have found an increased risk of lymph node metastasis and locoregional as well as distant recurrences, regardless of stage and histotype.TP53 mutations and abnormal p53‐IHC are shown in Figure A paradoxical scenario occurs when a morphological low‐grade endometrioid EC shows molecular characteristics associated with aggressive clinical behaviour, such as abnormal mutant‐type p53 staining. This is a rare finding in low‐grade EEC, as it is reported in 2–15% of glandular EEC with low nuclear grade,TP53 mutations in EC, with reported sensitivity and specificity of 0.96 and 1.00, respectively.p53 IHC has proved to be a very reliable surrogate marker for detecting underlying POLEmut and MMRd EC, due to their high mutational burden, obtain high levels of neoantigens and TILs, making them attractive candidates for immunotherapy such as anti‐PD1 immune check‐point blockade.POLEmut EC under current treatment regimens (independent of stage), however, argues against the rationale to the use of immunotherapy for this subpopulation. For MMRd EC, particularly when recurrent or in advanced metastatic stage (FIGO stage >III), immune check‐point inhibition may be an attractive option.ERBB2 gene, encoding for human epidermal growth receptor 2 (HER2) and homologous recombination deficiency (HRD), both occurring in p53mut EC, are being explored and show some promise.To date, treatment options for patients with advanced or recurrent EC are limited. In recent years several targetable pathways in the context of molecular EC subgroups have been investigated, resulting in novel treatment strategies with potential clinical benefit. ERBB2 gene and/or HER2 overexpression have been reported in serous EC and uterine carcinosarcomas with a serous carcinomatous component, albeit with a substantial variation in reported rates of 14–80% and 21–47%, respectively.ERBB2 by in‐situ hybridisation is shown in Figure P = 0.005) when trastuzumab was given in combination with carboplatin‐paclitaxel chemotherapy.ERBB2 gene amplifications and serous histology is of interest, as it suggests that this genomic alteration may be limited to p53mut EC. It would be informative to address this association in more detail in future studies, as it would provide a rationale for focused HER2 testing in the context of p53mut EC.Amplification of the BRCA1/2 mutations.Homologous recombination repair capacity has been successfully used as a marker to identify patients that benefit from poly (ADP ribose) polymerase inhibitors (PARPi) in high‐grade serous ovarian cancer (HGSOC).POLE gene and perform three immunohistochemical stains  in order to provide an integrated ‘histomolecular’ diagnosis. Next, the design of (adjuvant) treatment strategies incorporating this novel classification will become a priority. This shift towards a molecular driven EC classification is yet another illustration of how pathology is delivering the promise of precision medicine.The molecular subgrouping proposed by the TCGA has been fundamental in evolving the dualistic Bokhman model into a more refined and molecular‐based system. The subsequent development and validation of widely accessible surrogate markers for the four distinct EC subgroups has accelerated this field. This has led to a diagnostic algorithm that incorporated molecular characteristics, resulting in a novel, objective and clinically meaningful EC classification. Once regulatory authorities understand the potential clinical impact of this improved EC classification, financial aspects that come with the implementation of additional testing should be quickly resolved. Pathologists will be asked to assess the mutational status of the CTNNB1 (52%).CTNNB1 mutations in low‐grade early‐stage EC showed a significantly worse recurrence‐free survival.CTNNB1 exon 3 mutations were shown to have prognostic significance in patients with high–intermediate‐risk NSMP EC.CTNNB1 exon 3‐mutated EC are associated with low‐grade, low abundance of TILS and with squamous differentiation.CTNNB1 exon 3 gene sequencing in EC have reported varying concordance rates, resulting in the conclusion that β‐catenin IHC is insufficient to act as a surrogate marker for CTNNB1mut EC.CTNNB1 mutant EC as the fifth molecular EC subgroup, which may be referred to as CTNNB1mut EC (after excluding MMRd and POLE mutations). It is conceivable that CTNNB1mut EC will be incorporated into the diagnostic algorithm as a separate ‘histomolecular’ entity.The molecular EC classification follows the subclassification proposed by the TCGA; however, further refinement of this classification is to be expected. It would be particularly attractive to further refine the largest EC subgroup, NSMP EC, which can be regarded as a heterogeneous molecular rest group mainly consisting of alterations in PI3K‐Akt and Wnt‐signalling alterations.CTNNB1 2%.CTNNB1et al. showed, in an univariable analysis of 242 EEC with low‐risk features, a trend towards a higher rate of distant recurrences and lower overall survival  for patients with p53mut EC.POLEmut EC in the high‐risk EC population, which show an excellent prognosis when adjuvant treatment is limited to radiotherapy, strongly suggesting that these patients do not benefit from adjuvant chemotherapy.Most studies that report on the impact of adjuvant treatment in the context of the molecular EC classification have included high‐intermediate risk patients. For patients who would currently be regarded as ‘low‐’ or ‘high‐risk’, the current data on the impact of adjuvant treatment are limited. Stelloo POLE testing or that results from any of the required assays