Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. It has been proposed that epigenetic modification and transcriptional dysregulation may contribute to motor neuron death. In this study, we investigate the basis for therapeutic approaches to target lysine-specific histone demethylase 1 (LSD1) and elucidate the mechanistic role of LSD1-histone H3K4 signaling pathway in ALS pathogenesis.In order to examine the role of spermidine (SD), we administered SD to an animal model of ALS (G93A) and performed neuropathological analysis, body weight, and survival evaluation.Herein, we found that LSD1 activity is increased while levels of H3K4me2, a substrate of LSD1, is decreased in cellular and animal models of ALS. SD administration modulated the LSD1 activity and restored H3K4me2 levels in ChAT-positive motor neurons in the lumbar spinal cord of ALS mice. SD prevented cellular damage by improving the number and size of motor neurons in ALS mice. SD administration also reduced GFAP-positive astrogliogenesis in the white and gray matter of the lumbar spinal cord, improving the neuropathology of ALS mice. Moreover, SD administration improved the rotarod performance and gait analysis of ALS mice. Finally, SD administration delayed disease onset and prolonged the lifespan of ALS (G93A) transgenic mice.Together, modulating epigenetic targets such as LSD1 by small compounds may be a useful therapeutic strategy for treating ALS.The online version contains supplementary material available at 10.1186/s12929-022-00890-3. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a loss of upper and lower motor neurons resulting in progressive muscular atrophy and paralysis. ALS has an annual incidence of 1–2 per 100,000 and is usually fatal within 2–5 years of diagnosis –6. Benefl-arginine protects motor neurons from glutamate-induced toxicity in vitro and prolongs the survival of ALS (G93A) transgenic mice  and animal model of ALS [mSOD1 (G93A)]. Moreover, LSD1 is increased in NSC-34 cells treated with hydrogen peroxide to produce oxidative stress and in spinal motor neurons of ALS (G93A) mice, supporting the potential role of LSD1 in ALS pathogenesis. We further examined the therapeutic effect of spermidine (SD) on the modulation of LSD1, neuropathology, behavior, and survival rate of ALS transgenic mice.Male transgenic ALS mice of the G93A H1 high-expresser strain  are bred with females with a similar background (B6/SJLF1). Offspring were genotyped using a PCR assay on tail DNA. To ensure homogeneity of the cohorts tested, we have developed a standardized method to select mice. Mice were randomized from 24 litters all within 4 days of age from the same ‘f’ generation removed from the founding mice in our colony. Body weights were taken at 20 days and mice were equally distributed according to weight within each experimental cohort. Mice under 8 g at 20 days of age were excluded from the experiments. Only male mice were used in the treatment studies since there are gender differences in survival in the G93A transgenic ALS mouse model .SD and saline were separately administered  to groups of 6 wildtype and ALS (G93A) mice from 30 to 120 days of age 4 times per week. Injection begins 30 and 70 days of age, before the occurrence of symptoms in G93A mice. Control groups of G93A mice were injected with saline only.n = 10), from L3–L5 spinal cord segments were used for neuronal analysis. Spinal cord tissue sections were stained for Nissl substance and immunostained for glial fibrillary antigen protein (GFAP), LSD1, H3K4me2, SMI32, ChAT, etc., as previously described [Groups of 6 animals from saline and SD treatment paradigm were deeply anesthetized and transcardially perfused with 4% PFA at 120 days of age. Serial-cut lumbar spinal cord tissue-sections  was used to assess motor performance of mice. All mice were familiarized with the rotarod apparatus for 5 days prior to testing. Mice were subjected to the rotarod test with 3 trials per day. Mice were placed on the rod, and the apparatus was set to accelerating mode (4–40 rpm in 200 s). The latency to fall with upper limit of 200 s per trial was measured [The gait analysis was performed using a wheel test. All mice were familiarized with the wheel apparatus with exactly the same conditions as the real test right before test. Each mouse was placed inside of the wheel and the apparatus was to accelerating mode (4–15 rpm in 180 s). Both the bottom and front views of the wheel test were recorded by two cameras. For the footprint analysis, the hindlimbs and forelimbs of mice were dyed with different colors using non-toxic animal marking stick. The gait analysis was simulated by the EthoVision XT software (Noldus). Seven or eight continuous strides were analyzed for each mouse .Both body weight and survival were measured throughout the study. Body weights were recorded twice a week at the same time of day. G93A mice were assessed twice daily (mid-morning and late afternoon) for morbidity and mortality.The tissue sections were incubated with blocking solution containing 0.3% Triton X-100, 5% bovine serum albumin, and 3% goat serum for 1 h followed by incubation with SMI32 mouse monoclonal antibody , anti-LSD1 rabbit monoclonal antibody , anti-H3K4me2 rabbit monoclonal antibody , and ChAT antibody (BD Biosciences) overnight at 4 °C. After three washes with PBS, the cells were incubated for 1 h with FITC-conjugated goat anti-mouse IgG antibody  and Cy3-conjugated goat anti-rabbit IgG antibody . All antibodies were diluted in PBS. The slides were washed three times with PBS and mounted with fluorochrome mounting solution (Vector Laboratories). Moreover, images were taken by a spinning confocal microscopy , and the size of motor neuronal cell body was analyzed by AQI-X-COMBO-CWF program . Briefly, we took a series of 50 confocal layers representing fluorescence data from the ventral horn, then analyzed all layers by the software. Control experiments were performed in the absence of primary antibody.EX/590EM.LSD1 activity was determined from spinal cord lysates that had been administered with SD or vehicle . The spinal cord tissues were minced and homogenized in PBS containing protease inhibitors  with 23 full strokes by Dounce homogenizer on ice. The lysates were centrifuged at 4 °C at 3000 rpm for 10 min. The pellet (nuclear fraction) was resuspended in 350 μm NaCl  buffer with protease inhibitors to lyse nuclear membranes, forcing DNA into solution. The nuclear lysates were centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant was aliquoted and its protein concentration was measured by Bradford assay. The 10 µg of nuclear extract was reacted according to the protocol from EpiQuik Histone Demethylase LSD1 Activity Assay Kit . The LSD1 standards were included for the quantification of LSD1 activity. Finally, the endpoint result of LSD1 activity was measured and read on a 96 well fluorescence microplate reader at 5302. Cells were sub-cultured in 60 mm dishes at a density of 1 × 106 cells/well. After 80% confluence, cells were treated with 100 µM hydrogen peroxide (H2O2) for 12 h. For immunostaining experiments, cells were seeded in 6-well plates containing 13 mm round coverslips at a density of 5 × 105 cells/well. For drug treatment, NSC-34/WT-SOD1 cells and NSC-34/mSOD1(G93A) cells in six-well plates were treated with SD (5 µM) for 24 h.NSC-34 mouse motor neuron-like cell lines transfected with pCI-neo expression vector containing human WT-SOD1 and mSOD1(G93A) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were kept in a humidified incubator at 37 °C under 5% CORpl26 and 2810001G20Rik) was normalized to Gapdh levels in parallel reactions. The primer sequences used for qPCR are as follows: Rpl26, 5′-CGA AGC AAG AAC CGC AAA CGG C and 3′-ACC ACC TTG CCA ATC TGC TGG C; 2810001G20Rik, 5′-TGG GAA TGA ACC CTG GCG CTGA and 3′-TTG GGC ACA GCA TCC GTC TTG G; Gapdh, 5′-TTT CCT CGT CCC GTA GAC AAA A and 3′-CGT TGA ATT TGC CGT GAG TGG.qPCR was performed with ABI PRISM 7700 Sequence Detection System Instrument and software , using the manufacturer’s recommended conditions. Total RNA was isolated from transiently transfected cells , reverse transcribed , and subjected to quantitative PCR analysis using SYBER green master mix . The comparative threshold cycle (Ct) method was used to calculate the amplification factor, and the relative amount of targets . RNA was measured in a spectrophotometer at 260 nm absorbance. For the mRNA-Seq sample preparation, the Illumina standard kit was used according to the manufacturer’s protocol. Briefly, 3 µg of each total RNA sample was used for polyA mRNA selection using streptavidin-coated magnetic beads, followed by thermal mRNA fragmentation. The fragmented mRNA was subjected to cDNA synthesis using reverse transcriptase (SuperScript II) and random primers. The cDNA was further converted into double-stranded cDNA and, after an end repair process , was finally ligated to Illumina paired end (PE) adaptors. Size selection was performed using a % agarose gel, generating cDNA libraries ranging in size from 200 to 250 bp. Finally, the libraries were enriched using ten cycles of PCR and purified by the QIAquick PCR purification kit (Qiagen). The enriched libraries were diluted with Elution Buffer to a final concentration of 10 nM. Each library was run at a concentration of 8 pM on one Genome Analyzer (GAIIx) lane using 53 bp sequencing. Raw reads were aligned to the mouse genome  according to the STAR as an accurate alignment tool for high-throughput RNA-seq data . MoreoveN-ethylmaleimide, 2 μm Na3VO4, 20 μm sodium pyrophosphate, and 50 μm NaF. Lysates were centrifuged at 15,000 rpm at 4 °C for 30 min. The clear cytosol was separated from the insoluble pellet and immediately used for immunoblot. The supernatants were removed carefully and the protein concentration was quantified by Bradford Method . Lysates were mixed with 2× or 5× boiling Laemmli’s buffer . Then the samples were boiled for 10 min and then spun at 15,000 rpm for 10 s. In general, 30 µg of protein was electrophoresed on 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were blocked in 5% skim milk in TBST  for 30 min at room temperature. Blots were probed with primary antibodies overnight at 4 °C. This was followed by incubation with anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase  for 1 h. Signals were detected by using the ECL system .For immunoblot analysis, cells were harvested and washed with ice-cold PBS and then resuspended in an ice-cold cell extraction buffer containing 50 μm Tris-HCl, pH 7.4, 150 μm NaCl, 2 μm EDTA, 1% Triton X-100, 1 μm PMSF, 10 mg/ml leupeptin, 1 μm pepstatin, 1 μm The mouse H3K4me2 ChIP-seq data was downloaded from GSE123652. Sequence reads were trimmed and aligned to the Mus musculus genome version mm10 using Bowtie2 . Then, wp < 0.05 was considered significant.Survival data was analyzed by the Kaplan–Meier survival curves. Values were analyzed by one-way ANOVA and the Brown–Forsythe and Welch test followed by Kolmogorov–Smirnov multiple-comparison test, unless indicated otherwise. All statistics and graphs were performed using Prism 8 . In order to examine LSD1 expression in a cellular model of ALS, we used spinning disk confocal microscopy imaging and Western blot analysis to detect LSD1 immunoreactivity. Using motor neuronal cell line (NSC-34) as a model, we transfected wild type (WT)-SOD1 and mSOD1 (G93A) overexpression constructs and observed for LSD1 and H3K4me2 expression. Images and