BZRAP1-AS1 is unknown in lung cancer. We evaluated the clinicopathologic significance of BZRAP1-AS1, and its role in non-small-cell lung cancer (NSCLC) progression.The function of BZRAP1-AS1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR) in tumor tissues and adjacent normal tissues. Then, the clinicopathological significance and prognostic value of BZRAP1-AS1 were analyzed by using our cohort and TCGA cohort. Finally, the effect of BZRAP1-AS1 on proliferation and motility of NSCLC cell lines were evaluated by cell growth assay, colony formation assay, xenograft tumorigenesis experiment in nude mice and transwell assays respectively.Sixty-three NSCLC patients from Beijing Chest Hospital were included. The expression of BZRAP1-AS1 showed lower expression in NSCLC tumor tissues. As for the relationship between BZRAP1-AS1 and clinical characteristics, our results were consistent with those of TCGA data. BZRAP1-AS1 was lower in T1 than T2–T4 patients, N1-N3 than N0 patients. Low level BZRAP1-AS1 was related to shorter overall survival time (OS) in lung adenocarcinoma (LUAD), and poor first progression time (FP) in LUAD and lung squamous cell carcinoma (LUSC) patients. BZRAP1-AS1 was significantly associated with the prognosis of NSCLC patients. Overexpression of BZRAP1-AS1 inhibited proliferation and migration of H1299 and HCC827 cells.Compared with adjacent normal tissues, BZRAP1-AS1 expression decreases in tumor tissues with the increase of malignancy grades in NSCLC. BZRAP1-AS1 plays an anticancer role by inhibiting cell proliferation, invasion, and metastasis, and has a potential prognostic value in NSCLC. BZRAP1-AS1 may serve as a diagnostic marker and therapeutic target for NSCLC. Lung cancer is one of the malignant tumors with the highest morbidity and mortality. There were 2.2 million new cases diagnosed and 1.8 million deaths worldwide in 2020, as the latest Global Cancer Statistics has shown . In lungBZRAP1-AS1), also named TSPOAP1-AS1, was mentioned firstly in Alzheimer’s disease  cells inhibits HUVEC proliferation, migration, and angiogenesis, the lncRNA tends to be an oncogene : TGTCTGCATCCCACAACAGG, (R): GGACCAGCTTGGAGTTGTGT. GAPDH (F): ACTAGGCGCTCACTGTTCTC, (R): CGACCAAATCCGTTGACTCC (5′–3′).Total RNA was extracted from stored tissues and cells with the TRIzol reagent , according to the manufacturer’s instructions. NanoDrop 2000c  was used to measure the concentration of RNA. RNAs were reverse-transcribed into cDNA in a 20 µl system using the TransScrip First-strand cDNA Synthesis SuperMix (TRAN) according to the protocol. The quantitative PCR was performed according to the instructions of the SYBR®. Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) was used for qPCR with a 15-µl reaction system, including 7.5 µl of PowerUp™ SYBR™ Green Master Mix (applied biosystems), 2.5 µl of RNase-free water, 1.25 µl of upstream and 1.25 µl of downstream primers, and 2.5 µl of cDNA template. GAPDH was used as an internal reference gene. The primer sequences involved as follows. BZRAP1-AS1 overexpression (OE)  and negative control (NC) (LVCON238) both were purchased from the Shanghai Genechem Company. Lentivirus infection was performed in H1299 and HCC827 NSCLC cells following the manufacturer’s instruction. Firstly, H1299 and HCC827 cells were plated in 96-well culture plates at a density of 3–5 × 103/well. The next day, cells were infected with Lv-BZRAP1-AS1 and LVCON238 for 12 h. The infection efficiency was about 80%, 48–72 h after infection. Finally, the infected cells were screened with Puromycin (2 µg/mL) for 2–3 days.The lentiviruses for http://gepia.cancer-pku.cn/index.html) is an online tool for gene expression analysis based on TCGA and GTEx data . The expression of BZRAP1-AS in Lung normal tissues and cancer adjacent normal tissues were both downloaded from the GTEx database. Then the expression difference and OS survival analysis of BZRAP1-AS1 in NSCLC was analyzed by SPSS and GraphPad Prism. Additionally, FP and univariate survival analysis was made by KM Plotter, which is an online survival analysis tool based on data from GEO, EGA, and TCGA  (TEx data . In thisand TCGA .3/well) and HCC827 cell (3 ×103/well) were seeded in 96-well plates. Five repetitive wells were set for each experiment. Cell activity was tested by cell counting kit-8 (CCK-8)  for 4 days. 10 µl CCK-8 was added into each well and then incubated with the cells for 2 h at 37 °C. The absorbance was recorded at 450nm with Bio Tek Epoch Microplate Reader . The experiments were repeated 3 times.H1299 cells (4 ×10300 cells/well were plated into 6-well plates and routinely cultured for 7–14 days. Subsequently the cells were washed quickly with PBS buffer solution and stained with 0.2% gentian violet. The number of colonies (≥50 cells) was recorded under an optical microscope. The experiments were repeated three times.vs H1299 OE, HCC827 NC vs HCC827 OE) were collected and resuspended in 10% RMPI-1640 at a density of 1  × 107 cells/ml. 2 × 106 were injected into subcutaneously the right armpit of the mice, NC as control group. Then length (L) and width (W) sizes of tumors were measured regularly every 2–3 days by researchers who didn’t know the grouping. The mice were euthanized on the 30–40th day, and tumors were harvested and photographed. Tumor volume was analyzed by V = 1/2(L × W2). On the 40th day following tumor injection and the average tumor diameter does not exceed 20 mm, or if ulceration, infection, or necrosis occurs, the experiment was terminated and the mice were euthanized using a standard carbon dioxide method. Finally, tumors were harvested and weighed. Data was analyzed using GraphPad Prism, the tumor volume data was analyzed using ANOVA and weight differences by unpaired t-test.All animal experiments were approved by the Ethics Committee of the Beijing Chest Hospital, Capital Medical University, Beijing, China (No. 2021-059), in compliance with national or institutional guidelines for the care and use of animals. Female BALB/c-nu mice (aged 4–6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The average weight of mice was 17 g. All mice were housed in animal facility under pathogen-free conditions. The animal room was kept at 18–23 °C, 40%–60% humidity, and a 10 h–14 h light-dark cycle. In order to get the most scientific experimental results, at least 10 mice in each group, but according to the 3R-reduction principle, so six mice in each group, a total of 24 mice were randomly divided into four groups. All food, cages, water, and other items that contact mice were sterile and handled using aseptic technique within a certified biosafety cabinet. Cells (H1299 NC 6) and H1299 cells (1.5  × 106) were seeded in the upper compartment with serum free medium. The lower chamber was filled with RMPI-1640 medium containing 10% FBS. After 48 h, the chamber was taken out and stained with 0.2% gentian violet for 20min, then upper Matrigel was washed away the staining reagent and removed using cotton swab cautiously. Finally, metastatic cells were observed and pictures taken under a Nikon microscope. The experiments were repeated three times.Cell invasion was tested using Transwell chamber  pre-coated with Matrigel . Cells were cultured in serum-free RMPI-1640 medium for 24 h. HCC827 cells  enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis for up-regulated genes and down-regulated genes. Then, we selected genes with significant differences in expression and closely related to tumor proliferation, metastasis and signaling pathways, and predicted the genes affected by BZRAP1-AS1 based on the above results. Furthermore, the co-expression analysis between BZRAP1-AS1 and predicted genes was performed using Starbase Analysis platform (https://starbase.sysu.edu.cn/) (Subcutaneous tumors were RNA sequenced in H1299 NC group and H1299 edu.cn/) .t-test. Abnormal distribution data were presented as median (25–75 percentile), and compared using the nonparametric test. The log-rank test was used to compare Kaplan–Meier survival curves. Statistical methods used for RNA-Seq analysis and TCGA data analysis were described above. P < 0.05 was considered statistically significant.The experiments were repeated three times. Normal distribution data were presented as Mean  ± SD, and compared using a two-tailed BZRAP1-AS1 was significantly lower in tumor tissues than in adjacent normal tissues (P = 0.004). Then, further subgroup analysis exhibited that in >60 years old , male , smoker , LUSC , and stage II–IV subgroup , BZRAP1-AS1 was lower in tumor tissues than adjacent normal tissues. There was no expression difference in ≤60 years old, female, non-smokers, LUAD, and stage I subgroup  vs 1.528 (0.117–12.300), P = 0.040], and N1-N2 than N0 patients  (BZRAP1-AS1 was lower in N1+N2 than N0 patients (0.359 (0.038–1.413) vs 2.346 (0.216–14.076), P = 0.018). Whether tumor tissues or adjacent normal tissues was no difference in subgroups divided by smoking history, histologic type, pleura invasion, and vessel carcinoma embolus. Additionally, the analysis results in subgroups may be biased because of the limitation of sample size, which still need to be validated.To find the potential biological functions of ectively . In tumo= 0.032] –1F. And BZRAP1-AS1 in NSCLC, we analyzed TCGA data using the SPSS, and Graphpad Prism. The results were in accordance with our RT-qPCR data. BZRAP1-AS1 was low expressed in NSCLC cancer tissue compared with normal tissue (41.00 (24.00–72.00) vs 69.00 (37.00–95.00), P < 0.001)  vs 91.00 (76.00–114.00), P < 0.001], but no difference in LUAD (BZRAP1-AS1 was lower in T2–T4 than T1 patients (38.00 (22.00–63.00) vs 58.00 (32.75–91.25), P < 0.001), N1-N3 than N0 patients (38.00 (23.00–63.00) vs 43.00 (24.00–76.00), P = 0.018) (BZRAP1-AS1 was lower in T2–T4 than T1 patients (38.50 (21.00–62.00) vs 58.50(32.00–95.00), P < 0.001), N1-N3 than N0 patients (34.00 (22.00–57.00) vs 46.00 (27.00–83.00), P = 0.001) in LUAD (BZRAP1-AS1 was lower in T2–T4 than T1 patients (37.00 (22.00–63.00) vs 57.00 (33.75–82.00), P < 0.001), but no difference in N stages in LUSC  . In diff in LUAD –2C. In N= 0.018)  and 2G.  in LUAD –2F. BZRA in LUSC –2I. The N stages .BZRAP1-AS1 was lower in tumor tissues than adjacent normal tissues in smokers, smoking was more meticulously analyzed. Compared with the adjacent normal tissues of nonsmokers, BZRAP1-AS1 level reduced in tumor tissues of nonsmokers, lower in nonmalignant tissues of smokers, and the lowest in tumor tissues of smokers  and the first