Breast cancer is the most frequent cancer among women and the second highest mortality in female across the world. Recent studies have illustrated that sex-determining region Y (SRY)-box protein (SOX) family plays essential roles in regulating various cancers. Nevertheless, the detailed effects of SOX13 on breast cancer are still uncovered. In our present study, SOX13 protein level was measured by using western blot assay in tissues and cells, and the results showed that SOX13 was upregulated in breast cancer tissues and cells compared with normal samples. Moreover, silencing SOX13 inhibited breast cancer cell viability, arrested cell cycle at G1/S phase and suppressed glycolysis, while overexpression of SOX13 reversed these events. Additionally, SOX13 knockdown reduced the level of proteins related to Wnt/β-catenin signaling pathway, whereas overexpression of tripartite motif containing 11 (TRM11) efficiently attenuated the effects, indicating that SOX13 controlled Wnt/β-catenin pathway depending on TRIM11. Furthermore, the data gained from xenograft tumor model illustrated that silencing SOX13 suppressed the tumor growth in nude mice and the glycolysis of tissues. In conclusion, our investigation illustrated that SOX13 facilitated breast cancer cell proliferation and glycolysis by modulating Wnt/β-catenin signaling pathway affected via TRIM11.  This work elucidated the roles of SOX13 in breast cancer progression.This study uncovered that SOX13 promoted breast cancer growth and glycolysis.The finding proved that SOX13 regulated TRIM11/Wnt/β-catenin signaling pathway.More downstream genes of SOX13 involved in glycolysis will be explored.Breast cancer, a highly heterogeneous disease, is considered the leading malignancy in women all over the world and the second highest cause of mortality in female . NeitherEven though cancer cells are rich in oxygen, glucose metabolism is still largely dependent on glycolysis. Aerobic glycolysis, also known as the Warburg effect, is verified to be the leading outcome of oncogenic drivers . Wnt/β-cTRIM11, an E3 ubiquitin ligase, has been discovered to facilitate various cancers progress . For exaSex-determining region Y (SRY)-box protein (SOX) family, the testis-determining factor of mammal, consists of approximately 20 SOX proteins, which all have SRY-related high motility group (HMG) domain, the highly conserved DNA-binding sequences in mammals . Recent Here, we hypothesized that SOX13 is closely related to the development of breast cancer. Therefore, this work aimed to verify the essential roles of SOX13 in breast cancer progress and to explore the molecular mechanism of SOX13.Fifty paired breast cancer tissues and their adjacent tissues used in this investigation were collected from Taizhou Municipal Hospital. All patients received mastectomy at this hospital and did not receive chemoradiotherapy or other adjuvant treatment before surgery. All experiments in this study were approved by the Ethics Committee of Taizhou Municipal Hospital  and performed in accordance with the policy of the Declaration of Helsinki. All patients participating in this research have signed informed consent.2. MCF-10A cells were cultured according to a protocol provided in a previous reporter [MDA-MB-231, SK-BR-3, ZR-75-30 and BT-474 cells were incubated in Dulbecco’s modified eagle medium  added with 10% fetal bovine serum at 37°C incubator containing 5% COTo knockdown SOX13, small interfering RNA 5’-GAGAGUAGAUGUCCUAUAA-3’ was purchased from GeneChem Co. Ltd (China). Disordering edition of this sequence was used as control. The above-mentioned sequences were inserted into lentiviral plasmids (pLKO.1), which is operated by GeneChem Co. Ltd. For overexpressing SOX13, full-length sequence of human SOX13 was constructed into pcDNA plasmid.For transient transfection, plasmids were transfected into cells by using Lipofectamine 2000 . To establish stable cell lines, the lentiviral plasmid pLKO.1 with shRNA and 2 helper plasmids, psPAX2 and pMD2.G were transfected into HEK293T cells using Lipofectamine 2000. Then, the medium was replaced after 6 h and viral particles in the medium were harvested after 48 h. Next, a 0.45-μm filter was employed to screen the supernatant. Seventy percent confluence MDA-MB-231 cells were infected with the supernatant containing viral particles and 2 μg/ml polybrene . Finally, the positive cells were selected by 2 μg/ml puromycin  .3 cell/mL/well) and were transiently transfected with the indicated vectors for 24 h. Then, 20 μL 5 mg/mL MTT solution  was added into each well at 0, 24, 48 and 72 h and incubated with cells for 4 h at 37°C in the dark. Next, MTT solution was replaced with 150 μL dimethyl sulfoxide, and a microplate reader was introduced to record the 590 nm absorbance of cells [MDA-MB-231 cells were seeded in 96-well plates (5 × 10of cells .5 cells/well) overnight and were transiently transfected with indicated vectors for 24 h. Then, the cells were harvested in phosphate buffer saline (PBS) solution . Seventy percent ethanol was utilized to fix cells in PBS for 2 h on ice, and the cells were washed twice using PBS. Next, the mixture was centrifuged (2000 rpm/min for 5 min), and the supernatant was abandoned. The cells were re-suspended using 500 μl staining buffer  and were incubated at 37°C. After 30 min, cells were analyzed with the flow cytometer  immediately [MDA-MB-231 cells were grown in 6-well plates  lysis buffer , and the concentration was estimated utilizing bicinchoninic acid (BCA) protein assay kit  according to the manufacturer’s protocol. The total proteins were then isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.45 μm polyvinylidene fluoride (PVDF) membranes . Next, the membranes were treated with 3% defatted milk for 40–60 min at room temperature. Subsequently, membranes were incubated with primary antibody SOX13 , lactate dehydrogenase A , GLUT1 , hexokinase 2 (HK2) , β-catenin , Axin1 , CyclinD1 , c-Myc , TRIM11 , proliferating cell nuclear antigen  and glyceraldehyde-3-phosphate dehydrogenase  at 4°C for a night. After washing 3 times using Tris Buffered saline Tween  solution, membranes were incubated with Anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked Antibody  or Anti-mouse IgG, HRP-linked Antibody  for 1 hour and protein bands were detected with the Hesper chemiluminescence imaging system . The levels of proteins were quantified using an imageJ software v 1.8.0  .2O2 was employed to block the tissues. Subsequently, the tissue slices were incubated with anti-SOX13  antibody, while the control group was treated with immunoglobulin IgG  overnight at 4°C. Finally, the slices were counterstained with hematoxylin followed by visualizing with 3, 3’-diaminobenzidine [IHC assay was performed with the guidelines offered in the previous study . Brieflyenzidine .In this part, Glucose Assay Kit , Lactate Assay Kit  and ATP Assay Kit  were utilized to estimate the glucose consumption, lactate production and ATP level according to the detailed protocol of manufacturer .2/2) [All operations were approved by the Ethics Committee of the Taizhou Municipal Hospital and are in compliance with the policy of the Institutional Animal Care and Use Committee. Briefly, the stably transfected MDA-MB-231 cells were injected into the left flank of nude mice . The tumor size was recorded at day 7, and it was measured every 7 days for a total of 35 days (The tumorigenesis rate in nude mice was 100%). Finally, the volume of tumor was calculated in accordance with the formula: length × (width2/2) .t-test and one-way analysis of variance (ANOVA) were employed to analyze unpaired data and multiple comparisons, respectively, by using GraphPad Prism 6.0. Comparing mean ± deviation (SD) was used to determine statistical significance and P < 0.05 was identified significant  [Student’s  < 0.05) . All expIn this study, we assumed that SOX13 exerted crucial roles in breast cancer progression and aimed to verify this hypothesis. In summary, this investigation revealed the increased expression and tumor-stimulative effect of SOX13 on breast cancer progress. In addition, our study expounded that SOX13 was capable of mediating Wnt/β-catenin signaling pathway through TRIM11, which contributed to promote aerobic glycolysis and breast carcinogenesis.p < 0.001, p < 0.001, p < 0.001, **p < 0.01, *p < 