Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3′5′ hydroxylase_2 (F3′5′H_2) from a pigeonpea wild relative Cajanus platycarpus, possessing a robust chemical resistance response to H. armigera. Though F3′5′H_2 displayed a dynamic expression pattern in both C. platycarpus (Cp) and the cultivated pigeonpea, Cajanus cajan (Cc) during continued herbivory, CpF3′5′H_2 showed a 4.6-fold increase vis a vis 3-fold in CcF3′5′H_2. Despite similar gene copy numbers in the two Cajanus spp., interesting genic and promoter sequence changes highlighted the stress responsiveness of CpF3′5′H_2. The relevance of CpF3′5′H_2 in H. armigera resistance was further validated in CpF3′5′H_2-overexpressed transgenic tobacco based on reduced leaf damage and increased larval mortality through an in vitro bioassay. As exciting maiden clues, CpF3′5′H_2 deterred herbivory in transgenic tobacco by increasing total flavonoids, polyphenols and reactive oxygen species (ROS) scavenging capacity. To the best of our knowledge, this is a maiden attempt ascertaining the role of F3′5′H_2 gene in the management of H. armigera. These interesting leads suggest the potential of this pivotal branch-point gene in biotic stress management programs.Pod borer  Helicoverpa armigera or pod borer is one of the biotic stress factors responsible for major crop losses [Pigeonpea, commonly known as red gram, is an ancient and important legume crop cultivated in India . The seep losses  leading p losses ,6. Furthp losses .Cajanus platycarpus [Crop wild relatives have emerged as predominant players in several crop improvement programmes due to an amplitude of resistance traits bestowed to them ,9. Many tycarpus ,12. The tycarpus ,13,14 alC. platycarpus, it was observed that there was a structured reprogramming of flavonoid biosynthesis pathway leading to the hyper-accumulation of several flavonoids [chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), flavonoid 3′5′-hydroxylase_2 (F3′5′H_2), flavonol synthase (FLS), leucoanthocyanidin reductase (LAR), anthocyanidin synthase (LDOX/ANS) and production of their concomitant metabolites [flavonoid 3′5′ hydroxylase (CpF3′5′H_2) an important branch-point enzyme in the production of flavonoids from C. platycarpus.Plants respond to the attack by herbivorous insects through an amalgamation of several intricate molecular processes that include physical, chemical and molecular events. Chemical responses to feeding herbivores that include, signaling, transduction and production of secondary metabolites ,15 have avonoids . It was abolites . UnderstF3′5′H belongs to cytochrome P450 family and plays a vital role in the flavonoid biosynthesis pathway [F3′5′H for improving colour in ornamental flowers [F3′5′H has also shown increased flavonoids in many plant species [F3′5′H overexpression in pod borer management. pathway  as it ha flowers ,20. Addi species . Further species . HoweverCpF3′5′H_2 from pigeonpea wild relative C. platycarpus and assess its role in the management of H. armigera in a model plant, tobacco. Information stemming from the study can form a base for the inclusion of a novel plant-based gene in several crop improvement programmes targeting pod borer management.Therefore, the primary aim of this study was to characterize flavonoid 3′5′hydroxylase_2 (CpF3′5′H_2)  A during F3′5′H_2 to herbivory in the pigeonpea wild relative vis a vis the cultivated counterpart. To determine the expression levels of F3′5′H_2, 45 days old plants of C. cajan and C. platycarpus were challenged at different time intervals  with the second instar larvae of H. armigera  after the initiation of herbivory and was upregulated in the two later time points (48 and 96 h) to a maximum of 3-fold .Global sequence alignment of relative A, which tycarpus B. The auF3′5′H_2 gene in both the Cajanus spp. under study versus other closely related plant species disclosed amino acid variations within the two species. It was seen that Gly, Ile, Val, Leu and Met of C. cajan were replaced by Ala/Arg, Asp/Thr, Ile/Leu, Ser and Ile of C. platycarpus. Further, Leu was replaced by Pro in C. platycarpus  from the two species, cloned, and the findings were analysed to compare the promoter regions (C. cajan (C. platycarpus vis a vis C. cajan (2 in C. cajan and 4 in C. platycarpus). These Mybs were present at different positions within the F3′5′H_2 gene in both species, showing deletion, insertion and 1 or 2 bp mismatches. We have found that in C. platycarpus, Myb was present at the 748th location, but there was a deletion at the same place in C. cajan. However, the 180th position of C. platycarpus had an insertion of a few bases in C. cajan. In plants, Myb transcription factors play a cardinal role in the positive or negative regulation of anthocyanin synthesis [F3′5′H_2 during herbivory in the wild relative. Nucleotide and amino acid polymorphism, as well as expression level variation in  regions . The resC. cajan A. Intereynthesis ,27,28. TCpF3′5′H_2 gene by tagging to mGFP and validated the localisation using a CSP-mGFP construct  fragment and absence of the amplification of 1.5 Kb gene (CpF3′5′H_2) fragment  fragment A. Southel plants B. This aCpF3′5′H_2 gene in selected transgenic tobacco plants along with L25- ribosomal protein gene as an internal control . The relative fold change expression ranged between 3.15–16.43-fold in CpF3′5′H_2-overexpressed transgenic tobacco plants  further. One of the foremost markers used to assess the ability of redox management in plants is the assessment of DPPH activity ,46. ThisCpF3′5′H_2 overexpressed transgenic TE8 event vis a vis the vector control plants. The results revealed a significant increase in the flavonoid and polyphenol contents in TE8 compared to VC tobacco plants. While TE8 accumulated 18.1 and 164.8 mg/100 g of tissue, respectively, of flavonoids and polyphenols, the vector control accrued 13.3 and 150.4 mg/100 g tissue of the same  from C. cajan and CpF3′5′H_2 (QIJ55482.1) from C. platycarpus, identified in-house during the dynamic transcriptome analyses [H. armigera were retrieved from National Center for Biotechnology Information (NCBI) database [Nucleotide sequences (cDNA) of analyses  under chdatabase  and usedC. platycarpus (ICPW 068) and C. cajan (TTB7) were procured from ICRISAT, Hyderabad, India and UAS, GKVK, Bangalore, India, respectively. Healthy seeds were surface sterilized by initially washing thrice with sterile double-distilled water, once with 70% ethanol , followed by three washes with sterile double-distilled water. The seeds were later treated with 2% NaOCl (sodium hypochlrorite)  for 1 min followed by washing with sterile double-distilled water at least four times. The sterilized seeds were sown in soil and maintained under net house conditions, care was taken that the plants were not stressed prior to challenging.Seeds of H. armigera larvae were collected from pigeonpea-growing fields of IARI, New Delhi, India, reared on an artificial diet and maintained under controlled conditions of 25 ± 5 °C, 70 ± 10% RH and 16 h/8 h day and light photoperiod. For the herbivore challenge, five 2nd instar H. armigera larvae were released on 45-day-old confined plants of C. cajan and C. platycarpus, as developed and standardized earlier [ earlier . Leaves TM total RNA isolation kit . RNA was treated with DNaseI  to remove DNA contamination as per the manufacturer’s instructions. RNA was later quantified using NanoDrop 2000  Spectrophotometer and run on 0.8% agarose gel for quantity and quality assessment. Later, cDNA synthesis was carried out using 2.5 µg of total RNA .The total RNA was extracted from 100 mg of herbivore-challenged and control leaf samples by SpectrumF3′5′H_2 isoforms identified from the in-house developed transcriptome data were used for expression analysis using qRT-PCR . Gene-specific primers (initiation factor 4α (IF4α) as the reference gene (p ≤ 0.05) differences in gene expression between the two Cajanus spp., two-way ANOVA with post hoc Tukey test was performed by using the MULTCOMPVIEW package in R .The 2 selected  primers , along wnce gene , were usF3′5′H_2 in C. cajan and C. platycarpus, high-quality genomic DNA was isolated from young leaves of plants according to Cetyl trimethyl ammonium bromide  method [Cajanus spp. was digested overnight separately with BamHI and HindIII . Additionally, about 300 pg of pCambia 2300 vector consisting of CpF3′5′H_2 gene was linearized with HindIII  and used as the positive control. Overnight-restricted DNA samples were electrophoretically separated on a 0.8% agarose gel in Tris-acetate EDTA  buffer. The separated fragments were further blotted onto a positively charged nylon membrane (Millipore Sigma) and hybridized with a Digoxigenin (DIG)-labelled 543-bp F3′5′H_2 probe. Membrane washing and the development of X-ray film were carried out according to the manufacturer’s instructions .In order to identify the gene copy number of ) method . ApproxiF3′5′H_2 genes from both the Cajanus spp. were predicted using MEME online tool  with default parameters. The identified F3′5′H_2 protein sequences were separately aligned using the Clustal Omega server  and visualized using BioEdit 7.2 software [C. cajan, C. platycarpus, Phaseolus vulgaris, Spatholobus suberectus, Arbus precatorius, Vigna radiata, Trifollum pratense, Medicago trunculata, Vitis vinifera, Populus alba, Petunia scheideana and other closely related species was retrieved from NCBI database. The protein sequences were aligned in MEGA X software using the ClustalW algorithm. Phylogenetic trees were generated using the neighbour-joining method with the following parameters: pairwise deletion and 1000 bootstrap replicates.The conserved motifs present in software . For phyF3′5′H_2 in both the Cajanus spp., the sequences were retrieved from NCBI genome BLAST. The upstream regulatory regions of CpF3′5′H_2 and CcF3′5′H_2 were amplified using primers designed from the available C. cajan genome  from C. platycarpus cDNA using specific primers  sequence from C. cajan. CSP was amplified using specific primers from the C. cajan cDNA sample, cloned into pGEMT-easy vector and confirmed by Sanger