progression (FP) of NSCLC patients was analyzed by GraphPad Prism and Kaplan–Meier plotter in TCGA database. Quartile was selected as expression threshold to distinguish the high-expression and low-expression cohorts. Patients of low o groups –4C. In Kpatients –4F. Nextpatients .BZRAP1-AS1 in NSCLC, we firstly detected the level of BZRAP1-AS1 in NSCLC cell lines  by RT-qPCR. Except for A549 cells, the expression of BZRAP1-AS1 was very low in NSCLC cells , less than HCC827 NC  , and HCC827 OE than HCC827 NC group (P = 0.082) , and HCC827 OE less than HCC827 NC group  , similar to HCC827 OE cells compared with HCC827 NC cells . Thus, overexpression of BZRAP1-AS1 inhibited migration of H1299 and HCC827 cells  –5F. Thes= 0.082) –5H. The = 0.042) . Additio27 cells –5K.BZRAP1-AS1 correlated with tumor-associated signaling pathways including cell adhesion molecules (CAM), MAPK signaling pathway, mTOR signaling pathway, pathways in cancer , BZRAP1-AS1 is high expression and knockdown of BZRAP1-AS1 inhibits cell proliferation, migration, and angiogenesis in HUVEC . Also, smoking is a major high-risk factor for LUSC (BZRAP1-AS1 is lower in T2–T4 than in T1, and in N1-N2 than in N0. In other words, BZRAP1-AS1 level is high in small tumors and low in big tumors, and high in tumors without N2 lymph nodes metastasis and low in tumors with N2 lymph nodes metastasis. When TNM stage, tumor size, and N metastasis more meticulously analyzed, BZRAP1-AS1 expression reduces gradually from I to III-IV, from ≤ three cm to >5 cm, from N0 to N2. In other words, the levels of BZRAP1-AS1 associated with the malignancy of NSCLC. So, we deduce that BZRAP1-AS1 participates in the pathological progress of NSCLC. To validate the hypothesis, TCGA data is analyzed. The results are consistent with our data. In addition, the survival analysis results show BZRAP1-AS1 is related to OS in LUAD, and FP in LUAD and LUSC. Patients with high level of BZRAP1-AS1 have a better prognosis, which is consistent with the literatures (For lung cancer, the relationship between  limited . NSCLC pfor LUSC . Then sueratures .BZRAP1-AS1 in NSCLC cell lines. In six NSCLC cell lines, Except for A549 cells, the expression of BZRAP1-AS1 is very low in five cells and only high in A549 cells. This result is consistent with that of tumor tissues. That is to say that the expression of BZRAP1-AS1 is very low in tumors. As for A549 cells, we think it is just a one example. How does BZRAP1-AS1 affect pathological process of NSCLC? We overexpress BZRAP1-AS1 in NSCLC cells to study the effect of BZRAP1-AS1 on cell proliferation, invasion, and migration in vitro and in vivo. The data indicate that overexpressing BZRAP1-AS1 indeed suppresses proliferation, invasion, and metastasis of NSCLC cells. This further confirms BZRAP1-AS1 plays an anticancer role by inhibiting proliferation, invasion, and migration. This is consistent with the data in cervical cancer and pancreatic cancer (In addition, we study the expression of c cancer .BZRAP1-AS1 in NSCLC, transcriptome analysis was performed. By GO enrichment analysis and KEGG pathway enrichment analysis, we screened twelve potentially relevant genes: RPS6KA6, LUM, TWIST1, PRKACB, TWIST2, PPM1A, FGF14, CNTN1, L1CAM, VDR, DPYSL3, ARHGAP4. After co-expression verification, four genes were finally concerned, namely FGF14, VDR, DPYSL3 and ARHGAP4. According to available literatures, these four genes play a role in suppressing tumor (BZRAP1-AS1 exerts its tumor suppressive function by influencing above four genes. BZRAP1-AS1 is expected to become a new therapeutic target for NSCLC.To further explore which genes are affected by ng tumor . So we gBZRAP1-AS1 expression negatively correlates with malignancy grades of NSCLC. BZRAP1-AS1 plays an anticancer role by inhibiting cell proliferation, invasion, and metastasis, and has promising prognostic value in NSCLC. BZRAP1-AS1 may serve as a potentially diagnostic marker and therapeutic target for NSCLC.10.7717/peerj.13871/supp-1Figure S1Click here for additional data file.10.7717/peerj.13871/supp-2Figure S2Click here for additional data file.10.7717/peerj.13871/supp-3Figure S3Click here for additional data file.10.7717/peerj.13871/supp-4Table S1Click here for additional data file.10.7717/peerj.13871/supp-5Table S2Click here for additional data file.10.7717/peerj.13871/supp-6Supplemental Information 1Click here for additional data file.10.7717/peerj.13871/supp-7Supplemental Information 2Eexpression differential analysis for RNAseq of mice tumor.Click here for additional data file.10.7717/peerj.13871/supp-8Data S1Click here for additional data file.


Aspergillus fumigatus, is composed of α-(1→2)-/α-(1→6)-linked mannan and β-(1→5)-/β-(1→6)-linked galactofuran side chains. Recently, CmsA and CmsB were identified as the α-(1→2)-mannosyltransferases involved in the biosynthesis of the α-core-mannan. However, the α-(1→6)-mannosyltransferase involved in the biosynthesis of the α-core-mannan has not been identified yet. In this study, we analyzed 9 putative α-(1→6)-mannosyltransferase gene disruption strains of A. fumigatus. The ΔanpA strain resulted in decreased mycelial elongation and reduced conidia formation. Proton nuclear magnetic resonance analysis revealed that the ΔanpA strain failed to produce the α-core-mannan of fungal-type galactomannan. We also found that recombinant AnpA exhibited much stronger α-(1→6)-mannosyltransferase activity toward α-(1→2)-mannobiose than α-(1→6)-mannobiose in vitro. Molecular simulations corroborated the fact that AnpA has a structure that can recognize the donor and acceptor substrates suitable for α-(1→6)-mannoside bond formation and that its catalytic activity would be specific for the elongation of the α-core-mannan structure in vivo. The identified AnpA is similar to Anp1p, which is involved in the elongation of the N-glycan outer chain in budding yeast, but the building sugar chain structure is different. The difference was attributed to the difference in substrate recognition of AnpA, which was clarified by simulations based on protein conformation. Thus, even proteins that seem to be functionally identical due to amino acid sequence similarity may be glycosyltransferase enzymes that make different glycans upon detailed analysis. This study describes an example of such a case.Fungal-type galactomannan, a cell wall component of IMPORTANCE Fungal-type galactomannan is a polysaccharide incorporated into the cell wall of filamentous fungi belonging to the subphylum Pezizomycotina. Biosynthetic enzymes of fungal-type galactomannan are potential targets for antifungal drugs and agrochemicals. In this study, we identified an α-(1→6)-mannosyltransferase responsible for the biosynthesis of the α-core-mannan of fungal-type galactomannan, which has not been known for a long time. The findings of this study shed light on processes that shape this cellular structure while identifying a key enzyme essential for the biosynthesis of fungal-type galactomannan. Aspergillus fumigatus, the major pathogenic fungus causing invasive pulmonary aspergillosis, contains 2 types of GMs: fungal-type galactomannan (FTGM) and O-mannose-type galactomannan (OMGM) , composed of d-mannose (Man) and d-galactofuranose residues, is a component of the cell wall of filamentous fungi –4. Asper–n (OMGM) . FTGM han (OMGM) , 7. FTGMn (OMGM) , 7. FTGMn (OMGM) , 9, whern (OMGM) , while an (OMGM) , 12. LikcmsA and/or ΔcmsB strains, hyphal elongation was remarkably suppressed and conidium formation was defective -galactofuranosyl chains observed in both FTGM and OMGM are biosynthesized by the enzymes GfsA and GfsC -galactof, 14. Rec15–efective . Moreovel strain . These dSaccharomyces cerevisiae, except for the endoplasmic reticulum-localized glycosyltransferases (GTs) involved in N-glycan and GPI anchor biosynthesis, 3 GT families containing α-(1→6)-mannosyltransferases are known: GT32, comprising Och1p and Hoc1p; GT34, comprising Mnn10p and Mnn11p; and GT62, comprising Mnn9p, Van1p, and Anp1p -mannosyl residues of O-glycans -mannosyltransferases from A. fumigatus were isolated as S. cerevisiae homologs -mannosyltransferase activity in vitro. Molecular modeling, docking, and molecular dynamics (MD) simulations revealed that the structure of AnpA contributes to α-(1→6)-mannoside bond formation at the α-core-mannan of FTGMs, and the C6-hydroxyl group of α-methyl mannoside can act as a model acceptor substrate for GDP mannose (GDP-Man) at the active site of AnpA.The aim of this study was to identify the α-(1→6)-mannosyltransferases involved in synthesizing the α-core-mannan of FTGMs. We analyzed the 9 gene disruptants in A. fumigatus listed in the study by Henry et al. -mannosyltransferases of y et al. : Van1  Chromosomal maps of the gene-disrupted strains of mnn9, van1, anpA, och1, och2, och3, och4, mnn10, and mnn11. The positions of the primers are indicated by arrows. pyrG, orotidine 5′-phosphate decarboxylase gene from Aspergillus nidulans. (B) Confirmation of correct recombination of each strain using PCR analysis. Electrophoretic analyses of products amplified by PCR using primer pairs xxxX-F/xxxX-R. M, DNA size markers . Download FIG S1, PDF file, 0.6 MB.Construction of Copyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the anpA, Δmnn9, and Δvan1 strains. We observed their colony formation at 30°C, 37°C, 42°C, and 50°C for 2 days. Δmnn9 and Δvan1 strains formed colonies similar to those of the A1151 strain under these conditions but the ΔanpA strain showed growth defects at 37°C and 42°C (in vivo.AnpA is a member of the GT62 family that has an amino acid sequence homology of 40.0% (in the 120 to 300 amino acid region) and 51.5% (in the 150 to 291 amino acid region) with Mnn9 and Van1 (other members of the GT62 family), respectively . Becauseand 42°C , suggest10.1128/msphere.00484-22.2FIG S2FIG S2, PDF file, 0.7 MB.Multiple sequence alignment of Mnn9p, AnpA, Mnn9, and Van1 amino acid sequences. Well-preserved residues are highlighted by blue boxes. Fully conserved amino acid residues are indicated by white letters on a red background. Aligned amino acid residues with similar properties are indicated in red. Arrows indicate amino acid residues required for the coordination of manganese ions. Download Copyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00484-22.3FIG S3mnn9, Δvan1, and ΔanpA strains, cultured on MM agar at 30°C, 37°C, 42°C, 50°C for 2 days, respectively. The agar medium was inoculated with 1.0 × 104 conidiospores. Download FIG