0.05, First, the protein level of SOX13 in breast cancer and para-carcinoma tissues was measured, and the data illustrated that SOX13 expression was higher in tumor tissues than in para-carcinoma samples  and corresponding controls (si-NC) were transfected to establish SOX13-knockdown MDA-MB-231 cell lines, while SOX13-expressing plasmids (pcDNA-SOX13) or control plasmids (pcDNA) were introduced to overexpress SOX13. Besides, si-SOX13#1 was chosen for subsequent analyses for its better efficiency . The tumor formation was recorded at day 7, and it was measured every 7 days for a total of 35 days (The tumorigenesis rate in nude mice was 100%). As presented in p < 0.001, p < 0.01, *p < 0.05, Subsequently, nude mice were employed to determine whether SOX13 affected tumorigenesis Breast cancer that exhibits high level of heterogeneity is identified as the most frightening malignancy worldwide . Surgeryin vitro and in vivo were consistent, which confirmed the reliability of our conclusion.In the present study, we systemically summarized that SOX13 promoted glycolysis and viability of breast cancer cells by activating TRIM11-mediated Wnt/β-catenin signaling pathway. In addition, the results collected from SOX13 knockdown and SOX13 overexpression experiments SOX13 is an essential member of SOX family, a collection of transcription factors that play an important role in the progression of cell development. The disorders of SOX genes are verified to be associated with the multiple diseases as both oncogenes and anti-oncogenes . For exain vivo and reduced TRIM11 and glycolysis-associated proteins expression. These findings suggested that SOX13 exerted vital roles both in vitro and in vivo. However, how SOX13 mediated TRIM11 and the other downstream genes of SOX13 involved in glycolysis need more investigations in the future.Subsequently, we explored the potential mechanisms of SOX13 in breast carcinogenesis, and we found that silencing SOX13 significantly inhibited glycolysis, whereas overexpressing SOX13 accelerated the glycolysis of breast cancer cells. Previous studies have proved that glycolysis is needed for various cancers including breast cancer. It could offer the main energy source under different conditions. Scientists regard this phenomenon as the Warburg Effect, which is characterized by up-regulation of glucose consumption and lactate production . MoreoveIn conclusion, this study for the first time revealed that SOX13 promoted breast cancer progression by accelerating TRIM11/Wnt/β-catenin network-mediated glycolysis. These findings strongly suggest that SOX13 as a breast cancer oncogene has the potential to be developed as the biomarker and therapeutic target of breast cancer.Click here for additional data file.


Casimiroa edulis (C. edulis) and Glycosmis pentaphylla (G. pentaphylla), in addition to, mucilage and protein, the separated compounds from C. edulis fruits and seeds respectively in a dose of (500 mg/kg). Molecular, Biochemical and histopathological examinations were then conducted. Data recorded showed a significant elevation in hydrogen peroxide (H2O2) level and reduction in glutathione peroxidase (GPX) level post AgNPs intoxication. The oxidative stress occurred was modulated upon treatment regimens. Protein expression of C-reactive protein (CRP) showed a significant elevation and Molecular analysis recorded a significant up-regulation in the expression of both Bax and caspace-3 genes upon AgNPs intoxication in both particles size. On the contrary, both Bcl2 and P53 gene expression were shown to be significantly reduced. Treatment by C. edulis, G. pentaphylla, protein and mucilage extracts revealed modulation in apoptotic and pro-apoptotic biomarkers. Histopathological examination confirmed the obtained results. AgNPs exposure could induce neurotoxicity, genetic alternation and oxidative stress; the targeted extracts could be considered as a promising candidate in modulating apoptosis and neurotoxicity induced by AgNPs.Rapid progress in nano-scales and nanostructure extremely altered the way of diagnosing or preventing numerous diseases. One of the most important nano-medicines used in cancer treatment and diagnosis is silver nanoparticles (AgNPs). Regardless of their extensive utilization, their prospective neurotoxicity wasn’t studied yet. Herein, male Swiss Albino mice were intoxicated via two Nano-scales of AgNPs; (20 nm and 100 nm) for one month (100 mg/kg) then treated by leaves extracts of both   •Silver nanoparticles (AgNPs) are frequently employed in consumer and medicinal products.•Prolonged use of AgNPs poses harm to human health.•Investigation of its adverse consequence specifically neurotoxicity is urgent .•AgNPs intoxication declared an alteration in biochemical and molecular biomarkers.•Upon natural products treatment, the assessed parameters were modulated.•Nutraceuticals belong to rutaceae family could modulate neurotoxicity induced by AgNPs.•It could be promising therapeutic regimen against neurotoxicity induced by AgNPs. Previous studies reported that prolonged use of AgNPs can harm human health. Therefore, it is urgent to investigate its adverse impact specifically neurotoxicity.Nanoparticles are active molecules as they can path through cell membrane and interact with intracellular molecules. A former study highlighted the blood-brain barrier (BBB) and brain accumulation of oral AgNPs The mechanisms of toxic effect of nanoparticles especially AgNPs is still unclear despite the high vulnerability of brain tissue to various oxidative stresses. Several in vitro studies investigated AgNPs toxic effects on cellular system that disturb cellular respiration, mitochondrial function and elevate the production of free radical that increase oxidative stress leading to cellular death Previous studies reported that smaller nanoparticles relative surface induced more toxic effects. In regard, it has been reported that 50 and 20 nm AgNPs were less toxic compared with 5 nm particles. Furthermore, smaller sized AgNPs elevated interleukin- 8 gene expression as compared to larger nanoparticles. Furthermore, it has been reported that smaller and larger particles could alter cellular mechanisms leading to cellular apoptosis and then cell death.Although, AgNps neurotoxicity mechanism is still unclear, alteration in the gene expression, oxidative stress induced by free radical, and apoptosis are the most common underlying mechanisms Real-time polymerase chain reaction (RT-PCR), one of the most sensitive and responsible methods for analyzing gene expression, was used in the current study to investigate alternations in the expression of the Bcl-2, Bax, and P53 genes in a mouse model following exposure to Ag-NPs.Various investigations demonstrated plant-derived constituents as antioxidants for treating neurodegenerative disorders Casimiroa edulis (C. edulis) is a commonly used plant in wide range as a sedative folk medicine inducing sleep C. edulisNa, Na-dimethyl histamine, imidazole, zapoterin and 2 & 4-quinolinones C. edulis aqueous extract of leaves demonstrated anti-inflammatory as well as diuretic effects. Furthermore, the alcoholic leaves extract recorded anti-mutagenic, anticonvulsant and sedative activities C. edulis appears promising for its sedative efficiency in inducing sleep Numerous studies have supported the use of antioxidants derived from plants in the treatment of neurodegenerative illnesses. By reducing or reversing cellular damage by slowing the loss of neuronal cells, antioxidants may have neurodegenerative effects in addition to their neuroprotective effects (apoptosis prevention). Numerous phytochemicals are known to have neuroprotective effects, but further research is required to understand how they affect the blood-brain barrier. Many plants have diverse biological characteristics and have lately been employed in traditional medicine. Glycosmis pentaphylla (G. pentaphylla) is a flowering small plant or evergreen shrub belongs to Rutaceae family. It is mainly found in East as well as Southeast Asia G. pentaphylla such as arborine, flavonoids, terpenoids, alkaloids, coumarins, amides and imides G. pentaphylla were used in folk medicine in treating various diseases as hook worm infestation, cure boils, ureterolithiasis, chest pain, rheumatism, cough, bleeding and anemia Diverse parts of G. pentaphylla, sometimes known