sequencing. Similar to CpF3′5′H_2, the confirmed CSP sequence was sub-cloned into the 5′ region of mGFP in pCambia1302 vector by using the same restriction sites. pCambia1302 35S:CSP-mGFP (control) and 35S:CpF3′5′H_2-mGFP constructs were further digested with respective restriction enzymes to confirm the positive clones and then finally transformed into Agrobacterium tumefaciens (GV3101).To access the subcellular localisation of  primers , cloned 2 , 100 mM acetosyringone  solution and later incubated in dark for 3 h [600 of 0.5. For transient expression, one-month-old N. benthamiana plants were selected, and solutions with 35S:CSP-mGFP and 35S:CpF3′5′H_2-mGFP were infiltrated with the help of a syringe into green, fully expanded healthy leaves. After incubation for 24 h, the leaves were observed under the confocal microscope Leica SP5 with an excitation filter of 488 nm. LAS AF lite software was used for processing confocal images.For Agroinfiltration , the res for 3 h  up to anC. platycarpus was extracted using SpectrumTM total RNA isolation kit . The extracted RNA was qualitatively and quantitatively analyzed by using 1.2% agarose gel and Nanodrop®2000 , respectively. Around 2.5 µg RNA was used for cDNA synthesis using SuperScript™ VILO™  kit. The target gene (CpF3′5′H_2) was amplified from this cDNA using gene-specific primers  to confirm the cloned product, sub-cloned into a binary vector, pCambia 2300 and transformed into A. tumefaciens (Gv3101).The total RNA from young and healthy leaves of  primers . The PCRin planta validation of CpF3′5′H_2, transgenic tobacco explants were raised with the engineered construct. For vector control, tobacco explants were transformed with an empty pCambia 2300 vector without the CpF3′5′H_2 gene. All the steps of cutting and stabilizing leaf discs, A. tumefaciens infection and co-cultivation were carried out as per the standardized protocol [−1 NAA , 2.5 mgL−1 BAP , 100 mg L−1 kanamycin , 500 mg L−1 cefotaxime  and 500 mg L−1 carbenicillin . To maintain growth and avoid false positives, the explants were sub-cultured after 15 days onto fresh MS selection media. After attaining a height of up to 4–5 cm, the explants were transferred to a rooting medium (½ MS media) harbouring 100 mgL−1 kanamycin for plant selection, 500 mg L−1 cefotaxime and 500 mg L−1 carbenicillin as antibiotics. After sufficient root formation, each plant was sub-cultured to produce three replicates. Once the plants were well established, they were transferred onto soilrite. The plants were finally shifted to the net house for hardening and analyses.For the protocol . Leaf diCpF3′5′H_2 overexpressed transgenic events against H. armigera infestation. Laboratory-reared five-second instar larvae that were previously starved for 16 h were loaded onto healthy and fully expanded leaves of 40 days old transgenic tobacco plants harbouring the CpF3′5′H_2 gene. For comparison, vector control plants were collected, and the experiment was continued up to 96 h. The percentage of leaf damage and rate of larval mortality was recorded to judge the performance of transgenic plants. Since three clones for each transformed plant were maintained, accordingly three biological and two technical replicates were used in the experiment. For statistical analysis, a one-way ANOVA post hoc Tukey test was executed to determine significant (p ≤ 0.05) differences in the performance of CpF3′5′H_2 transgenics and vector control tobacco plants by using the MULTCOMPVIEW package in R .A detached in vitro leaf bioassay was performed to check the efficacy of CpF3′5′H_2 gene-specific and nptII gene-specific primers , and all the necessary steps were carried out as mentioned earlier. DNA from pCambia 2300: CpF3′5′H_2 (300 pg) and vector control plants were considered positive and negative controls. The DIG-labelled hybridization probe was prepared by amplifying 1.5 Kb PCR product with CaMV 35S promoter forward primer and CpF3′5′H_2 gene reverse primer  method and used for PCR and Southern analyses. Genomic DNA was isolated as mentioned earlier and 100 ng was used for PCR analyses using  primers . The reae primer .CpF3′5′H_2 and cDNA synthesis were carried out as mentioned earlier. Expression analyses of the CpF3′5′H_2 gene in tobacco transgenics were performed by qRT-PCR with gene-specific primers .Total RNA extraction from leaves of transgenic tobacco overexpressing  primers  as standcA = Absorbance of control; sA = Absorbance of sample.About 100 mg each of leaf tissues from transgenic event (TE8) and vector control tobacco were extracted in 2 mL methanol  by placing it overnight on a shaker. The extracts were later centrifuged at 10,000 rpm for 15 min. The assay was carried out following the method of Mellors and Tappel  by usingp < 0.05, 0.01, 0.001) difference, an unpaired t-test was performed for radical (DPPH) scavenging activity between the control and transgenic event.To determine the significant  was added, allowing the reaction mixture to react for 6 min. Further, 0.5 mL of 10% AlCl3.6H2O  was added to the reaction mixture and again allowed for 6 min to react. Later, 4 mL of 1 M NaOH  and 3 mL of distilled water were added to the reaction mixture and incubated at room temperature for 15 min. Absorbance was recorded at 510 nm and a standard curve of Quercetin  was prepared. Results were expressed as mg Quercetin equivalent per 100 g tissue (mg RE/100 g tissue). To determine significant  differences, an unpaired t-test was performed on total flavonoid content between the vector control and transgenic plants.The estimation of total flavonoid content was followed as elaborated by Tohidi et al. . Briefly2CO3  was added. This reaction mixture was then incubated at room temperature for 90 min, and absorbance was recorded at 760 nm. A standard curve of gallic acid  was prepared to calculate total phenolic content, and results were expressed as mg gallic acid equivalent per 100 g of tissue (mg GAE/100 g tissue). To determine significant  differences, an unpaired t-test was performed on the total phenolic content between the control and transgenic event.The total phenolic content of transgenic and vector control tobacco plants was estimated using previously frozen leaf tissues. Accordingly, phenolic samples (250 µL methanol extract) and distilled water (1 mL) were mixed in a test tube, to which Folin–Ciocalteu reagent (2N) (250 µL)  was added. After 6 min, 2.5 mL 7% Na


The Traditional Chinese medicine, Guizhi Fuling , has been confirmed in meta-analysis studies to reduce recurrence of endometriosis and improve pregnancy outcomes; however, the possible use of Fuling as a fertility-preserving treatment in endometrial cancer has not previously been tested. Results here are the first to demonstrate dose-dependent inhibition of cell motility by Fuling in two endometrial cancer cell lines, classified as Grade I which is responsive to progesterone treatment, and Grade III (MFE-280) which is resistant. The major outcome of this study was the novel demonstration that Fuling (30-80 µg/ml) significantly inhibits invasiveness in both high and low grades of EC cells, achieving 70-80% block of trans-barrier migration without cytotoxicity. This effective dose range is estimated to be comparable to that used in human clinical trials and traditional practice. Results here further show that clinically relevant doses of Fuling override the motility-promoting effects of estradiol in endometrial cancer cell lines. Medroxyprogesterone acetate has to date been the standard therapy to treat metastatic or inoperable endometrial cancers; however, success rates are low with high rates of recurrence, due in part to acquired resistance to medroxyprogesterone acetate therapy. The discovery here that Fuling appears to control the spread of treatment-resistant advanced cancers is an exciting prospect. Endometrial cancer (EC) arises from over-proliferation and spreading of cells from the wall of the uterus, constituting the most common gynecological malignancy worldwide and the sixth most common cancer among females, affecting approximately 17 per 100,000 women . These cPTEN, PIK3R1, PIK3CA, FBXW7 and KRAS genes, and associated with more favorable prognoses; Class 2: MSI [Microsatellite-instable (hypermutated)] which comprise low grade endometroid tumors showing high mutation rates, microsatellite instability, and KRAS and PTEN mutations; Class 3: CNL (copy-number low) tumors also known as the ‘endometroid sub-group’ which are low grade tumors with low mutation rates and frequent CTNNB1 mutations; Class 4: CNH (copy-number high) tumors also referred to as the ‘serous sub-group’ which include a combination of serous, mixed, as well as some grade 3 endometroid tumors, characterized by recurrent TP53 mutations and poor outcomes. A meta-analysis study by Travaglino et al.  in 2013  support o et al.  indicate(i) Ishikawa with properties of FIGO grade I (low grade) endometroid cancer cells; and (ii) MFE-280 with properties of FIGO grade III (high grade) endometroid cancer cells. The Ishikawa cell line originally was established from an endometroid cancer from a 39-year-old pre-menopausal woman, and has been shown to express estrogen and progesterone receptors  , all potOne promising candidate for pharmaceutical intervention is the Traditional Chinese Medicine, Guizhi Fuling (here referred to as Fuling), an herbal extract with medicinal properties first noted in the third century by Han Dynasty physician Zhang Zhongjing who used Fuling for treatment of dysmenorrhea . Fuling in vitro proliferation and invasiveness of ovarian cancer cells (2) assays of gene expression after exposure to Fuling suggested possible involvement of PI3/Akt  . The trein vivo human clinical studies of endometriosis; the high doses are equivalent to those shown in prior work to kill a variety of reproductive cancer cell types. Results here are the first to show that Fuling at low non-cytotoxic doses is highly effective as an inhibitor of invasiveness in high- and low-grade endometrial cancer cells. These effects are consistent with the suggested clinical benefits of Fuling in patients with endometriosis.Endometriosis and endometrial cancer share common features in terms of molecular pathogenesis, overgrowth, and resistance to apoptosis –37. Surpin vivo.Fuling shows promise as a novel adjunct for the clinical management of both low grade and high grade endometrial cancers, but confirmation of this idea awaits efficacy testing Dulbecco’s Modified Eagle’s Medium , fetal bovine serum (FBS), GlutaMAX™ (#35050061), penicillin-streptomycin (#15070-063), phenol red-free DMEM/F12 (#21041025) were purchased from Merck . 