S3, PDF file, 0.6 MB.Phenotypic analysis of GT62 family mutants. The colony morphology of A1151, ΔCopyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the anpA phenotype was truly due to the disruption of the anpA gene (anpA gene complementation strain (ΔanpA+anpA) and measured the hyphal elongation rate and the conidia number per colony area   , panel b6 M KCl) , panel b10.1128/msphere.00484-22.4FIG S4anpA for ΔanpA. (A) Schematic representation of ΔanpA complementation with anpA. Primer positions are indicated by arrows. pyrG, orotidine 5′-phosphate decarboxylase gene from Aspergillus nidulans; hph, hygromycin B phosphotransferase gene. (B) Confirmation of the correct recombination of anpA using PCR analysis. Results of electrophoretic analysis of products amplified by PCR using the primer pairs anpA-F/anpA-R are shown. M, DNA size markers (Gene Ladder Wide 2). Download FIG S4, PDF file, 0.1 MB.Complementation of Copyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the anpA disruption on cell wall integrity by testing the drug sensitivities of A1151, ΔanpA, and ΔanpA+anpA strains to the cell wall stress inducers Congo red   , panel bhibitor) , panel chttps://www.ncbi.nlm.nih.gov/) or FungiDB (https://fungidb.org) using the amino acid sequences of S. cerevisiae Van1p, Mnn9p, and Anp1p as search queries. The Mnn9p homologous protein from a rhizobia Mesorhizobium opportunistum WSM2075(T)  , presumaM2075(T) . Some GTM2075(T) . S. cerepA group . The dat1H-NMR spectroscopy. The 4 signals of A1151, Δmnn9, Δvan1, and ΔanpA+anpA strains indicate the H-1 signal of the chemical shift of the α-(1→2)-/α-(1→6)-mannan -/β-(1→6)-galactofuran side chains from FTGM and OMGM. OMGMs were removed from the total GM fraction using the β-elimination method to obtain the purified FTGM fraction, whose α-core-mannan structures were analyzed using )-mannan  15). Ho. Ho1H-NMA strain , which w or mnn9 , establiA. fumigatus  was overexpressed using the pPTR-II-SucA plasmid in the A1151 and ΔanpA strains. Extracted proteins were immunoblotted using anti-FLAG polyclonal antibodies. Download FIG S5, PDF file, 0.07 MB.Detection of invertase expression in A1151 and ΔCopyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ (1.0 mM) as a cofactor. The reaction mixtures were separated and analyzed by reverse-phase HPLC using a C18 column and an UV detector. The fraction without AnpA exhibited only α-Man-pNP peak at 18.2 min (product AnpA) at 16 min , α-Man-pNP (1.5 mM) as a sugar acceptor, GDP-Man (5 mM) as a sugar donor, and Mn18.2 min , panel at 16 min , panel bXanthomonas manihotis. The product was completely digested and converted to α-Man-pNP (in vitro. These results strongly support the hypothesis that AnpA is responsible for transferring the α-(1→6)-mannosyl residue in the FTGM α-core-mannan.To determine the chemical structure of the product, it was collected , panel aTo explore the functional differences between AnpA and the other α-(1→6)-mannosyltransferases, we measured its activity on 2 acceptor substrates: α-(1→6)-mannobiose and α-(1→2)-mannobiose. AnpA’s mannosyltransferase activity toward α-(1→6)-mannobiose was 16.9% relative to that toward α-(1→2)-mannobiose (100%) , panel c20–S. cerevisiae – has been reported -mannosyltransferase homologs, only 1 crystal structure – Mnn9p from reported . The stroutliers  30). An. AnS. ceoutliers , just liae Mnn9p  29). In. InS. cep of GDP . The fir GDP (2+ . In the h His395 . In addih His395  and E.10.1128/msphere.00484-22.6FIG S6S. cerevisiae. (D) The metal and GDP-Man binding site of Mnn9p from S. cerevisiae. The Mn2+ ion (magenta), GDP, and the binding residues are shown by ball-and-stick models and labeled. (E) The putative metal and GDP-Man binding site of AnpA. The residues are shown by stick models and labeled. Download FIG S6, PDF file, 1.1 MB.The dimeric structure of the catalytic domain of AnpA as predicted by AlphaFold2. (A) A ribbon representation of the AnpA dimer is colored from blue (high confidence) to red (low confidence) as per the pLDDT score. The N-termini and C-termini are labeled. Except for the C-termini, the model had high pLDDT score (mean = 91.2). (B) The predicted overall structure of the AnpA dimer. Secondary structures are color-coded red:helix, yellow:strand, and green:loop. AnpA was predicted to have a mixed α/β fold with 13 β-strands and 11 helices in a monomer. (C) The crystal structure of Mnn9p from Copyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ and the substrates to AnpA and the mechanism with which it forms an α-(1→6)-glycosidic bond, we performed MD simulations analyses using its substrates and the predicted structure of AnpA. The structure of AnpA, in complex with the Mn2+ ion, the donor substrate GDP-Man, and the acceptor substrate α-Man-OMe, was generated by docking simulations using the GNINA program , GDP-Man (gray), and α-Man-OMe (green) are shown by ball-and-stick models. (B) The energy-minimized conformation of α-Man-(1→2)-α-Man-(1→2)-α-Man-(1→2)-α-Man-OMe was superimposed on the simulated complex structure. When the nonreducing terminal Man was superimposed on the α-Man-OMe of the complex, the reducing terminal residue reached the molecular surface. Therefore, glycans as long as α-Man-(1→2)-α-Man-(1→2)-α-Man-(1→2)-α-Man would be required as the physiological acceptor substrates of AnpA in vivo. Download FIG S7, PDF file, 0.4 MB.AnpA requires longer sugar chains as its physiological acceptor substrate. (A) In the complex obtained by MD simulation, the position of α-Man-OMe, which was used as a acceptor substrate model, was about 10 Å inside from the molecular surface of AnpA. The MnCopyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the CmsA/ktr4 has been reported to reduce the virulence of A. fumigatus -mannosyltransferase responsible for biosynthesis of the FTGM α-core-mannan structure in mycotina . Fungal mycotina , suggestgatus 15–, 28, 32.mnn9 and van1 did not affect the FTGM α-core-mannan structure -mannosyltransferase responsible for synthesizing the FTGM α-core-mannan structure in A. fumigatus. Du et al. reported that Mnn9 was important for cell wall mannan synthesis, although it did not majorly affect N-glycan structure  was characterized as a α-glucosaminide α-(1→6)-mannosyltransferase responsible for synthesizing Af3c, a zwitterionic glycosphingolipid  have been shown to be free of hyphal growth defects . In addition, the disruption of cmsA/ktr4 and cmsB/ktr7 increases the amount of unidentified glycosyl inositol phosphoceramides, thereby activating the synthesis of non-FTGM glycolipids by an unknown compensatory mechanism  other than FTGM . Fi. FianpA han FTGM . The inc2+ and GDP, but does not provide information on the acceptor substrate -mannosyltransferases, only the crystal structure of yeast Mnn9p has been published . The strubstrate . Recentlubstrate . Van1p ain vitro , 22. On in vitro  and B, aomodimer . The fachis idea . In thisstituted  and C.in vivo. Recognizing the acceptor substrates of AnpA probably contributes to specifying the structure of the core-mannan of FTGM.Therefore, AnpA is considered to have diversified into fungal enzymes while retaining the structural basis for their α-(1→6)-mannosyltransferase activities. Docking and MD simulations further verified the predicted structure of AnpA as an α-(1→6)-mannosyltransferase and showed that the donor substrate GDP-Man and the acceptor substrate α-Man-OMe could be retained at the active site in the appropriate orientations  and 6C. 35–In conclusion, we identified an α-(1→6)-mannosyltransferase responsible for the biosynthesis of the α-core-mannan of FTGM. Our findings are expected to enhance the understanding of fungal cell wall structures and inform the development of new drugs against fungal pathogens in medicine and agriculture.A. fumigatus strains used in this study are listed in A. fumigatus strains were grown on Aspergillus minimal medium (MM).The 10.1128/msphere.00484-22.9TABLE S1Table S1, PDF file, 0.05 MB.Strains used in the present study. Download Copyright © 2022 Kadooka et al.2022Kadooka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the AnpyrG was amplified by PCR using A. nidulans A4 genomic DNA as the template and the primer pair pHSG396-AnpyrG-IF-F and pHSG396-AnpyrG-IF-R , and the homology arm at the AnpyrG were amplified by fusion PCR using A1151 genomic DNA, pHSG396-hygB, and pHSG-AnpyrG as templates and the primer pairs anpA-1/anpA-comp-1, pHSG396-F/pHSG396-R, and anpA-comp-2/anpA-comp-3, respectively. The DNA fragment, which was amplified using the primer pair anpA-1/anpA-comp-2 was used to substitute the ΔanpA locus with a complementary sequence, thereby yielding ΔanpA+anpA. MM agar plates containing 200 μg/mL hygromycin B were used to select transformants. The correct replacement of the DNA fragments for gene complementation was confirmed by PCR using the primer pair anpA-F/anpA-R  from d method . A β-elid method . To remo1H-NMR was performed according to a previously described method (1H-NMR were exchanged twice in D2O with intervening lyophilization and then dissolved in D2O (99.97% 2H). 1H-NMR spectra were measured using a JNM-LA600 spectrometer (JEOL) at 45°C. Proton chemical shifts were referenced relative to internal acetone at δ 2.225.The d method . SamplesE. coli codon-optimized anpA (AFUB_063940) fragments from A. fumigatus A1163 were synthesized as gBlocks (Integrated DNA Technologies). The anpA fragments were cloned into the NdeI and NotI sites of pET15b-KAI  was purchased from the Tokyo Chemical Industry Co., Ltd. Mannosyltransferase assays were performed as described previously . By excluding the N-terminal region containing the transmembrane helix, the amino acid sequence from residue 43 to the C-terminus, which corresponds to the catalytic domain of AnpA, was submitted for prediction. Of the 25 predicted structures from the 5 AlphaFold-Multimer models, the structure with the highest confidence according to the predicted LDDT (pLDDT) score  score  in additS. cerevisiae Mnn9p in complex with one GDP molecule and one Mn2+ ion at the active site  was administered via intraperitoneal injection (10 mL/kg). The survival rate in mice was plotted against time. Differences between groups were compared by one-way analysis of variance.The virulence assays in the mouse model (C57BL/6) were performed as previously described with a slight modification . BrieflyThe materials and data generated in this study will be made available upon reasonable request to the corresponding author.


Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters.Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance. Neisseria gonorrhoeae, the best-characterized and most clinically important multidrug efflux system is the multiple transferrable resistance (MtrCDE) tripartite efflux pump  essential for drug expulsion from the bacterial cell. This multidrug efflux pump couples with the MtrC periplasmic membrane fusion protein and the MtrE outer membrane channel to actively extrude antimicrobials and mediate drug resistance    . We also CR.103)  carrying CR.103) . These scationic antibiotic-sensitizing peptide (CASP) that interacts with the MtrD multidrug efflux pump using amino acid contact sites distinct from those of two other cationic peptides that possess antibacterial activity but can be exported by MtrD (–N. gonorrhoeae susceptible to an RND antibiotic substrate (novobiocin [Nov]), suggesting that it could serve as an adjuvant for antibiotic treatment regimens that have been thwarted by multidrug RND efflux pump systems similar to the gonococcal MtrCDE pump.With this structural knowledge in mind, we are interested in designing peptide-based inhibitors to hinder the function of multidrug RND efflux pumps. In particular, we have sought to discover novel peptide-based antimicrobial adjuvants that have the potential to allow the return of antibiotics previously withdrawn from treatment regimens. Here, we identify a cyclic  by MtrD –17. CritN. gonorrhoeae MtrD protein (strain FA19). Each heptapeptide in the library is flanked by a pair of cysteines located at the N and C termini. Under nonreducing conditions, the cysteines spontaneously form a disulfide cross-link, resulting in the phage display of cyclized peptides suitable for peptide-based therapeutics  ΔacrB/pACYCΩmtrCDE cells, which express the MtrCDE tripartite efflux complex (strain FA19). We used Nov as a test antimicrobial substrate for MtrD as the loss of this protein in N. gonorrhoeae due to the genetic inactivation of mtrD results in increased susceptibility to this antibiotic (Table S2).We then tested the growth of E. coli BL21(DE3) ΔacrB/pACYCΩmtrCDE cells in the presence of 75 μg/mL of Nov (the MIC for Nov was 125 μg/mL) and 2.0 mg/mL of ADP1, ADP2, ADP3, ADP4, or ADP5 were lower by 34.4%, 49.8%, 6.5%, 1.2%, or 47.6%, respectively, than the levels of growth of BL21(DE3) ΔacrB/pACYCΩmtrCDE cells in the presence of 75 μg/mL Nov only  ΔacrB/pACYCΩmtrCDE cells in the presence of Nov (E. coli BL21(DE3) ΔacrB/pACYCΩmtrCDE cells . Furthermore, CASP did not render E. coli BL21(DE3) ΔacrB/pACYCΩmtrCDE cells more susceptible to an antibiotic (streptomycin) not exported by the gonococcal MtrCDE efflux pump  ΔacrB cells and purified this protein using a Ni2+ affinity column. We reconstituted the purified MtrDCR103 pump into lipidic nanodiscs. We then incubated 2 μM the MtrDCR103-nanodisc sample with 100 μM CASP for 2 h to form the MtrDCR103-CASP complex and determined the MtrDCR103-CASP structure using single-particle cryo-EM . The reconstituted sample led to a cryo-EM map at a nominal resolution of 2.95 Å , particularly the structural elements making up the periplasmic entrance (formed by the PC1 and PC2 subdomains), where this entrance can be open or closed to help advance the transport cycle.Superimposition of the binding, extrusion, and access protomers of MtrDCR103 in complex with other cationic peptides.Structures of MtrD The structures of RND multidrug efflux pumps bound with small molecules have been studied extensively  and the human cathelicidin LL-37 (residues 17 to 32) peptide (LL).ensively , 42–48. lymyxins –17. How CR103-Col.(i) Structure of MtrD To study MtrDCR103-Col interactions, we incubated 2 μM the MtrDCR103-nanodisc sample with 20 μM Col for 2 h to form the MtrDCR103-Col complex. We obtained a cryo-EM map of this complex at a 3.08-Å resolution  Structure of MtrD As the MtrD multidrug efflux pump recognizes and confers resistance to the human antimicrobial peptide LL-37  that contains 16 amino acids of LL-37 (residues 17 to 32)  values within 0.6 Å, suggesting that the conformations of these three trimers are more or less identical. Again, within the binding protomer of MtrDCR103, the cryo-EM images depict an additional large density corresponding to the bound α-helical LL peptide located inside the open cleft of the periplasmic domain . The majority of these two cyclic peptides are located at the distal drug binding pocket and immediately behind the G-loop. However, the binding mode of the elongated, α-helical LL is quite distinct from those of Col and CASP. LL is found to span both the distal and proximal drug binding sites as well as part of the entrance site. The differences in these binding modes may be attributed in part to the fact that both Col and CASP are cyclic in form, but LL is an all-α-helical peptide.The chemical structure of LL is depicted in rmations  and C. Sc domain  to E. Thc domain , where tand R767 . In partN. gonorrhoeae strain FA1090 with CASP would render gonococci susceptible to sublethal levels of Nov (0.06 and 0.125 μg/mL). We found that CASP at 2.0 mg/mL in GCB  broth did not impede the growth of FA1090. However, CASP also did not sensitize FA1090 to the lethal effects of Nov, although Nov is a substrate for the MtrCDE efflux pump . Similarly, CASP could not sensitize two different gonococcal strains (FA19 and CDC2) to Nov (data not shown). Based on these results, we concluded that while CASP could sensitize E. coli cells producing the gonococcal MtrCDE efflux pump, it did not have a similar effect on N. gonorrhoeae.We tested whether incubation of Neisseria sp. (N. subflava [ATCC 49275]) to Nov . As E. coli is a Gram-negative bacillus, but N. gonorrhoeae and N. subflava are Gram-negative cocci, we hypothesized that these heptapeptides may be more specific for Gram-negative bacilli. Since N. gonorrhoeae MtrD is homologous to the E. coli AcrB, Pseudomonas aeruginosa MexB, and Salmonella enterica serovar Typhimurium AcrB multidrug efflux pumps, we rationalized that this cyclic peptide could be capable of inhibiting the growth of these Gram-negative bacilli in the presence of Nov.Furthermore, we also found that CASP could not sensitize a nonpathogenic commensal E. coli AcrB and P. aeruginosa MexB, we used the program AutoDock Vina .To elucidate if the CASP peptide is capable of binding other RND multidrug efflux pumps such as ock Vina  to studyE. coli K-12 ΔtolC::kan cells (strain JW5503), which have a deletion of the tolC gene expressing the TolC outer membrane channel. E. coli harbors seven RND membrane proteins of the hydrophobe-amphiphile efflux (HAE)  and heavy metal efflux (HME) (CusA) types (–E. coli K-12 ΔtolC::kan cells in the presence of 2 μg/mL Nov (the MIC of Nov for E. coli K-12 ΔtolC::kan was found to be 4 μg/mL in this study), supplemented with different concentrations of CASP . We found that CASP could not sensitize E. coli K-12 ΔtolC::kan cells to Nov  types –59. All s to Nov .E. coli K-12 (BW25113), Salmonella enterica subsp. enterica (ATCC 53648), and Pseudomonas aeruginosa (ATCC 39327). We grew E. coli K-12 cells in the presence of 75 μg/mL Nov (the MIC of Nov for E. coli K-12 was found to be >128 μg/mL in this study), supplemented with 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL CASP , supplemented with 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL CASP , supplemented with 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL CASP  (We previously solved cryo-EM structures of MtrDpicillin . It was ce (AMR) , 17. In ce (AMR) . TherefoN. gonorrhoeae or N. subflava. We hypothesize, however, that differences in the permeability barrier of the outer membranes of rod-shaped bacilli versus coccoid-shaped Neisseria may, in part, determine the ability of CASP to enter and reach MtrD-like transporter proteins harbored by these different bacteria. Despite this, we hope that this study may help initiate structure-guided and peptide-based antimicrobial design for obstructing these critical multidrug RND (and other) efflux pumps that help mediate AMR, allowing the return of previously used antibiotics. Thus, CASP-like compounds could serve as an adjunctive therapy to combat the increasing threat of multidrug-resistant (MDR) pathogens.Presently, we cannot explain why CASP treatment of Gram-negative bacilli can sensitize a panel of bacteria to Nov but fails to do so against mtrD gene, encoding the MtrD multidrug efflux pump, from N. gonorrhoeae strain FA19 was cloned into the pET15b expression vector in frame with a 6×His tag at the C terminus. The resulting pET15bΩmtrD plasmid was confirmed by the Sanger method of DNA sequencing. The MtrD protein was overproduced in E. coli BL21(DE3) ΔacrB cells, which harbor a deletion in the chromosomal acrB gene. Cells were grown in 6 L of Luria broth (LB) medium with 100 μg/mL ampicillin at 37°C. When the optical density at 600 nm (OD600) reached 0.5, the culture was treated with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to induce CR103mtrD expression. The cells were then harvested within 3 h of induction. The collected bacteria were resuspended in low-salt buffer  and then disrupted with a French pressure cell. The membrane fraction was collected and washed twice with high-salt buffer  and once with 20 mM HEPES-NaOH buffer (pH 7.5) containing 20 mM NaCl and 1 mM PMSF. The membrane protein was solubilized in 2% (wt/vol) n-dodecyl-β-d-maltoside (DDM), and the insoluble material was subsequently removed by ultracentrifugation at 100,000 × g. The extracted protein was then purified with a Ni2+ affinity column. The purity of the MtrDCR103 protein (>95%) was judged using an SDS-PAGE gel stained with Coomassie brilliant blue. The purified protein was dialyzed against 20 mM Na-HEPES (pH 7.5) and concentrated to 5 mg/mL in a buffer containing 100 mM NaHCO3 (pH 8.6) and 0.05% DDM.The 3 (pH 8.6) and 0.05% DDM and incubated overnight at 4°C. The coating solution was poured off, and blocking buffer  was added for 1 h at 4°C. After washing 6 times with TBST , 100 μL (2 × 109 PFU) of the library was added to the plate well for 2 h at room temperature, with gentle agitation. The plate well was washed 10 times with TBST to remove unbound phages. The bound phages were eluted with 0.2 M glycine-HCl (pH 2.2) and neutralized with 1 M Tris-HCl (pH 9.1). Eluted phages were amplified in 20 mL LB inoculated with the E. coli ER2738 strain, purified by polyethylene glycol (PEG)-NaCl precipitation, and titrated according to the NEB standard protocol to be used for the next round of selection. The two next rounds of selection were performed under more stringent conditions as the concentration of Tween 20 was increased (0.5%). After the third round of selection, eluted phages were plated onto NZ-amide and yeast extract (NZY) plates containing 20 μg/mL tetracycline. Individual randomly selected bacterial colonies were cultured and used for the isolation of phage DNA according to standard phage DNA purification procedures. The −96 gIII sequencing primer (CCCTCATAGTTAGCGTAACG) was used for sequencing to determine the amino acid sequences of inserts in the selected phage.The PhD-C7C phage display peptide library (New England BioLabs [NEB]) was used for screening antimicrobial peptides specific for the MtrD pump (strain FA19). The randomized segment of the PhD-C7C library is flanked by a pair of cysteine residues that are oxidized during phage assembly to a disulfide linkage, resulting in the displayed peptides being presented to the target as cyclized peptides. Peptide-harboring phage selection was performed as described below. A 96-well plate was coated with 1 mg/mL of the purified MtrD protein in a buffer containing 100 mM NaHCOE. coli