densitometry results demonstrate increase of LSD1 immunoreactivity  to motor neuronal cells (NSC-34) for 12 h  elevated the level of LSD1 while it reduced the level of H3K4me2 in the NSC34 cells -induced oxidative stress in NSC-34/mSOD1 cells  mouse to that of WT mouse. Using spinning disk confocal microscopy, we examined SMI32-positive motor neuron sections of the lumbar spinal cord. The Western blot results indicate increasing LSD1 and decreasing H3K4me2 protein level at both 90 and 120 days of age in ALS (G93A) mice compared to WT  and G93A ALS mice + SD GSE213091). After we identified SD-modulated targets from the spinal cord RNA-seq, we retrieved the public H3K4me2 ChIP-seq (GSE123652) and analyzed whether H3K4me2-landscaped genes are associated with our RNA-seq data. Lastly, H3K4me2-associated target genes were verified by qPCR on NSC-34 motor neuronal cells Fig. A. Consis13091. Af neurons . Moreove neurons , whereasmutation . TherefoDNA methylation, histone modification, and microRNAs are the major epigenetic mechanisms in the etiology of ALS. Histones are substrates for a number of covalent modifications that control chromatin state and gene expression. The core histones  are small basic proteins that form nucleosomes or the fundamental building blocks of chromatin structure . Their fRecently, researchers work on dosage-sensitive genes which gain and loss of the same gene involve in neurodevelopmental disorders . ChristoThe potential pharmacological value of LSD1 as a drug target stems from it being a flavin-dependent demethylase, rather than an iron-dependent enzyme of the Jumonji class . TherefoSD, a natural polyamine presented widely in mammalian living cells, has crucial roles in various cellular processes, and extends the lifespan of several model organisms by inducing autophagy . HoweverRpl26 and 2810001G20Rik as the candidate genes targeted by H3K4me2 and recovered by SD. Even though the cell line model of ALS did not exactly mimic gene changes in mouse model of ALS, SD treatment affected the expression of motor neuronal genes in both ALS models. Researchers have identified 60 S ribosomal subunits as a major feature of the tau interactome [C9orf72 mutation [Hezroni et al. demonstrated the decrease of nuclei count, neuron count and the total neurite length in 2810001G20Rik knock-down on embryonic cell derived neurons [2810001G20Rik and Rpl26 expression level in a cell line model of ALS.In summary, the alteration of LSD1 activity causes a reduction of histone H3K4me2 levels in the ALS mouse model. Our data indicates that polyamines are a negative regulator for LSD1 activity. Indeed, polyamine administration modulated chromatin remodeling and gene expression via the LSD1-H3K4me2-dependent pathway in ALS. Therefore, SD improved neuropathology and extended the survival of ALS mice Fig. . Togetheeractome . Moreovemutation . On the  neurons . We showALS is a neurodegenerative disease characterized by progressive paralysis due to motor neuron degeneration. It has been known that epigenetic modifications and its dysfunction contribute to motor neuron death. In this study, we focused on therapeutic approaches to target LSD1 by polyamines and elucidated the mechanistic role of LSD1-H3K4me2-dependent pathway in ALS pathogenesis. Our data indicates that the alteration of LSD1 activity leads to a reduction of histone H3K4me2 levels in the cell line and the mouse model of ALS. Based on our data, we expect that antioxidant may also ameliorate LSD1-H3K4me2 pathway in ALS mice. The effect of antioxidant on epigenetic modulation of ALS pathology and symptoms remains to be examined in the future study. SD (spermidine) administration modulated chromatin remodeling and gene expression via the LSD1-H3K4me2-dependent pathway in ALS. SD, as a negative regulator for LSD1 activity, ameliorated neuropathology and extended the survival of ALS mice. Together, modulating epigenetic targets by small compounds may be a useful therapeutic strategy for treating ALS.Additional file 1: Fig. S1. LSD1 is induced in a cellular model of ALS (N2a cell line). A, LSD1 immunoreactivity (red) was increased in the nucleus of mSOD1 (G85R) N2a cell line. The nucleus was counterstained with DAPI (blue). B, The densitometry analysis shows that LSD1 was significantly increased in G85R N2a cells. The number of cell counting: 35 cells/group. C, Western blot analysis shows that LSD1 protein level was highly induced in mSOD1 (G85R) N2a cell line compared to normal and WT-SOD1 overexpression cell lines. D, Densitometry analysis indicated a significant increase of LSD1 protein level in mSOD1 (G85R) N2a cells. Significantly different at *p < 0.05, ** p < 0.01, and ***p < 0.001. Fig. S2. Antioxidant modulates oxidative stress-induced LSD1-H3K4me2 pathway in a cell line model of ALS. A, A scheme illustrating procedure of cell experiment. B, Western blot analysis showed decrease of LSD1 and elevation of H3K4me2 by antioxidant (deferoxamine: DFO) in hydrogen peroxide (H2O2)-treated NSC-34/mSDO1 cells. C, Densitometry analysis of LSD1 and H3K4m2 levels from Western blot analysis (originated from B). D, Western blot analysis showed the LSD1 and H3K4me2 level maintained by DFO in H2O2-treated NSC-34/WT-SDO1 cells. E, Densitometry analysis of LSD1 and H3K4m2 levels from Western blot analysis (originated from D). F, Western blot analysis confirmed that DFO elevated the level of H3K4me2 in H2O2-treated NSC-34 motor neuronal cells.G, Densitometry analysis of LSD1 and H3K4m2 levels from Western blot analysis (originated from F). LSD1 and H3K4me2 were normalized to actin and histone H3 (H3), respectively. Significantly different at *p < 0.05 and **p < 0.01.Additional file 2: Table S1. Integrated analysis of RNA-sequencing and H3K4me2 ChIP-sequencing to identify spermidine-modulated gene signatures in ALS (G93A).


Rational: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease and is associated with high mortality due to a lack of effective treatment. Excessive deposition of the extracellular matrix by activated myofibroblasts in the alveolar space leads to scar formation that hinders gas exchange. Therefore, selectively removing activated myofibroblasts with the aim to repair and remodel fibrotic lungs is a promising approach. Stromal-derived growth factor (SDF-1) is known to stimulate cellular signals which attract stem cells to the site of injury for tissue repair and remodeling. Here, we investigate the effect of overexpression of SDF-1β on lung structure using the bleomycin-injured rat lung model. Methods: Intratracheal administration of bleomycin was performed in adult male rats (F344). Seven days later, in vivo electroporation-mediated gene transfer of either SDF-1β or the empty vector was performed. Animals were sacrificed seven days after gene transfer and histology, design-based stereology, flow cytometry, and collagen measurement were performed on the tissue collected. For in vitro experiments, lung fibroblasts obtained from IPF patients were used. Results: Seven days after SDF-1β gene transfer to bleomycin-injured rat lungs, reduced total collagen, reduced collagen fibrils, improved histology and induced apoptosis of myofibroblasts were observed. Furthermore, it was revealed that TNF-α mediates SDF-1β-induced apoptosis of myofibroblasts; moreover, SDF-1β overexpression increased alveolar epithelial cell numbers and proliferation in vivo and also induced their migration in vitro. Conclusions: Our study demonstrates a new antifibrotic mechanism of SDF-1β overexpression and suggests SDF-1β as a potential new approach for the treatment of lung fibrosis. Idiopathic pulmonary fibrosis (IPF) is an age-related progressive lung disease with no curative therapy available . IPF is Human lung tissue was obtained with the approval of the Human Ethics Committee of the University of Basel (Ref. Nr. EK: 05/06), Switzerland, and with the written informed consent of each patient.Animal experiments were performed after approval from the local ethical committee, University and Cantone of Bern, Switzerland (Ref. Nr. BE/136-17).IPF or control lung tissue pieces were placed into pre-wetted cell culture dishes containing DMEM/10%FCS . The cells grew by sprouting as described before  and wereSDF-1 (CXCL 12) transcript variant II (SDF-1β)  was obtained and was inserted into minicircle plasmid (pSDF-1) obtained from Plasmid Factory GmbH, Bielefeld Germany. Subsequently, pSDF-1β was dissolved in endotoxin-free water and used for in vitro and in vivo application as described below.TM 3000  according to the manufacturer’s instructions. For TNF-α CRISPER/Cas9 KO plasmid ((sc-400124), Santacruz biotechnology, Dallas, TX, USA) transfection the cells were transfected using Nucelofactor  following the parameters for fibroblast transfection as suggested. Supernatants were collected for cytokine measurements and cells were harvested for flow cytometry analysis or protein extraction. IPF lung fibroblast transfection in vitro lung fibroblasts were transfected with either pSDF-1β or empty vector (EV) control plasmids via LipofectamineHuman alveolar adenocarcinoma cells (A549)  from passages 5–15 were cultured in 6-well plates with fully supplemented RPMI1640 medium . When confluent, with the use of a 200μL pipette tip, cells were gently scratched in the middle of the well. A549 cells were then treated with 10, 25, 50 and 100 ng/mL SDF-1β  with or without the presence of 6 μg/mL AMD 3100 octahydrochloride , a highly selective CXCR4 receptor antagonist. Prior to SDF-1β treatment, the cells were treated with AMD 3100 for 20 min in order to inhibit the potential migratory effect of SDF-1. Light microscopy images were taken immediately after scratching (0 h) and after 24 h. The wound area was calculated with ImageJ software. Results are expressed as % of covered area. All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Laboratory Animal Research. All experiments were performed in accordance with the standards of the European Convention of Animal Care.n = 15) for stereological analysis n = 10 animals in each group were used and for other analysis n = 15 were used, 1 animal in the h-SDF-1 group died therefore n = 14 are reported.Schematic representation of the experimental setup is elaborated in n = 15 each). The first group received pSDF-1 plasmid 100 μg (concentration of 1 μg/mL) intratracheally and the second group received plasmid without insert (empty vector (EV)) followed by extracorporeal electroporation to both the lungs as described before [Seven days after administration of bleomycin, the animals were randomly divided into two groups ; animals were pre-anesthetized by inhaling 4% isoflurane, followed by the intraperitoneal administration of thiopental (50 mg/kg body weight). The heart–lung block was explanted, and tissue samples were collected for further analysis. Organs were harvested after a median thoraco-laparotomy, followed by flushing the pulmonary vasculature via the right ventricle with 0.9% sodium chloride solution. The right hilum was ligated, and the left lung was fixed by vascular perfusion fixation after two recruitment maneuvers at an airway opening pressure of 10 cm HResected lungs were weighted and frozen. The technique and all required reagents are as described before . ResultsLung volume was determined by fluid displacement, followed by a systematic uniform random sampling process . Lung piMasson–Goldner staining was used to highlight connective tissue. The staining was performed on 4 µm thin tissue