17β-estradiol , synthetic progesterone (P0130-25G), estrogen receptor antagonist, ICI182,780 (129453–61–8), and the progesterone receptor antagonist mifepristone, referred to here as RU486 (M8046) were obtained from Merck. Insulin from bovine pancreas (I6634-25mg) also was from Merck.https://www.cellbankaustralia.com; Cancer Institute NSW, Australia). The cell cultures were maintained < 6 months after re-animation and confirmed to be negative for mycoplasma using the MycoStrip™ test kit. The T47D breast cancer cell line (an estrogen receptor (ER) and progesterone receptor (PR) positive cell line originally obtained from pleural effusion from 54 yr old patient with infiltrating breast cancer  and MFE-280  cell lines were obtained from CellBank Australia . MFE-280 cells were maintained in DMEM/F12 supplemented with 10% FBS, 100 units/ml each penicillin and streptomycin, and 0.005 mg/ml insulin. 24 hr prior to treatment with steroid hormones, the culture medium was changed to phenol red free DMEM/F12  supplemented with 10% heat-inactivated charcoal-stripped FBS (CS-FBS) prepared as described previously  to minimCarthamus tinctorius (Safflower), Cinnamomum cassia (Chinese cinnamon), Poria cocos (Hoelen), Paeonia suffriticosa (Tree peony), Paeonia lactiflora (Peony) at 92 mg each and Salvia miltiorrhiza (Red sage) at 90 mg, in approximately equal proportions by dry weight (Capsules of Fuling (trade name Gynoclear) purchased from Metagenics , were opened in a sterile environment. The powdered Fuling content was dissolved in the vehicle dimethylsulfoxide (DMSO) to create a series of 2500x stock solutions (0.25 to 7.5 g/ml in DMSO), which in turn were diluted 1:2500 into culture media at final doses for experimental testing. Control groups were untreated and vehicle-treated (0.04% DMSO). Gynoclear capsules contain y weight .(ESR1), estrogen receptor-β gene (ESR2) and progesterone receptor gene (PGR) transcript levels. Hormone receptor positive breast cancer cell line T47D served as a positive control, whereas PSMC4 and IPO8 were used as housekeeping genes for 2ddct standardization  as per manufacturer’s instructions. RNA was eluted in 30 µl of RNase free water. Quantification of total RNA was done by Nanodrop spectrophotometry, and integrity was checked via 260/280 and 260/230 wavelength ratios. The integrity of 18S and 28S bands was assessed by electrophoretic separation of RNAs on 2% agarose gel. 1.5 µg RNA was reverse transcribed to cDNA using QuantiTect cDNA conversion kit (Cat. 205331) as per manufacturer’s protocol. Briefly, RNA was heated with gDNA wipe buffer for 3 min at 42°C. After adding RT and RT primer to final volume of 20 µl, the RNA was incubated at 42°C for 15 min and 95°C for 2 min and immediately placed on ice. qPCR was done with the Rotor Gene 3000. Reactions were carried out in final volumes of 10 µl with 5 µl Syber Green , 1 µl (1:100 dilution) cDNA, 1 µl of 10 mM forward and reverse primer mixture and 3 µl RNase free water. The qPCR reaction involved initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 3 sec and combined annealing extension at 60°C for 20 s. Analysis was done using the standard 2ddCt method. IPO8 and PSMC4 served as reference genes. Reactions were carried out in triplicate in two independent experiments.Real time quantitative polymerase chain reaction (RT-qPCR) was used to quantify estrogen receptor-α gene dization . Two ind® was done for 1 h. α- tubulin  was used as loading control on the same membrane. Secondary antibody used for α- tubulin was . Specificity of PR antibody was validated by PR negative breast cancer cell line, MDA-MB-231 (Protein was extracted from T47D (positive control), MDA-MB-231 (negative control) , 45, Ish3 cells per well in 100 µl of DMEM with 10% FBS. After 24 h growth, media with no drug, with vehicle only, or with Fuling (doses ranging from 100 to 3000 µg/ml) were added. After 24h incubation, 100µl of MTT reagent  was added to each well. Cells were incubated for 4.5 h , then the MTT solution was removed and the formazan product was dissolved in 100 µl DMSO per well. Foil covered plates were agitated on a shaker at room temperature for 15 min, then assessed on a plate reader (absorbance 570 nm). Three independent experiments were done with 3 to 4 replicates each.Cell viability was assessed by measuring metabolic activity using established methods . Ishikaw4 cells per well and incubated for 24h to allow growth approaching full confluency prior to wound generation. The circular wounds were created by brief suction with a sterile micropipette tip as per published protocols (Ishikawa and MFE-280 cells were plated at 3 x 10rotocols . Wells wrotocols . Three i5 per well) were added to the upper chamber; the lower chamber contained 700ul of media with 10% FBS as a chemoattractant. After 24 h incubation , non-invaded cells on the cis side were removed gently with a cotton swab, and remaining invaded cells on the trans side were fixed with 70% ethanol, stained with 0.4% crystal violet, and imaged. Numbers of invaded cells per standardized field of view were counted (with treatment blinded) by two independent researchers and averaged. For the tests of hormonal effects, cell lines were incubated with optimal doses of E2 (1nM for Ishikawa cells and 100 pM for MFE-280), or P4 (100nM) for 48 h. Fuling (80µg/ml) was added at the start of the invasion assay with the inoculation of cells into the upper chamber. ICI182,780 (100nM) and RU486 (1µM) were added 2hr prior to treatments with E2 or P4. Two independent experiments were done with two replicates each.Transwell invasion was carried out using 8µm pore filter Boyden chambers (Corning #3422) prepared with 40µl per well of 0.025 mg/ml Matrigel  . On the 3 cells/ml in full DMEM, and the plates were centrifuged at 600 rpm for 3 min, then incubated at 37°C in 5% CO2 for 48hrs, and cooled on ice for 15 min. 50 µl of 250mg/ml Matrigel in DMEM was added to each well; plates were centrifuged (600 rpm at 4°C for 5 min) and incubated 1h to allow the Matrigel to set. At time zero, media containing no drug, an equivalent dose of vehicle, or Fuling at 100 µg/ml were added  coating solution was made with 0.3g/ml of polyhema crystals dissolved in 95% ethanol, shaken 48 h at 37°C. 96-well round bottom plates (Corning-3799) were coated with 30 µl of polyhema per well in the dark, then exposed to UV light for 2 hrs prior to seeding cells. To produce spheroid cancer cell masses, Ishikawa and MFE-280 cell lines were seeded into wells at a density of 5x10post-hoc comparisons . In box plots, rectangles contain half the data values, whiskers show the full range, and horizontal lines indicate median values. Averaged data (in x-y plots) are mean ± standard deviation (SD).Distribution normality was tested by the D’Agostino-Pearson test. One-way ANOVA was used to assess significant differences across multiple groups , followed with ESR1 and ESR2) and progesterone receptors (PGR) were determined in both cell lines using real-time quantitative polymerase chain reaction ESR1  in high grade EC is related to shorter disease-free survival and poor prognosis  inhibitor ICI182,780 at 100nM .Images illustrating the invasion responses  at 24 h  for 24 h. Fuling significantly inhibited migration, slowing the closure of wounds in both cell lines as compared to the control (vehicle-treated) group  doses. The effective doses for preventing cancer cell invasiveness are comparable to those being taken by humans as medicinal supplements. Preventing cancer cell spread could enhance the success of parallel standard clinical treatments which are aimed at removing primary tumors.via synergistic combinations of pharmacological agents that appear to improve outcomes in disease conditions including cancers  has potential as an adjunct therapy which could restrain EC invasive spread prior to and during clinical interventions aimed at eradicating primary tumors. If successful, using Fuling as an adjunct therapy would be predicted to reduce rates of cancer cell escape and EC recurrence. Prior ll death , and migResults here indicate the mechanism by which Fuling blocks invasiveness appears to be independent of the estrogen and progesterone pathways. Fuling consistently inhibited invasion in all conditions, overriding the potentiating effects of estradiol in both cell lines. The estrogen receptor antagonist ICI 182,780 added a small but discernable level of inhibition to the effect of Fuling, suggesting an additive effect. Progesterone (100 nM) inhibited invasiveness in Ishikawa cells as expected, but the magnitude of inhibition of invasion by Fuling was greater, and not reversed by co-application of the progesterone receptor inhibitor RU486. The MFE-280 cell line used in our study did not respond to progesterone treatment, supporting other work showing that progesterone receptors are downregulated or develop resistance progressively with increasing grades of EC and poorer prognoses , 70, 71.Fuling merits exploration as an adjunct treatment in combination with other EC therapeutics . For exaMedroxyprogesterone acetate has to date been the standard therapy to treat metastatic or inoperable endometrial cancers, and to preserve fertility; however, success rates can be low (15-25%) with high rates of recurrence, due in part to acquired resistance to progesterone therapy , 77, 78.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Conceptualization and methodology, SK, AV. Investigation, SK, AV. Writing—original draft preparation, SK, AY. Writing—review and editing, AY, CR. Supervision, AY, CR. Funding acquisition, AY. All authors contributed to the article and approved the submitted version.