as the gin berry, contains large amounts of phyto-carbazole alkaloids. Numerous pharmacological effects, including anticancer, antioxidant, anti-inflammatory, antibacterial, antifungal, antidiabetic, and neuroprotective activity have been discovered in derivatives of carbazole alkaloids C. edulis or G. pentaphylla leaf extract can treat brain damage brought on by AgNPs. In light of this, the main goal of the current study is to investigate the biochemical, histopathological, and molecular changes related to AgNPs induction using high and low particle size in mice model. Furthermore, the therapeutic potential of both G. pentaphylla and C. edulis leave extracts after neurotoxicity induced by AgNPs was also investigated. The research was also expanded to explore the effectiveness of mucilage and protein, the active components that were extracted from C. edulis fruits and seeds respective extract to mitigate the neurotoxic effect induced by AgNPs.However, there is no evidence in the prior literature that 22.1Two particle sized of AgNPs were purchased from Sigma-Aldrich CO. . Kits for biochemical analysis were provided from biodiagnostic company, Egypt. ELISA Kit for CRP measurement was obtained from . Kits for mRNA extraction, primers specific for Bax, Bcl2 and P53 and one step RT-PCR SYBR green were gained from Qiagen . All other used chemicals in the current work are of highly analytical grade.2.2G. pentaphylla and C. edulis were obtained from Mohammed Ali museum and garden botany, Egypt, respectively. Plant taxonomy was identified with Voucher specimens: No: 31–3–2015I & 2015II).Leaves of 2.3G. pentaphylla and C. edulis were carried out according to the previous published article using air-dried powdered of plant leaves. Firstly powder was defatted in petroleum ether then soaked in methanol at 25 C°. The methanol extract was then evaporated and then, fractionated. Flavonoids were extracted with ethyl acetate at 35 °C under reduced pressure Briefly, extraction of both 2.4C. edulis plant fruits were used to make the mucilage, and the sugar from the hydrolysate's sugar was derivatized and evaluated by GLC under the same circumstances as previously described Five hundred grams of powdered 2.5Defatted dried powdered seeds (200 g) were mixed three times (100 ml each) in 10 % sodium chloride solution before being filtered. The experiment was carried out by adding 10 % trichloroacetic acid (TCA) equal volume solution to the filtrate in order to precipitate the protein as a white flocculent amorphous precipitate (2016). Using the previously specified circumstances, the previously synthesized protein (10 mg) was hydrolyzed, and the amino acids of the protein hydrolysate were studied 2.62.6.1Male Swiss Albino mice (22–25 g/each) were gained from the animal house of our institute . Then they were housed in cages, ten of each allowing standard chow diet and water. According to the institutional animal ethics committee of NRC, the animals used in this investigation received acceptable care and handling [ethical approval No: 16154]. One week post acclimatization, animals were randomly subdivided into the following groups (10 /each):Group 1: Healthy animals.Groups from group 2–6: mice that were orally administered 20 nm sized of AgNPs in a size of 100 mg/kg for one month Groups from group 7–11: Animals were administered an oral dose (100 mg/kg) of AgNPs in a size of 100 nm for one month After that the following regimen was applied:Groups 2 & 7: Intoxicated animals with AgNPs 20 nm and 100 nm respectively in a dose of (100 mg/kg) and served as positive control C. edulis (500 mg/kg) for three weeks; 5 days/week Groups 3 & 8: intoxicated group which treated with G. pentaphylla (500 mg/kg) for three weeks; 5 days/week Groups 4 &9: AgNPs intoxicated mice which were treated with Groups 5 &10: AgNPs intoxicated animals which were then administered mucilage (500 mg/kg) for three weeks; 5 days/week Groups 6 &11: AgNPs intoxicated animals which were then administered protein (500 mg/kg) for three weeks; 5 days/week At the end of the experiment, blood samples were collected from the sublingual vein. Sera were separated by centrifugation at 4000 rpm for 15 min and were kept at − 80 °C for subsequent assessment of biochemical parameters.2 asphyxiation. After that, brain tissue was carefully separated for molecular and histopathological examination and kept in − 80 °C and 10 % formaldehyde respectively.Mice were then euthanized by Co2.72O2) and Glutathione Peroxidase (GPx) were estimated according to Aebi, 1984 Antioxidant activity was estimated for all groups. Hydrogen peroxide (H2.8C-reactive protein (CRP) was measured in serum samples using ELISA technique according to the instructions of manufacturer then read the absorbance in spectrophotometer at 450 nm 2.9ΔΔCT) methodology Total RNA was isolated from the brain tissue using Qiqamp mini kit  according to the manufacturer’s instructions. Then subjected to RT-PCR analysis to quantify mRNA gene expression of Bax, Bcl2, P53 and caspases-3 using one step QuantiTecht SYBR green  RT-PCR reaction was performed according to our previously published article 2.10Brain tissues were cut into representative slices, which were subsequently fixed in 10% formalin buffer. After fixation, 4-mm thick paraffin-embedded slices were obtained, and hematoxylin and eosin (H&E) slide staining was done to analyze each sample under a light microscope 2.11Version 16 of SPSS software program was used to analyze the obtained data. The mean ± SE was used to express all values. Turkey's multiple comparisons post hoc test was used to statistically examine significant differences between the groups after one-way analysis of variance (ANOVA). Significant differences were defined as those with p.05.33.1The size of the two used nanoparticle was elucidated using transmission electron microscopy (TEM) was 20 ± 3 nm and 100 ± 11 nm recording a hydrodynamic diameter potential .Fig. 1Tr3.22O2 levels besides an obvious relief was recorded in GPX values against the control group . Whereas intoxication by particle size 100 nm declared a non-significant reduction in Bcl2 gene expression (0.98 fold change).C. edulis treatment declared a significant modulation of Bax gene expression in 20 nm intoxicated group (1.09 fold changes) as well as 100 nm intoxicated group (1.2 fold change) to be near the normal value. In addition, a significant reduction in Bax gene expression was observed upon G. pentaphylla treatment (1.1 & 1.54 fold changes for 20 nm and 100 nm respectively). Upon G. pentaphylla treatment, Bcl2 gene expression was elevated to be near the normal healthy group in 20 nm intoxicated group (0.91 fold change). In contrast, a non-significant elevation was declared in 100 nm intoxicated groups (0.86 fold change).Furthermore, Treatment by mucilage extract declared significant reduction of Bax gene expression in 100 nm intoxicated group (1.51 fold change). Otherwise, non-significant reduction was recorded in the expression of Bax gene to be near the normal value (2.05 fold change).Mucilage 100 nm intoxicated group treatment could reduce Bcl2 gene expression but not statistically significant (0.78 fold change). Moreover, a significant reduction was declared in the expression of Bcl2 gene to be near the normal value (0.8 fold change).Protein extract treatment declared non-significant reduction in Bax gene expression in both intoxicated silver nanoparticles sized (1.72 & 2.1 fold changes). Bcl2 gene expression declared a significant reduction in 20 nm as well as 100 nm intoxicated groups  & 5.Fig.3.5Data recorded in the present research declared an obvious elevation in P53 gene expression induced by silver nanoparticles intoxication in both 20 nm and 100 nm (1.7 &1.8 respectively).C. edulis declared a significant modulation of P53 gene expression in 20 nm intoxicated group (1.1 fold change) whereas non-significant improvement was declared in 100 nm intoxicated group upon C. edulis treatment (1.65 fold change). In addition, a significant reduction in P53 gene expression was observed upon G. pentaphylla treatment (1.11 & 1.52 fold changes for 20 nm and 100 nm respectively). Mucilage extract treatment showed an obvious reduction of the expression of P53 in 20 nm intoxicated group (1.4 fold change). Otherwise, non-significant reduction in the expression of P53 was declared in 