slices embedded in paraffin. For deparafinization slides were incubated in Xylol and the tissue was rehydrated in a descending ethanol concentration. The slides were incubated in hematoxylin followed by two washing steps in water and 1% acetic acid and transferred to azophloxin solution and washed again with 1% acetic acid. After incubation in phosphotungstic acid orange the slides were washed again with 1% acetic acid, followed by staining in light green solution and washing in 1% acetic acid. Finally, the slides were dehydrated using an ascending sequence of ethanol followed by a final incubation in Xylol and mounted.g/15 min/4 °C). Protein content was measured with Bradford assay , and western blot was performed, using 7.5% gels . Gels were run using Mini-Gel Trans Blot cell  for 30 min at 60 mV and for 60 min at 120 mV and were transferred to Nitrocellulose membranes  with the use of the Trans-Blot Turbo Transfer System . The membranes were blotted using the PAK sampler kit . Visualization was done with a LI-COR Odyssey scanner. Western blot results were analyzed using Image Studio Lite Version 5.2 (LIQOR). Information regarding the antibodies used is given on A549 cells were grown to confluence and treated with different concentrations of recombinant human SDF-1   in the absence or presence AMD (6 µg/mL). Either 15 min or 24 h after treatment cells were washed with ice-cold 1xPBS and lysed with Pierce lysis buffer . The lysis procedure was performed on ice. Cell lysates were collected and centrifuged (300× g/5 min/4 °C) and placed in 12 × 75 polypropylene tubes  at a seeding density of 500,000 cells/tube. Cells were re-suspended in 200 μL flow cytometry buffer  and stained with surface or intracellular antibodies. A series of different antibody panels with different flourochroms was used as described in the Resected lungs were finely chopped into tiny pieces under sterile conditions with scissors and scalpel, and digested in collagenase solution as described in . Cell suHuman SDF-1 ELISA  was used to measure human SDF-1 levels in rat lung homogenates. The rat SDF1/CXCL12 ELISA kit  was used to measure SDF-1 levels in rat serum and lung homogenates; to measure TGF-β in rat lung homogenate, rat TGF-β ELISA  was used. To study the anti-fibrotic mechanism of SDF-1 in vitro, TNF-α was measured in the supernatants of human IPF fibroblasts using TNF-α ELISA . To measure TNF-α levels in rat lung homogenates and Broncho Alveolar Lavage (BAL), rat TNF-α ELISA  was used. All assays were performed following manufacturer protocol.t-test was used.Prism 7 software  was used for statistics and for designing of the graphs. A parametric non-paired Seven days after electroporation-mediated pSDF-1β gene transfer 34.04 ± 3.36% of total rat lung cells stained positive for hSDF-1β compared to 0.48 ± 0.11% in the Empty Vector group (EV) as measured by flow cytometry A. This cAreas of collagen deposition were reduced after pSDF-1β gene transfer as compared to the EV group, as observed by Masson–Goldner staining A. Fibros3 vs. 0.1267 ± 0.01054 cm3) or ECM (0.07948 ± 0.007051 cm3 vs. 0.07570 ± 0.003996 cm3). The volumes of AEC1 and AEC2 cells also showed no significant difference between the EV and the pSDF-1β groups (0.01331 ± 0.001659 cm3 vs. 0.01992 ± 0.003144 cm3 and 0.01176 ± 0.001860 cm3 vs. 0.01218 ± 0.001089 cm3). No changes were observed in the volume of the endothelium or the capillary lumen between the EV and pSDF-1 (0.02220 ± 0.002397 cm3 vs. 0.02657 ± 0.003397 cm3 and 0.05484 ± 0.01424 cm3 vs. 0.07521 ± 0.01472 cm3).The samples were studied at electron microscopic level using design-based stereology to investigate the relative and absolute volumes of the different components forming lung parenchyma or interalveolar septa, respectively. + collagen secreting cells were reduced in pSDF-1 (16.59 ± 3.81% vs. 21.93 ± 1.92%) compared to EV-treated animals (Flow cytometry data shows a reduced percentage of myofibroblasts in pSDF-1β (36.09 ± 8.36% vs. 41.96 ± 6.62%), compared to EV-treated animals A. Simila animals B. These  animals C. Howeve animals . Caspase 3-dependent apoptosis in myofibroblasts is induced in pSDF-1 treated group compared to the EV group (60.83 ± 11.92% vs. 50.6 ± 10.49%), A. Intere2 vs. 41.87 ± 8.47 cm2),  compared to EV group A. This i47 cm2), B. Comple47 cm2), C,D. + cells    B compare7 pg/mL) C. Furthe7 pg/mL) D.In vitro wound-healing experiments were performed on A549 cells. Recombinant SDF-1 was used at different concentrations. Improved wound-healing in a concentration-dependent manner was observed with the strongest effect at 100 ng/mL (100 ± 0% vs. 14.60 ± 2.59%) compared to control group A. IntereSDF-1β overexpression attenuated lung fibrosis, reduced total collagen content, induced apoptosis of collagen-producing myofibroblast and increased proliferation of alveolar epithelial cells in the bleomycin-injured rat lung model. We also observed that myofibroblast apoptosis was mediated via TNF-α, and in vitro alveolar epithelial cell migration was regulated via PAK 2 after SDF-1β treatment. We have recently reported the presence of stem cells of mesenchymal origin (MSC) in fibrotic lung ,30, and


K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression.Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics.  Klebsiella pneumoniae is a Gram-negative rod belonging to the family Enterobacteriaceae [eriaceae  and a maeriaceae ,3,4. Seperiaceae . The cyteriaceae . Excessieriaceae ,6.The exact pathogenesis of sepsis and septic shock is still unclear. Analyses of differentially expressed genes in sepsis have shown that the Interleukin-1 receptor associated kinase 3 (IRAK3) is among the genes whose expression is increased in Toll-like receptor (TLR) signalling pathways . TLR sigWhile elimination of bacteria causing sepsis remains fundamental for its treatment, therapies for sepsis can be problematic . SpecifiK. pneumoniae to cause sepsis infections in humans, in this study we isolated two lytic bacteriophages against this bacteria, one of which was a novel jumbo bacteriophage. Using these, we aimed to further assess the role of bacteriophages in modulating cytokine production (TNF-α and IL-6), by using IRAK3 knockdown, knockout and wildtype THP-1 monocytes as a model.Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. They are bacterial predators that have co-evolved with their prey and are equipped with the capacity to negate some of the bacterial resistance through mechanisms developed in an evolutionary arms race over millennia . Bacteriv/v) foetal bovine serum  in 5% CO2 at 37 °C. The cells were grown to 70–80% confluence before being passaged, approximately 80% of the volume of cell medium was removed and replaced with fresh RPMI media. The cells were then split for three passages before they were confirmed to be mycoplasma free using the PCR Mycoplasma Detection Kit .The cell lines utilised in this study were wild type THP-1 human monocytes, IRAK-3 knockdown THP-1 human monocytes, and IRAK3 knockout THP-1 human monocytes as described previously . THP-1 cK. pneumoniae strain KLEB009 (NCBI: WGS Project: JAERPY01) was cultured in Tryptone Soy Broth (TSB) and on 1% Tryptone Soy Agar (TSA)  culture plates before incubation at 37 °C aerobically for 24 h. Lytic bacteriophages for K. pneumoniae were screened from wastewater samples collected from Victoria, Australia as described previously [6 CFU mL-1 before adding 1 mL of 0.2 µm cellulose acetate filtered wastewater. This enrichment was incubated at 37 °C for 4 days before filtering using 0.2 µm Advantec cellulose acetate filters  and spotting 10 µL of the filtrate onto a freshly prepared lawn of KLEB009 on TSA. These were then incubated for 24 h at 37 °C and examined for clearance zones or plaques caused by bacteriophages. Wastewater samples from two different sites  were shown to produce plaques on KLEB009. Isolated bacteriophages vB_KpnP_KPN7 (KPN7) and vB_KpnM_KPN8 (KPN8), were then purified from their single plaques and concentrated as described previously [11 PFU mL−1) was treated with 10 µg/mL of DNase I and RNase A for 30 min at room temperature before incubating at 4 °C in 10% w/v polyethylene glycol (PEG) 8000 and 1 mol/L NaCl for 15 min. As described previously [v/v Triton® X-100  and washed 3 times in phosphate-buffered saline  by centrifugation at 12,000× g for 15 min. The precipitation, Triton® X-100 treatment and PBS (pH 7.4) wash steps were repeated 3 times before precipitated bacteriophages were filtered using 0.2µm cellulose acetate filters and resuspended in 5 mL RPMI 1640.To isolate bacteriophages, eviously . Brieflyeviously . This pr7 PFU/mL) were allowed to adsorb to the grid for one min before excess solution was removed using filter paper. The grids were then negatively stained three times with 2% (w/v) uranyl acetate for 20 s. Excess stain was removed by filter paper and grids were air-dried for 20 min before examination under a JEOL JEM-2100 transmission electron microscope. This was operated at an accelerating voltage of 200 kV and high-resolution digital images were recorded on a Gatan Orius SC200D 1 wide angle camera with Gatan Microscopy Suite and Digital Micrograph (Version 2.32.888.0) imaging software. Virions were measured using ImageJ software [Transmission electron microscopy (TEM) was performed using 400-mesh formvar and carbon copper grids  as described previously . Bacterisoftware  , 10 µg/mL RNAse A, and 10 µg/mL DNAse I  for 30 min at room temperature. Using 10% (w/v) PEG-8000 and 1 g/L sodium chloride , the bacteriophages were then precipitated at 4 °C and resuspended in 50 mL nuclease-free water . Viral proteins were then digested with 50 µg/mL of proteinase K, 20 mmol/L EDTA  and 0.5% (v/v) of sodium dodecyl sulfate  for 1 h at 55 °C before an equal volume of phenol-chloroform-isoamyl alcohol (29:28:1)  was added to the DNA/bacteriophage protein mixture. The DNA and protein were separated by centrifugation at 12,000× g for 10 min to collect DNA in the aqueous phase before incubating it with 200 µL of absolute isopropanol-2-propanol  at −20 °C overnight. Following centrifugation for 10 min at 12,000× g, the supernatant was taken and discarded, and the DNA pellet washed with 200 µL 70% ethanol  before 2 min centrifugation at 12,000× g. The 70% ethanol  was discarded then the DNA pellet resuspended in 30 µL nuclease-free water .Bacteriophage DNA was extracted as previously described . Ten mL ® technology. DNA libraries were prepared using the DNA Library Prep Kit (NEB) and a MiSeq® V3 600 cycle reagent kit  according to manufacturer’s instructions. Generated reads were trimmed using Trim Galore v0.6.4 with the default settings  and assembled using the Unicycler de novo assembly pipeline [https://www.geneious.com/ (accessed on 10 October 2022)) employing the bacterial translation Table 11 including codon start ATG, TTG, and GTG. To predict taxa, bacteriophage whole genomes were uploaded to the Viral Proteomic Tree (ViPTree) webserver to construct a viral proteomic tree with other related bacterial viral genomes in the reference database [https://www.r-project.org/ (accessed on 10 October 2022)).The extracted bacteriophage DNA was then sequenced using the Illumina MiSeqpipeline . The bacpipeline  and qualdatabase . To assedatabase . Based odatabase  and annodatabase  in the p4 cells in 300 µL media per well in Corning® Costar cell culture clear flat bottom 96 well plates . Cells were treated with 1 × PBS as the negative control, 1 µg/mL