BL21(DE3) ΔacrB/pACYCΩmtrCDE cells, as a surrogate for the N. gonorrhoeae strain FA19 mtrCDE genes, in the presence of each peptide with 75 μg/mL novobiocin. Of the peptides, only peptide 6 (ADP6) showed significant inhibition of the MtrCDE efflux system. To improve the stability of ADP6, the head-to-tail cyclic peptide ADP6 was synthesized  to produce the CASP peptide.The third-round-screened peptides listed in Table S1 in the supplemental material were subsequently synthesized by solid-phase peptide synthesis (GenScript). As a first screen to assess their activity, we studied the growth curve of mtrD::kan transformants was described previously  of pACYCDuet, whereas mtrD was cloned into MCS2 of pACYCDuet, to generate the pACYCΩmtrCDE expression vector. Each expressed protein has an in-frame 6×His tag at the C terminus. Next, E. coli BL21(DE3) ΔacrB cells harboring the plasmid pACYCΩmtrCDE were grown in LB medium containing 12.5 μg/mL chloramphenicol at 37°C to an OD600 of 0.4 and induced with 0.2 mM IPTG for 1 h at 37°C. Cells were collected and washed with LB three times to remove chloramphenicol. The cells were then resuspended using LB supplemented with 75 μg/mL Nov. The OD600 was adjusted to 0.1. One hundred microliters of the cell culture was aliquoted into each well of a 96-well plate. Two hundred fifty micrograms of different peptides screened from the phage display library was added to each well containing 100 μL of the cell culture. The experiments were performed at 37°C on a microplate reader (BioTek) by monitoring the OD600 value, which was recorded every 30 min for 24 h. Each experiment was repeated at least 3 times in duplicate.The construction and expression of the tripartite efflux pump system MtrCDE (strain FA19) were described previously by Janganan et al. . BrieflyE. coli BL21(DE3) ΔacrB/pACYCΩmtrCDE cells were grown in LB medium at 37°C to an OD600 of 0.6. Cells were collected, washed with LB once, resuspend with LB to an OD600 of 1.0, and then further diluted to an OD600 of 0.1 with LB supplemented with 4 μg/mL streptomycin. Cells were inoculated into a 96-well plate with a total volume of 100 μL. A final concentration of 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL of the CASP peptide was added to each well. The experiments were performed at 37°C on a microplate reader (BioTek). The OD600 value was monitored and recorded every 30 min within a 24-h period. Each experiment was repeated a minimum of three times.For cell growth in the presence of the antibiotic streptomycin, CR103mtrD gene, encoding the MtrDCR103 multidrug efflux pump, from N. gonorrhoeae strain CR.103 was cloned into the pET15b expression vector in frame with a 6×His tag at the C terminus. The resulting pET15bΩCR103mtrD plasmid was confirmed by the Sanger method of DNA sequencing. The procedures for expressing and purifying the MtrDCR103 protein were described previously by Lyu et al. (CR103 protein (>95%) was judged using an SDS-PAGE gel stained with Coomassie brilliant blue. The purified protein was dialyzed against 20 mM Na-HEPES (pH 7.5) and concentrated to 8.5 mg/mL (78 μM) in a buffer containing 20 mM Na-HEPES (pH 7.5) and 0.05% DDM.The u et al. . The purCR103 into nanodiscs, a mixture containing 20 μM MtrDCR103, 45 μM membrane scaffold protein (MSP) (1E3D1), and 930 μM E. coli total lipid extract was incubated for 15 min at room temperature. Afterwards, 0.8 mg/mL of prewashed Bio-beads (Bio-Rad) was added. The resultant mixture was incubated for 1 h on ice, followed by incubation overnight at 4°C. The protein-nanodisc solution was filtered through a 0.22-μm nitrocellulose filter tube to remove the Bio-beads. To separate free nanodiscs from MtrDCR103-loaded nanodiscs, the filtered protein-nanodisc solution was loaded into a Superose 6 column  equilibrated with buffer containing 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. Fractions corresponding to the size of the trimeric MtrDCR103-nanodisc complex were collected for cryo-EM studies.To assemble MtrDCR103-nanodisc sample was incubated with 100 μM CASP, 20 μM Col, or 50 μM LL for 2 h to form the MtrDCR103-peptide complex. The samples were applied to glow-discharged holey carbon grids , blotted for 17 s, and then plunge-frozen in liquid ethane using a Vitrobot instrument (Thermo Fisher). The grids were then transferred to cartridges.A 2 μM MtrDCR103-CASP complex data set, the images were recorded at a −0.75- to −1.75-μm defocus on a K3 summit direct electron detector (Gatan) with counting mode at a nominal magnification of ×81,000, corresponding to a sampling interval of 1.08 Å/pixel . Each micrograph was exposed for 2.6 s at a dose rate of 18.0 e−/s/physical pixel , and 40 frames were captured per specimen area using Latitude.For the MtrDCR103-Col complex data set, the images were recorded at a −1.75- to −2.5-μm defocus on a K3 summit direct electron detector (Gatan) with counting mode at a nominal magnification of ×81,000, corresponding to a sampling interval of 1.12 Å/pixel . Each micrograph was exposed for 2.8 s at a dose rate of 17.98 e−/s/physical pixel , and 40 frames were captured per specimen area using Latitude.For the MtrDCR103-LL complex data set, the images were recorded at a −1.0- to −2.25-μm defocus on a K3 summit direct electron detector (Gatan) with counting mode at a nominal magnification of ×81,000, corresponding to a sampling interval of 1.08 Å/pixel . Each micrograph was exposed for 2.6 s at a dose rate of 18.035 e−/s/physical pixel , and 40 frames were captured per specimen area using Latitude.For the MtrDCR103-CASP, the image stacks in the superresolution model were aligned using cryoSPARC  reconstruction. Two rounds of heterogeneous refinement were used, where 269,948 particles were used for further processing with nonuniform refinement using cryoSPARC  cells were grown overnight at 37°C under a 5% (vol/vol) CO2-enriched atmosphere on GCB agar  plates containing Kellogg’s supplements I and II (7 CFU from cultures grown overnight on GCB agar plates were resuspended in 100 μL GC broth containing Kellogg’s supplements I and II and 0.042% (wt/vol) sodium bicarbonate in 96-well plates. A total of 107 CFU of gonococcal strain FA1090 was seeded into 100 μL of GCB broth alone and GC broth containing Nov at sublethal levels (0.06 or 0.125 μg/mL), with or without 2.0 mg/mL of CASP. Plates were incubated statically at 37°C in a CO2 incubator, and the OD600 was read every hour using a VictorX3 2030 multilabel reader (PerkinElmer).I and II . For groNeisseria subflava (ATCC 49275) cells were grown in GCB broth containing 0.042% sodium bicarbonate at 37°C. Cells were harvested at an OD600 of 0.6, washed with GCB medium once, resuspended in GCB medium to an OD600 of 1.0, and then further diluted to an OD600 of 0.1 with GCB medium supplemented with 0.5 μg/mL novobiocin and 0.042% sodium bicarbonate. Cells were inoculated into a 96-well plate with a total volume of 100 μL. A range of final concentrations (0 to 2.0 mg/mL) of the CASP peptide with or without a sublethal level of Nov was added to each well. The experiments were performed at 37°C on a microplate reader (BioTek). The OD600 value was monitored and recorded every 30 min within a 24-h period. Each experiment was repeated a minimum of three times.N. gonorrhoeae MtrDCR103, E. coli AcrB, and P. aeruginosa MexB. The binding protomers of MtrDCR103 (from the MtrDCR103-CASP structure from this study), AcrB (PDB accession number 2DRD) (2V50)  , and Mex) (2V50)  were useE. coli K-12 ΔtolC::kan cells were grown in LB medium containing 25 μg/mL kanamycin at 37°C to an OD600 of 0.6. Cells were collected, washed with LB three times to remove the antibiotic kanamycin, resuspend in LB to an OD600 of 1.0., and then further diluted to an OD600 of 0.1 with LB supplemented with 2 μg/mL novobiocin. Cells were inoculated into a 96-well plate with a total volume of 100 μL. A final concentration of 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL of the CASP peptide was added to each well. The experiments were performed at 37°C on a microplate reader (BioTek). The OD600 value was monitored and recorded every 30 min within a 24-h period. Each experiment was repeated a minimum of three times.E. coli K-12 (BW25113) cells were grown in LB medium at 37°C to an OD600 of 0.6. Cells were collected, washed with LB once, resuspend with LB to an OD600 of 1.0, and then further diluted to an OD600 of 0.1 with LB supplemented with 75 μg/mL Nov. Cells were inoculated into a 96-well plate with a total volume of 100 μL. A final concentration of 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL of the CASP peptide was added to each well. The experiments were performed at 37°C on a microplate reader (BioTek). The OD600 value was monitored and recorded every 30 min within a 24-h period. Each experiment was repeated a minimum of three times.Salmonella enterica subsp. enterica (ATCC 53648) cells were grown in LB medium at 37°C to an OD600 of 0.6. Cells were collected, washed with LB once, resuspended with LB to an OD600 of 1.0, and then further diluted to an OD600 of 0.1 with LB supplemented with 128 μg/mL Nov. Cells were inoculated into a 96-well plate with a total volume of 100 μL. A final concentration of 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL of the CASP peptide was added to each well. The rest of the experimental procedures were the same as the ones described above for E. coli K-12 (BW25113) cells. Each experiment was repeated a minimum of three times.Pseudomonas aeruginosa (ATCC 39327) cells were grown in LB medium at 37°C to an OD600 of 0.6. Cells were collected, washed with LB once, resuspended with LB to an OD600 of 1.0, and then further diluted to an OD600 of 0.1 with LB supplemented with 512 μg/mL Nov. Cells were inoculated into a 96-well plate with a total volume of 100 μL. A final concentration of 0, 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 mg/mL of the CASP peptide was added to each well. The rest of the experimental procedures were the same as the ones described above for E. coli K-12 (BW25113) cells. Each experiment was repeated a minimum of three times.CR103-CASP, MtrDCR103-Col, and MtrDCR103-LL have been deposited under PDB accession numbers 8DEU, 8DEV, and 8DEW and EMDB accession numbers EMD-27399, EMD-27400, and EMD-27401, respectively.Atomic coordinates and EM maps of MtrD