100 nm intoxicated group upon mucilage treatment (1.61 fold change). Upon protein administration, P53 gene expression was reduced significantly in 100 nm intoxicated group (1.5 fold change) further, non-significant reduction was observed in P53 gene expression in 20 nm intoxicated group that treated by protein extract (1.67 fold change) (Treatment by  change) a & 6b.Fi3.6The current study investigated a remarkable elevation in the expression of caspace-3 induced by silver nanoparticles intoxication in both 20 nm and 100 nm (2.67 &3.22 respectively).C. edulis declared a significant downregulation in the expression of caspace-3 in 20 nm intoxicated group (1.07 fold change) to be near the normal value. Furthermore, a significant improvement was declared in 100 nm intoxicated group upon C. edulis treatment (1.81 fold change). Upon G. pentaphylla treatment, a significant reduction in the expression of caspace-3 was recorded of 20 nm as well as 100 nm groups (1.2 & 1.04 respectively). Mucilage extract treatment declared more significant improvement in the expression of caspace-3 in 20 nm intoxicated group (1.62 fold change) than those in 100 nm (2.13 fold change). Otherwise, non-significant reduction was observed in the expression of caspace-3 in 100 nm intoxicated group upon mucilage extract (1.61 fold change). Meanwhile, protein extract treatment was significantly reduced in 100 nm intoxicated group (1.5 fold change). Otherwise, non-significant reduction was recorded in the expression of caspace-3 in both 20 nm and 100 nm intoxicated groups , human colon adenocarcinoma, and hamster kidney upon AgNPs intoxication The binding of Bax to mitochondrial membrane leads to p53-mediated apoptosis. Then caspases are activated in numerous cells playing an essential role in the initiation as well as the accomplishment of apoptosis. The activated caspase-3 was previously reported to be necessary in DNA damage and apoptosis C. edulis leaves extract, G. pentaphylla, mucilage and protein declared a significant mitigation in all biochemical, oxidative as well as molecular measured parameters. These findings suggested that the above mentioned extracts possess neuroprotective, and anti-apoptotic potential against AgNPs induced cerebral damages and can induce beneficial therapeutic effects by suppressing the process mediated with free radical formation; an action that could be related to the antioxidant properties of these extracts.In the current study, treatment by G. pentaphylla displayed several activities. Furthermore, the glucoside casimirosine was present in the seed extract and other substances, including coumarins, flavonoids, and limonoids, which are known to have anti-inflammatory actions, were also discovered Due to the presence of various biologically active compounds like isoimpinellin, casimiroin, skimmianine, 1-methyl-2-phenyl-4-quinol, edulein, and scopoletinmethyl ether 5In conclusion, the current study demonstrated that intoxication by AgNPs in murine model could promote apoptosis of nerve tissue through pro-apoptotic Bax mRNA overexpression and reduction of anti-apoptotic Bcl-2 mRNA gene expression in nanoparticle size-dependent manner in addition to a notable elevation of caspace-3 as well as P53 apoptotic biomarker. Furthermore, histopathological examination confirmed all the obtained results. However, treatment by natural products extracts investigated a significant modulation of all biochemical, oxidative stress as well as molecular parameters.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The compound exhibits a variety of biological activities. This study details the evaluation of the HOO• antiradical activity of 7OGT by quantum chemistry calculations. The HOO• trapping activity of 7OGT in the gas phase (reference state) was discovered to follow the formal hydrogen transfer mechanism with a rate constant of k = 4.58 × 108 M−1 s−1. In physiological environments, 7OGT is predicted to be an excellent HOO• radical scavenger with koverall = 2.65 × 108 and 1.40 × 104 M−1 s−1 in water and pentyl ethanoate solvents, respectively. The HOO• antiradical activity of 7OGT in water at physiological pH is approximately 2000 times that of Trolox and substantially higher than that of other well-known natural antioxidants such as trans-resveratrol or ascorbic acid. Thus, 7OGT is an excellent natural antioxidant in polar environments.7-O-Galloyltricetifavan ( The al media ,14,15. S. 2kcalc/kexp ratio = 1–2.9) [The density functional theory-based quantum chemical calculations used here had been outlined in a range of former works for modelling antioxidant activities of various compounds –20. In b= 1–2.9) ,26,36, a= 1–2.9) ,20,37,38k) was computed as follows [σ is the reaction symmetry number [κ contains the tunnelling corrections calculated using the Eckart barrier [Bk is the Boltzmann constant, h is the Planck constant and ΔG≠ is the Gibbs free energy of activation.Using the transition state (TS) theory at 298.15 K, 1 M standard state, the rate constant  reactions in media were determined using the Marcus theory ,41. The reaction ,43.kapp) for an irreversible bimolecular diffusion-controlled reaction were computed using the Collins–Kimball theory in solvents at 298.15 K [kD) was estimated using the literature [A correction was applied to rate constants that were close to the diffusion limit . The app298.15 K ; the steterature ,45,2.4kABD = AD + BD  [AD or BD is determined using the Stokes–Einstein formulation (2.6) [η is the viscosity of the solvents (i.e. η(pentyl ethanoate) = 8.62 × 10 −4 Pa s, η(H2O) = 8.91 × 10 −4 Pa s) and a is the radius of the solute.A and B) ,46, wheron (2.6) ,48.2.6DTo avoid over-penalizing entropy losses in solution, the solvent cage effects were added using Okuno's adjustments , which wFor species with numerous conformers, all of them were energy minimized, with the lowest electronic energy conformer being included in the study. The existence of only one single imaginary frequency was a defining feature of all transition stages. To verify that each TS is accurately related to the pre-complex and post-complex, intrinsic coordinate calculations were completed. The calculations were carried out using Gaussian 09 software .. 3. 3.17OGT was first evaluated according to the three main radical scavenging mechanisms: sequential electron transfer followed by proton transfer (SETPT), formal hydrogen transfer (FHT) and sequential proton loss followed by electron transfer (SPLET). In the two-step reactions such as the SETPT and SPLET pathways, the first step reaction (i.e. SET and proton loss (PL) for the SETPT and SPLET pathways, respectively) normally has the higher activation energy, with the exception of the proton dissociation of acidic moieties in water that is considered separately. Therefore, the thermochemical parameters i.e. proton affinity (PA), bond dissociation energy (BDE) and ionization energies (IE) that characterize the PL, FHT and SET reactions, respectively, were computed for all relevant bonds of 7OGT with the M06-2X/6-311++G//M06-2X/6–31+G(d) method in the gas phase [−1, while the BDEs are 73.9–85.3 kcal mol−1 for O-H bonds. This suggests that the hydroxyl groups are the thermodynamically preferred sites of activity via the hydrogen transfer reaction. The lowest BDE values were presented at the O4' − H bond  and the O13−H bond . These sites are believed to play a key role in 7OGT's radical scavenging activity via the FHT mechanism. According to this data, 7OGT has lower BDE(O−H) values than e.g. vanillic acid , [−1), [−1), [−1) [7OGT could exhibit faster antiradical activity (following the FHT mechanism) than these natural antioxidants.Following the established protocol ,54, the as phase . The res mol−1),  puerarin), [−1),  or vinif1), [−1) . This su7OGT is expected to favour the FHT mechanism in lipid media. The calculated Gibbs free energy changes (ΔG°) of the 7OGT + HOO• reaction via the main mechanisms: FHT, PL, which is the first step of SPLET, and SET as the first step of the SETPT suggest that the FHT reaction is spontaneous (ΔG° < 0) for most of the sites, apart from the C3(4)H−bonds; however, the PL and SET reactions are not spontaneous (ΔG° > 0) in any cases. The Marcus theory was also used to estimate the reaction barriers of the SET reaction in the gas phase [G≠(SET) = 155.6 and λ = 21.2 kcal mol−1). Thus kinetics of HOO• + 7OGT reaction were computed following the FHT mechanism at the positions that yielded negative ΔG°.As shown in as phase ,41; howe• + 7OGT reaction were performed following the QM-ORSA protocol with the M06-2X/6-311 ++ G method [Kinetic studies of the HOO) method ,28, and 7OGT + HOO• reaction following the FHT pathway are within the range of −2.0 to 7.3 kcal mol−1 . The activation Gibbs energies (ΔG≠) range 7.4−16.6 kcal mol−1, while the κ values vary 19.3−53.3. Thus, the κ values play an important role in the rate constants of the hydroperoxyl antiradical activity of the 7OGT. This result is consistent with previous studies on phenolic compounds [• trapping activity of 7OGT is defined by the FHT reaction at the O4′ position; therefore, this reaction will be further analysed in physiological media.The energy barriers for the al mol−1 . The O4'ompounds ,27. The . 