lipopolysaccharide  as the positive control, or 3.0 × 106 plaque forming units (PFU) per mL of crudely purified or highly purified (see above) suspensions of bacteriophages KPN7 or KPN8 for 24 h. 100 µL of cell supernatants were then collected and measured for concentrations of human cytokines (TNF-α and IL-6) using BD OptEIA™IL-6 and OptEIA™TNF-α ELISA kits  according to the manufacturer’s instructions.Wild type and IRAK3 knockout/knockdown THP-1 cells were split at approximately 2 × 10https://www.r-project.org/ (accessed on 10 October 2022)).Experimental data were collected as above to assess whether there were differences in cytokine production between THP-1 monocytes with varying expression levels of IRAK3, and treated with different preparations of bacteriophages, LPS or PBS. Using the Shapiro–Wilk normality test, data was found to be significantly skewed, hence, the difference in the medians of IL-6 and TNF-α production between the treatment groups was compared using the Kruskal–Wallis non-parametric test. All data were visualised as boxplots comprising the five-member statistic median, 25th percentile, 75th percentile and the upper (Q3 + 1.5 × IQR) and the lower limit (Q1 − 1.5 × IQR). Bacteriophage morphology characteristics were normally distributed and summarised as standard error of mean. All statistical analyses were performed in R version 2022.07.1+554 . A summary of their characteristics is highlighted in Autographiviridae, with GC% content of 52% and 57 open reading frames (ORFs) for the bacteriophage KPN7. Of the 57 annotated ORFs in the bacteriophage KPN7 genome, approximately two-thirds (66.7%) were denoted as hypothetical proteins  (Klebsiella phage vB_KpnP_ZK1 (accession number: OK625527) (query cover of 94% and identity of 97.3%) with which it formed a monophyletic clade in the phylogenetic analysis of the putative terminase genes  A. Using se genes B. Phylogse genes C.Klebsiella jumbo phage Miami (MT701590) [Myoviridae family of bacteriophages while KPN7 formed a clade with bacteriophages from the Autographiviridae family  . When ane family D.p < 0.001) (p < 0.001). IL-6 production stimulated by purified KPN7 and KPN8 was lower (less than 2 pg/mL) across all cell lines used. In the wild-type THP-1 monocytes, IL-6 was at a mean (Q1–Q3) of 0.3 (0.2–0.5) pg/mL and 0.2 (0.1–0.7) pg/mL for KPN7 and KPN8, respectively. In the IRAK3 knockdown THP-1 monocytes, the IL-6 mean (Q1–Q3) concentrations for KPN7 and KPN8 were 0.04 (0.007–0.09) pg/mL and 0.01 (0.007–0.03) pg/mL, while in the IRAK3 knockout cells, the mean (Q1–Q3) was 1.1 (0.01–2.2) and 0.05 (0.03–0.08), for KPN7 and KPN8, respectively. Similarly induced TNF-α were also lower. Mean (Q1–Q3) of the TNF-α levels for the purified KPN7 and KPN8 in the wild-type THP-1 monocyte cell lines were 0.02 (0.01–0.06) pg/mL and 1.44 (1.1–1.67) pg/mL, respectively. In the IRAK3 knockdown THP-1 monocytes, the mean (Q1–Q3) TNF-α levels induced by KPN7 were 0.93 (0.26–1.61) pg/mL while those produced by the KPN8 were 5.93 (5.87–5.98) pg/mL. When monocytes had no functional IRAK3, the purified bacteriophages stimulated mean (Q1–Q3) TNF-α levels that were 11.37 (5.8–16.95) pg/mL and 4.56 (2.4–5.78) pg/mL for KPN7 and KPN8, respectively. The data on the mean and quantiles across all cell lines are summarised in KPN7 and KPN8 were tested for their capacity to stimulate the production of IL-6 and TNF-α in monocyte cell lines, including those with an IRAK3 knockdown  and a complete IRAK3 knockout (lacking IRAK3 expression) phenotype. In this analysis, the crude bacteriophage preparations involving only the 0.2 µm filtration process , and the highly purified bacteriophage suspensions  were assessed. Both KPN7 and KPN8 revealed relatively similar results with the highly purified preparations, showing similar levels of IL-6 stimulation as the PBS control. The crude preparations induced a significantly higher IL-6 level in comparison, in all monocyte cells regardless of the IRAK3 functionality (< 0.001) A. The IL< 0.001) A. This o< 0.001) B. CombinK. pneumoniae bacteria strain KLEB009 (NCBI: WGS Project: JAERPY01). The GC% content of both bacteriophages KPN7 and KPN8 were lower  than the 57.5% of the host KLEB009. Bacteriophage GC% content has been known to be close to that of their host, and in cases where they have lower GC% content than their host, it has been suggested that bacteriophages may carry tRNAs to complement potential biochemical shortfalls [Klebsiella phage vB_KpnP_ZK1 (accession number: OK625527), and with the bacteriophage MAG TPA_asm: Caudovirales sp. isolate ct60g1 (Accession number: BK056972), when using the capsid proteins. Phylogenetic analysis using the capsid protein showed that KPN8 differed markedly from most of the bacteriophages with which it clustered, and formed a monophyletic clade with the novel Klebsiella phage Miami, another Jumbo bacteriophage.This study isolated and characterised two bacteriophages targeting the ortfalls ,43. In-sK. pneumoniae bacteriophages into cultures of wild type THP-1 monocytes had little effect; levels of TNF-α and IL-6 cytokines were similar to the low levels produced by negative control PBS. Other studies using monocytes and purified bacteriophages showed similarly low levels of cytokines in their models [We demonstrated that introducing purified r models . The levr models ,45. In tThe tests using crudely prepared bacteriophage suspensions highlight the importance of removing endotoxin and other bacterial cell debris to prevent immune stimulation. That the purified bacteriophages did not elicit an extreme immune response may be due to the fact that these viruses do not target cells of higher organisms, and that humans have had extensive exposure to them  throughout their evolution . Others Escherichia coli) failed to induce expression of TLR2 and TLR4 in human monocytes [The low levels of TNF-α and IL-6 in both the wild type and the IRAK3 knockdown THP-1 monocytes suggest that the pure bacteriophages are not recognised by PRRs such as TLRs on human monocytes. Studies exploring this relationship are very limited, however, one study suggests that T4 bacteriophage (lytic for onocytes . Therefoonocytes ,50,51, wonocytes . ResearcThe IRAK3 knockdown and knockout THP-1 monocytes were included in this study as a model to evaluate if individuals with dysregulated immune response have increases in inflammatory cytokines induced by bacteriophages. To our knowledge, this is the first study to assess the effects of these viruses on monocytes where the negative attenuation of IRAK3 is diminished or abolished. What was interesting in this investigation was that there was little difference when the purified bacteriophage was added to the IRAK3 knockdown THP-1 monocytes or the wild type monocytes. Despite the reduced levels of IRAK3 in the knockdown monocyte cell line and an absence of detectable IRAK3 protein in the knockout monocyte cell line , the purTherefore, our work has verified that some form of endotoxin removal needs to occur before application of bacteriophages clinically or in veterinary applications. Bacteriophage therapy is being applied clinically in humans in isolated cases, especially on compassionate grounds, where antibiotics and other treatments have failed ,54. In t


Artemisia herba-alba (L), Matricaria pubescens (L), Juniperus phoenicea (L), and Rosmarinus officinalis (L), and their extract pHs were respectively 5.25, 5.05, 4.63, and 3.69. The four greenly synthesized samples of magnetite were characterized by XRD, SEM, ATR-FTIR, and UV-Vis techniques. The novelty of this paper lies in highlighting the influence of the mediating plant extract's acidity on the greenly synthesized magnetite surface reactivity towards the preferential attachment of chromophore and auxochrome functional groups in azo dye adsorption, where obtained results show that the mediating plant extract's acidity has a clear effect on the preferential attachment of chromophore and auxochrome groups on magnetite surfaces as well as on azo dyes' adsorption yields and capacities. Indeed, the decrease in the plant extract's acidity leads to an increase in the attachment of chromophore groups and a decrease in the attachment of auxochrome groups. So, it leads to an increase in Lewis acid site density and a decrease in Brønsted acid site density of magnetite surfaces. Also, the decrease of the plant extract's acidity leads to an increase in the studied dye adsorption yields, and this is because the majority of functional groups of MO and EB dyes are chromophores that attach to Lewis acid sites. The difference found in adsorption yields of EB and MO on all four magnetite samples is due to the fact that the ratio of chromophore/auxochrome groups in EB is remarkably greater than that in MO. The linear and non-linear pseudo-first-order and pseudo-second-order kinetics of the adsorption as well as the intra-particle diffusion mechanism have been analyzed. Obtained results indicate that in all adsorption processes the adsorption kinetics followed a linear pseudo-first-order kinetic model, and film diffusion is the step that controlled adsorption mechanisms. The thermodynamic studies of EB and MO adsorption processes on the four magnetite surfaces have been analyzed in the temperature range of 303.15–318.15 K. Obtained results reveal the endothermic nature of the adsorption in all cases.In this paper, the adsorption of Evans blue (EB) and methyl orange (MO) azo dyes on four greenly synthesized magnetite nanoparticles has been studied to investigate the effect of the mediating plant extract's acidity on magnetite surface reactivity in azo dye adsorption. Magnetite surface reactivity has been studied through the analysis of preferential attachment of dye chromophore and auxochrome groups on magnetite nanoparticles, and adsorption yields. According to the contents of chromophore and auxochrome groups in dye structures, the mediating plant extract's acidity effect on acid site types and densities was also deduced. Used plants for the green synthesis were:  The decrease in plant extract's acidity leads to the increase in Lewis acid site densities of greenly synthesized magnetite surfaces. The increase in Lewis acid site densities leads to the increase in azo dye adsorption yields. Thus, one of the major environmental problems related to the numerous industrial applications of dyes is their removal from effluents.3Dyes are present in the effluent water of several industries, including textile, leather, paper, rubber, plastics, printing, cosmetics, pharmaceutical, and food industries. Dyes contribute to water toxicity and represent an increasing danger for the environment, humans, and animals.4 The most important chromophores, as defined in this way, are: N PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N, CO, –CHN, NO2, NO, NOH, CN, C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N, CC, and CC groups and ionizing auxochromes mainly include: SO3H, OH, COOH, NH2, NH3, NHCH3, and N(CH3)2 groups.4,5Dyes are composed of chromophores that are commonly electron withdrawing and auxochromes that are usually electron-releasing groups.6–9 Nanomaterials are widely used in the purification of aqueous media due to their advantages, such as high surface area and increased number of active sites.10–13 They therefore allow a rapid thermodynamic equilibrium between adsorbent and adsorbate during the adsorption process and selective removal of pollutants.7–9 Several factors influence the