may not be conclusive. To molecularly classify an individual EC, following the diagnostic algorithm provided in Figure POLE testing is a prerequisite. This is best illustrated by POLEmut EC that carry TP53 mutations, which would be falsely classified as p53mut EC when POLE‐testing is not performed. With this in mind, we propose that a ‘not otherwise specified’ (NOS) diagnostic designation is used when there is insufficient information to assign a specific molecular entity. This is in contrast to ‘NSMP EC’, which defines EC that have been completely profiled, but do not carry a specific classifying feature. Our proposed ‘NOS’ designation is used in combination with a specified histological subtype  to ensure that the information of histological subtyping is not lost in these cases. We believe that the introduction of this NOS designation will provide necessary clarity in translational research and in reporting to clinicians.This review shows that changing the EC classification to include molecular characteristics provides exciting opportunities, but will also create certain challenges with respect to testing and reporting. These challenges include the availability and choice of assays for surrogate markers, as well as the management of patients in centres that do not have access to these assays. Immunohistochemical surrogate markers, such as p53 and MMR‐protein immunohistochemistry, are readily available, but it is recognised that some centres may not have the ability to carry out The scenarios presented illustrate that the molecular EC classification provides clinically relevant and prognostic information, with the potential to influence (adjuvant) treatment strategies. When clinical trials confirm the added value of integrated molecular diagnostic entities, we envision that this will become the new standard in EC reporting. Independent of ‘histomolecular’ type, certain histopathological characteristics, such as (the extent of) LVSI and stage, do not have a molecular surrogate and will remain essential in the pathological assessment of a hysterectomy with EC. The value of histological type and FIGO grade is less certain, but it is recommended to still report on these in the years to come. The precise weight of all these intrinsic tumour variables should be evaluated to design a novel risk stratification scheme for adjuvant treatment that includes some of the most relevant molecular characteristics. Finally, the molecular classification provides a framework for subclass‐specific trial designs that should be developed over the coming years to examine the potential benefit of subclass‐specific targeted therapy.All authors declare no conflicts of interest.


Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure.The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as  The ductus arteriosus (DA), an essential embryonic blood vessel between the pulmonary artery (PA) and the descending aorta, exists in all terrestrial vertebrates. The DA works as a right-to-left shunt in these animals to run the oxygenated blood from the embryonic gas exchanger directly into the descending aorta. Mammals use the placenta from maternal circulation for the embryonic gas exchanger system, whereas oviparous animals such as birds and reptiles utilize the chorioallantoic membrane. Despite the differences in the gas exchanger systems, the DA shares similar physiological functions in all terrestrial vertebrates. For example, the DA constricts after birth or hatching due to an increase in the oxygen tension associated with the first breath, and it eventually closes to terminate embryonic circulation. , 2.In addition to the differences in the embryonic gas exchanger systems, the anatomy of the DA in oviparous animals is different from that in mammals. Most studies on oviparous ductus arteriosi have been conducted using chick embryos. Chicks have two, long ductus arteriosi that branch off from the right and left pulmonary arteries, whereas mammals have a single, short DA . Both thTo date, the characteristic features of DA closure are known to occur by a combination of two events: functional constriction and morphological remodeling in all terrestrial vertebrates. These characteristics are based on the specific transcriptional profiles of the DA. Several studies, including ours, have identified a unique transcriptional profile of the mammalian DA in humans , 9, baboFertilized Hypeco Nera chickens  were obtained from Shiroyama Keien Farms . Eggs that were incubated at 37.5°C at a relative humidity of 60–75% were turned every hour. The DA and its connecting arteries including the pulmonary arteries and the descending aorta from five different ages of developing chickens were isolated: prepipped embryonic day 16 (ED16), day 19 (ED19), internal pipping (IP), external pipping (EP), and one day after birth (AB), which were used within 24 hours of hatching. At the IP stage, embryos break into the air cell with their beaks and still breathe hypoxic air, but after the EP phase, embryos break the eggshell and start to breathe normoxic air. Animals were euthanized with isoflurane, and all efforts were made to minimize suffering. The protocol of animal experiments was reviewed and approved by the Institutional Animal Care and Use Committee of The Jikei University (No. 2015–143) and conformed to the Guidelines for the Proper Conduct of Animal Experiments of the Science Council of Japan (2006).In order to histologically analyze the chicken DA at ED16, ED19, IP, EP, and AB with an optical microscope BZ-9000 (KEYENCE Inc.), they were fixed with 10% neutral