The standard preoperative treatment for resectable locally advanced esophageal squamous cell carcinoma (LAESCC) in Japan is docetaxel, cisplatin (CDDP), and 5-fluorouracil. However, patients with renal or cardiac dysfunction and elderly patients are ineligible for a CDDP-containing regimen because of toxicities. Oxaliplatin, leucovorin, and 5-fluorouracil (FOLFOX) therapy has less renal toxicity than CDDP-containing regimens and does not require hydration. However, there are limited data on preoperative FOLFOX therapy in these patients.This retrospective study analyzed patients with resectable LAESCC who were aged ≥ 75 years or had renal or cardiac dysfunction and received preoperative FOLFOX between 2019 and 2021. FOLFOX was administered every 2 weeks for 3 or 4 cycles and was followed by surgery. Adverse events associated with chemotherapy, the complete resection (R0) rate, relative dose intensity (RDI), and histopathological response were evaluated.Thirty-five patients were eligible. Median age was 77 (range 65–89) years; 68.6% were aged ≥ 75 years, 74.3% had renal dysfunction, and 17.1% had cardiac dysfunction. The RDI was 70.2% and 87.1% for bolus and continuous intravenous 5-fluorouracil, respectively and 85.2% for oxaliplatin. The most common grade ≥ 3 adverse events were neutropenia (60.0%) and leucopenia (28.6%). Two patients (5.7%) had febrile neutropenia and grade 3 pneumonia. Thirty-one patients underwent surgery. The R0 resection rate was 87.1%, and there was no histopathological evidence of residual tumor in 16.1%. There were no treatment-related deaths.Preoperative FOLFOX had a manageable safety profile and showed favorable short-term efficacy in patients with resectable LAESCC who were ineligible for CDDP-containing treatment. Esophageal cancer (EC) has the sixth highest mortality rate of all cancers worldwide . EC is dIn Western countries, the standard treatment for resectable locally advanced EC is preoperative chemoradiotherapy followed by surgery plus postoperative nivolumab based on the results of the CROSS and CheckMate 577 trials , 4. HoweOxaliplatin, leucovorin, and 5-fluorouracil (FOLFOX) therapy is an optional regimen for EC. FOLFOX is less emetogenic and has less renal toxicity than CDDP-containing regimens and does not require hydration –19. In tThis study assessed the safety and efficacy of preoperative FOLFOX in patients with resectable LAESCC who were not eligible for CDDP.2, leucovorin 200 mg/m2, and a bolus of 5-fluorouracil 400 mg/m2 on day 1 followed by 5-fluorouracil 2400 mg/m2 over 46 h) was administered intravenously every 2 weeks for 3 or 4 cycles according to the patients’ overall condition as determined by the attending oncologist. The study was approved by the Institutional Review Board of the National Cancer Center, Japan  and conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. Because of the retrospective nature of this study, informed consent was not obtained from each patient. Patient consent was obtained using an opt-out method.This retrospective study analyzed patients with advanced EC who received preoperative FOLFOX at the National Cancer Center Hospital between October 2019 and October 2021. The main selection criteria were as follows: histologically proven esophageal squamous cell carcinoma; cT1N1-3M0, cT2-3N0-3M0, or cT1-3N0-3M1 (M1 disease limited to supraclavicular lymph node metastasis) ; age ≥ 75 years, renal dysfunction (creatinine clearance < 60 mL/min), or cardiac dysfunction ; and no prior therapy for EC. Preoperative FOLFOX  scans were obtained before initiation of preoperative FOLFOX, before cycle 2, and before surgery. The CT schedule after surgery was determined by the surgeons. Laboratory investigations were performed at the initiation of each treatment cycle and before surgery.The primary outcomes were adverse events during preoperative chemotherapy and histopathological response. Adverse events were graded according to the Common Terminology Criteria of Adverse Events (CTCAE) version 5.0. Histopathological response was classified according to the proportion of tumor tissue that degenerated or became necrotic using the grading system outlined in the Japanese Classification of Esophageal Cancer, 11th edition : grade 0The secondary outcomes were the relative dose intensity (RDI) of preoperative chemotherapy, response to preoperative chemotherapy, complete resection (R0) rate, progression-free survival (PFS), and overall survival (OS). The RDI was calculated on the scheduled dose intensity divided by the actual delivered dose intensity. The scheduled period in patients received 3 cycles was 6 weeks and in patients received 4 cycles was 8 weeks. The response to chemotherapy was evaluated by CT based on the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 . PFS wasn = 6), distant lymph node metastasis (n = 2), or prior therapy for EC (n = 1), leaving 35 patients eligible for inclusion in the study. The characteristics of the study participants at the start of chemotherapy are shown in Table Forty-four patients received preoperative FOLFOX. Nine patients were excluded because they had histology other than squamous cell carcinoma , grade 3 neutropenia (n = 1), or disease progression (n = 1). One patient discontinued preoperative chemotherapy after 2 cycles because of disease progression. Thirty-one patients (88.6%) received 3 cycles and 1 received 4 cycles. Three of the patients who received three or 4 cycles of preoperative chemotherapy did not undergo esophagectomy . Three of 4 patients who discontinued preoperative chemotherapy underwent esophagectomy and 1 declined surgery. Finally, 31 patients (88.6%) underwent esophagectomy.The treatment details are shown in Fig. n = 2), renal dysfunction (n = 1), or a low platelet count (n = 1). The dose was reduced between the second and third cycles in 13 patients (37.1%) because of adverse events  or because it was considered necessary in the opinion of the attending oncologist (n = 1). The RDI was 70.2% and 87.1% for bolus and continuous intravenous 5-fluorouracil, respectively and 85.2% for oxaliplatin.Four patients (11.4%) had a dose reduction during the initial cycle because of decision by attending oncologist , recurrent laryngeal nerve paralysis , anastomotic leakage  pleural effusion , and acute heart failure . There were no surgery-related deaths.Thirty-one of the 35 patients underwent esophagectomy. Two-stage surgery was performed in 7 patients. Open esophagectomy was performed in 1 patient (3.2%), laparoscopic esophagectomy in 22 (71.0%), mediastinoscopic esophagectomy in 5 (16.1%), and robot-assisted esophagectomy in 3 (9.7%). Postoperative complications were pneumonia (The R0 resection rate was 87.1% 27/31). The histopathological response rate was 0% for grade 0, 64.5% (20/31) for grade 1a, 0% for grade 1b, 19.4% (6/31) for grade 2, and 16.1% (5/31) for grade 3. The baseline distributions of clinical and pathological stages are shown in Fig. 7/31. TheIn this study, patients with resectable LAESCC who were not eligible for CDDP showed a favorable histopathological response to preoperative FOLFOX therapy. Although there was a high frequency of severe neutropenia, non-hematological adverse events were mild. In particular, any renal or gastrointestinal toxicity was tolerable.Based on the results of the JCOG1109 trial, the standard treatment for LAESCC is DCF chemotherapy followed by surgery . In otheIn the present study, adverse events that occurred during preoperative FOLFOX therapy were manageable. Nausea of any grade was observed in 28.6% of patients and grade ≥ 3 nausea was observed in 2.9%. This mild emetogenic toxicity was also reflected in the low frequencies of decreased appetite of any grade 25.7%) and grade ≥ 3 (2.9%). Decreased appetite and nausea can lead to malnutrition, and weight loss before esophageal surgery is associated with a higher risk of mortality .7% and g. TherefoIn this study, the grade 3 histopathological response rate was 16.1% for preoperative FOLFOX, which is higher than the rate of 2.2% reported for preoperative CF , althougTo our knowledge, this study is the first to evaluate the safety and efficacy of preoperative FOLFOX therapy. However, it had some limitations. First, the study was performed retrospectively at a single institution. Second, although we defined age ≥ 75 years, renal dysfunction (creatinine clearance < 60 mL/min), and cardiac dysfunction  as criteria for selecting patients who were ineligible for CDDP in this study, the treatment strategy was decided at the discretion of each medical oncologist without clear criteria for eligibility for CDDP. Third, the survival data are immature because of the short follow-up duration and occurrence of few events. However, we found a high histopathological complete response rate, which is associated with a good prognosis . FurtherIn conclusion, preoperative FOLFOX was well tolerated and demonstrated favorable short-term efficacy in patients with LAESCC who were not eligible for CDDP.