adsorption process, mainly the solution chemistry,7,14 the characteristics of the dye (adsorbate)7,15 and the adsorbent surface properties.16–22 Saha et al.7 studied the preferential adsorption of seven different dyes on magnetite NPs. They reported that the magnetite surface preferred adsorbing dyes containing higher OH content. Xiao et al.15 studied the preferential adsorption of different cationic and anionic dyes on iron nanoparticles. They reported that iron NPs preferred removing cationic dyes more than anionic dyes.Adsorption is one of the most effective processes for the removal and recovery of colored materials and dyes from effluents.et al.17 found that the use of amine-functionalized cobalt–iron NP surface enhanced the removal of anionic azo dyes. Mahmoodi et al.22 synthesized a titania/silica nano-hybrid (TSNH) and an amine-functionalized titania/silica nano-hybrid (AFTSNH) to use them in Reactive Red 198 and Acid Red 14 removal from wastewater. They found that the AFTSNH adsorbent showed high dye adsorption capacities compared to the TSNH adsorbent. The authors of ref. et al.24 reported that magnetite-modified activated carbon preferred adsorbing cationic dyes than anionic dyes. Moreover, a comparative study on adsorption of methylene blue on sericin-modified and unmodified magnetite NPs25 reported that sericin-modified magnetite NPs were approximately 40% more effective than the unmodified magnetite NPs. Furthermore, in the study of the preferential adsorption of magnetite NP loaded fig leaves (MNLFL) and magnetite NP loaded azolla (MNLA) to remove crystal violet and methylene blue,26 the authors found that MNLFL preferred adsorbing crystal violet more than MNLA.Other authors studied the effect of changing the adsorbent surface properties by binding ligands on the adsorbent surface. Khurshid 20 found that the adsorption on natural magnetite was more efficient than that on the synthesized one, and this is because of its higher surface site density. Moreover, Gogoi et al.27 studied the degradation of catechol using an Fe3O4–CeO2 nanocomposite as a Fenton-like heterogeneous catalyst. They reported that the increase of Brønsted acid site density of this nanocomposite increased the degradation of catechol.Surface acid site types and densities were also found to impact the adsorption process. Indeed, a study of the adsorption of organic contaminants on both natural and synthesized magnetiteet al.16 They reported that the plant extract had an effect on the adsorption yields of methyl green dye on the three iron oxide NPs, where yields varied from 81.2% to 75.6 to 67.1%. Duyen et al.28 synthesized metal oxide NPs using the extracts of flowers, bark, and leaf of Tecoma stans in order to use them in the removal of Congo red (CR) and crystal violet (CV) dyes. They reported that the adsorbent derived from flower extract gave better adsorption efficiency than those derived from other extracts. Islam et al.29 synthesized magnetite NPs using six plant extracts in order to use them in the removal of methyl orange (MO) and crystal violet (CV) dyes. They reported that plant extract had an effect on magnetite NP surface reactivity in the adsorption, where magnetite NPs synthesized using tea extract showed the highest performance . Ahmouda et al.30 used different greenly synthesized magnetite NPs in the removal of methyl green (MG) dye via the adsorption process. They reported that the mediating plant extract's acidity had an effect on the preferential adsorption of MG on the magnetite NP surface. Indeed, magnetite NPs synthesized using plant extract having the lowest acidity exhibited the highest acid site density (OH groups) and hence the highest performance in the removal of MG.The impact of changing the mediating plants on the behavior of greenly synthesized metal oxide NPs in dye adsorption was studied in several works. The effect of three different tea extracts on the capacity of greenly synthesized iron oxide surfaces in the removal of methyl green dye from aqueous solution has been studied by Huang As it is known that the adsorption of dyes from wastewater is directly affected by the reactivity of the adsorbent surface towards the attachment of dyes' functional groups, looking for parameters that could control the greenly synthesized magnetite NP surface reactivity is always of importance. In this way, this paper looks for mediating plant extract parameters that could, in the case of green synthesis, impact the magnetite NP surface reactivity in azo dye adsorption.31 These dyes contain one or more azo bonds (NN). The complex aromatic structures of azo dyes make them more stable and more difficult to remove from the effluents discharged into water bodies.31 The used dyes in this study are: methyl orange (MO) and Evans blue (EB) azo dyes, and their chemical structures are illustrated in N, CC, and CN groups linked to the benzene ring, and of auxochrome groups such as sulphonic acid (SO−3), phenol, and aniline groups. Meanwhile, the MO molecule is composed of chromophore groups such as benzene, phenyl, phenyldiazonium, NN, CC, and CN, and of auxochrome groups such as sulphonic acid (SO−3) and dimethylamine (N(CH3)2).Azo dyes are the largest and most versatile class of organic dyes.−3,32 dimethylamine,33 phenol,34 and aniline35 auxochrome groups are electron donating, and their attachment on the adsorbent surface is based on their ionic interaction with the developed positively charged Brønsted acid sites of the surface. Meanwhile, the chromophore groups phenyldiazonium, phenyl,36 benzene, NN, CC, CN,32 and toluene37 are electron withdrawing, and their attachment on the adsorbent surface is based on their interactions with the Lewis acid sites of the adsorbent surface.The attachment of these functional groups on the adsorbent surface is based on functional group properties (chromophore or auxochrome) towards the surface acid site type (Lewis or Brønsted). In the case of EB and MO dyes, SOArtemisia herba-alba (L), Matricaria pubescens (L), Juniperus phoenicea (L), and Rosmarinus officinalis (L). Their extract pHs are respectively 5.25, 5.05, 4.63, and 3.69. The synthesized Fe3O4 samples are respectively denoted in this paper by ARM–Fe3O4, ROS–Fe3O4, MAT–Fe3O4 and JUN–Fe3O4. They were characterized by XRD, SEM, FTIR-ATR, and UV-Vis techniques.In this paper, the effect of the mediating plant extract's acidity on the greenly synthesized magnetite surface reactivity in the adsorption of methyl orange (MO) and Evans blue (EB) azo dyes has been investigated through the analysis of MO and EB adsorption on four greenly synthesized magnetite samples, with the aim of studying the effect of the mediating plant extract's acidity on the preferential attachment of the studied dye chromophores and auxochromes  on greenly synthesized magnetite NPs, and adsorption yields. For this purpose, after the accomplishment of adsorption experiments, the free functional groups that are not attached on magnetite surfaces have been deeply analyzed in all dye residual solutions using ATR-FTIR spectroscopy, so as to perceive their preferential attachment on the four magnetite surfaces. Based on the analysis of preferential attachments of chromophore and auxochrome groups, it was possible to compare between Brønsted and Lewis acid site densities on each magnetite surface and their influence on adsorption yields and capacities. To the best of our knowledge, there is no study in the literature that has dealt with the influence of the mediating plant extract's acidity on magnetite surface reactivity towards the preferential attachment of dye functional groups, and thus on surface acid site type and density. The used plants are 2This section focuses on listing needed materials and used apparatuses. It also provides methods utilized to perform adsorption experiments and characterization of iron oxide NPs.2.12.1.13O4, MAT–Fe3O4, ROS–Fe3O4 and ARM–Fe3O4 nanoparticle powders were greenly synthesised using iron salt (FeCl3·6H2O)  as precursor and Artemisia herba-alba (L) (Asteraceae family), Matricaria pubescens (L) (Asteraceae family), Juniperus phoenicea (L) (Cupressaceae family), and Rosmarinus officinalis (L) (Lamiaceae family) plants as reducing agents. Magnetite samples were obtained after 4 months of storage of the synthesized iron oxides under ambient conditions. Characterization of the freshly synthesized iron oxides samples is presented in Ahmouda et al.38Evans blue, methyl orange dyes, NaCl salt, HCl acid, and NaOH base are purchased from Sigma-Aldrich. JUN–Fe2.1.2λ = 1.54056 Å). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR): Shimadzu IR-Infinity. Ultraviolet-visible spectroscopy (UV-Vis): Shimadzu UV-Vis spectrophotometer apparatus Model 1800 operating in the range of 200–900 nm. SEM-TESCAN VEGA3 Model XMU, LMH  for speeding up the voltage of 15–20 kV in order to capture the scanning electron micrograph (SEM) of greenly synthesised iron oxide NPs.XPERT-PRO X-ray diffractometer (XRD) (Rigaku Miniflex 600) with 30 keV and 30 mA as conditions of X-ray generation and Kα radiation of copper . A dilute solution of HCl was used to adjust the dye/Fe3O4 solution pH to 4. This protocol is used to prepare, in total, 88 experiment sets (11 with each dye/magnetite sample). In addition, control experiment sets (without NPs) were also prepared.First, the prepared standard aqueous solutions of EB and MO dyes were diluted several times as required. Then, 0.0015 g of JUN–FeAll experiment sets are sonicated in an ultrasonic bath for 15 minutes and they were then stirred continually for 60 minutes until a steady state was reached. All adsorption experiments were carried out under ambient conditions in batch mode, and they were performed in triplicate for data consistency.3O4 solutions at a regular time interval to obtain, after centrifugation, adequate aliquots for the purpose of quantifying residual dye concentrations and the adsorbed amounts. The concentrations of residual dye aqueous solutions were quantified using a UV-Vis spectrophotometer at absorbance maxima of EB (λmax = 602 nm) and MO (λmax = 463 nm). Furthermore, the adsorbed amounts of EB and MO molecules are calculated from the calibration curve for all adsorption experiments . On another side, after the adsorption was accomplished and steady state reached, the aliquots were centrifuged to separate liquid solutions and solid phases. The liquid solutions, which represent MO/magnetite and EB/magnetite residual solutions, were then analyzed using ATR-FTIR spectroscopy.Kinetic experiments were performed by withdrawing samples of dye/Feqe in mg g−1) and the amount of MO and EB ions adsorbed per unit mass (qt in mg g−1) of JUN–Fe3O4, MAT–Fe3O4, ROS–Fe3O4 and ARM–Fe3O4 at equilibrium contact time, the following equations were used:To calculate the adsorption capacity , liquid phase dye concentration at the steady state (mg mL−1), liquid phase dye concentration at time t (mg mL−1), volume of dye solution used (mL), and the amount of adsorbent (g).Adsorption yield was calculated using the following equation:2.2.239 and linear , adsorbed dye quantity at instant t (mg g−1) and adsorbed dye quantity at thermodynamic equilibrium (mg g−1).The linear  and non-d linear  and non-d linear  pseudo-s44If the active surface of the adsorbent is regarded as invariable, the reaction could be treated as pseudo-first-order kinetic. However, once the active sites have been saturated, the transfer at the adsorbate/adsorbent particle interface may be