3.27OGT were investigated at physiological pH to discover potential radical scavenging mechanisms [Ka1 and pKa2 values were 6.87 and 8.29, respectively (2A: 20.7%), anion (HA−: 70.2%) and dianion (A2−: 9.1%) will be used for studying the radical scavenging activity, whereas the neutral state will be considered in the lipid medium (pentyl ethanoate solvent).Previous research has shown that the antiradical activity of phenolic compounds in aqueous solutions is dominated by anion states ,38. The chanisms ,38,57. Bchanisms , pKa1 anectively . Therefo• antiradical activity in non-polar media follows the hydrogen transfer mechanism at the O4′ − H bond. Thermodynamic evaluation in pentyl ethanoate and water  did not differ at the most likely site of activity  for the neutral state (H2A), however, the PL and SET pathways of this state are not spontaneous in either media (ΔG° > 0). Previous studies showed that for a compound containing acidic moieties the SET reaction of the dissociated states should be also considered in the aqueous solution [7OGT against HOO• radical in physiological media were carried out following equations (3.1) and (3.2) below, and the results are presented in The calculated results in the vacuum suggest that the HOOsolution ,37,54,57Lipid medium,Water at physiological pH,• + 7OGT reaction in the aqueous solution  is approximately 104 times faster than that  in the lipid medium. The SET of dianion A2− plays a principal role  in the HOO• antiradical activity of 7OGT in the aqueous solution. Compared with typical antioxidants indicated that the HOO• scavenging activity of 7OGT is faster than those of Trolox , [trans-resveratrol  [k = 1.00 × 108 M−1 s−1) [k = 1.56 × 105 M−1 s−1) [k = 1.69 × 108 M−1 s−1) [k = 5.80 × 107 M−1 s−1) [7OGT is one of the most excellent natural antioxidants in polar environments.According to the results , the HOO−1 s−1),  trans-reM−1 s−1) , ascorbiM−1 s−1) , ramalinM−1 s−1) , deoxyniM−1 s−1)  and 8-hyM−1 s−1) . Hence, . 47OGT was successfully evaluated by computational chemistry. The results showed that the antiradical activity of the 7OGT in non-polar media such as gas phase and pentyl ethanoate follows the FHT reaction . 7OGT also presented excellent antiradical activity  in the aqueous solution. The HOO• radical scavenging of 7OGT is faster than that of typical antioxidants such as trans-resveratrol, Trolox, deoxynimbidiol, ramalin, 8-hydroxyconiothyrinone B and ascorbic acid. Thus, 7OGT is one of the most potent natural antioxidants identified thus far in polar environments.The hydroperoxyl antiradical activity of


Tg) values, mechanical properties, crosslinking density (ve), and thermal stability of disulfide linkers networks were higher than those of diselenide linkers networks, according to our research. The activation energies of disulfide linkers were higher than those of diselenide linkers (up to 14 kJ/mol), but their healing efficiency was lower than that of the diselenide network. These findings demonstrate the advantages of diselenide and disulfide dynamic linkers in epoxy networks systems, as well as a method for designing and preparing the appropriate diselenide dynamic linkers or disulfide dynamic linkers incorporated into epoxy networks for the appropriate application and processing technology.Over the last decade, there has been a lot of interest in incorporating dynamic covalent bonds (DCBs) into epoxy resins. Because diselenide and disulfide bonds have similar properties, they are frequently used as DCBs in self-healing epoxy networks. In this paper, we present diselenide and disulfide dynamic linkers containing epoxy networks by analyzing the effects of mechanical properties, thermal stability, activation energies, and self-healing properties. The glass transition temperature ( Because of their excellent chemical resistance, thermal stability, and mechanical properties, epoxy resins are widely used in coatings, adhesives, electronics industry machinery, and other fields . These bBoth diselenide bonds and disulfide bonds are DCBs, and they are frequently used as self-healing, dynamic covalent bonds . Much reTg) values, mechanical properties, crosslinking density (ve), thermal stability, and activation energies of disulfide linkers networks were higher than those of diselenide linkers networks, but the healing efficiency was lower than diselenide linkers networks.The effect of dynamic disulfide bonds and dynamic diselenide bonds containing epoxy networks is investigated in this study. The glass transition temperature (Bisphenol A epoxy resins (128), 2-aminophenyl disulfide (2-DS), 4-Aminophenyl disulfide (4-DS), 2-Iodoaniline and 4-Iodoanline were purchased from Energy Chemical . CuO nanopowders, KOH and selenium powder were provided by Aladdin Bio-Chem Technology. Poly (ethylene glycol) diglycidyl ether (DER736) was obtained by Macklin Biochemical. Petroleum ether, dimethyl sulfoxide (DMSO), ethyl acetate and dichloromethane were supplied by Guangzhou reagent factory. All the materials were used as received.wt CuO nanoparticles and stirred for another 30 min. Finally, the above reactions were transferred to an oil bath at 100°C in a N2 atmosphere for 24 h. The reaction was monitored by TLC. Following the completion of the reaction, the crude products were extracted by vacuum, and the pure diselenides linkers were purified by column chromatography separation  with the yield of 52% (4-DSe linker) and 47% (2-DSe linker). 4-DSe: 1H NMR : δ(ppm) = 7.38 , 6.57 , 3.50 ; 13C NMR : δ(ppm) = 146.93, 136.06, 134.51, 115.45; 2-DSe: 1H NMR : δ(ppm) = 7.35 , 7.16–7.12 , 6.73 , 6.58–6.54 , 2.95 ; 13C NMR : δ(ppm) = 148.12, 137.75,130.97, 117.77, 114.10.A solution of 1 equiv p-Iodoaniline and 5 equiv selenium powder in dry DMSO (100 ml) was stired for 30 min at room temperature. The mixture solution was then supplemented with 2 equiv KOH and 10% 3 as the solvent. A Bruker-Veretex70 spectrometer was used to obtain the FTIR spectra. The differential scanning calorimeter (DSC) measurements were carried out on TA-Q200 (United States) from −20°C to 110°C at a heat rate of 10°C/min. Tensile tests were carried out using a dynamic mechanical thermal analysis  under 50 mm min−1 of the cross-head rate with a range of −10°C to 130°C at a heating rate of 5°C min−1. Thermogravimetric analysis  was performed under nitrogen from 25°C to 700°C at a heat rate of 10°C/min. The dynamic mechanical analysis (DMA) machine was also used for stress-relaxation experiments and activity energy experiments . The stress-relaxation curves of the film were tested at different temperatures  with a constant strain of 2% (at appropriate size). The gel fraction (W) was calculated using the following formula: W = M2/M1 × 100%. M1 represents the weight of the original sample, while M2 represents the weight of the dried samples .NMR measurements were carried out at 400 MHz on a Bruker AV-400 NMR instrument with CDCl−1 and 909 cm−1 (128 and DER736) vanished, while benzene ring absorption peaks at 1,584, 1,506 and 1,460 cm−1 and the hydroxyl groups at 3,390 cm−1 appeared in dynamic epoxy resin networks. These findings were consistent with previous findings . As shown in findings . Furtherfindings , the gelTg) was determined using DSC. Tg values for BED4DS, BED4DSe, BED2DS, and BED2DSe, as shown in Tg values of p-phenylamino networks (BED4DS and BED4DSe) were higher than o-phenylamino networks (BED2DS and BED2DSe), which may be attributed to the steric hindrance of o-phenylamino linkers. And the gT values of disulfide linkers networks (BED4DS and BED2DS) were slightly higher than those of diselenide linkers networks (BED4DSe and BED2DSe), which could be attributed to the lower bond energies of diselenide linkers networks (The glass transition temperature (networks .Tg(ve) was calculated using the equation  was performed on the samples  and the results are shown in Tg. In prevequation .