Escherichia coli strains DH5α and RB791, Salmonella enterica sv. Typhimurium LT2, and Bacillus subtilis 168. The minimal bactericidal concentration (MBC) was determined by plating cells treated with different concentrations of PEI and chlorophyllin on agar and monitoring their growth after 24 h. All tested combinations of PEI and chlorophyllin were lethal for S. enterica after 240 min of incubation in light, whereas PEI alone (<100 µg mL−1) was ineffective. In the darkness, complete inhibition was noted with a combination of ≥2.5 µg mL−1 chlorophyllin and 50 µg mL−1 PEI. If applied alone, PEI alone of ≥800 µg mL−1 of PEI was required to completely inactivate E. coli DH5α cells in light, whereas with ≥5 µg mL−1 chlorophyllin, only ≥100 µg mL−1 PEI was needed. No effect was detected in darkness with PEI alone. However, 1600 µg mL−1 PEI in combination with 2.5 µg mL−1 resulted in complete inactivation after 4 h dark incubation. PEI alone did not inhibit E. coli strain RB791, while cells were inactivated when treated with 10 µg mL−1 chlorophyllin in combination with ≥100 µg mL−1 (in light) or ≥800 µg mL−1 PEI (in darkness). Under illumination, B. subtilis was inactivated at all tested concentrations. In the darkness, 1 µg mL−1 chlorophyllin and 12.5 µg mL−1 PEI were lethal for B. subtilis. Overall, PEI can be used as an antimicrobial agent or potentiating agent for ameliorating the antimicrobial activity of chlorophyllin.Polyethylenimines (PEIs), a group of polycationic molecules, are known to impair the outer membrane of Gram-negative bacteria and exhibit antimicrobial activity. The outer membrane of Gram-negative strains hinders the uptake of photosensitizer chlorophyllin. In this study, we report chlorophyllin and branched PEI combinations’ activity against  The discovery of antibiotics in the last century and their subsequent application against pathogens resulted in a significant reduction in infectious diseases and an increase in the average life expectancy . However2O2) or hydroxyl radicals (•OH). The energy forms singlet oxygen (1O2) in type II reactions, a highly reactive ROS [4 showed potent activity against Gram-negative bacteria with negligible effects on mammalian cell lines [Escherichia coli and Staphylococcus aureus [Ichthyophthirius multifiliis or Ergasilus sp. [Photodynamic inactivation of bacteria, also known as antimicrobial photodynamic therapy (Apdt) or photodynamic antibacterial chemotherapy (PACT), is gaining interest in mitigating bacterial pathogens ,8,9,10. tive ROS ,12. Thesll lines . Chen etll lines . By anchs aureus . Promisiilus sp. ,16,17. Iilus sp. ,19,20.Bacillus subtilis [E. coli strains (NR698) exhibit higher sensitivity to chlorophyllin than Gram-positive bacteria [Gram-negative strains are less susceptible to photodynamic treatment with chlorophyllin than planktonic Gram-positive bacteria, such as subtilis . This disubtilis ,10. The rains NR68 exhibitbacteria ,22. As abacteria  and the rains NR68 exhibitSimilarly to colistin, polyethylenimines (PEIs) are a group of polycationic molecules. PEIs have branched or linear configurations due to numerous amino groups . PEIs exS. enterica sv. Typhimurium considerably fast in light (12.7 mW m−2 blue-red) and dark incubation. In light incubation, the inhibitory effect was observed at the 0 min analysis (corresponding to 15 min considering the time from sample preparation to the first sample point) at concentrations of 200–1600 µg mL−1 and 1.25–20.0 µg mL−1 of PEI and chlorophyllin, respectively (−1) eradicated the cells. After 240 min of incubation, all combinations of chlorophyllin and PEI were lethal for the cells, and colonies could only be recovered from cells treated with PEI alone at concentrations <100 µg mL−1. Similarly, in dark incubation, chlorophyllin/PEI combinations were highly effective against this strain  than cells in light (0–100 µg mL−1). PEI alone did not exhibit any lethal effect on the strain RB791, even after incubation for 240 min in light and dark conditions. However, in combination with 10 µg mL−1 chlorophyllin, apparent eradication was observed under light and dark incubation conditions  and PEI concentrations of 100 µg mL−1 and above in light incubated cells. At 0 min, chlorophyllin alone did not exhibit an immediate effect. However, 60 min sample analysis showed that ≥0.004 µg mL−1 of chlorophyllin alone completely eradicated the cells in light conditions (−1) and PEI (>12.5 µg mL−1). The cells were killed within 120 min of incubation in light at all the tested concentrations of PEI (12.5–800 µg mL−1) and chlorophyllin (0.002–2.000 µg mL−1) combinations. Only control cells (no chlorophyllin and PEI) showed growth. Pronounced synergistic effects were detected above all at 0 min. Due to the strong effect of both components on bacteria survival, Combenefit analysis can hardly detect synergism at later time points. Complete (100%) inhibition of cells was observed at 2 µg mL−1 of chlorophyllin alone and >12.5 µg mL−1 of PEI alone at 120 min of incubation in darkness (The Gram-positive model bacterium inations . The sennditions . Light idarkness .600nm) of 0.01. The MBC of different concentrations of PEI (1.95–250 µg mL−1) + chlorophyllin (0–30 µg mL−1) was investigated against different initial cell densities (OD600nm = 0.0003–0.73). For this study, freshly grown E. coli DH5α was employed, and the experiment was performed in 96-well microtiter plates. In light, higher concentrations of PEI-chlorophyllin showed stronger eradication effects on higher cell concentrations. Cell densities ≥ 0.0007 (OD600nm) incubated in light conditions were completely eradicated at the highest tested concentration of PEI (250 µg mL−1) (−1) and under illumination, a strong effect was noticed at 30 µg mL−1 chlorophyllin for the tested highest cell density . Dark controls, in contrast, were hardly affected at the applied concentration range.All the above experiments were performed with a defined culture density (ODµg mL−1) . ChloropE. coli DH5α and S. enterica, as well as Gram-positive B. subtilis. However, combinations of PEI and chlorophyllin exhibited considerably stronger effects than either of these compounds. Similarly to colistin, PEI interacts with and destabilizes the outer membrane lipopolysaccharides of Gram-negative bacteria [x is the distance, t is the lifetime, and D is the diffusion coefficient of 1 × 10−5 cm2 [2O2) can affect cellular targets at a longer distance. This led to the assumption that chlorophyllin present outside the cell is less effective in producing cell damage than chlorophyllin accumulated in the cytoplasm. The outer membrane of Gram-negative bacteria was found to be a barrier for chlorophyllin, significantly reducing its efficiency [Destabilizing the outer membrane of Gram-negative bacteria with substances such as lactic acid, EDTA, and PEI is a promising tool against pathogenic bacteria, because the outer membrane is an effective barrier against xenobiotics and other harmful substances ,29,30. Tbacteria ,27,28. Cbacteria ,27,28,31bacteria ,31. Cellbacteria . The lifbacteria , ~50–700bacteria , and ~10bacteria . During 10−5 cm2 ). ConseqE. coli DH5α was eradicated after 4 h of incubation with concentrations of 0.5 µg/mL colistin and 5 µg/mL chlorophyllin compared to 100 µg/mL PEI in combination with the same concentration of chlorophyllin. The difference is probably due to the different structures of the molecules. Colistin is smaller (1155.4 g/mol) compared to the branched PEI (about 25 kDa) employed in this study. In addition, the colistin molecule contains hydrophilic as well as lipophilic components, while the polybasic amine polymer branched PEI (B-PEI) is water soluble and lacks lipophilic components [Pseudomonas aerguinosa 902, as well as membrane depolarization ). All quaternized PEI800-polymers exhibited increased cytotoxicity against bacteria and induced significant membrane depolarization compared to unmodified PEI800. PEI800 FQ C6, a PEI800 fully quaternized with short alkyl groups, was the most effective PEI polymer. In another study, Roest et al. (2015) examined the charging properties of hyperbranched polyurea-polyethyleneimine coatings [Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii) and the yeast Candida albicans as well as their ability to form biofilms in a polyurethane catheter-model system [S. aureus and E. coli [Our data show that branched PEI has significant potential as synergistic or potentiating agents against bacteria. However, in the course of experiments using the cell wall destabilizing agent colistin in combination with chlorophyllin, the necessary concentration (weight per volume) of colistin for bacterial eradication was found to be far below that required by branched PEI. mponents . PEI is mponents . They teEI about  kDa empll system . PEI, in E. coli . The add E. coli . Followi E. coli  and bone E. coli .B. subtilis was severely affected by combinations of chlorophyllin and PEI. Under illumination, a complete loss of viability was found after 60 min at all tested concentrations of PEI, chlorophyllin, or their combinations, respectively. Interestingly, the highest tested concentration of chlorophyllin (2 µg/mL) alone did not completely eradicate the cells in darkness; however, combination with 12.5 µg/mL PEI led to complete eradication of the cells. This result comes in agreement with Foxley et al., (2016), who found that susceptibility of methicillin-resistant S. aureus (MRSA) strains against ampicillin and vancomycin can be restored in the presence of branched PEI because of its interaction with the wall teichoic acids (WTA) without entering the cells [The Gram-positive model bacterium he cells . Generalhe cells .From the above-mentioned data, we can conclude that PEI can be used as an antimicrobial agent or potentiating agent for ameliorating the antimicrobial activity of chlorophyllin or other antibiotics.E. coli DH5α, E. coli RB791, S. Typhimurium LT2, and B. subtilis 168. Cells were grown overnight (16 ± 2 h) in Luria Bertani (LB) medium at 37 °C. Cell concentration was determined by measuring the culture’s optical density (OD) at 600 nm, and the initial cell concentration was adjusted to OD600nm 0.01 for antimicrobial assay.Experiments were performed with bacterial strains 3 to avoid phaeophytin formation, and pressed through a cotton tissue to remove water. In the darkness, the leaves were extracted in 500 mL methanol for 12 h at 4 °C. After filtration, 300 mL petroleum benzene was added, forming a hydrophobic phase on top of the methanolic phase. Subsequent addition of small aliquots of saline water (5% NaCl) forced the chlorophyll into the benzene layer. After the complete transfer of chlorophyll into the benzene phase, phases were separated using a separating funnel. A defined small sample volume was withdrawn, the benzene was evaporated, and the residual chlorophyll was dissolved in a specified volume of methanol. The chlorophyll concentration in the benzene phase was determined according to Lichtenthaler and Wellburn (1983) [Chlorophyllin was prepared from spinach, as described previously . In brien (1983) . Finally600nm) of 0.01. For each experiment, two plates were prepared. One plate was exposed to light at an intensity of 12.7 mW cm−2, and the other plate was wrapped with aluminum foil, which was used as a dark control. A spectrum of the light source is shown elsewhere [The minimum bactericidal concentration (MBC) of PEI  and chlorophyllin combination was determined as described . In brielsewhere . At defihttps://www.cruk.cam.ac.uk/research-groups/jodrell-group/combenefit (accessed on 23 July 2022). The dose-response matrix shows the eradication percentage . Loewe synergy model was used to determination of the synergism and the antagonism between the tested combinations. In the Loewe synergy and antagonism matrix, N represents the number of biological replicates.The data were analyzed by Combenefit software (version 2.021) E. coli DH5α (OD600nm 2.92). Cell density gradients were prepared in 96-well plates in a horizontal direction (left to right of the plate), with two-fold serial dilution of cells in LB medium from one column to the next. Subsequently, two-fold serial dilution of PEI was in the vertical direction (top to bottom of the plate). The last column represents growth control. Defined chlorophyllin concentrations  were added separately. After incubation for 24 h under light (12.7 mW cm−2) or darkness, 3 µL from each well were plated on LB agar and incubated overnight at 37 °C as described in Experiments were performed with overnight (16 ± 2 h) grown cultures of Combinations of chlorophyllin and PEI exhibit strong antibacterial effects, above all under illumination but to a lower extent also in darkness. The results of this study are in context with observations of other research groups and suggest that the potential for synergistic effects of cell wall destabilizing substances  and photosensitizers may exist. The chemical structures can be modified to improve antimicrobial activity further. Furthermore, the determination of antimicrobial properties and cell toxicity of different PEIs or PEI-nanoparticles are of utter interest. At least under light conditions, antimicrobial effects of chlorophyllin are most likely caused by induction of excessive oxidative stress, which makes the formation of resistant strains very unlikely.