limited by mass transfer.40 It is based on the adsorption capacity expressed as follows:K2, qt and qe are the pseudo second order kinetic constant (mg g−1 min−1), adsorbed dye quantity at instant t (mg g−1) and adsorbed dye quantity at thermodynamic equilibrium (mg g−1), respectively.The pseudo second-order (PSO) model is proposed by Ho and McKay.2.2.3et al.,45 is used to describe the diffusive adsorption process. In this model, the rate-limiting step is usually described by either an intra-particle diffusion or a film diffusion mechanism.F(t) is the fractional attainment at time t, i.e., F(t) = qt/qe, Dp (m2 s−1) is the effective diffusion coefficient, r0 is the radius of Fe3O4 particles assumed to be spherical, and Z is an integer. For 0 < F(t) < 1, a simplified equation can be obtained for the adsorption on spherical particles:In order to gain insights into the adsorption mechanisms involved, a homogeneous particle diffusion model (HPDM) as shown in kp is the diffusion rate constant (1/s) and kp = Dpπ2/r20. Dp (m2 s−1)) from the experimental data. In the first step, a graph of −ln(1 − F2(t)) vs. t is developed. The values of kp of EB and MO for JUN–Fe3O4, MAT–Fe3O4, ROS–Fe3O4 and ARM–Fe3O4 adsorption processes are obtained from the slopes of the fitted lines (plots of −ln(1 − F2) vs. time), and the values of effective diffusion coefficient, Dp (m2 s−1), can then be obtained from Dp = kpr20/π2.A further formula manipulation gives the following:46Df is the film diffusion coefficient (m2 s−1) in the liquid phase, and Ce (mol L−1) and Cr (mol L−1) are respectively the equilibrium concentrations of dye in solution and in solid phases. δ is the thickness of the liquid film which was assumed to be 10−5 m according to Yu and Luo.47 A further formula deformation of kf is the diffusion rate constant (1/s).Additionally, kf = 3DfCe/r0δCr for the adsorption of MO and EB on ARM–Fe3O4, ROS–Fe3O4, MAT–Fe3O4, and JUN–Fe3O4 are obtained from the slopes of the fitted lines (plots of −ln(1 − F) vs. time), and the values of effective diffusion coefficient, Df (m2 s−1), can then be obtained from Df = kfr0Cr/3Ce.The values of F) and −ln(1 − F2) versus time plots is employed to distinguish between the film diffusion and particle diffusion-controlled adsorption mechanism. If the plot of −ln(1 − F) versus time is a straight line passing through the origin, then the adsorption rate is governed by the particle diffusion mechanism, otherwise if −ln(1 − F2) versus time is a straight line passing through the origin then the adsorption is governed by film diffusion.The linearity test of Boyd plots −ln were also prepared. After centrifugation, adequate aliquots were taken for the purpose of quantifying desorbed dye concentrations and the desorbed amounts from magnetite surfaces. The concentrations of desorbed dye from magnetite surfaces were quantified using a UV-Vis spectrophotometer at absorbance maxima λmax = 602 nm for EB and λmax = 463 nm for MO. Furthermore, the desorbed amounts of EB and MO molecules are calculated from the calibration curve for all adsorption experiments .In order to regenerate used magnetite samples after the end of dye adsorption, a dilute solution of NaOH was used to adjust the dye/FeR%) of MO and EB ions at contact time t = 60 min from JUN–Fe3O4, MAT–Fe3O4, ROS–Fe3O4 and ARM–Fe3O4 surfaces, the following equation was used:C0 and Ct are respectively: initial dye concentration adsorbed on the solid phase (magnetite) (mg mL−1) and liquid phase desorbed dye concentration at contact time t = 60 min (mg mL−1).To calculate the desorption yields .In order to study the thermodynamics of EB and MO adsorption processes, all sets of experiments are sonicated in an ultrasonic bath for 15 minutes. Then, they are stirred continually for 20 minutes at temperatures ranging from 303.15 to 318.15 K. The concentrations of residual EB and MO dyes in liquid phases are quantified using a UV-Vis spectrophotometer at absorbance maxima qTe (mg g−1), was calculated using the following equations:The adsorption capacity, C0, CTe, V and m are respectively: initial dye concentration without any treatment (mg mL−1), residual dye concentration in the liquid phase (mg mL−1) at a given temperature T, the volume of dye solution (mL), and the amount of magnetite NPs (g).Adsorption yield was calculated using the following equation:H0) of EB and MO adsorption processes on the magnetite NP surface was determined using the Arrhenius equation as follows:R  is the universal gas constant, T is the absolute solution temperature (K), and kd is the distribution coefficient which was calculated by:CTae (mg mL−1) and CTe (mg mL−1) are respectively the concentration of adsorbed dye on the solid (magnetite) and dye residual concentration in the liquid phase at a given temperature T.The activation enthalpy  and entropy ΔS0  were respectively calculated from the slope and intercept of plots between ln kd and 1/T. ΔG0  was then calculated using the following equation:The values of activation enthalpy ΔEa , of EB and MO adsorption processes on magnetite surfaces was determined using Arrhenius's equation:k2 is the distribution coefficient which can be calculated by:qTe (mg g−1) and CTe (mg mL−1) are respectively the adsorption capacity of dye on the solid and dye residual concentration in the liquid phase at temperature T.The free energy change indicates the degree of spontaneity of the adsorption process. The higher negative value reflects more energetically favorable adsorption. The activation energy, Δ33.1θ = 16.20°, 20.30°, 22.39°, 25.60°, 29.72°, 32.30°, 41.05°, 41.39°, 42.48°, and 52.69°. All Bragg peaks are in agreement with orthorhombic Fe3O4 powder and correspond to Miller indices 021, 212, 030, 400, 314, 001, 250, 251, 522, and 644, respectively (JCPDS file 01-076-0958).X-ray patterns of all synthesised samples are presented in θ = 16.20°, 16.70°, 20.39°, 22.42°, 29.75°, 30.80°, 32.30°, 41.10°, 42.53°, 49.82°, and 52.72°. All Bragg peaks are in agreement with orthorhombic Fe3O4 powder and correspond to Miller indices 021, 210, 212, 030, 400, 041, 106, 251, 522, 534, and 644, respectively (JCPDS file 01-076-0958).The X-ray diffraction pattern in θ = 16.20°, 22.56°, 26.04°, 32.28°, 37.11°, 41.59°, 49.98°, and 52.69°. All Bragg peaks are in agreement with orthorhombic Fe3O4 powder and correspond to Miller indices 021, 030, 400, 106, 404, 251, 534, and 644, respectively (JCPDS file 01-076-0958).The X-ray diffraction pattern in θ = 16.35°, 20.58°, 22.60°, 25.77°, 29.94°, 32.47°, 41.59°, 42.69°, 49.98°, and 52.69°. All Bragg peaks are in agreement with orthorhombic Fe3O4 powder and correspond to Miller indices 021, 212, 030, 400, 001, 106, 251, 522, 534, and 644, respectively (JCPDS file 01-076-0958).The X-ray diffraction pattern in 48D, β, λ, and θ are the crystallite size, the full width at half-maximum (FWHM) of the most intense diffraction peak, the X-ray wavelength (1.54056 Å), and Bragg angle, respectively.The average diameters of different magnetite samples, presented in 3.23O4 nanoparticle powders recorded between 4000 and 500 cm−1 are presented in −1 correspond to the O–H stretching vibration, while the peaks at 2930.18–2932.06 cm−1 correspond to C–H vibrations. The peaks at 1590.07–1594.63 cm−1 correspond to CC stretching in aromatic rings and anti-symmetric stretching of the carboxylate group (COO−), whereas peaks at 1033.45–1044.36 cm−1 are assigned to the symmetric stretching vibration of the C–O–C functional group of the phenolic groups.49 The peak at around 592 cm−1 corresponds to the Fe–O stretching band of Fe3O4 NPs.50FTIR spectra of the synthesized Fe3O4 surface, next on ROS–Fe3O4, then on MAT–Fe3O4, and finally on JUN–Fe3O4. This reveals that the density of functional OH groups is higher on the ARM–Fe3O4 surface, next on ROS–Fe3O4, then on MAT–Fe3O4, and finally on JUN–Fe3O4.3.33O4 are measured in the wavelength range of 200–900 nm, to deduce their band gap energies. The band gap Eg and the optical absorption coefficient (α) of a direct band gap semiconductor are related through the known following equation:51α is the linear absorption coefficient of the material, hν is the photon energy, A is a proportionality constant, and the exponent n depends on the nature of electronic transition; it is equal to 1/2 for a direct allowed transition and 2 for an indirect allowed transition.The optical absorbance spectra of all the synthesized FeEg of the direct transition of all samples were obtained from plotting (αhν)2 as a function of αhν by the extrapolation of the linear portion of the curve 1/2 as a function of αhν by the extrapolation of the linear portion of the curve  and pseudo-second-order (PSO) kinetics analysis of MO and EB adsorption processes on the four magnetite surfaces  and 9 in3.5.2.2F) and −ln(1 − F2)) versus time are presented in R2 values. The results of linear regression analysis for Df are found to be in the order of 10−11 m2 s−1, while intra-particle diffusion coefficients Dp are found to be in the order of 10−20 m2 s−1. Michelson et al.55 reported that the adsorption mechanism is controlled by film diffusion and that film diffusion is in control at Df ranging from 10−10 to 10−12 m2 s−1, while intra-particle diffusion is the rate-limiting step at Dp in the range of 10−15 to 10−18 m2 s−1. Indeed, the obtained results indicate that film diffusion is the step that controls the adsorption mechanism of MO and EB on magnetite surfaces, which is in agreement with the pseudo-first-order kinetic model.The linearity tests of Boyd plots  and MO/JUN–Fe3O4  systems are the highest ones, and those of EB/ARM–Fe3O4  and MO/ARM–Fe3O4  systems are the lowest ones. This indicates that the bonds between EB and MO molecules and active site hydroxyl groups on the JUN–Fe3O4 surface are the strongest ones, followed by those on MAT–Fe3O4, then on ROS–Fe3O4, and finally on ARM–Fe3O4 surfaces.The calculated activation enthalpy Δrsus 1/T . Likewisrsus 1/T . Activat3O4 and MO/plant–Fe3O4 systems are positive, which reveals the affinity of Fe3O4 surfaces for EB and MO molecules. The increasing randomness at the EB/plant–magnetite and MO/plant–magnetite solution interfaces indicates that significant changes in the number of surface active hydroxyl groups occurred in the internal structure of Fe3O4 surfaces. However, activation entropies of EB/JUN–Fe3O4  and MO/JUN–Fe3O4  systems are the highest ones, and those of EB/ARM–Fe3O4  and MO/ARM–Fe3O4  systems are the lowest ones. This indicates that the changes occurring in the structure of the JUN–Fe3O4 surface are the greatest ones, followed by those of MAT–Fe3O4, then ROS–Fe3O4, and finally ARM–Fe3O4 surfaces.56,57The activation entropies in all EB/plant–Fe3O4 , EB/ROS–Fe3O4 , EB/MAT–Fe3O4 , and EB/JUN–Fe3O4  systems are negative. However, activation energies of the EB/ARM–Fe3O4 system are more negative than those of EB/ROS–Fe3O4, EB/MAT–Fe3O4 and EB/JUN–Fe3O4 systems, which indicates the feasibility of the EB adsorption process and its spontaneous nature with more EB adsorption on ARM–Fe3O4, then on ROS–Fe3O4, next on MAT–Fe3O4, and finally on JUN–Fe3O4 surfaces.The activation free energies of EB/ARM–Fe3O4 , MO/ROS–Fe3O4 , and MO/MAT–Fe3O4  systems are negative. However, activation energies of the MO/ARM–Fe3O4 system are more negative than those of MO/ROS–Fe3O4 and MO/MAT–Fe3O4 systems, which indicates the feasibility of the MO adsorption process and its spontaneous nature with more MO adsorption on ARM–Fe3O4 than on ROS–Fe3O4 surfaces. In the