νe=E′/3Tg+50°C, R the gas universal constant, and T the Tg temperature. Tg and ve, while BED2DSe has the lowest Tg and ve. These results could be attributed to the low steric hindrance and the fact that the selenium atom has a larger radius than the sulfur atom (E′ denotes the storage modulus at fur atom .ve).  was used to characterize the mechanical properties of these samples, as shown in ve). .(t)/G0 = 0.37  was calculated to be 71, 57, 84, and 62 kJ/mol, respectively, using the equation. When comparing the activation energies of disulfide and diselenide linkers in the epoxy network, it is clear that the activation energies of disulfide linkers were higher (up to 14 kJ/mol) implying that diselenide bonds are more dynamic. The activation energy of p-phenylamino networks (BED4DS and BED4DSe) was lower than that of o-phenylamino networks (BED2DS and BED2DSe), which could be attributed to the steric hindrance of o-phenylamino linkers.  = linkers. To verify the self-healing ability of the samples, two dumbbell samples (with different colors) were cut into two pieces using BED4DSe as an example. One of the dumbbell samples was stained with black dye, which is clearer to observe. The two halves were rejoined together using external forces  and the sample was kept at 90°C for 24 h. As shown in The sample was subjected to a stress-strain test to further study the self-healing performance. We cut the sample in half and rejoined it with our hands. The cracked sample was then placed in a 90°C oven for varying times . The healing efficiency (η) was able to calculated by the following equation :η = ε hhealed and origianl, respectively. The healing efficiency (η) is also shown in Where the tensile strength of the healed and the original sample samples was represented by Tg) values, mechanical properties, crosslinking density (ve), and thermal stability of disulfide linkers networks were higher than those of diselenide linkers networks, and the p-phenylamino networks were higher than the o-phenylamino networks. The activation energies of disulfide linkers were higher than those of diselenide linkers (up to 14 kJ/mol) indicating that diselenide bonds may be more dynamic. Finally, the self-healing properties of these networks were investigated and the healing efficiency of diselenide linkers was found to be significantly higher than that of disulfide linkers networks. These advantageous properties of diselenide and disulfide dynamic linkers in epoxy networks system will provide a method for designing and preparing the right self-healing epoxy networks for the right application and processing technology.In conclusion, four self-healing epoxy networks were created through the reaction of diselenide and disulfide dynamic linkers reaction with bisphenol A epoxy resins (128) and poly (ethylene glycol) diglycidyl ether (DER736). FTIR confirmed the chemical structure, and DSC, DMA, and TGA were used to characterize the thermal and mechanical properties. The results show that the glass transition temperature (


Rhodococcus qingshengii PM1 was isolated from selenium-rich carbonaceous mudstones in Enshi, Hubei, China. Here, we report the complete genome sequence of this strain, which was obtained by combining Illumina and Nanopore sequencing. Rhodococcus qingshengii was first isolated from a carbendazim-contaminated soil sample  were quality controlled and trimmed using Guppy v5.0.16 with the high-accuracy base-calling mode (implemented in the MinKNOW interface). De novo assembly and circularization were performed in a hybrid genome assembly and the assembly was rotated to start with dnaA using Unicycler v0.5.0 . A ~1-g soil sample was vortex-mixed in 50 mL sterile distilled water, and 100 μL of the slurry was plated at 28°C on Luria-Bertani (LB) agar for 3 to 5 days. The colonies were picked up and restreaked on LB agar at least five times, to ensure that the cultures were pure, and then were stored at −80°C. Strain PM1 was recovered from storage, grown at 160 rpm and 28°C in LB medium for 2 days, and harvested for genomic DNA extraction. Genomic DNA was extracted using the QIAamp DNA minikit  according to the manufacturer’s protocol. For Illumina sequencing, 100 ng DNA was randomly sheared to ~300 bp by Covaris (USA), and end-repair, dA-tailing, and adapter ligation procedures were performed using the NEBNext Ultra II DNA library preparation kit for Illumina . Paired-end sequencing (2 × 150 bp) was performed on the NovaSeq 6000 platform . For Nanopore sequencing, the genome library was produced with no shearing using the ligation sequencing kit 1D (SQK-LSK109) with the native barcoding expansion kit . Then, the genome libraries were loaded and sequenced using a PromethION sequencer (ONT). All software was used with default parameters unless otherwise noted. Illumina raw reads  were cleaned and trimmed using fastp v0.23.2 (R. qingshengii based on its average nucleotide identity (ANI) of 98.46% (R. qingshengii JCM 15477 (GenBank accession number CP096563.1). The genome size of PM1 is 6,373,845 bp, with a GC content of 62.84% and high average coverage levels (326× for Illumina sequencing and 721× for Nanopore sequencing). A total of 5,893 genes were annotated, including 5,802 coding genes, 70 RNA genes, and 21 pseudogenes.PM1 was identified as f 98.46%  and its CP104782, BioProject accession number PRJNA881770, and SRA accession numbers SRR21679470 (Illumina sequencing) and SRR21679469 (Nanopore sequencing).The complete genome sequence has been deposited in GenBank under accession number


Hordeum brevisubulatum (wild barley) was used as material in the greenhouse to study the effects of endophyte infection on plant growth, Cd absorption and transport, subcellular distribution, and Cd chemical forms under CdCl2 stress. Endophytic fungi respond positively to chlorophyll content and photosynthetic efficiency under Cd stress. The order of Cd absorption in different parts of the plant was: roots > stems > leaves. Endophyte infection increased the plant’s absorption and transport of Cd while causing a significant difference in the stem, which was associated with the distribution density of endophyte hyphae. The proportion of organelle Cd in endophyte-infected wild barley was significantly higher, which facilitated more Cd transport to aboveground. Cd stress showed a slight effect on the chemical forms of Cd in leaves. The proportion of phosphate, oxalate, and residual Cd increased in the stem. Cd existed in the form of inorganic salt, organic acid, pectin, and protein in roots. Endophyte infection reduced the Cd content of the more toxic chemical forms to protect the normal progress of plant physiological functions. Therefore, the isolation of cell walls and vacuoles is a key mechanism for plant Cd tolerance and detoxification. As endophyte infections have more ability to absorb Cd in plants, H. brevisubulatum–Epichloë bromicola symbionts can improve heavy metal contaminated soil and water.Hydroponic  In recent years, due to the continuous increase of agricultural and industrial activities, a large number of heavy metals have been released into the atmosphere, water, and soil . Cd is a2+, Fe2+, and Zn2+ transporters are used by Cd2+ to enter the cell, which is the molecular basis for plant cells to absorb Cd2+ [2 levels can effectively alleviate Cd-induced growth inhibition [Symplastic and apoplastic refer to the ways for plant roots to absorb Cd, which are distributed aboveground through various transport pathways . Simultaorb Cd2+ ,14. The hibition ,17,18,19hibition ,21.Epichloë [Epichloë endophytes often form symbionts with cool-season grasses (subfamily Poöideae) [Epichloë endophytes, including pathogens, insects, waterlogging, drought, cold, salt, and heavy metals, etc. [Epichloë endophytes that produce alkaloids or secondary metabolites to the host [Festuca pratensis) and tall fescue (Festuca arundinacea) infected by Epichloë endophytes (E+) can significantly