Brain cortical levels of ASCT1 substrate, serine, were increased in 5xFAD mice compared to WT animals. LAT1 (encoded by Slc7a5) and 4F2hc (encoded by Slc3a2) protein expressions were significantly altered in the isolated brain microvessels of 5xFAD mice compared to WT controls . Overall, the study provides important information, which is crucial for the optimal use of the 5xFAD mouse model in AD drug development and for investigating novel drug delivery approaches. In addition, the findings of the study shed light on the novel potential mechanisms underlying AD pathogenesis.Membrane transporters such as ATP-binding cassette (ABC) and solute carrier (SLC) transporters expressed at the neurovascular unit (NVU) play an important role in drug delivery to the brain and have been demonstrated to be involved in Alzheimer’s disease (AD) pathogenesis. However, our knowledge of quantitative changes in transporter absolute protein expression and functionality in vivo in NVU in AD patients and animal models is limited. The study aim was to investigate alterations in protein expression of ABC and SLC transporters in the isolated brain microvessels and brain prefrontal cortices of a widely used model of familial AD, 5xFAD mice (8 months old), using a sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic approach. Moreover, we examined alterations in brain prefrontal cortical and plasmatic levels of transporter substrates in 5xFAD mice compared to age-matched wild-type (WT) controls. ASCT1 (encoded by Slc1a4) protein expression in the isolated brain microvessels and brain prefrontal cortices of 5xFAD mice was twice higher compared to WT controls (The online version contains supplementary material available at 10.1007/s12035-022-03111-y. Alzheimer’s disease (AD) is a prevalent neurodegenerative disease, which is the most common cause of dementia. AD is biologically defined by the presence of extracellular β-amyloid (Aβ) plaques and intraneuronal neurofibrillary tangles (NFTs) composed of aggregated hyperphosphorylated tau protein in the brain . The amyThe transporters expressed at the brain capillary endothelial cells and other cells of NVU including glial cells, such as astrocytes, microglia, as well as pericytes and neurons mainly refer to either Solute carrier family transporters (SLCs) or adenosine triphosphate (ATP)-binding cassette transporters (ABCs) . ABC andThe majority of AD cases refer to late-onset AD which occurs after the age of 65 years, while the rarer form of AD, early-onset AD, accounts for less than 5% of all cases and manifests by age 60 . Most paThe aim of the present study was to characterize a mouse model of familial AD, 5xFAD mice , which i2PO4 (#P5655), KCl (#P3911), HEPES (#H3375), CaCl2·2H2O , MgSO4·7H2O , dextran , non-labelled amino acids L-serine (#PHR1103), L-alanine (#PHR1110), L-leucine (#PHR1105), L-isoleucine (#PHR1099), L-tyrosine (#PHR1097), L-phenylalanine (#PHR1100), histidine (#H0750000), and protease inhibitor cocktail  were purchased from Sigma-Aldrich . The stable isotope-labelled peptides (absolutely quantified) were provided by the JPT Peptide Technologies GmbH, Berlin, Germany. ProteoExtract® Subcellular Proteome Extraction Kit  was purchased from Merck KGaA, Darmstadt, Germany. Tosylphenylalanylchloromethyl ketone-treated trypsin (VA9000), Protease-Max surfactant (V2072), and lysyl endopeptidase  were purchased from Promega . A mixture of standards (product number MSK-A2-1.2) containing stable isotope-labelled amino acids was obtained from Cambridge Isotope Laboratories, Inc.Dithiothreitol (#D9760), guanidine hydrochloride (#G3272), acetonitrile (#1.00029), formic acid (5.33002), ethylenediaminetetraacetic acid (EDTA) (#E9884), Tris–HCl , bovine serum albumin , NaCl (#S9888), KHn = 35)  carrying human APP with the APP Swedish, Florida, and London mutations as well as human PSEN1 with the M146L and L286V mutations, which were driven by the mouse Thy1 promoter [n = 35) on the C57BL/6 J background . All mice were 8-month-old female mice as at this age 5xFAD female mice have been shown to develop a full range of AD pathological characteristics, such as amyloid pathology, presence of phosphorylated tau aggregates in the brain, cognitive impairment, long-term potentiation and synaptic transmission deficits, and astrocyte reactivity [t-test with 5% significant level and 80% power resulted in 6 observations per group.The study was performed according to the Council of Europe Legislation and Regulation for Animal Protection. The approval for all the experiments  was obtained from the Animal Experiment Board in Finland . The animal experiments complied with the ARRIVE guidelines and were conducted according to EU Directive 2010/63/EU for animal experiments. In the present study, we used transgenic hemizygous 5xFAD mice  and aged until the study age.Transgenic 5XFAD mice (RRID:MMRRC_034848-JAX) and C57BL/6 J mice  were purchased from The Jackson Laboratories . For maintaining the 5xFAD and non-transgenic WT colonies on site, male transgenic hemizygous 5xFAD mice were crossed with female C57BL/6 J dams at sexual maturity age (approximately 6 weeks of age). Due to the congenic background of 5xFAD mice, non-transgenic littermates (WT) were used as a control in the study. After pups were weaned, they were ear-punched for subsequent identification and genotyping. Mice were housed together with their littermates  at 95 °C for 1 h followed by vortexing. Samples were then cooled to room temperature and 1 M Tris, pH 8.0 , was added to neutralize the lysis. Samples were centrifugated for 6 min at 13000 g at room temperature. The standard polymerase chain reaction (PCR) assay , Jackson Laboratories, Bar Harbor, ME, USA) was performed with obtained DNA samples and primers for the PSEN1 transgene . All the primers are listed in Table 2PO4, 4.6 mM KCl, 15 mM HEPES, 5 mM CaCl2·2H2O, and 1.2 mM MgSO4·7H2O. Prefrontal cortex of about 15 mg was snap frozen, and stored at −70 °C until the quantitative real-time polymerase chain reaction (qRT-PCR) experiment, Aβ ELISA analysis, and LC–MS/MS-based proteomic analysis and amino acid analyses.On the day of tissue collection, at light time, mice were killed using carbon dioxide asphyxiation, as it has been used in previous studies to which the results of the present study are compared. In addition, the use of anaesthetics might affect the expression of transporters. Transcardial perfusion using heparinized 0.9% saline  was performed to remove the blood. After that, mouse brains were dissected out of the skull, excised, and the cerebrums were extracted. For the immediate microvessel isolation, the brains were placed on ice cold buffer A (pH 7.4) consisting of 101 mM NaCl, 1.2 mM KH1–40 and Aβ1–42 were measured in mouse brain cortical tissues as described previously [40 and Aβ42 ELISA Kits purchased from Thermo Fisher Scientific (KHB3481 for Aβ40 and KHB3544 for Aβ42), following the manufacturer’s instructions. The concentrations of Aβ1–40 and Aβ1–42 were normalised to total protein levels , which were measured using Bio-Rad DC Protein Assay .Total concentrations of Aβeviously  using HuExpression of inflammation markers, such as a marker of microglial activation, allograft inflammatory factor 1 (Aif1), and a marker of abnormal activation and proliferation of astrocytes, glial fibrillary acidic protein (Gfap), as well as a pro-inflammatory cytokine, interleukin-1 beta (Il1b), were quantified by qRT-PCR analysis in brain cortices of WT and 5xFAD mice. For this purpose, RNeasy Mini Kit , Qiagen, Stockach, Germany, was used for total RNA extraction from mouse brain cortices according to the manufacturer’s protocol. Total RNA was quantified by NanoDrop, Thermo Scientific, Dreieich, Germany. After that, cDNA was synthetized with Biozym cDNA synthesis Kit (#331475S), Oldendorf, Germany, according to the manufacturer’s protocol. The obtained cDNA was mixed with the PowerUp ™ SYBR ™ Green Master Mix (#A25741) purchased from Thermo-Fischer, Waltham, USA, and different gene-specific primers Table , which wg at 4 °C. The pellet was suspended in Buffer B, which consisted of Buffer A with 16% dextran, followed by centrifugation for 15 min at 4500 × g at 4 °C. After that, the supernatant was removed to a new tube, and centrifuged again for 15 min at 4500 × g at 4 °C. The pellets obtained after two centrifugation steps were suspended in Buffer C consisting of Buffer A with 5 g/L BSA and combined, followed by passing through a pre-wet nylon mesh of 200 µm . Consequently, the mesh was washed with 10 mL of Buffer C. The suspension, which passed through the filter, was then loaded onto a pre-wet nylon mesh of 100 µm . The nylon mesh was washed with 10 mL of Buffer C, and the obtained suspension was transferred to a pre-wet nylon mesh of 20 µm . The 20 µm mesh was washed with 40 mL of Buffer C, and the cerebral microvessels retained on the mesh were immediately collected after diverting the nylon mesh and washing it with 30 mL of Buffer C. The resulting suspension of the isolated cerebral microvessels was centrifuged for 5 min at 1000 × g at 4 °C. The obtained pellet consisting of the isolated brain microvessels was suspended in 1 mL of Buffer A. Consequently, the microscopic evaluation of the purity of the brain microvessels was performed. Finally, the suspension was centrifuged for 5 at 1000 × g min at 4 °C, and the supernatant was completely removed, while the resulting pellet was processed for crude membrane isolation using ProteoExtract® Subcellular Proteome Extraction Kit  according to the manufacturer’s protocol. Subsequently, total protein levels were measured in the crude membrane fractions of the isolated cerebral microvessel samples using the Bio-Rad DC Protein Assay. The crude membrane fractions were stored at −80 °C until LC–MS/MS-based QTAP analysis.Mouse cerebral microvessels were isolated using the method combining a dextran density gradient separation with size filtration in accordance with the previously validated protocol , which h+/K+-ATPase, and the endothelial luminal membrane marker γ-glutamyltransferase (γ-Gtp), between the crude membrane fractions of isolated cerebral microvessels and corresponding mouse brain cortical tissue [In addition to microscopic examination, the purity of the isolated cerebral microvessel preparations was assessed by comparing the absolute protein expression of the plasma abluminal membrane marker, such as Nal tissue .