MO/JUN–Fe3O4 system, the values of activation free energy are negative only at 313.15 K and 318.15 K , while positive values are found at 303.15 K and 308.15 K  revealing that activated MO/Fe3O4 complexes are in an excited form in the transition state.56 This leads to the spontaneity of MO adsorption at 313.15 K and 318.15 K.The activation free energies of MO/ARM–FeEa) for EB adsorption on ARM–Fe3O4, ROS–Fe3O4, MAT–Fe3O4, and JUN–Fe3O4 surfaces are respectively: 2.79, 3.21, 5.59, and 6.29 kcal mol−1. Ea is calculated from the slopes of the Arrhenius linear plots ln K2versus 1/T (Ea) for MO adsorption on ARM–Fe3O4, ROS–Fe3O4, MAT–Fe3O4, and JUN–Fe3O4 surfaces are respectively: 3.27, 3.66, 6.28, and 8.45 kcal mol−1. Ea is calculated from the slopes of Arrhenius linear plots ln K2versus 1/T , the highest adsorption capacities were on ARM–Fe3.67,14,19To study the adsorption of MO and EB on these four magnetite samples, all adsorption experiments were conducted under the same conditions including solution pH = 4 so as to eliminate solution pH effect on the adsorption.3O4 NPs according to the pH of plant extracts used in magnetite sample synthesis. EB and MO anions were highly adsorbed on the ARM–Fe3O4 surface with achieved adsorption yields and capacities of 86.05%, 25.39 mg g−1 and 70.31%, 20.82 mg g−1, respectively, then on the ROS–Fe3O4 surface with achieved adsorption yields of 77.71%, 22.92 mg g−1 and 66.88%, 19.80 mg g−1, respectively, next on the MAT–Fe3O4 surface with achieved adsorption yields of 61.98%, 18.03 mg g−1 and 49.94%, 14.78 mg g−1, respectively, and finally, on the JUN–Fe3O4 surface where the adsorption yields and capacities of EB and MO achieved values of only 56.88%, 16.83 mg g−1 and 39.61%, 11.73 mg g−1, respectively.When magnetite is immersed in an aqueous acidic solution, it develops its surface charge via the protonation and deprotonation of FeOH sites on its surface according to the following equation:59FeOH+2 and FeOH0 are respectively the protonated positively charged surface group with two dissociable H+, and the neutral surface group with one dissociable H+. pKa1 = 5.1 is the intrinsic acidity constant determined by Davis et al.59 for magnetite. FeOH+2 known as the Brønsted acid site is an electron-pair acceptor and it is able to transfer H+ from the solid to the adsorbed molecule, implying an H-bond with the surface. Meanwhile, FeOH0 known as the Lewis acid site is an electron-pair acceptor from the adsorbed molecule, implying a coordinated bond with the surface.59In order to study how the plant extract's acidity impacted preferential attachment of dyes' chromophore and auxochrome groups on synthesized magnetite surfaces, the free chromophore and auxochrome groups that were not attached to magnetite surfaces have been deeply analyzed in all dyes' residual solutions using FTIR spectroscopy, so as to perceive preferential attachment of chromophore and auxochrome groups on each surface and hence determine Brønsted and Lewis acid site densities. Based on this analysis, it was possible to infer Brønsted and Lewis acid site densities on each magnetite surface. For this purpose, after the accomplishment of the adsorption in all experiments, the solid and liquid fractions were separated using a centrifuge. In the next sections, the analysis of residual dye chemistry changes of MO and EB will be studied in detail by comparison between FTIR spectra of MO and EB reference solution chemistry, and the chemistry of their residual solutions.3O4, MO/ROS–Fe3O4, MO/MAT–Fe3O4, and MO/JUN–Fe3O4. Meanwhile, EB residual solutions will be denoted as EB/ARM–Fe3O4, EB/ROS–Fe3O4, EB/MAT–Fe3O4, and EB/JUN–Fe3O4.In the rest of this paper, MO residual solutions will be denoted as MO/ARM–Fe3.6.1−1, aromatic CH ring stretching at 2915.93 cm−1,60 CC at 2300–2100 cm−1, NCN at 2100.95 cm−1,61 aromatic CC ring stretching at 1655.87 cm−162 and at 1522.60 cm−1,63 NN at 1569.87 cm−1 and at 1416.02 cm−1,62,64 C–N at 1355.01 cm−1 and 1160.08 cm−1,62,64 CN at 1304.74 cm−1,64 SO at 1112.02 cm−1,63 and C–H bending vibrations of the aromatic ring at 1047.03 cm−164 and at 1000–550 cm−1.65The FTIR spectrum of the MO reference solution  shows a 3O4, MO/MAT–Fe3O4, MO/ROS–Fe3O4, and MO/ARM–Fe3O4 residual solutions, respectively. The disappearance of certain peaks and the appearance of new ones indicate that the MO/plant–magnetite residual solution chemistry was changed as a result of MO adsorption on magnetite surfaces. The newly appeared peaks correspond to SO−3, , asymmetric CH, phenyl, and the ring skeleton of benzene groups (RSB). More details about the identification of these peaks in MO/plant–magnetite residual solutions will be given in the next paragraphs, for the purpose of investigating the preferential attachment of chromophore and auxochrome groups on all four magnetite surfaces.In 66The decomposition of the dye is significantly accelerated by the presence of acidic centers at the surface.3.6.1.1−1 to around 3308 cm−1 in all four residual solutions , then on ROS–Fe3O4 (66.98%), next on MAT–Fe3O4 (49.94%), and finally on JUN–Fe3O4 (39.61%). This is due to the fact that most of the MO functional groups are chromophores.The functional group attachment analysis results are consistent with MO adsorption yields, being the highest on ARM–Fe3.6.1.23O4 and MO/ROS–Fe3O4 show new peaks at 1367.14 and 1202.44 cm−1, and at 1365.24 and 1203.61 cm−1, respectively. These peaks correspond to SO−3 sulfonic acid groups.62 It is remarked that these peaks are slightly more intense in the FTIR spectrum of MO/ARM–Fe3O4 than in that of MO/ROS–Fe3O4. However, these peaks do not appear in the spectra of MO/MAT–Fe3O4 and MO/JUN–Fe3O4. This indicates that SO−3 groups were completely attached on MAT–Fe3O4 and JUN–Fe3O4 surfaces, whereas they were more attached on ROS–Fe3O4 compared to the ARM–Fe3O4 surface.As illustrated in 3 in ionized dimethylamine (DMA)  groups appear at around 2342 cm−1,68 and their NCN and  stretching bonds at around 2090 cm−1 and 1739.94 cm−1, respectively, with however remarkably different areas. The broadest peak area appears in the FTIR spectrum of MO/ARM–Fe3O4, next in that of MO/ROS–Fe3O4, then in that of MO/MAT–Fe3O4, and finally, the narrowest ones are in the FTIR spectrum of MO/JUN–Fe3O4. This leads to the conclusion that  groups were more attached on JUN–Fe3O4, then on MAT–Fe3O4, next on ROS–Fe3O4, and finally, on ARM–Fe3O4.Furthermore, FTIR spectra of all four residual solutions  show thaThe analysis of preferential attachment of auxochrome groups shows that:3O4, all MO auxochrome groups were less attached compared to on the three other magnetite NPs.• On ARM–Fe3O4, sulphonic acid and dimethylamine groups were more attached compared to on ARM–Fe3O4.• On ROS–Fe3O4, sulphonic acid groups were completely attached; however, dimethylamine groups were less attached compared to on JUN–Fe3O4.• On MAT–Fe3O4, sulphonic acid groups were completely attached, and dimethylamine groups were more attached compared to on MAT–Fe3O4.• On JUN–Fe3O4 is the highest one, followed by that of MAT–Fe3O4, then that of ROS–Fe3O4, and finally, that of ARM–Fe3O4.This leads to the conclusion that the density of Brønsted acid sites of JUN–Fe3.6.2−1,60 aromatic CH ring stretching at 2920.97 cm−1,60 CC at 2300–2100 cm−1,61 aromatic CC ring stretching at 1943.10 cm−1, 1658.11 cm−1,62 and 1519.25 cm−1,63 NN bond at 1569.75 cm−1 and 1418.76 cm−1,62,64 C–N at 1351.98 cm−1 and 1164.01 cm−1,62,64 CN at 1306.03 cm−1,64 SO at 1114.67 cm−1,63,64 and C–H bending vibration of the aromatic ring at 1042.40 cm−164 and 1000–550 cm−1.65The FTIR spectrum of the EB reference solution presented in 3O4, EB/MAT–Fe3O4, EB/ROS–Fe3O4, and EB/ARM–Fe3O4 are presented in 3O4, ROS–Fe3O4, MAT–Fe3O4, and JUN–Fe3O4 surfaces. The newly appeared peaks correspond to not attached  SO−3, phenol, aniline, and phenyl groups, and the ring skeleton of benzene groups. More details about the identification of these peaks in EB/plant–magnetite residual solutions will be given in the next paragraphs, for the purpose of investigating the preferential attachment of the EB chromophore and auxochrome groups on all four magnetite surfaces.FTIR spectra of EB/JUN–Fe3.6.2.1−1, with however different peak bond areas. Additionally, new peaks, with different areas, appear at around 1634 cm−1 which correspond to the vibration of CC of phenyl groups.67 Furthermore, the peaks of the ring skeleton of benzene groups appear, with different areas, in all four FTIR spectra at around 642 cm−1.21 It is worth noting that peak areas of NH of phenyldiazonium, CC of phenyl, and CH of benzene groups vary in the same manner, where the peak area in the FTIR spectrum of EB/ARM–Fe3O4 is the narrowest one, followed by that of EB/ROS–Fe3O4, then that of EB/MAT–Fe3O4, and finally, the broadest one is that of EB/JUN–Fe3O4. This indicates that phenyldiazonium, phenyl, and benzene groups were more attached on ARM–Fe3O4, next on ROS–Fe3O4, then on MAT–Fe3O4, and finally, on the JUN–Fe3O4 surface.The spectra of all four EB/plant–magnetite residual solutions  show thaC bond appears with different peak areas in the FTIR spectra of EB/MAT–Fe3O4 and EB/JUN–Fe3O4 (in the FTIR spectrum of EB/JUN–Fe3O4 it is broader than that of EB/MAT–Fe3O4 and attached on MAT–Fe3O4 more than in JUN–Fe3O4). In contrast, no peak corresponding to the CC bond appears in the FTIR spectra of EB/ROS–Fe3O4 and EB/ARM–Fe3O4 which confirms that CC bonds were completely attached on ROS–Fe3O4 and ARM–Fe3O4 surfaces, and almost completely attached on MAT–Fe3O4; however, these groups were less attached on the JUN–Fe3O4 surface.3O4 shows minor peaks of NN, CC, and CN (linked to the benzene ring) groups, whereas no peak corresponding to them appear in other FTIR spectra. This reveals that these groups were completely attached on MAT–Fe3O4, ROS–Fe3O4, and ARM–Fe3O4; however, these groups were less attached on the JUN–Fe3O4 surface.Furthermore, the spectrum of EB/JUN–Fe3 of toluene groups appear at around 2343.82 cm−168 in the FTIR spectrum of EB/JUN–Fe3O4 and a minor peak of toluene groups appears in the FTIR spectrum of EB/MAT–Fe3O4. Meanwhile, no peak corresponding to toluene groups appears in the FTIR spectra of EB/ARM–Fe3O4 and EB/ROS–Fe3O4. This reveals that toluene groups were completely attached on ROS–Fe3O4 and ARM–Fe3O4, and almost completely attached on MAT–Fe3O4; however, these groups were less attached on the JUN–Fe3O4 surface.It is also remarked that new peaks of the CH bond of CHThe analysis of preferential attachment of chromophore groups on magnetite surfaces shows, as summarized in 3O4, all EB chromophore groups were less attached compared to on the three other magnetite NPs.• On JUN–Fe3O4, NN, CC, and CN were completely attached, whereas CC and toluene groups were almost completely attached while other groups were more attached compared to on JUN–Fe3O4 and less attached compared to on ARM–Fe3O4 and ROS–Fe3O4.• On MAT–Fe3O4, NN, CC, CN, toluene, and CC were completely attached, while other groups were less attached than on ARM–Fe3O4 and more attached than on MAT–Fe3O4 and JUN–Fe3O4.• On ROS–Fe3O4, NN, CC, CN, toluene, and CC were completely attached, while phenyldiazonium, phenyl, and benzene groups were more attached than on other magnetite surfaces.