buffered formalin fixative, and embedded with paraffin. Finally, the 4-μm serial paraffin sections were stained with hematoxylin-eosin (HE) staining and Elastica van Gieson (EVG) staining. The vessel wall thickness was measured at four different parts in each vessel by using the ImageJ 1.51 program, and results were obtained from the average of four parts in each vessel. The vessel luminal area was measured in each vessel by using ImageJ 1.51 program.in vitro transcription reaction. Sense-strand cDNA containing dUTP was synthesized by amplified cRNA. The Affymetrix GeneChip Whole Transcript Terminal Labeling Kit  was used to recognize the dUTP and to fragment the cDNA with uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1. These fragmented cDNAs were then labeled through a terminal deoxy-transferase reaction and hybridized to the Affymetrix GeneChip Chicken Gene 1.0 ST-v1 Array (Affymetrix). The arrays were incubated in a 45°C hybridization oven, at 60 rpm, for 17 hours. After incubation, the arrays were washed, stained, and scanned using an Affymetrix GeneChip Scanner. The data were analyzed with Affymetrix GeneChip Command Console Software version 2.0 and then exported as Affymetrix CEL files. Normalization of the raw data, quality control checks, summarized to probeset expression estimates through robust multi-array average (RMA) were performed using the oligo package (v1.40.1) in Bioconductor (v3.5). The hybridization experiments were performed in duplicate, and the intensities in the proximal DA, the distal DA, and the descending aorta were averaged.Pooled tissues from ten embryos of the proximal and distal DA of the right-sided DA and the descending aorta at the IP stage were collected separately for each sample of RNA isolation. Sepasol-RNA I Super G  was used as recommended by the manufacturer. RNA purity of the samples was confirmed to have an OD260/280 ratio between 1.8 and 2.0 by UV spectrophotometry. Total RNA was converted to cDNA using Ambion Whole Transcript Expression Kit for Affymetrix GeneChip Whole Transcript Expression Arrays. cDNA was then biotin-labeled and hybridized to a GeneChip Chicken Gene 1.0 ST Array . Briefly, a total of 100 ng of total RNA was reverse-transcribed to cDNA, which was subsequently used as a template for an The similarity matrix of normalized data described above was computed using the Dist function from amap package (v0.8–14) using centered Pearson’s correlation coefficient and clustered using the hclust function from stats package (v3.5.0) using Ward’s method for agglomeration.p-value lower than 0.05 were considered to be significantly enriched.We took the top 0.5% (88 genes) in the ratio of signal intensities of the proximal DA to the distal DA and asked which GO terms in the KEGG pathway were enriched in the genes using DAVID knowledgebase v6.8 . Only pagapdh mRNA.Pooled tissues from the proximal and distal DA of the right pulmonary artery side and the descending aorta were collected separately at five different developmental stages: ED16, ED19, IP, EP, and AB. Total RNA was extracted from these pooled tissues using Sepasol-RNA I Super G  and then reverse-transcribed to cDNA using a PrimeScript™ RT Master Mix  as recommended by the manufacturer. cDNA was amplified using TB Green™ Premix Ex Taq II for intercalator-based quantitative polymerase chain reaction (qPCR), or Probe qPCR Mix for TaqMan probe-based qPCR . Sequences for PCR primers and probes are listed in post hoc comparisons using GraphPad Prism version 8.0.1 for Windows . Statistical differences between groups are not being performed by two-way analysis of variance in post hoc comparisons using GraphPad Prism version 8.0.1 for Windows. Statistical differences were considered significant at p-values < 0.05.All data are presented as the mean ± standard deviation. Statistical differences between groups were analyzed by two-way analysis of variance to evaluate the interaction between the developmental stage and the vessel type in Figs As described in the introduction, the right and left ductus arteriosi in chicks exhibit two distinct parts: the proximal and distal ductus arteriosi that are separated in their middle portions. The morphology of the right and left chicken ductus arteriosi is known to be very similar during development , and we The wall thickness of the proximal DA was significantly thinner than that of the distal DA at ED16 and ED19 . At ED19We performed microarray analysis with the proximal and distal DA of the right-sided DA at the IP stage. The degree of similarity of the transcriptional profiles of the proximal DA, distal DA, and the descending aorta at IP was calculated, and the graphical representation showed that the profiles of gene expression are grouped into two distinct clusters: a DA-related cluster and an aorta-related cluster . AlthougBecause it has been known that, in addition to the structural difference, the proximal and distal ductus arteriosi have distinct functional characteristics such as oxygen responsiveness, the difference of the transcriptional profiles should be of interest. ada, chrna4, kdr, rora) were identified from the top 0.5% in the ratio of signal intensities of the proximal DA to the distal DA or proximal DA to the descending aorta . It has been known that mutations in tfap2b cause Char syndrome, a familial form of patent ductus arteriosus in humans [tfap2b cause patent ductus arteriosus in mice [tfap2b is very well conserved among species and plays a critical role in the development of the DA. In addition to tfap2b, tnc [des [foxf1 [ankrd1 [mgp [atp2a3 [fhl2 [col8a1, mamdc2, hpse2, and fam19a2 by RT-PCR analyses during development. These proximal DA-dominant genes of which expression is conserved well in many species may play an important role in the regulation of DA development, although the specific function of these genes in the DA has not yet been determined. Interestingly, the transcription factor foxf1 is known to bind to serum response factor and myocardin to regulate gene transcription in visceral smooth muscle cells [foxf1 is a candidate gene for familial patent ductus arteriosus [foxc1 gene is known to cause failure of DA closure in mice [Our microarray analyses uncovered that the gene expression profiles between the proximal and distal ductus arteriosi differed, although they were much closer than that of the aorta during chick development. To our knowledge, this is the first study examining the transcription profiles of the chicken DA. Importantly, the proximal DA is morphologically and functionally distinct from the distal DA, which more resembles the descending aorta , 7, 20. n humans –26 and t in mice , 28. Thep2b, tnc , des [11tnc [des , 15, 29,s [foxf1 , ankrd1 rd1 [mgp , atp2a3  [atp2a3 , and fhla3 [fhl2  are alsole cells . In additeriosus . Moreove in mice . Therefohif2a, plays an important role in DA development in mice [We assume that the genes distinctly expressed between the proximal and distal ductus arteriosi should be involved in generating the morphological and functional differences between the proximal DA and the distal DA. Interestingly, gene ontology analysis revealed that the differentially expressed transcripts were involved in melanogenesis and tyrosine metabolism of the chicken DA. In mice, tyrosinase promoter is transiently active from day 9 of gestation in common precursors of melanocytes and smooth muscle cells in the DA . Further in mice , examiniThe role of most of the genes in The present DNA microarray analysis newly found genes that were distinctly expressed in the chicken proximal DA. Furthermore, cluster analysis found that the expression pattern of the proximal DA was closer to that of the distal DA than that of the aorta, although the proximal and distal ductus arteriosi in chicks have distinct characteristics such as oxygen responsiveness. The transcription profiles of the chick DA would provide a new insight in investigating the mechanism of DA closure.In the present study, we only compared the gene profiles of chick DA at the IP stage. The main aim of the present study is to examine comprehensive transcriptional profiles of chick DA because no study has been conducted to examine a transcriptional profile of the DA of oviparous animals. We thought that the DA at the IP stage is rapidly differentiated and that the functional and morphological differences are becoming apparent. Therefore, we chose the IP stage for our first investigation. It would be interesting, however, to compare the transcriptional profiles of the chicken DA during development.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Mikania micrantha H.B.K. (a vine of Asteraceae) is one of the top 10 most invasive weeds worldwide and causes serious damage to agroforestry ecosystems. Water is an important environmental factor that affects plant growth; however, the relationship between water conditions and the rapid growth of M. micrantha is not clear. In this study, 162 M. micrantha population sizes were investigated in dry, wet and aquatic habitats in the Pearl River Delta region of Guangdong, China. In addition, the seed germination and seedling growth characteristics of M. micrantha were determined by submerging tests. The results showed that the population size of M. micrantha was the largest in aquatic habitats, and the soil moisture content was positively correlated to the population size in dry and wet habitats. Furthermore, M. micrantha seeds could germinate underwater and grow out of the water surface at a depth of 6 cm with a survival rate of 7.4%. Aquatic habitat promoted vine elongation, whereas dry habitats resulted in the reverse pattern. After 8 weeks of water treatments, the vine stem length was 2 and 3 times longer in the aquatic habitat than the wet and dry habitats, respectively. The total root length, root volume, and root tip number increased significantly in the aquatic habitat when compared to those in the wet habitat; however, these parameters exhibited the opposite pattern in the dry habitat. The results showed that flooding with shallow water is conducive to the invasiveness of M. micrantha, suggesting that water is the key determinant during the intrusion process of M. micrantha populations.The invasive ability of alien plants is not only affected by their biological characteristics but also by environmental factors. Therefore, investigating the relationship between plant growth and environmental factors is helpful for predicting the invasive potential of alien species. https://osf.io/ksz2f/?viewonly=30b6fec21f0447edbdfc9cebe2b01065, https://osf.io/a5ymf/ and https://osf.io/ksz2fl?viewonly=cfcbfOfc829c402fb22deb3be801dffcThis article has been awarded Open Data, Open Materials and Preregistered research design Badges. All materials and data are publicly accessible via the Open Science Framework at  Second, the seed germination and seedling growth characteristics were determined by different submerging tests to further elucidate the specific effects of water conditions. Lastly, the survival rates of M. micrantha were examined at different water‐drown depths to reveal the effects of flooding. Our aim is to provide a scientific basis