+/K+-ATPase, and γ-Gtp was quantified in crude membrane fraction of the isolated cerebral microvessels and brain prefrontal cortices in WT and 5xFAD mice. The investigated transporters have been selected previously [S-carbamoylmethylation was performed by adding iodoacetamide. The precipitation with methanol and chloroform was performed on the samples. The resulting precipitates were dissolved in 6 M urea in 0.1 M Tris–HCl (pH 8.5). The samples were diluted 5 times by adding 0.1 M Tris–HCl (pH 8.5) containing internal standard peptides  coupled to an Agilent 1290 Infinity LC  system. The Advance Bio Peptide Map column  was used for HPLC separation and elution of the peptides as reported previously . SimultaThe target protein expression quantitation was performed using one unique peptide Table , which hMouse plasma samples were prepared in accordance with previously reported protocols with minor modifications , 46. BriThe brain prefrontal cortical tissue samples were prepared in accordance with the previously reported protocol with small modifications . BrieflyFor relative quantification of serine, alanine, phenylalanine, tyrosine, leucine, isoleucine, and histidine, an Agilent 1200 Series Rapid Resolution LC System  coupled with Agilent 6410 Triple Quadrupole Mass Spectrometer with an electrospray ionization source ESI  was used. The separation was performed using hydrophilic interaction chromatography (HILIC) in positive mode ESI in accordance with the previously described method , 47 usin1–40 and Aβ1–42, normalized fold expression of Gfap, Aif1, and Il1b, and amino acid levels in plasma and brain cortical tissue of 5xFAD mice are presented as percentage of control in WT mice (mean ± SD). Statistical significance of differences in absolute protein expression, amino acid levels, total concentrations of Aβ1–40 and Aβ1–42, and normalized fold gene expression between WT and 5xFAD mice was analyzed using an unpaired t-test. A p-value of less than 0.05 was considered to be statistically significant. A ROUT test was used to identify statistical outliers which were removed from the analysis. To confirm the normal distribution, the data were tested against the null hypothesis. Data analysis was done using GraphPad Prism, version 5.03, GraphPad Software, San Diego, CA.The absolute protein expression levels are expressed as mean ± standard deviation (SD) as well as a ratio of protein expression between WT and 5xFAD. The total concentrations of Aβ1–40 and Aβ1–42 concentrations in the brain cortices of 5xFAD mice were 1.0 ± 0.67 mg/g total protein and 0.24 ± 0.098 mg/g total protein, respectively, while in WT mouse cortical tissue, the Aβ1–40 and Aβ1–42 were not detected (ULQ < 1.56 pg/mL for Aβ1–42 and ULQ < 7.81 pg/mL for Aβ1–40). In addition, the mRNA expression of inflammation markers, such as a marker of abnormal activation and proliferation of astrocytes, Gfap, a marker of microglial activation, Aif1, and pro-inflammatory cytokine interleukin-1 beta (Il1b), was significantly increased in the brain cortex of 5xFAD mice compared to WT controls . In addition, the mean protein expression of 4F2 cell-surface antigen heavy chain (4F2hc) in isolated brain microvessels was 1.4-fold higher in 5xFAD mice compared to WT animals (p = 0.05). The protein expression of large neutral amino acids transporter small subunit 1 (LAT1) in the isolated brain microvessels of 5xFAD mice was significantly lower than that in WT animals (p = 0.008) . The mean protein expression levels of other SLC transporters, i.e., GLUT1, 4F2hc, LAT1, and FATP1, as well as all investigated ABC transporters, did not differ between the study groups  and humans  . Thus, Lp = 0.005). In addition, brain prefrontal cortical levels of phenylalanine and histidine were 13% (p = 0.003) and 30% (p = 0.0005) higher, respectively, in 5xFAD mice compared to WT animals. The concentrations of other amino acids in the brain prefrontal cortical tissue did not differ significantly between the study groups , leucine , and phenylalanine  were observed in 5xFAD mice compared to WT controls  the transporter expression characterization of one of the most commonly used familial AD mouse model, 5xFAD, reproducing Aβ pathology and inflammation as confirmed in the present study; (2) investigation of the transporter expression in both the isolated brain microvessels and the brain prefrontal cortices representing brain parenchyma in the same animals; (3) investigation of changes in the brain and plasmatic levels of the transporter substrates, for which altered protein expression was observed, in the same animals; and (4) providing information on quantitative absolute protein expression of the transporters using the state-of-the-art highly selective, sensitive, and reproducible LC–MS/MS-based QTAP approach .+-dependent neutral amino acid transporter [The study revealed significant alterations in protein expression of amino acid transporters at the NVU cells of 5xFAD mice compared to WT controls. Thus, twice higher expression of alanine/serine/cysteine/threonine transporter 1 , a Nansporter , 54, wasnsporter , capillansporter , and rodnsporter , 56, 57,nsporter  indicatiN-methyl-D-aspartate (NMDA) receptors playing a key role in neurodevelopment, synaptic plasticity, learning, and memory [0AT2 (encoded by SLC6A15) and LAT2 (encoded by SLC7A8) mediating its uptake to neurons and astrocytes [ASCT1 is responsible for the efflux of L-serine and other neutral amino acids from astrocytes , 52. Cond memory . The actd memory . D-serind memory . Interesd memory , such astrocytes . Overall+- and pH-independent exchange of large branched-chain and aromatic neutral amino acids such as leucine, isoleucine, phenylalanine, tyrosine, histidine in antiport with histidine and tyrosine [The protein expression of large neutral amino acids transporter small subunit 1  was significantly decreased, while the expression of heavy chain subunit 4F2hc (encoded by Slc3a2) was elevated in the isolated brain microvessels of 5xFAD mice compared to WT controls. The light chain subunit  is a functional subunit, which mediates Natyrosine , 67. It tyrosine –70. Moretyrosine . LAT1 prtyrosine . Similartyrosine . As LAT1tyrosine , 72. Thetyrosine , future The changes in 4F2hc expression and function in AD patients have not been investigated. In our previous report, no changes in 4F2hc protein expression in the isolated brain microvessels of TgF344-AD rats were found when compared to WT controls . In 5xFAInterestingly, we have not observed changes in protein expression of other investigated SLC transporters as well as ABC transporters in the NVU cells of 5xFAD mice, while altered expression of FATP1, Abcg2, and Abcc1 in the isolated brain microvessels of TgF344-AD rats compared to WT controls was reported in our previous study . AlthougOverall, we found model-specific changes in protein expression of amino acid transporters in isolated brain microvessels. Moreover, changes in transporter protein expression in the isolated brain microvessels observed in our study did not always represent the changes reported in sporadic AD patients . These dSome additional limitations and future perspectives should be discussed. First of all, the study focused on the investigation of changes in transporter protein expression in female mice, while we previously demonstrated evidence for sex-specific changes in transporter protein expression in the AD rat model . Thus, fIn conclusion, we quantified absolute protein expression of ABC and SLC transporters in the isolated brain microvessels and the brain prefrontal cortices of 5xFAD mouse model and age-matched WT controls using LC–MS/MS-based QTAP approach. Here, we revealed significant upregulation of ASCT1 transporter in both isolated brain microvessels and brain prefrontal cortices of 5xFAD mice. These findings, along with the elevated brain levels of ASCT1 substrate, serine, provide evidence of the involvement of this amino acid transporter in AD pathogenesis. Importantly, we found altered protein expressions of other amino acid transporters such as LAT1 and 4F2hc in the isolated brain microvessels of 5xFAD mice, as well as changes in the brain levels of LAT1 substrates, phenylalanine and histidine. As LAT1 is extensively investigated as a target for drug delivery to the brain, the present study provides important information for optimal use of the 5xFAD model while testing new drug delivery strategies. All in all, the study gives insights not only into the potential role of the transporters in molecular mechanisms underlying AD, but into the optimal use of the 5xFAD mouse model during AD drug development and investigation of drug delivery strategies in AD.Supplementary file1 (DOCX 1499 KB)Below is the link to the electronic supplementary material.