increase under Cd stress (p < 0.05) in comparison with endophyte-free   and Elymus dahuricus  significantly increase under high concentrations of Cd stress. In addition, the antioxidant enzyme activity (AEA), proline content, malondialdehyde (MDA) content, H2O2 concentration, and chlorophyll level also increase significantly (p < 0.05) [Epichloë endophytes also enhanced the zinc tolerance of perennial ryegrass (Lolium perenne) and aluminum tolerance of fescues (Festuca spp.) [Epichloë endophyte symbionts have great potential for the remediation of heavy metal contaminated soil and water.According to the principle of “one fungus, one name”, the asexual (anamorphic) taxa and the sexual (teleomorphic) taxa are collectively referred to as Epichloë . EndophyEpichloë . EndophyEpichloë ,25. Epicoöideae) . The graoöideae) ,27 but als, etc. ,29, whicthe host . The remolutions ,32. Endo < 0.05) ,34. Meanca spp.) ,36. TherHordeum brevisublatum) is a universal high-quality forage that is widely distributed in the lightly salinized meadows of northern China. It is well known for its high resistance to abiotic stress, including drought, salt, and alkali tolerance [Epichloë endophytes in wild barley make Hordeum brevisubulatum–Epichloë bromicola symbionts more competitive, and the salt tolerance is the most prominent [H. brevisubulatum–E. bromicola symbionts demonstrate a certain absorption of metal (Na). Therefore, as an ecological grass, the wild barley endophyte symbionts play a vital role in improving the environment and repairing contaminated soil. However, the adsorption capacity of H. brevisubulatum–E. bromicola symbionts to heavy metals needs to be further explored.Wild barley  and KU365152-154 (actG). A phylogenetic tree reference was adopted from the work of Chen [2 was gradually applied in half Hoagland’s nutrient solution, and the concentration gradients were set as 0, 50, 100 µM. The treatment solution was changed at every 3 d to maintain the precise Cd concentrations in the solution.The seeds of  of Chen . The see2 stress, the plant roots were soaked in 20 mM ethylenediamine tetraacetic acid disodium salt (EDTA-2Na) for 15 min to remove ions from the root surface and then repeatedly washed with distilled water for 3 times. The roots, stems, and leaves of the plant were harvested separately.After 6 weeks of exposure to CdClPlant photosynthetic indexes were measured before harvesting using a GFS-3000  portable photosynthesis-fluorescence measurement system. The SPAD-502 Plus  chlorophyll meter was used to determine the chlorophyll content of plants. At the same time, the plant height, root length, and tiller number were measured and the fresh weight of roots, stems, and leaves was recorded. All tissues were dried to a constant weight at 65 °C and used for the determination of Cd content after grinding into powder.V:V:V) was added to the solution and placed on the graphite digestion instrument for digestion at 370 °C. After complete digestion and cooling, the tube wall was repeatedly washed and the volume adjusted in a 50 mL volumetric flask. The flame atomic absorption spectrometer  was used to determine the Cd content in the solution.Chen’s  method wThe subcellular extraction and separation in plant tissues were carried out by Weigle and Jager’s differential centrifugation method . BrieflyEthanol).80% ethanol, extracting inorganic Cd, giving priority to nitrate/nitrite, chloride, and aminophenol cadmium, i.e., 80% ethanol extraction state (F2PO4)2, i.e., deionized water extraction state (FH2O).Deionized water, extracting water-soluble Cd, Cd-organic acid complexes, and Cd(HNaCl).1 M NaCl, extracting pectates and protein-integrated Cd, i.e., 1 M NaCl extraction state 2, and other Cd-phosphate complexes, i.e., 2% HAc extraction state (FHAc).2% acetic acid, extracting undissolved cadmium phosphate, including CdHPOHCl)0.6 M HCl, extracting cadmium oxalate, i.e., 0.6 M HCl extraction state (FR).The remainder is the residual state  and Cd stress (S) on plant growth and development, photosynthesis, Cd distribution in different parts of plants, subcellular distribution, and Cd chemical forms. Statistical significance was defined at the 95% confidence level. All measurements were shown as mean ± standard errors.SPSS statistical software  was used for the analysis of variance (ANOVA) and Duncan’s multi range test. Two-way analysis of variance was applied to determine the effects of p < 0.05) reduced the plant height, root length, dry weight, and fresh weight, tiller number  difference between them (p < 0.05) higher than E+ under control condition (p > 0.05) effect on plant growth under other CdCl2 concentration gradients (p < 0.05) reduced with increased CdCl2 stress, and no significant (p > 0.05) difference was observed between E+ and E− under the treatment of 0 and 50 µM CdCl2 concentrations. The chlorophyll content of E+ plants under 100 µM CdCl2 stress was significantly (p < 0.05) higher than E− plants at the same concentration  reduced the transpiration rate (E), stomatal conductance (Gs), and net photosynthetic rate (Pn) of the plants and increased the concentration of intercellular CO2 (Ci) (p < 0.05) higher than E− (p < 0.05) lower than E−, and no significant (p > 0.05) difference was observed between them with the aggravation of stress (p < 0.05), but had no significant effects on other growth and photosynthetic indexes (CdClCO2 (Ci) . Among t than E− A–C, wherf stress D. In two indexes .2 treatment (p < 0.05) differences  with the increase of CdCl2 concentration, while the transport index  was to the contrary. However, the above indicators all showed that E+ plants were higher than E−, but only with significant (p < 0.05) differences in the Cd concentration of stems , and E+ was lower than E− but the difference was not significant (p > 0.05). The results for stems and leaves were to the contrary, as the increase in the CdCl2 concentration resulted in a significant (p < 0.05) decrease in the distribution proportion of Cd compared with the control. Moreover, E+ plants were higher than E−, but did not show significant (p > 0.05) differences (p > 0.05) effects on Cd concentration in different parts of plants and transport index   .2 stress did not significantly (p > 0.05) affect the distribution proportion of Cd in the cell wall fraction and soluble fraction (p < 0.05) lower in E+ plants compared with the control under Cd stress, while E− plants had no significant (p > 0.05) difference  lower than that in E− (2 treatment significantly (p < 0.05) increased the Cd concentration in the cell wall and soluble fraction, but had no significant (p > 0.05) effect on the organelle fraction (p > 0.05) effect on the subcellular distribution of Cd in leaves  effect of CdCl2 stress was detected on the distribution proportion (2 condition was significantly (p < 0.05) lower than that of the control, and that of E+ plants under the 100 µM CdCl2 condition was significantly (p < 0.05) higher than that of the control  increased (p < 0.05) differences in the 100 µM CdCl2 cell wall fraction, 0 µM CdCl2 organelle fraction, and 50 µM CdCl2 soluble fraction (2 stress was not significantly different (p > 0.05) in two-way ANOVA  significantly (p < 0.05) and E+ plants were lower than E−, showing a significant (p < 0.05) difference at 50 µM CdCl2. However, the organelle fraction was significantly (p < 0.05) decreased (from 30% to 10%) and E+ plants were significantly (p < 0.05) higher than E− (2 treatment was significantly (p < 0.05) increased (2 concentration did not significantly (p > 0.05) affect the concentration of Cd in organelle fraction, but significantly (p < 0.05) increased the concentration of Cd in the soluble fraction (p < 0.05) lower than that in E+ plants under 0 µM CdCl2 conditions, but it increased significantly (p < 0.05) as Cd stress worsened in E− plants and was significantly higher than that of E+ plants (The subcellular distribution of Cd in roots was significantly affected by Cd stress . The sub than E− . The Cd ncreased C. The infraction C. The Cd+ plants C.Epichloë endophyte and Cd stress had no significant (p > 0.05) effects on the subcellular distribution of wild barley roots, stems, and leaves  with increasing CdCl2 concentration (Ethanol (CdCl2 at 50 and 100 µM) and FNaCl (CdCl2 at 50 µM) forms increased significantly (p < 0.05) in E+ plants (p > 