for predicting the invisibility or susceptibility of ecosystem and setting up an effective management strategy for M. micrantha in the field.Here, we hypothesize that 22.1M. micrantha population sizes were randomly investigated in the field across agricultural systems in the Pearl River Delta in Guangdong, China in April 2015  solution, with 0, 85.4, 160.6, 212.6, 255.0, or 291.7 g of PEG6000 dissolved in 1 L of distilled water to obtain aqueous solutions with osmotic potentials of 0, −0.1, −0.3, −0.5, −0.7, or −0.9 MPa, respectively, according to Wei et al.  at 30/25°C (day/night) under a 12‐hr photoperiod. Each treatment set consisted of eight replicates. Germinated seeds were counted after 14 days, and the seed germination rates under the different treatments were calculated . The roots, stems, and leaves of M. micrantha were dried , and their biomasses were determined. In addition, the relative growth rate , Guangdong Province, China from April to June 2016. Seedlings of similar sizes were transplanted to plastic pots with sandy soil  at the 2‐leaf stage. Water treatments were applied when the seedlings were at the 6‐leaf stage. Three water treatments were established: soil with a 40% water‐holding capacity as the dry habitat; soil with a 90% water‐holding capacity as the wet habitat Burns, ; and flo2.4M. micrantha seedlings were at the 4‐leaf stage with heights of ~6 cm. The tested flooding depths were 0, 2, 4, 6, and 8 cm. The M. micrantha seedlings were planted in soil with a 90% water‐holding capacity as the control treatment. To calculate the survival rate, each treatment had eight replicates, each with 32 plants. Seedlings that grew above the water surface were regarded as surviving plants (preliminary experiments showed that M. micrantha would survive as long as it grew above the water surface). When all the M. micrantha seedlings underwater had died (after 4 weeks), the experiment was terminated, and the survival rates of M. micrantha at different water depths were calculated.The experiment was conducted in Guangzhou , Guangdong Province, China from April to May in 2016. Four seedlings at the 2‐leaf stage were transplanted to one plastic pot with a diameter of 12 cm containing sandy soil. Flooding treatments were applied when the 2.5M. micrantha under different water conditions were compared using a one‐way ANOVA followed by Tukey's test at p < .05. All observations were independent of one another, and the scores in groups were normally distributed. A univariate F test for each variable was used to interpret the respective effects. The equality of error variances was tested by using Levene's test, and the error variance of the dependent variable was considered to be equal across groups when p > .05. A linear regression analysis was used to assess the response of the population size of M. micrantha (as the dependent variable) to relative soil humidity (as the independent variable). Figure All statistical tests were performed using SPSS 16.0 (SPSS Inc.). The population size, germination rate, growth index, and root morphology of 33.1M. micrantha population size in the aquatic habitat was the largest, followed by wet habitat, and the smallest one was found in dry habitat. The M. micrantha population size in the aquatic habitat was 3 and 38 times larger than that in the wet and dry habitats, respectively . The germination rate was not significantly different between the flooding and the lower osmotic potential treatments (0 to −0.3 MPa). With an increase in the osmotic potential, the seed germination rate decreased significantly , most slowly in the dry habitat (soil with 40% water‐holding capacity), and at an intermediate rate in the wet habitat (soil with 90% water‐holding capacity). Eight weeks after the treatments, the main stem length in the aquatic habitat was 2 times longer than that in the wet habitat and 3 times longer than that in the dry habitat.In the water‐submerging test, the speed at which the main stem of s Figure . M. micrM. micrantha in the aquatic and wet habitats was much higher than that in the dry habitat . Compared with the wet habitat, the aquatic habitat significantly promoted the stem biomass of M. micrantha while the dry habitat significantly inhibited it (p < .05). The leaf biomass of M. micrantha in the aquatic and dry habitats was significantly lower than that in the wet habitat, while the leaf biomass in the aquatic habitat was significantly higher than that in the dry habitat . The root/shoot ratio in the aquatic habitat was similar to that in the wet habitat. Compared with the wet habitat, the aquatic and dry habitats inhibited the leaf area of M. micrantha (p < .05), resulting in areas that were 51% and 65% smaller compared with the wet habitat, respectively. The specific leaf area and branch number of M. micrantha in the aquatic and dry habitats were less than those in the wet habitat (p < .05). Compared with the wet habitat, the aquatic habitat promoted the stem node length while the dry habitat inhibited it . With an increase in water depth, the survival rate decreased significantly (p < .05). When the water depth was 6 cm, the survival rate was only 7.4%. None of the M. micrantha plants survived at a water depth of 8 cm  are also very tolerant to waterlogging  and concentrated from April to September (Yu, M. micrantha by more than 8 cm, it will be fatal to the plant. In South China, the rainy season often deepens the river, which is unfavorable for M. micrantha growing along the riverbank.The m Figure . Similarm Figure  found thmber Yu, ; therefoM. micrantha. Because