Recovery of phosphate (P) from sludge, domestic wastewater,andindustrial wastewater is beneficial for overcoming the problem ofshortage of P rock resources. In this study, Mg-functionalized peanutshell-derived biochar was prepared by ultrasound-assisted impregnation.The obtained Mg-laden biochar had a higher content of Mg, a largerspecific surface area, and more porosity. The prepared Mg-modifiedbiochar exhibited excellent adsorption properties of phosphorus. Modifiedbiochar has a higher amount of adsorbed P than raw biochar. The capacityof P adsorption by modified biochar was 30.48–114.24% higherthan that by raw biochar. Moreover, the Mg-laden biochar can be appliedin a wide working environment . This study not only develops a new strategyfor the preparation of high-capacity P adsorbents but also providesa new green use for agricultural peanut shells. Jung and Ahn prepared porosity-enhanced biocharcontaining periclase (MgO) nanocomposites (PE-MgO/biochar) using anovel combined electrochemical modification method.28 Takaya et al. reported that biochar P adsorption can beenhanced from relatively low levels  to relativelyhigh levels (66.4–70.3%) by impregnation with magnesium.29 In recent years, substances used for modifyingbiochar are mainly MgO, MgCl2, La (OH)3, LaCl3, Na2CO3, NaOH, and FeCl3.33Adsorption is a low-cost and easy-to-operate technology that canhelp remove and recover P from water.34 Mostof the peanut shells are disposed by incineration or landfill, whichcauses a waste of resources and a huge impact on the environment.Preparation of biochar from peanut shells is a green, sustainable,low-cost, win–win production method, which can not only reducethe pollution caused by peanut shells but also be used to remove Pfrom wastewater. Ultrasound is a type of energy that can enrich thepores of biochar and increase the functional group content of thesurface.36 In this experiment, peanut shells were usedto prepare biochar. In addition, we evaluated the P adsorption propertiesof Mg-loaded biochar prepared under different ultrasound waves.The annual output of peanut shells (appendages in peanut production)is about 5.2 million tons in China.22.1Peanut shells were providedby Shijiazhuang Dasong Agricultural Planting Co. Ltd. .All chemicals used in the present study were of analytical reagentgrade (>99.0% purity).2.2–1 and a holdingtime of 2 h under a N2 atmosphere. Thereby, peanut shell-derivedbiochar samples were obtained and denoted as PSB. The element contentsof C, N, and O are shown in Table S1.Peanut shellswere washed with deionized water 3 times, then naturally dried, crushed,and sieved through a 100-mesh sieve. The peanut shell powder was driedat 60° for 24 h. Then, the peanut shell powder was placed ina programmable tube electric furnace and heated up to 550 °Cwith a heating rate of 15 °C min2 solution, and the pH was adjustedto around 8.0 with 0.1 M HCl or 0.1 M NaOH. The mixture was incubatedwith constant stirring at 25 °C for 24 h and filtered; then,the solid was dried at 105 °C and named MPSB. In addition, weevaluated the performance effect of MPSB prepared under differentultrasound interventions. In brief, PSB was pretreated in an ultrasoundenvironment before incubating the mixture of MgCl2 solutionand PSB. According to the different ultrasound healing times , three samples were obtained and named UMPSB1, UMPSB2,and UMPSB3, respectively.The Mg-laden biochar was prepared as follows: 25 g of PSB was addedto 500 mL of 0.5 M MgCl2.32PO4) in deionized water. The detailed steps of the P adsorption experimentof the sorbent were as follow: (1) 0.05 g of the sorbent was addedto 50 mL of phosphate solution with 10–200 mg L–1 P in 100 mL containers, respectively. (2) The containers were thenshaken using a shaker with a rate of 180 rpm at 25 ± 0.5 °Cfor 24 h. (3) The mixtures were filtered through a 0.22 μm filtermembrane, and the concentration of P in the filtrate was determinedusing ammonium molybdate and ascorbic acid.P solutions were preparedby dissolving monopotassium phosphate (KH–1 P)in 100 mL containers. Then, these containers were shaken using a shakerwith a rate of 180 rpm at 25 ± 0.5 °C for 0.5–24h. Afterward, the mixtures were filtered, and the P concentrationin the filtrate was determined. The P adsorption capacity of biocharcould be characterized according to the P concentration differencein solution before and after adsorption experiments.The P adsorptionkinetics of the adsorbent were addressed by adding 0.05 g of adsorbentinto 50 mL of phosphate solution (200 mg LQe is theamount of adsorption (mg P g–1), C0, Ce, V,and m are the initial P concentration(mg L–1 P), equilibrium solution (mg L–1 P), the volume of solution (L), and the weight of adsorbent (m),respectively.The adsorptioncapacity of the adsorbent was calculated using thefollowing formulaQ is the Langmuir maximumcapacity (mg g–1), Ce is the equilibrium solution concentration (mg L–1), and K and K2 representthe Langmuir bonding terms related to interaction energies and constants,respectively. tq and qe are the adsorption capacity at time t and at equilibrium, respectively (mg g–1).The Langmuir equations 2 were em–1 P).To investigate the effect of ambient temperature on the P adsorptionperformance of the adsorbents, a series of adsorption experimentswith different ambient temperatures from 15 to 40 °C were performedby mixing 0.05 g of adsorbent and 100 mL of phosphate solutions (200mg L–1 P) with differentinitial pH values from 2 to 12. The initial pH was adjusted with 0.1M HCl and 0.1 M NaOH solutions.To explore the effects of initialpH values on the P adsorptioncapacity of adsorbents, a series of adsorption experiments were performedin phosphate solutions (200 mg L2.4–1. The P-laden biochar samples were then addedto 100 mL of NaOH solution (3 M) and shaken for 12 h. The regeneratedadsorbents were washed with deionized water and then dried at 80 °Cfor 12 h. The obtained samples were directly reused in the next adsorptionexperiment. Finally, the adsorption–regeneration experimentswere repeated for five cycles following the same process.The recyclingability of adsorbents was explored in this study. The adsorption experimentswere carried out by mixing 0.1 g of adsorbents and 200 mL of 200 mgL2.5–1. The Brunauer–Emmett–Teller (BET)surface area, total pore volume, and pore diameter of adsorbents weremeasured using a N2 absorption-adsorption spectrometer. The elemental compositionof samples was determined by energy-dispersive X-ray spectroscopy(EDX) and X-ray photoelectron spectroscopy (XPS). X-ray diffraction(XRD) analyses of adsorbents before and after adsorbing phosphatewere performed .Elemental analysis was performed using a Vario-EL Elemental Analyzer. The morphologies of PSB and modified PSBs wereinvestigated by scanning electron microscopy . Functionalgroups of the samples were analyzed by Fourier transform infrared(FTIR) spectroscopy (Nicolet IS10) in the scanning range of 4000–500cm2.6p < 0.05.All experimentswere conducted in triplicate, and the average values are reported.Analysis of variance among treatments and mean separation tests (theDuncan’s multiple range test and least significant differencetest) were performed using SPSS 22.0. Differences among means andcorrelation coefficients were considered significant when 33.138The physicochemical properties of different biochar samples are presentedin –1 were associatedwith the −OH, −CH2, −CHO, and C=Cgroups, respectively. The disappearance of the peak at 1050 cm–1 indicated the rupture of the ether bond during pyrolysis.In addition, the peak at about 576 cm–1 was assignedto the Mg–O composite  suggests that the prepared samples have high activity.To confirm the formation of Mg-loaded biochar, FTIRspectra 1were obomposite 1L-C.39 T spectra 2R. The p biochar1R. As sh biochar1R-B,whi biochar2L show t2 in an ultrasonic environment, the surface structureof the biochar became looser   3. The P 3.3Figure S2A). Thismay be related to the strong obligate adsorption of P by the adsorbentin a neutral or acidic solution.44 TheP adsorption capacity of the adsorbent decreased obviously when thepH increased to 10 . The P adsorptioncapacity increased slightly with an increase in the ambient temperaturefrom 15 to 40 °C; however, no obvious difference was observed.These results indicate that these adsorbents show excellent P adsorptioncapacity at room temperature.Effects of ambient temperature on P adsorptionwere investigated ) appearedafter adsorption of P, suggesting that phosphate was successfullyadsorbed on raw and modified biochar.47 The XRD spectra 2, respectively.48 The results indicatedthe Mg3(PO4)2 was formed during phosphateadsorption.To further investigate the adsorption mechanism,XPS analysis 8 was perFigure S1A), and strong competitiveadsorption of OH– existed in the system when the pH was higherthan 10. According to the study of Jiao et al., abundant OH–may be generated during phosphate adsorption.45In addition, the effects of the initial pH valueson P adsorptionshowed that there was strong obligatory adsorption during the P adsorptionprocess  were evaluated for their adsorption performance ofphosphate. Low-frequency ultrasound can increase the content of Mgloading on biochar, specific surface area, and total porosity. Thenovel preparation method contributes to the formation of the skeletonwith more abundant pores. The prepared Mg-laden biochar exhibitedexcellent adsorption properties in a wide operating pH range (2–10).In addition, the Mg-laden biochar could be regenerated for at leastfive cycles, and each cycle showed good phosphate adsorption capacity.In summary, we believe the Mg-laden biochar is an excellent adsorbentfor removing and recovering phosphate from wastewater.