Previous studies have revealed that the side effects of anticancer drugs induce a decrease of neurogenesis. Methotrexate (MTX), one of anticancer drugs, can induce lipid peroxidation as an indicator of oxidative stress in the brain. Melatonin has been presented as an antioxidant that can prevent oxidative stress-induced neuronal damage via the activation of antioxidant enzymes associated with the increase of neurogenesis. The aims of the present study are to examine the neuroprotective effect of melatonin on the neurotoxicity of MTX on neurogenesis and the changes of protein expression and antioxidant enzyme levels in adult rat hippocampus and prefrontal cortex (PFC). Male Sprague-Dawley rats were assigned into four groups: vehicle, MTX, melatonin, and melatonin+MTX groups. The vehicle group received saline solution and 10% ethanol solution, whereas the experimental groups received MTX  and melatonin  treatments. After the animal examination, the brains were removed for p21 immunofluorescence staining. The hippocampus and PFC were harvested for Western blot analysis and biochemical assessments of malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD). The immunofluorescence result showed that coadministration with melatonin diminished p21-positive cells in the hippocampal dentate gyrus, indicating a decrease of cell cycle arrest. Melatonin reduced the levels of MDA and prevented the decline of antioxidant enzyme activities in rats receiving MTX. In the melatonin+MTX group, the protein expression results showed that melatonin treatment significantly upregulated synaptic plasticity and an immature neuron marker through enhancing brain derived neurotrophic factor (BDNF) and doublecortin (DCX), respectively. Moreover, melatonin ameliorated the antioxidant defense system by improving the nuclear factor erythroid 2-related factor 2 (Nrf2) in rats receiving MTX. These findings suggested that the effects of melatonin can ameliorate MTX toxicity by several mechanisms, including an increase of endogenous antioxidants and neurogenesis in adult rat hippocampus and PFC. The anticancer agents have been found to have various adverse effects in several organs –4. PreviVarious studies have found that antioxidant exposure can stimulate adult neurogenesis in animal models –21. MelaIn addition to the interesting studies in the effects of melatonin on memory and neurogenesis, the present study focused on neuroprotective effects of melatonin on molecular mechanism related to neurogenesis in the MTX-induced rats. Therefore, the present study examined that melatonin administration mitigated MTX-induced cell cycle arrest, oxidative stress, and a decline of antioxidant enzymes and levels of DCX, Nrf2, and BDNF protein expression in hippocampus and PFC. However, these side effects of MTX were alleviated by cotreatment with melatonin.Fifty-six male Sprague-Dawley rats  were acquired from Nomura Siam International, Bangkok, Thailand. Rats were acclimatized in cages and freely accessed water and food during the experiment. Rats were housed under humidity control (30%-60%) in 12 h light/12 h dark cycle (06 : 00 am.-06 : 00 pm.) at room temperature (23 ± 2°C). The experimental procedures were performed in accordance with the Khon Kaen University Ethics Committee in Animal Research .Melatonin  was immediately dissolved in 10% ethanol solution  and kept in a falcon tube wrapped with aluminum foil to prevent light-induced melatonin deterioration. 10% ethanol solution was chosen according to previous studies without any toxicity , 37, 38.n = 14 animals/group): vehicle, MTX, melatonin, and melatonin+MTX. In the vehicle group, 10% ethanol solution  was given by intraperitoneal (i.p.) injection to rats at 7 : 00 pm. from day 1 to day 15, and a dose of saline solution  was administered by intravenous (i.v.) injection at 10 : 00 am. on days 8 and 15 of the experiment. In the melatonin group, melatonin (8 mg/kg) was given by i.p. injection to rats at 7 : 00 pm. from day 1 to day 15. In the MTX group, a dose of MTX (75 mg/kg) was given by i.v. injection at 10 : 00 am. on days 8 and 15 of the experiment. After each MTX injection, LCV  was administered to rats, whereas rats in the vehicle group received an identical volume of saline solution by i.p. injection. Rats in the melatonin+MTX group received equal volumes of melatonin and MTX at the corresponding time points  for 3 h at 4°C . Next, aThe cold deionized water was used to homogenize the hippocampal and PFC tissues, and the tissue homogenates were centrifuged at 13,000 rpm at 4°C for 10 min. The supernatant was gently collected, and the protein concentrations were analyzed using the NanoDrop spectrophotometer . All biochemical assessments were detected using the microplate reader  and examined according to the procedures reported previously .The evaluation of MDA was carried out to determine lipid peroxidation. The standard solution was prepared using 1,1,3,3-tetraethoxypropane  to produce a calibration line. The supernatant was mixed with 20% acetic acid solutions , 8.1% sodium dodecyl sulfate , and 0.8% thiobarbituric acid . The reaction mixture was boiled in a water bath at 95°C for 1 h before mixing with pyridine  and n-butanol . After centrifugation at 4,000 rpm for 10 min, each sample was evaluated at 540 nm, and the measurement was repeated in triplicate.A standard curve was prepared using 100 units/mL of SOD enzyme solution  as a standard compound. SOD levels were determined by adding the supernatant into the reaction cocktail (pH 7.8) containing 0.108 mM xanthine solutions , 216 mM potassium phosphate buffer , 1.1 mM cytochrome C , and 10.7 mM ethylenediaminetatraacetic acid . After that, the reaction mixture was reacted with 0.1 units/mL of xanthine oxidase enzyme solution  before analyzing the absorbance at 540 nm at 0 and 5 min in triplicate.1000 units/mL of CAT enzyme solution  was prepared as a standard compound. The supernatant was added to the solutions such as 50 mM potassium phosphate buffer, pH 7.0 , 30% hydrogen peroxide , and sulfuric acid . The reaction compound was incubated in the dark condition for 10 min before reacted with potassium permanganate solution . The concentration of CAT was detected by evaluating the absorbance at 540 nm, and each sample was performed in triplicate.β-nicotinamide adenine dinucleotide phosphate , sodium azide , L-glutathione oxidized , glutathione reductase solutions , and sodium phosphate buffer, pH 7.0 . The reaction mixture was mixed with 5,5′-dithiobis-(2-nitrobenzoic acid)  before incubating in the dark for 10 min. After that, 30% hydrogen peroxide solution and the reaction compound were mixed together, and the absorbance was detected at 405 nm in triplicate.The solution of GPX standard was performed by 20 units/mL of GPX enzyme solution . GPX levels were examined by mixing the supernatant with the reaction cocktail (pH 7.0), including, μg) per lane were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were blotted onto nitrocellulose membranes . Membranes were blocked with 5% bovine serum albumin  in Tris-buffered saline/0.1% Tween 20 (TBST) for 1 h and incubated at 4°C overnight with the following primary antibodies: anti-Nrf2 (1 : 1000), anti-BDNF (1 : 1000), anti-GAPDH (1 : 20000) , and anti-DCX (1 : 200) . Then the membranes were washed with TBST before probed with anti-mouse (1 : 2000), anti-goat (1 : 2000) and anti-rabbit (1 : 2000)  for 1 h and examined by enhanced chemiluminescence (ECL) using ECL reagents . The images of blots were developed with an Image Quant ECL Imager , and analyzing the images of protein bands was performed using Image J 1.53a software . Each sample was done in triplicate.The hippocampus and PFC were homogenized on ice with cold lysis buffer contained protease inhibitors  . After cμm along the coronal plane to get the whole dentate gyrus (bregma point -2.3 to -6.3 mm) [The frozen brains were fixed with 30% sucrose in 0.5% paraformaldehyde solution overnight before serially sectioning into 40 -6.3 mm)  using a -6.3 mm) . The p21p < 0.05 being considered statistically significant. One-way ANOVA with Bonferroni post hoc test were utilized to compare differences among groups in the analysis of immunofluorescence study, Western blotting, and biochemical assessments.GraphPad Prism 5.0  was carried out for statistical analysis in this experiment. The results are displayed as mean ± SEM with p < 0.05, p < 0.01, p < 0.001, To determine cell cycle arrest induced by MTX, immunofluorescence staining of p21 was performed. The p21-positive cells are detected in the SGZ of the dentate gyrus as shown in Figures p < 0.05, p < 0.001, p < 0.001, A significant increase of the MDA level in the hippocampus was detected in the MTX group in comparison with the vehicle group. A significant decline in the MDA level was found in the MEL+MTX group in comparison with the MTX group. Data represents mean ± SEM of the MDA levels in the hippocampus, vehicle: 11.88 ± 0.81 nmol/mg protein; MTX: 15.04 ± 0.38 nmol/mg protein; MEL: 9.47 ± 0.68 nmol/mg protein; and MEL+MTX: 12.05 ± 0.56 nmol/mg protein (p < 0.01, p < 0.001, p < 0.05, p < 0.01, p < 0.001, A significant decline of the CAT level in the hippocampus was revealed in the MTX group in comparison with the vehicle group. However, melatonin treatment significantly increased CAT levels in the MEL+MTX group in comparison with the MTX group. Data represents mean ± SEM of the CAT levels in the hippocampus, vehicle: 338.30 ± 6.34 unit/mg protein; MTX: 283.10 ± 10.71 unit/mg protein; MEL: 342.80 ± 4.22 unit/mg protein; and MEL+MTX: 326.40 ± 10.50 unit/mg protein (p < 0.001, p < 0.001, Melatonin treatment mitigated MTX-induced reduction in the GPX level in the hippocampus. In the MEL+MTX group, the levels significantly increased in comparison with the MTX group. Data represents mean ± SEM of the GPX levels in the hippocampus, vehicle: 5.52 ± 0.20 unit/mg protein; MTX: 3.14 ± 0.42 unit/mg protein; MEL: 5.99 ± 0.30 unit/mg protein; and MEL+MTX: 5.09 ± 0.26 unit/mg protein (p < 0.05, p < 0.01, p < 0.001, p < 0.001, MTX treatment induced the decline of SOD levels in the hippocampus. SOD levels in the MTX group were significantly lower than the vehicle group. Data represents mean ± SEM of the hippocampal SOD levels, vehicle: 24.55 ± 2.69 unit/mg protein; MTX: 14.49 ± 1.57 unit/mg protein; MEL: 27.66 ± 1.41 unit/mg protein; and MEL+MTX: 23.64 ± 1.23 unit/mg protein (p < 0.01, p < 0.001, A significant decrease of DCX expression in the hippocampus was demonstrated in the MTX group in comparison with the vehicle group, while melatonin treatment relieved MTX-suppressed DCX expression. A significant decline of DCX expression in the MEL+MTX group was not found in comparison with the MTX group. Data represents mean ± SEM of DCX levels, vehicle: 99.82 ± 4.076; MTX: 75.14 ± 3.461; MEL: 108.8 ± 3.314; and MEL+MTX: 95.09 ± 4.155 (p < 0.001, p < 0.01, p < 0.001, Nrf2 expression of the hippocampus was markedly upregulated in the MEL+MTX group in comparison with the MTX group. Data represents mean ± SEM of Nrf2 levels in the hippocampus, vehicle: 100.80 ± 2.40; MTX: 75.56 ± 3.19; MEL: 112.50 ± 4.47; and MEL+MTX: 101.60 ± 2.02 (p < 0.001, p < 0.05, p < 0.01, p < 0.001, The expression of BDNF level in the hippocampus in the MTX group was significantly less than the vehicle group. However, melatonin treatment significantly restored MTX-induced reduction of BDNF levels in the MEL+MTX group. Data represents mean ± SEM of BDNF levels in the hippocampus, vehicle: 99.76 ± 3.10; MTX: 72.84 ± 3.95; MEL: 110.50 ± 4.26; and MEL+MTX: 103.70 ± 4.33 (Chemotherapy-induced deterioration of memory and hippocampal neurogenesis has been recently reported . MTX is Numerous studies suggest that the SGZ of dentate gyrus in the hippocampus is where neurogenesis occurs throughout life , 48. EarFurther study in the present work confirms one of the negative consequences of MTX on neurogenesis in the SGZ of the dentate gyrus is cell cycle arrest using p21 immunofluorescence examination . The p21Associations between neurogenesis and BDNF have been found in several studies , 58. BDNSeveral researches have reported the function of oxidative stress in neurodegenerative disorders –68. MTX Melatonin has antioxidant capability and acts as a free radical scavenger , which dIn conclusion, these findings highlighted the side effects of MTX that are strongly related to oxidative stress-suppressed neurogenesis and induced an increase of cell cycle arrest in the hippocampal dentate gyrus. Here, toxicity of MTX also promoted lipid peroxidation and restrained antioxidant enzyme levels and protein expression of DCX, Nrf2, and BDNF. From further investigations, we found that melatonin had a potential to relieve the side effects of MTX by enhancing the antioxidant enzymes and Nrf2 expression to reduce the cell oxidative stress for maintaining the ROS balance and redox homeostasis in the hippocampus and PFC. Moreover, an impairment of neurogenesis and memories was restored by increasing BDNF expression that regulated cell survival and synaptic plasticity via the neuroprotective effect of melatonin.