the vine stems of M. micrantha can grow infinitely as long as there is adequate water, we should prioritize the management of M. micrantha populations in aquatic and wet habitats with shallow water or riverbanks over those in terrestrial habitats.In the agroecosystem, the aquatic and wet habitats should be eliminated around the farmland and orchard sits since these two types of habitats are more conducive to the growth of M. micrantha prefers aquatic habitats and flooding with shallow water promoted its stem elongation, which makes M. micrantha form largest population size under waterlogging conditions. Thus, flooding with shallow water could be conducive to the invasiveness of M. micrantha, which implies that water is the key determinant during the intrusion process of M. micrantha populations. Future research is needed to do with regard to the ecological adaptability mechanism of M. micrantha to waterlogging.In conclusion, None declared.MY and XT designed the study; MY and WL wrote the manuscript; AY conducted most of the experimental work; HY and CY analyzed the data; and all co‐authors contributed to writing the manuscript and discussions.


The binary catalyst system included nonbridged half-titanocene catalyst, Cp*TiCl2 (Cat A) and bis(phenoxy-imine) zirconium, {η2-1-[C(H)=NC6H11]-2-O-3-tBu-C6H3}2ZrCl2 (Cat B), as well as co-catalyst methylaluminoxane (MAO). In contrast to dual-catalyst system in the absence of CSA, the blocky structure was obtained in the presence of CSA and rationalized from rheological studies. The binary catalyst system could cause the CSP reaction to occur in the presence of CSA ZnEt2, which yielded broad distribution ethylene/1-octene copolymers (Mw/Mn: 35.86) containing block polymer chains with high Mw. The presented dual-catalytic system was applied for the first time in CSP and has a potential to be extended to produce a library of olefin block copolymers that can be used as advanced additives for thermoplastics.In this contribution, olefin block copolymers were produced via chain shuttling polymerization (CSP), using a new combination of catalysts and a chain shuttling agent (CSA) diethylzinc (ZnEt CSP is based on reversible chain transfer reactions, including catalyzed chain growth ,2 and coet al.  and Zincet al. , CSP catThe above-mentioned selection of catalysts could be explored in catalytic production of ethylene-based block copolymers. For example, ethylene/1-octene block copolymers were prepared via CSP using bis(phenoxy-imine) Zr and pyridylamide-Hf catalyst system ,9, whereA catalytic system can define the microstructure of the resulting polymers, enabling access to the macromolecular structures with new properties. For example, CSP of propylene using either the pyridylamide-Hf catalyst or the binary zirconocene-based catalyst system realized copolymer structures with a broad range of molecular weights ,20. MeanThe core of the CSP method is to find a catalytic system where catalysts and chain shuttling agents are well matched. However, to date, there is no specific theory to reach this goal, and a large number of experiments are still required to find an efficient catalyst system for CSP.A) and bis(phenoxy-imine) zirconium (Cat B) catalysts in a CSP reaction of ethylene block copolymers. The binary catalyst system was used to perform ethylene homopolymerization and ethylene/1-octene copolymerization in the presence of co-catalyst methylaluminoxane (MAO) and CSA diethylzinc (ZnEt2). The polymers produced by the dual-catalytic system (Cat A/Cat B/MAO/ZnEt2) have remarkable characteristics, including broad molecular weight distribution and containing high molecular weight block polymer chains. The block copolymers are capable of improving compatibility of the blends, and thus polymer mixtures containing block polymer chains have superior physical and mechanical properties to general blends [Therefore, in this work, we first explored a binary catalytic system, composed of nonbridged half-titanocene  was obtained from Acros Organics and used as received. Toluene was purchased from Hangzhou Chemical Reagents Company of China and purified by MBRAUN SPS-800X solvent purification system before use. 1-Octene and 1-Hexane were supplied by J&K Chemical Ltd and dried over 4 Å molecular sieves. Polymerization-grade ethylene was obtained from the Sinopec Yanshan Petrochemical Company. All other chemicals were commercially available and used as received.Methylaluminoxane  was purchased from Albemarle Corporation and used directly without further purification. Diethyl zinc 2 with two equivalents of lithium salt of N- cyclohexylamine according to the literature [Complex A Cat A,  was syntterature . HoweverB Cat B,  was syntterature .Scheme 2.2.1.1H NMR : 1.38 2CH-), 2.00 5), 3.51 2CH-), 7.11–7.16 .2.2.2.1H NMR : 1.60 , 1.15–2.10 , 3.83–4.07 , 6.90–7.58 , 8.23 .2.3.2 (if needed) were injected into the reactor. Ethylene was fed continuously to maintain the required 1.0 MPa pressure during the reaction. After 30 min, the polymerization was terminated, the resulting mixtures were immediately poured into acidified alcohol (5 vol% of hydrochloric acid). The polymers were washed with an excess of alcohol, and dried in vacuum at 50°C for 4 h until constant weight was attained.The ethylene polymerization was carried out in a jacketed 1 l high-pressure polymerization reactor (Julabo ACE). The reactor was dried for at least 1 h at 120°C under vacuum. Then the reactor was repeatedly evacuated and purged with nitrogen gas three times, followed by a final purge with ethene at 60°C. Then 500 ml of dried toluene was introduced into the reactor, and the stirring speed was controlled at 300 r.p.m. At the same time, the toluene solutions of catalysts, co-catalyst MAO and CSA ZnEt2.4.