• On ARM–Fe3O4 is the highest one, followed by that of ROS–Fe3O4, then that of MAT–Fe3O4, and finally that of JUN–Fe3O4.As it is known that chromophore groups prefer to attach to Lewis acid sites, it is possible to infer that the density of Lewis acid sites of ARM–Fe3O4 (86.05%), then on ROS–Fe3O4 (77.71%), next on MAT–Fe3O4 (61.98%), and finally on JUN–Fe3O4 (56.88%). This is due to the fact that most of the EB functional groups are chromophores.The functional group attachment analysis results are consistent with EB adsorption yields, being the highest on ARM–Fe3.6.2.23O4 and EB/ROS–Fe3O4 show that new peaks corresponding to SO−3,62 to the bending vibration of OH of phenol69 and to the bending vibration of NH of aniline groups70 appear at 1099.04 and 1204.58 cm−1, respectively , next on ROS–Fe3O4 , then on MAT–Fe3O4 , and finally on JUN–Fe3O4 , respectively. Accordingly, the adsorption yields and Lewis acid site densities varied in the same manner. Seeing that plant extracts used in the synthesis of ARM–Fe3O4, ROS–Fe3O4, MAT–Fe3O4 and –Fe3O4 NPs have respectively pH 5.25, 5.05, 4.63, and 3.69, one can conclude that plant extract pH has a clear effect on the preferential attachment of dye chromophore and auxochrome groups, magnetite nanoparticle acid sites, and adsorption yields. Indeed, the decrease of the mediating plant extract's acidity leads to the increase of Lewis acid site densities and the decrease of Brønsted acid site densities on magnetite NPs and hence an increase in the attachment of chromophore groups and a decrease in the attachment of auxochrome groups of dyes. As most of the MO and EB functional groups are chromophores, the decrease of the mediating plant extract's acidity also leads to an increase in adsorption yields. The remarked difference in adsorption yields of EB and MO on all four magnetite NPs is due to the fact that the ratio of chromophore/auxochrome groups in EB is remarkably greater than that in MO.The analysis of the preferential attachment of chromophore and auxochrome groups in EB and MO adsorption leads to the conclusion that the Lewis acid site density is the highest on ARM–FeThus, the plant extract's acidity could provide a preconceived idea about the densities of Brønsted and Lewis acid sites of magnetite NPs to be greenly synthesized and therefore about azo dye adsorption yields. Dye adsorption yield can be predicted according to the content of chromophore and auxochrome groups in the azo dye structure.3.73.7.13O4, ROS–Fe3O4, MAT–Fe3O4 and JUN–Fe3O4 NPs were respectively 69.33, 64.77, 61.69, and 59.78%, where desorption efficiencies of MO on ARM–Fe3O4, ROS–Fe3O4, MAT–Fe3O4 and JUN–Fe3O4 NPs were respectively 62.83, 61.71, 60.31, and 57.88%. However, at higher pH (pH = 12), almost 100% desorption was achieved from the magnetite surfaces in each case. This is mainly due to the electrostatic repulsion between the negatively charged functional groups of EB and MO molecules and the negatively charged active sites of magnetite surfaces.Desorption yields of EB and MO from magnetite surfaces (adsorbed at pH = 4) in the pH range of 8–12 are calculated using 3.7.23O4 samples  after the second and third cycles of reuse, where the average adsorption efficiencies of the first cycle were found to be 100% in all plant–Feples see . This in4The adsorption of methyl orange (MO) and Evans blue (EB) azo dyes on four greenly synthesized magnetite NPs has been studied. The pHs of plant extracts used for the green synthesis were 5.25, 5.05, 4.63, and 3.69. The aim of the study was the investigation of the plant extract's acidity effect on the magnetite surface reactivity through the analysis of the preferential attachment of the dyes' chromophore and auxochrome groups on magnetite nanoparticles, and adsorption yields, and therefore determination of the plant extract pH effect on acid site types and densities. To do so, the free chromophore and auxochrome groups that are not attached to magnetite surfaces have been deeply analyzed in all dye residual solutions using FTIR spectroscopy, so as to perceive preferential attachment of dye chromophore and auxochrome groups on the four magnetite surfaces.Obtained results show that the mediating plant extract's acidity has a clear effect on preferential attachment of dye chromophore and auxochrome groups, magnetite nanoparticle acid sites, and adsorption yields. Indeed, the decrease of plant extract acidity leads to the increase of Lewis acid site densities and the decrease of Brønsted acid site densities on magnetite NPs and hence an increase in the attachment of chromophore dye groups and a decrease in the attachment of auxochrome dye groups. As most of the MO and EB functional groups are chromophores, the decrease of the mediating plant extract's acidity also leads to an increase in adsorption yields.The linear and non-linear pseudo-first-order and pseudo-second-order kinetics of the adsorption as well as the intra-particle diffusion mechanism have also been analyzed. Obtained results indicated that the adsorption kinetic followed a linear pseudo-first-order kinetic model. Meanwhile, film diffusion was found to be the step that controlled the adsorption mechanism of MO and EB adsorption processes. The thermodynamic studies of EB and MO adsorption processes have been analyzed in the temperature range of 303.15–318.15 K. They reveal the physical and endothermic nature of the adsorption in all cases.Kaouthar Ahmouda: writing – original draft, resources, investigation, visualization, methodology, and revision. Moussa Boudiaf: visualization and revision. Boubaker Benhaoua: visualization.There are no conflicts to declare.


NH3 exists in two different chemical forms, namely the weak base (free molecule: NH3) and the weak acid (ammonium: NH4+), which are generally in equilibrium with each other in plants. However, the effect of NH3 on seed germination, seedling growth, and thermotolerance acquirement in maize remains unclear. Here, maize seeds were imbibed in the different concentrations of NH3·H2O (NH3 donor), and then germinated and calculated seed germination rate at the various time points. Also, the 60-h-old seedlings were irrigated in the different concentrations of NH3·H2O, and then subjected to heat stress and counted survival rate. The data implied that the appropriate concentrations  of NH3·H2O accelerated seed germination as well as increased seedling height and root length compared with the control without NH3 treatment. Also, the suitable concentrations (2 and 4 mM) of NH3·H2O improved tissue vitality, relieved an increase in malondialdehyde content, and enhanced survival rate of maize seedlings under heat stress compared with the control. These results firstly suggest that NH3 could accelerate seed germination, seedling growth, and thermotolerance acquirement in maize.Ammonia (NH NH3, similar to other gaostransmitters H2S, nitric oxide (NO), carbon monoxide (CO), ethylene (ETH), methane (CH4), hydrogen gas (H2), sulfur dioxide (SO2), and hydrogen cyanide (HCN), plays a crucial physiological role in plant growth, development, and response to environmental stresses.1 Meanwhile, NH3 exhibits a dual role during performing its physiological processes, that is, as gasotransmitter at low concentration and toxic agent at high dosage.4 Therefore, NH3, the same as signaling molecules calcium ion (Ca2+), hydrogen peroxide (H2O2), and other gasotransmitters, must be maintained homeostasis in plant cells. The homeostasis of NH3 in plant cells is minutely governed by its production, transport, and scavenging via enzymatic and non-enzymatic pathways, such as nitrate reduction, photorespiration, amino acid catabolism, and phenylpropanoid metabolism.5 Similar to other signaling molecules, NH3 signaling can be triggered when its active production exceeds scavenging, and then cascades downstream signaling pathways, thus regulating many physiological processes in plants.7Ammonia  signaling pathways, promotes lateral root branching by auxin signaling pathways, and regulates root hair development by signaling network involved in Ca2+, ROS, and pH oscillations.3 Similarly, in rice seedlings, NH3 could enhance drought tolerance by interplaying with abscisic acid and aquaporins via facilitating water uptake by roots.5 Also, NH3 was able to increase the resistance of tomato plants to pathogen infection by triggering ROS burst.8 However, in maize, the effect of NH3 on seed germination, seedling growth, and thermotolerance acquirement remains indistinct.NH3 on seed germination, seedling growth, and thermotolerance acquirement of maize (Zea mays L.), the maize (Qiuqing No 88) seeds with the same size and health were sterilized in 5% (v/v) sodium hypochlorite solution for 15 min, and then washed cleanly and imbibed in the different concentrations  of ammonia water (NH3·H2O) for 12 h. Afterward, the soaked seeds were sown on the wetted filter paper (with six-layer) in culture bottles and germinated at 26°C in the dark in climatic chamber. Germination rate was calculated at the indicated time points. Similarly, the soaked seeds by distilled water were germinated at 26°C for 60 h in the dark in climatic chamber. The 60-h-old seedlings were irrigated with 100 mL of the different concentrations  of NH3·H2O for 4 h, and then rinsed in 100 mL of distilled water to remove NH3 residue. The control seedlings (irrigated with distilled water) and NH3-treated seedlings were transferred into another climatic chamber with 47°C in the dark for 16 h to subject heat stress. After heat stress, the heated seedlings were recovered at 26°C, 100 μmol m−2 s−1, and 16/8 h (light/dark) photoperiodism for a week in climatic chamber. Finally, the survival rate was counted according to the previous methods.9 Experiments were repeated at least three times, and the data was assessed as per one-way analysis of variance and the least significant difference. Asterisk (*) and double asterisks (**) in figures represent significant difference (P < .05) and very significant difference (P < .01) compared with the control without NH3.H2O treatment, respectively.To investigate the effect of NH3·H2O accelerated seed germination as well as increased seedling height and root length in a concentration-dependent manner. The germination rate of seeds imbibed with 3, 6, 9, 12, and 15 mM reached 38% (62% and 92%), 45% (P˂ 0.05) [66% (P˂ 0.05) and 90%], 46% (P˂ 0.05) [68% (P˂ 0.01) and 91%], 55% (P˂ 0.01) [75% (P˂ 0.05) and 93%], and 33% (54% and 90%) at 36 h (48 and 60 h), respectively, compared with the control 37% (60% and 92%) . Among the concentrations, the concentration of 12 mM was the most effective concentration for the germination of maize seeds . For seedling growth, the seed-imbibition with 6 mM significantly increased the height of maize seedlings (P˂ 0.05), but other concentrations  had no significant effect on seedling height and root length compared with the control . Similarly, the root-irrigation with the different concentrations of NH3·H2O enhanced survival rate  and tissue vitality (485 and 4.1 nmol g−1 fresh weight FW) (P˂ 0.05), 85% (1.15 A485 and 3.2 nmol g−1 FW) (P˂ 0.01), and 35% (0.6 A485 and 5.3 nmol g−1 FW), respectively, compared with the control 45% (0.8 A485 and 6 nmol g−1 FW) .5 Also, NH3 was able to improve salt tolerance in citrus plants by modulating abscisic acid, hydrogen peroxide, polyamines, and proline.12 However, the underlying mechanism of NH3-accelerated seed germination, seedling growth, and thermotolerance acquirement needs to be further explored in the coming days.The results show that seed-imbibition with the different concentrations of NHvitality , as wellvitality  in maize g−1 FW) . Among t g−1 FW) . These dClick here for additional data file.