Dipterocarpus intricatus Dyer fruit at pH 5.5 in the presence of Triton X-114. Reagent extraction, complexation, and preconcentration were performed simultaneously using ultrasound-assisted extraction at 45 °C. The surfactant-rich phase was diluted with ethanol and loaded through a syringe barrel packed with cotton that acted as a filter to trap the reagent powder. Analyte-entrapped on cotton was eluted using 0.1 mol·L−1 nitric acid solution. Filtrate and eluate solutions were measured absorbance of the dark-blue product at 575 nm. Influential parameters for the procedure were investigated. Under the optimum experimental conditions, the calibration curve was linear, ranging from 0.1 to 1.0 mg·L−1 with r2 = 0.997. Limits of detection and quantification were 0.03 and 0.09 mg·L−1, respectively while precision values of intra-day and inter-day were less than 5%. Recovery at 0.5 mg·L−1 ranged from 89.0 to 99.8%, while iron content in vegetable samples ranged from 2.45 to 13.36 mg/100 g. This method was cost-effective, reliable, eco-friendly, and convenient as a green analytical approach to determining iron content.An ultrasound-assisted, one-pot cloud point extraction was developed for the determination of iron in vegetable samples by UV-Visible spectrophotometry. This method was based on the complexation of iron with an environmentally-friendly natural chelating agent extracted from  Iron is the most abundant essential trace element in the human body. Iron is necessary for the synthesis of hemoprotein that functions as oxygen transport and oxidative metabolism. The median dietary intake of iron is 16 to 18 mg/day for men and 12 mg/day for women [UV-Visible spectrophotometry based on commercially available synthetic reagents such as 2- hydrozinecarbothiamide ,6, 2- was introduced in 1995 as a clean, environmentally-friendly analytical method. GAC has now been widely accepted by researchers and technicians as a pragmatic approach to reducing expenses while maintaining environmental sustainability. GAC has been implemented in many fields including spectroscopy (atomic and molecular) and electroanalytical and separation methods covering chromatography and electrophoresis techniques ,11.Curcuma longa L.) for acetic acid assay [Ulmus rubra) leaf to determine trace amounts of molybdenum(VI) [Platanus orientalis) for the determination of zirconium [Smilax china root [In molecular spectroscopic fields especially UV-Visible spectrophotometry, many publications have reported natural plant extractions as alternatives to commercially available reagents for the development of environmentally-friendly analytical methods, such as pumpkin for benzoyl peroxide assay , turmeriid assay , Indian id assay , heartwoid assay  and Indiid assay  for alumenum(VI) , and Oriirconium . Naturalirconium , guava lirconium , green tirconium , betel nirconium ,23, leadirconium , and Smiina root .p-chlorophenyl)-1-(pyridin-2-yl)thiosemicarbazide [A cloud point extraction method was employed to increase sensitivity and eliminate background interference before analysis . Cloud parbazide , and pyrarbazide  have beearbazide . Cucurbita pepo seed [Eryngium caucasicum leaves [Trigonella foenum-graecum L. seed [Ultrasound-assisted extraction (UAE) was utilized to enhance extraction efficiency through the cavitation phenomenon. The implosion of cavitation bubbles increased pressure near the solid surface and allowed extraction solvents to penetrate solid matrices ,35. UAE epo seed , Eryngium leaves , and Tri L. seed .Dipterocarpus intricatus Dyer was introduced from Thailand [Dipterocarpus intricatus Dyer stem including (Z)-ε-viniferin, (–)-4′-Omethylepigallocatechin 3-gallate, 11-Ogalloylbergenin, bergenin, 4-hydroxybenzal-dehyde, vanillin, vanillic acid, and syringic acid [Dipterocarpus intricatus Dyer [Dipterocarpus intricatus Dyer fruit.Thailand  and is ngic acid . Tannic gic acid . Antioxitus Dyer , with noDipterocarpus intricatus Dyer fruit, consisting of phenolic compounds as a natural chelation agent, was used for the determination of iron. No previous studies have reported using crude natural reagent extract from Dipterocarpus intricatus Dyer fruit as a chelating ligand to form a complex with iron coupling by micellar phase extraction. Crude reagent extract was compared with standard tannic acid to evaluate the chemical composition. Reagent extraction, complexation, and preconcentration require many types of glassware, with increased analysis time. Thus, to overcome these steps, a one-pot operation for reagent extraction, complexation, and preconcentration by cloud point extraction was developed for the determination of iron quantity in vegetables. A simple aqueous extract of 2+, Fe3+, Mn2+, Mo(VI), Pb2+, Cr(VI), Ni2+, Cd2+, Hg2+, Ca2+, Cu2+, Al3+, Na+, Mg2+, and K+ was investigated using 1.0 mL of natural reagent. A total of 1.0 mL of 100 mg·L−1 of each metal ion with final concentration was 10 mg·L−1 and 5.0 mL of 1.0 mol·L−1 acetate buffer pH 5.5. Digital image results are presented in 3+) form.The ability of the natural reagent to associate with metal ions such as Fe−1, (C-C) stretch and (C-H) deformation in the plane of the benzene ring at 1620.57 cm−1, (C-C) stretch and (C-H) deformation in the plane of the benzene ring, (C-O) stretch of phenolic at 1571.27 cm−1, (C-O) stretch and (O-H) deformation of phenolic, (C-C) stretch and (C-H) deformation in the plane of the benzene ring at 1383.91 cm−1, (O-H) deformation in the plane of phenolic and carboxylic acid, (C-C) stretch and (C-H) deformation in the plane of the benzene ring at 1174.82 cm−1, (C-O) stretch of phenolic at 1080.35 cm−1, (C-H) deformation out of the plane of the benzene ring at 834.01 cm−1, and (C-H) torsion of the benzene ring at 763.51 cm−1.Absorption spectra of the natural reagent extracted using deionized water and reaction with iron(III) are presented in Dipterocarpus intricatus Dyer fruit may include tannin or tannic acid, theogallin, gallic acid, and ellagic acid that act as metal chelators [Major chemical substances in helators ,48 in thDipterocarpus intricatus Dyer fruit was simultaneously operated with cloud point extraction of the iron-tannic complex under ultrasonication. The surfactant-rich phase was diluted and loaded into a syringe column packed with cotton before spectrophotometric determination. Various parameters affecting the proposed method including the amount of natural reagent, pH, buffer concentration, surfactant concentration, vortex time, incubation temperature, irradiation time, centrifugation speed and time, ethanol volume, and elution solvent were investigated using the univariate method at 1.0 mg·L−1 of iron(III) standard.The developed method for the extraction of tannic acid from natural Dipterocarpus intricatus Dyer on the absorption value of the complex between iron(III) and the reagent was investigated at 0.005, 0.01, 0.02, 0.03, and 0.05 g under 5.0 mL of 1.0 mol·L−1 acetate buffer pH 5.0, 0.03 g Triton X-114, 1 min of vortex, incubation at 45 °C for 10 min, centrifugation at 5000 rpm for 10 min, 0.05 g of cotton, 1.0 mL of ethanol, and 1.0 mL of 0.1% nitric acid. The absorbance signal at 575 nm increased with an increasing amount of reagent powder from 0.005 to 0.02 g  using the reagent powder at 0.02 g, 3.0 mL of 1.0 mol·L−1 acetate buffer pH 5.5, 1 min of vortex, incubation at 45 °C for 10 min, centrifugation at 5000 rpm for 10 min, 0.05 g of cotton, 1.0 mL of ethanol, and 1.0 mL of 0.1% nitric acid. Results are shown in w/v) and then the sensitivity declined due to dilution of the surfactant phase. Hence, concentration of Triton X-114 was optimized at 0.3% (w/v). Cloud point extraction offers high efficiency as a simple operation with rapid extraction involving small volumes of dilution solvent. Therefore, this method is inexpensive and friendly to both the operator and the environment [Triton X-114 is often used as the surfactant in cloud point extraction to preconcentrate the complexation product between iron(III) and the natural reagent to increase sensitivity with low cost, minimal hazard, high density, and low cloud point temperature . Triton ironment ,27,49.Dipterocarpus intricatus Dyer fruit powder, 3.0 mL of 1.0 mol·L−1 acetate buffer pH 5.5, 0.03 g Triton X-114, incubation at 45 °C for 10 min, centrifugation at 5000 rpm for 10 min, 0.05 g of cotton, 1.0 mL of ethanol, and 1.0 mL of 0.1% nitric acid. Results of absorbance obtained by varying the vortex time were not different (data not shown). Therefore, vortexing for 30 s was adopted for the proposed method to reduce analysis time.To homogenize the solution mixture and increase extraction efficiency, vortexing was investigated at 30 s, and 1, 2, 3, and 5 min by employing 0.02 g of −1 acetate buffer pH 5.5, 0.03 g Triton X-114, 30 s of vortex, incubation of 10 min, centrifugation at 5000 rpm for 10 min, 0.05 g of cotton, 1.0 mL of ethanol, and 1.0 mL of 0.1% nitric acid. Absorption value was highest when incubating the mixed solution at 45 °C. For incubation temperatures at 50 and 60 °C, the absorbance decreased slightly because of the unstable reaction complex (Triton X-114 clouds at room temperature (25 °C) and increasing temperature above the cloud point of the surfactant increased extraction efficiency. Cloud point temperature was examined between 35 and 60 °C by fixing 0.02 g of natural reagent powder, 3.0 mL of 1.0 mol·L complex d. Theref−1 acetate buffer pH 5.5, 0.03 g Triton X-114, 30 s of vortex, incubation at 45 °C, centrifugation at 5000 rpm for 10 min, 0.05 g of cotton, 1.0 mL of ethanol, and 1.0 mL of 0.1% nitric acid. Results are presented in Dipterocarpus intricatus Dyer fruit powder into the liquid phase through the acoustic cavitation phenomena and chemical effects [The effect of ultrasonic irradiation time was studied from 1 to 30 min under 0.02 g of natural reagent powder, 3.0 mL of 1.0 mol·L effects ,52. Ultr effects . −1 acetate buffer pH 5.5, 0.03 g Triton X-114, 30 s of vortex, incubate at 45 °C for 10 min, centrifugation for 10 min, 0.05 g of cotton, 1.0 mL of ethanol, and 1.0 mL of 0.1% nitric acid. The signal achieved from different speeds was not different. Therefore, centrifugation speed was selected