−1 and temperature of 150°C.Gel permeation chromatography (GPC) was performed with a Polymer Laboratories PL-220 GPC instrument using 1,2,4-trichlorobenzene as an eluent and operated at a flow rate of 1.0 ml min−1 and remained at 160°C for 1 min. Then, the samples were cooled to 0°C at the rate of 10°C min−1 and held at 0°C for 3 min. Finally, the polymers were reheated to 160°C at the rate of 10°C min−1. Peak melting temperatures of the polymers were determined from the second heating curves.Differential scanning calorimetry was performed on a Perkin-Elmer 8500 thermal analyser under a nitrogen atmosphere. The polymers of 10 mg were heated to 160°C at the rate of 10°C min1H NMR spectra were recorded on a Bruker TopSpin 300 MHz spectrometer using chloroform-d as a solvent at 25°C. 13C NMR spectra of the polymers were recorded with a Bruker TopSpin 300 MHz spectrometer using o-dichlorobenzene-d4 (o-C6D4Cl2) as a solvent at 100°C.−1 with a strain amplitude of 1.25% at 200°C.The linear viscoelastic properties of all samples were measured using the MCR 302 rotational rheometers with 25 mm parallel disc geometry and a gap of 1 mm. The dynamic frequency sweep tests were conducted between 0.1 and 500 rad s3.3.1.A/Cat B/MAO/ZnEt2). The properties of the resulting polymers are summarized in The effect of CSA on the molecular weight and molecular weight distribution of the polymers was analysed to investigate CSP reaction of the dual-catalytic system  yielded polyethylene with high molecular weight and polydispersity, which was consistent with the previous report [The results of ethylene polymerization using either Cat s report .2, there was a significant decrease in molecular weight and a little decrease in molecular weight distribution compared to the dual-catalyst system (Cat A + Cat B)/MAO /MAO, the amount of ZnEt2 had a significant effect on molecular weight and molecular weight distribution of the obtained polymers (Runs 5–7 in A/Cat B/MAO/ZnEt2) was broadened with increasing the amount of ZnEt2. The fractions with lower Mw showed bimodal distributions, while the fragments with higher Mw had unimodal distributions . Therefore, it would be possible that macromonomers with vinyl group as comonomer were inserted into growing polymer A. However, this process would be difficult due to the large steric hindrance of the comonomer. On the other hand, the growing polymer chains could be easily displaced with an ethyl group from ZnEt2 rather than terminated by the β-elimination process. However, the content of higher molecular weight components rose with increasing the ZnEt2 amount . The amount of ZnEt2 had a negligible impact on the thermal behaviour of the polymers obtained by dual-catalytic system .It would be reasonable to conclude that the fractions with lower from CSP  carried A + Cat B)/MAO in the presence of ZnEt2 could cause the occurrence of ethylene CSP, which produced polymers that had a broad molecular weight distribution and contained high molecular weight chains.In summary, using the dual-catalyst system (Cat 3.2.A/Cat B/MAO/ZnEt2) was investigated by introducing 1-octene in order to prepare block copolymers containing branching structure.The ethylene/1-octene copolymerization with the dual-catalytic system (Cat 3.2.1.A/Cat B/MAO/ZnEt2) and the architecture (linear/branched) of resulting copolymers was investigated. The results, summarized in 2 affected molecular weight, polydispersity, melting point, melting enthalpy and rheological properties of the copolymers.CSP was carried out with the dual-catalytic system  was decreased to 0.042 (Run 14) compared with EEO content got from Cat A . Herein it indicated that the dual-catalyst system (Cat A+ Cat B)/MAO yielded new polymer chains in the presence of ZnEt2. The detailed NMR analysis of samples is shown in the electronic supplementary material, figures S2, S3 and S4.The ternary sequence structure distribution of the ethylene/1-octene copolymers are listed in A, the vinyl group-terminated macromonomers produced by Cat B was possible to be inserted into growing polymer A chains, which generated the polymers with macromolecular long-chain branches. Hence, it would be reasonable to conclude that the fractions with high Mw were either polymers with macromolecular long-chain branches or block copolymers originated from CSP. The viscosity of polymers obtained in Run 14 was higher than Run 12 in the low angular frequency range. In the meantime, the shear thinning behaviour of polymers obtained in Run 14 was more pronounced than Run 12 with increasing angular frequency. Hence, it indicated that it was impossible for a large number of polymers with macromolecular long-chain branches to exist in the polymers obtained in Run 14. However, the viscosity of polymers obtained in Run 14 was lower than Run 12 in the high angular frequency range. It indicated that the number of hexyl-branches of polymers obtained from Run 14 was lower than Run 12. Thus, I further believed that the polymers obtained from Run 14 existed as block copolymers [Based on polymers  rather t2 . After adding 20 ml 1-octene (Run 8), the molecular weight of copolymers obtained with Cat A was significantly decreased (Mw: 451 800 g mol−1 → 63 200 g mol−1), and the molecular weight distribution became narrowed (Mw/Mn: 5.88 → 2.60) compared to ethylene homopolymers (Run 1). When catalyst A was used, the melting point and melting enthalpy