at 5000 rpm. Centrifuge time was tested from 5 to 30 min. Absorbance values were not different. Hence, 10 min of centrifugation was chosen.Centrifugation speed to aggregate phase separation was examined between 3000 and 6000 rpm by employing 0.02 g of natural reagent powder, 3.0 mL of 1.0 mol·L−1 acetate buffer pH 5.5, 0.03 g Triton X-114, 30 s of vortex, incubation at 45 °C for 10 min, centrifugation at 5000 rpm for 10 min, 0.05 g of cotton, and 1.0 mL of 0.1% nitric acid). The surfactant-rich phase remained at the bottom of the centrifuge tube and was not appropriate for measuring the absorbance by a spectrophotometer due to its high viscosity. Using ethanol to dissolve the surfactant phase provided the highest absorbance, as presented in Ethanol and methanol were tested as organic solvents to dissolve the extraction phase to reduce the viscosity  nitric acid, and 0.1% (v/v) hydrochloric acid were used to elute the iron(III)-natural reagent complex adsorbing on the cotton by employing the other parameters at 0.02 g of natural reagent powder, 3.0 mL of 1.0 mol·L−1 acetate buffer pH 5.5, 0.03 g Triton X-114, 30 s of vortex, incubation at 45 °C for 10 min, centrifugation at 5000 rpm for 10 min, 0.05 g of cotton, 1.0 mL of ethanol, and 1.0 mL of elution solvent. Results  and S is the slope of the calibration graph at 0.03 and 0.09 mg·L−1. RSD values of the proposed method tested at 0.1 and 0.5 mg·L−1 of iron(III) were 1.5% and 0.7%, respectively for intra-day (n = 10) and 2.3% and 0.9% for inter-day (n = 10 × 5), indicating that this method provided good precision. To prevent experimental inherent variability using the natural reagent, the calibration curve for the determination of iron by the proposed method should be constructed daily, with absorbance measured against the blank solution. Analytical figures of merit for the proposed method were compared with other natural reagents in Dipterocarpus intricatus Dyer to determine iron.Method validation as linearity range, limit of detection (LOD), limit of quantification (LOQ), and precision represented in terms of relative standard deviation (RSD) were examined under the optimal/selected conditions for the determination of iron. A calibration graph for iron determination was obtained in the range from 0.1 to 1.0 mg·L−1. Tolerance limits, defined as the interferent concentration, caused an error of absorbance ±5% at 0.5 mg·L−1 of iron(III). The ions Pb2+, Cr(VI), Ni2+, Cd2+, Hg2+, and Cd2+ also interfered at 10 mg·L−1. Tolerance limits of Al3+ and Cu2+ were 15 and 20 mg·L−1, respectively while Na+, Ca2+, Mg2+, K+, and Cl− interfered at 50 mg·L−1 and Zn2+, and Mn2+ interfered for iron assay at 100 mg·L−1. Anions such as CO32- and NO3− provided tolerance limits at 200 mg·L−1. The tolerance concentration of iron(II) was 5 mg·L−1.Some metal elements and anions interfered in the analysis of iron by our proposed method. This may lead to incorrect concentrations of iron in real samples. The interferences were investigated by increasing the concentration of the interferent with the analyte of interest at 0.5 mg·LTiliacora triandra leaf, and Siamese neem flower were selected as representative samples to study the accuracy of this method by spiking standard iron at 0.5 mg·L−1. Recoveries ranged from 89.0 to 99.8%, indicating that the proposed method provided high accuracy. Our method was also applied to assay iron content in eight vegetables. Iron was detected in all samples between 2.45 and 13.36 mg/100 g, as shown in t-test at 95% confidence level . This green alternative method to quantify iron concentration in vegetable samples was cost-effective, reliable, and convenient, with high precision, accuracy, and environmentally friendly.Our developed method quantified iron in green vegetable samples. Local vegetables such as Chinese kale, sweet basil, All chemicals and reagents used were analytical reagent grade, with solutions prepared by dissolution with deionized water .−1 iron(III) for atomic absorption spectrometry (AAS) was obtained from Merck, Germany. A working solution of iron(III) standard in the range from 0.1 to 1.0 mg·L−1 was generated by diluting 1000 mg·L−1 iron(III) with deionized water.A standard stock solution of 1000 mg·L2+, Mo(VI), Pb2+, Cr(VI), Ni2+, Cd2+, Hg2+, Cu2+, Al3+, Ca2+, Mg2+, K+ and Na+ (1000 mg·L−1) for AAS was purchased from Merck, Germany. Fe2+ stock solution at 1000 mg·L−1 was prepared from iron(II) sulfate heptahydrate .Stock solution of Mn−1 was prepared by weighing 110 g of sodium acetate trihydrate  and then mixing with 8.45 mL of 98% acetic acid . The final pH was adjusted using 1.0 mol·L−1 sodium hydroxide  and the volume was made up to 1000 mL with deionized water.Acetate buffer pH 5.5 at 1.0 mol·LTriton X-114 non-ionic surfactant  was employed to preconcentrate the reaction product between iron(III) and the natural chelating agent by cloud point extraction (CPE) before spectrophotometric analysis at 575 nm. Tannic acid standard, nitric acid, and 95% ethanol were purchased from Merck .Dipterocarpus intricatus Dyer and compare results with the tannic acid standard. An ultrasonic bath  was utilized to assist the extraction. A benchtop centrifuge  was used to separate the surfactant-rich phase, and a cooking blender  was employed to grind the natural reagent and vegetable samples. A furnace  was utilized to incinerate vegetable samples. All pH measurements were made using a 713-pH meter . A flame atomic absorption spectrometer  was used to detect iron concentration as the standard method, with results compared to the developed method.UV-Visible spectroscopy  was employed to scan spectra and measure the absorption values of the extraction phases. FT-IR  was used to confirm the functional groups of the natural reagent extracted from Dipterocarpus intricatus Dyer is a local plant found in Thailand. The fruit was harvested between February and May from Rio-Et and Maha Sarakham Provinces. The dry fruit was cleaned with tissue to remove the dirt and then desiccated in an oven at 60 °C for 24 h before grinding to powder using a cooking blender. The reagent powder was packed in polyethylene zip bags and kept at room temperature in a desiccator.Dipterocarpus intricatus Dyer powder 0.02 g into a 15 mL centrifuge tube. Then, 0.03 g of Triton X-114 was added. Various volumes between 0.1 and 1.0 mL of 10 mg·L−1 iron(III) standard solution  or 1.0 mL of sample solution were transferred into a centrifuge tube. After that, 3.0 mL of 1.0 mol·L−1 of acetate buffer pH 5.5 was added to control the pH of the solution. The volume was then adjusted to 10 mL with deionized water and vortexed for 30 s before the solution was transferred into an ultrasonic bath at 45 °C for 10 min (37 kHz of ultrasonic frequency and 80 W of ultrasonic power effective) for simultaneous one-pot cloud point extraction including natural reagent extraction, complexation, and preconcentration. The dark-blue cloudy solution was cooled to room temperature and then centrifuged at 5000 rpm for 10 min. The surfactant-rich phase was obtained and the upper supernatant solution was withdrawn using a long-tubed needle before dilution with 1.0 mL ethanol 95% to decrease the viscosity. The solution was then passed through a syringe barrel containing 0.05 g cotton to filter and trap the Dipterocarpus intricatus Dyer powder. Reaction products adsorbed on the cotton were eluted by 1.5 mL of 0.1% nitric acid. The mixed solution (filtrate and eluate) was measured for absorbance at 575 nm by a spectrophotometer. The operational steps are illustrated in Determination of iron by the proposed method followed a one-pot reaction involving natural reagent extraction, complexation reaction of iron between the natural chelating agent, and cloud point preconcentration. Ultrasonication was utilized to assist and increase extraction efficiency. The determination steps involved weighing Tiliacora triandra leaf, Siamese neem flower, wildbetal leafbush, Thai copper pod, peppermint leaf, and Turkey berry fruit were purchased from a local market in Maha Sarakham Province, Thailand. Sample preparation was adopted and modified from previous procedures for iron determination [−1 iron standard for recovery study. Then, 1.0 mL of 65% nitric acid was added and the samples were incinerated at 450 °C for 16 h in a furnace. Each dry ash sample was redigested with 1.0 mL of concentrated nitric acid and incinerated at 450 °C for 6 h. Next, the residual dry ash sample was added with 1.0 mL of concentrated nitric acid and 5.0 mL of deionized water. The solution was filtered through filter paper into a 25 mL volumetric flask, and the volume was adjusted to 25 mL with deionized water. The samples were diluted with deionized water before analysis by the proposed method to obtain a final iron concentration within the calibration range.Green vegetable samples including Chinese kale, sweet basil, mination ,55,56. B−1, acetylene flow 2.00 L min−1, burner height 13.5 mm, burner length 10 cm, and detection wavelength 248.3 nm. A calibration graph was plotted for concentrations ranging from 0.2-10.0 mg·L−1 to determine the total iron contents in the samples.The FAAS method was employed for quantification of total iron concentration in real samples as the standard method to compare results with our proposed method. The FAAS conditions were set as airflow 13.50 L minDipterocarpus intricatus Dyer fruit as a natural chelating agent for the determination of total iron was developed. Ultrasonic radiation was used to assist a one-pot reaction comprising reagent extraction and preconcentration based on cloud point to enhance sensitivity. This developed method was successfully applied to quantify iron content in vegetable samples. Results of iron concentration by the proposed method were not significantly different from those achieved using the FAAS standard method. Our proposed method using ultrasound-assisted one-pot cloud point extraction with natural reagent from Dipterocarpus intricatus Dyer fruit was cost-effective, reliable, eco-friendly, and convenient as an alternative green analytical approach for the determination of iron contents in vegetable samples.Green analytical chemistry using