of copolymers were significantly reduced compared to ethylene homopolymers. , which indicated that the catalyst A had a high copolymerization activity [B, adding 20 ml 1-octene had little effect on molecular weight, polydispersity, melting point and melting enthalpy of the polymers (Run 3 versus Run 10), indicating that the catalyst B was poor effective for copolymerization [The effectiveness of copolymerization reaction produces copolymers with significantly decreased molecular weight compared with homopolymers . The addactivity ,35. Howerization .2 reduced molecular weight and polydispersity of the polymers obtained with Cat A, in contrast to increased molecular weight distribution in the case of Cat B (Mw/Mn: 2.14 → 5.32). The addition of ZnEt2 also had a different influence on the thermal behaviour of the polymers obtained by either Cat A or Cat B respectively . Based on GPC data (B (Run 11) were bimodal and incorporated low molecular weight peak. It would be reasonable to conclude that the low molecular weight peak was polymers produced by Cat B/MAO/ZnEt2 system.According to Runs 8–11 in GPC data , the pol2 causes molecular weight and polydispersity of polymers to decrease. When ethylene/1-octene copolymerization was carried out in the presence of the dual-catalyst system (Cat A + Cat B)/MAO, the effect of amount of ZnEt2 on molecular weight and polydispersity of the polymers was significantly different from previous reports [2 increased, the molecular weight distribution of polymers gradually widened  and (Cat B/MAO/ZnEt2). While the fractions with higher Mw were produced by dual-catalytic system (Cat A/Cat B/MAO/ZnEt2) via CSP (Runs 13–14 in 2 (Run 15), lowered the molecular weight of the polymers. This result is consistent with previous reports [2 in the presence of dual-catalyst system, led to the increased number of produced polymer chains. It would lead to significant decrease in molecular weight of the polymers.The chain transfer reaction reduced the molecular weight of the polymers obtained with a single catalyst system. Therefore, in general, the addition of CSA ZnEt reports . As the  reports ,38, wherA + Cat B)/MAO in ethylene homopolymerization produced polymers with large molecular weight and broad molecular weight distribution  (Run 7) at the Zn/(Cat A + Cat B) molar ratio of 450. However, ethylene/1-octene copolymerization under the same catalytic conditions led to lower molecular weight and relatively narrower molecular weight distribution of resulting polymers  (Run 15). These findings were reinforced by previous reports [Using the binary catalyst system  molar ratio was increased to 450 (Run 15), a new melting peak appeared in the polymer melting curve at 115°C. This peak indicated that increasing amount of CSA led to the enhancement of soft segment content in the block copolymers.For random copolymers, the melting point is inversely proportional to the comonomer content. As the amount of ZnEtA + Cat B)/MAO was used in ethylene/1-octene copolymerization in the presence of CSA ZnEt2, which produced the polymers with weight-average molecular weight up to 119 300 g mol−1 and molecular weight distribution up to 35.86. At the same time, the high molecular weight block polymer chains were contained in the ethylene/1-octene copolymers.In summary, the dual-catalyst system  was investigated by comparing molecular weight, polydispersity, melting point and melting enthalpy of the resulting polymers. The findings are summarized in The effect of Cat A/Cat B in the binary catalyst system increased, the molecular weight of polymer mixtures rose and then decreased. The molecular weight of block copolymers followed the same trend (A/Cat B was 2/2 (Run 14), the molecular weight of block copolymers was high. This outcome was rationalized by the improvement of the soft segment content of block copolymers [A and ZnEt2 perhaps occurred. Therefore, reducing the average block length in the resulting polymers [As the molar ratio of the Cat me trend . When thpolymers . The catpolymers , which lA/Cat B in the dual-catalytic system increased, the melting point and melting enthalpy of polymers decreased (A/Cat B = 3/1 (Run 17), the polymer melting curve emerged with a new melting peak at 108°C. This occurrence indicated that the soft segment content in the block copolymers is high at the high molar ratio of Cat A/Cat B.When the molar ratio of the Cat ecreased . When thIn summary, the molecular weight, melting point and melting enthalpy of the polymers prepared by the binary catalytic system could be adjusted by changing the catalyst molar ratio. The molecular weight and thermal properties of block copolymers contained in the polymer mixtures also could be adjusted by varying the catalyst molar ratio.4.A) and bis(phenoxy-imine) zirconium catalyst (Cat B) was used in ethylene/1-octene copolymerization in the presence of co-catalyst MAO and CSA ZnEt2. The obtained polymers had a weight-average molecular weight up to 119 300 g mol−1 and molecular weight distribution up to 35.86, containing block polymer chains with high Mw.The dual-catalyst system composed of nonbridged half-titanocene catalyst (Cat A/Cat B/MAO/ZnEt2) will be further expanded to CSP of ethylene and other α-olefins, to prepare a series of broad distribution polymers containing blocky structure. These polymers are planned to be used as additives to thermoplastics, improving the physical and mechanical properties of the blends. Herein this work provides a new platform for the development of advanced functional materials.The use of the dual-catalytic system (Cat