0.05) effect on the concentration and percentage of different chemical forms of Cd in leaves , FHAC (from 2.7% to 15.5%), FHCl (from 2.3% to 17.5%), and FR (from 3.8% to 18.8%) forms of Cd were significantly (p< 0.05) higher than in leaves and roots (Ethanol and FH2O decreased more significantly (p< 0.05) than in leaves and roots, from 43.4% to 15.9% and from 42.8% to 13.6%, respectively (HCl forms Cd under 100 µM CdCl2 stress showed that E+ was significantly (p< 0.05) higher than E−  lower than E− plants  compared with the control (H2O and FNaCl forms of Cd in E+ plants was significantly higher than that in E− (p< 0.05), while the proportion of FHCl and FR forms of Cd was significantly lower than that in E− (p< 0.05). Under the 50 µM CdCl2 condition, the proportion of FNaCl and FHAC forms of Cd in E+ plants was significantly (p< 0.05) higher than that in E−, and the proportion of FH2O form of Cd was significantly (p< 0.05) lower than that in E−  higher than that in E−, and the proportion of FH2O and FNaCl forms of Cd was significantly (p< 0.05) lower than that in E− (2 stress had no significant (p> 0.05) effect on the FHCl and FR forms of Cd concentration, and there was no significant (p> 0.05) difference between E+ and E− (Ethanol and FNaCl forms of Cd concentrations at high concentration (CdCl2 at 100 µM) showed a significant (p< 0.05) difference between E+ and E−  lower than E− in the FH2O form of Cd concentration. The FHAc form of Cd concentration was significantly (p< 0.05) higher than the control under 100 µM CdCl2 stress, and there was no significant (p> 0.05) difference between E+ and E−  , and TIsts-stems . The ass2 stress, and both showed that E+ was higher than E−  have strong migration ability and the greatest toxicity to plants, followed by pectinate and Cd bound to protein or adsorbed (extracted by 1 M NaCl), while oxalate bound Cd (extracted by 0.6 M HCl) and residual Cd are the least harmful to plants [Ethanol and FH2O Cd were dominant in leaves, but CdCl2 stress had no significant effect on them. The distribution proportion of Cd in FHAc, FHCl, and FR with low toxicity in stems was significantly higher than that in leaves and roots, and the damage was less, which might be one of the mechanisms of stem resistance against heavy metal stress. The FEthanol, FH2O, and FNaCl forms were used to adapt to adverse conditions  and organic acid (extracted by d-Ho plants ,58. The nditions .2 environment represents the key mechanism for the tolerance and detoxification of wild barley [Previous studies have shown that Cd stress can increase Cd in pectin and protein binding forms in stems and leaves, while water-soluble inorganic salts and organic acid Cd are mainly in roots ,60. The d barley ,61,62. T2 stress. Endophytes increased the chlorophyll content and photosynthetic efficiency of plants and improved the ability of plants to accumulate more Cd in roots, stems, and leaves, indicating that H. brevisubulatum–Epichloë bromicola symbionts had a strong ability to remove Cd from an aqueous solution. The order of root Cd content in subcellular fractions was: soluble fraction > cell wall fraction > organelle fraction. The isolation of cell wall and vacuole represents a double barrier to protect plant cells. The higher organelle fraction in the roots of E+ plants makes it transport more Cd to the aboveground part, which is of great significance to improve the remediation of heavy metal polluted soil and polluted water. Inorganic salt and organic acid forms accumulated Cd in the leaves and inorganic salt, organic acid, pectin, and protein forms accumulated Cd in roots, while the proportion of phosphate, oxalate, and residual Cd in stems increased to reduce plant damage. The endophytes protect themselves by reducing Cd, which is more toxic to plants.Plant growth and photosynthesis were inhibited under CdCl


Furthermore, parallel reaction monitoring analysis validated these results. Meanwhile, we confirmed that the levels of NADH, ROS, H2O2, and iron ions were induced by CFDC, and the sensitivity of Escherichia coli to CFDC was inhibited by the antioxidant vitamin C, N-acetyl-l-cysteine, and deferoxamine. Moreover, deferoxamine also suppressed the H2O2 stress induced by CFDC. In addition, knockout of the NADH-quinone oxidoreductase genes  in the respiratory chain attenuated the sensitivity of E. coli to CFDC far beyond the effects of cefepime and ceftazidime; in particular, the E. coli BW25113 ΔnuoJ strain produced 60-fold increases in MIC to CFDC compared to that of the wild-type E. coli BW25113 strain. The present study revealed that CFDC exerts its antibacterial effects by inducing ROS stress by elevating the levels of NADH and iron ions in E. coli.This study aimed to investigate the antibacterial mechanism of cefiderocol (CFDC) using data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays. Numerous differentially expressed proteins related to the production of NADH, reduced cofactor flavin adenine dinucleotide (FADHIMPORTANCE CFDC was the first FDA-approved siderophore cephalosporin antibiotic in 2019 and is known for its Trojan horse tactics and broad antimicrobial activity against Gram-negative bacteria. However, its antibacterial mechanism is not fully understood, and whether it has an impact on in vivo iron ion homeostasis remains unknown. To comprehensively reveal the antibacterial mechanisms of CFDC, data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays were performed in this study. The findings will further facilitate our understanding of the antibacterial mechanism of CFDC and may provide a theoretical foundation for controlling CFDC resistance in the future. Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii and third-generation cephalosporin-resistant Enterobacterales as “priority 1: critical” pathogens, which emphasizes the need for comparatively more potent antibiotics  Gram-negative pathogens, which have challenged the clinical efficacy of many antibiotics –4. The W–ibiotics . HoweverSiderophores, including enterobactin (catecholate), desferrioxamine (hydroxamate), and pyoverdine (mixed type), are small organic iron-chelating molecules secreted by bacteria –8. All s–9–13–Enterobacterales and nonfermenters  (Cefiderocol (CFDC) is the first FDA-approved parenteral siderophore cephalosporin antibiotic with a broad range of antimicrobial activity against Gram-negative bacteria, including CR olderia) , 23. Theolderia) . This prE. coli.Quantitative proteomics provides a systemic tool to investigate the antibacterial mechanisms of antibiotic and natural products –27. In t–E. coli, we monitored the MIC and the growth curves of E. coli treated with different concentrations of CFDC. The MIC of CFDC against E. coli was 0.8 μg/mL, and the growth curves of E. coli treated with 0, 1/8× MIC, 1/4× MIC, 1/2× MIC, 1× MIC, and 5/4× MIC CFDC is shown in E. coli in the logarithmic phase for the proteomics assay in this study. DIA-based quantitative proteomics was applied to analyze the global protein alteration of E. coli from the 0 h (control) and 2 h CFDC-treated groups . As shown in the volcano plots, 194 DEPs, including 114 upregulated proteins and 81 downregulated proteins, were identified in the samples with CFDC-2 h treatment  and KEGG analysis for all DEPs. As shown in To explore the pathways and molecular functions involved in the antibacterial effects of CFDC in 2) biosynthesis , fatty acid degradation, and amino acid metabolism   and B. Nynthesis . Convers2, and NADPH biosynthesis; iron binding; penicillin binding; and cell motility in E. coli.Taken together, the bioinformatics analysis indicated that CFDC exerts its antibacterial effect by affecting energy synthesis; oxidation-reduction processes; NADH, FADH2, and NADPH can serve as electron donors and elevate NADH levels to increase reactive oxygen species (ROS) production in cells  and Fe2+ were significantly induced by CFDC , which has been reported to protect cells from iron overload toxicity, was significantly upregulated and validated by PRM after CFDC treatment , 38 Fig. Therefo by CFDC  but not ol group  and D. T by CFDC . Based oE. coli, and the molecular structure of CFDC is similar to that of CPM and CAZ except for its unique catechol moiety. These unique structures might mediate the production of ROS stress by iron overload in CFDC-treated groups.As mentioned above, CFDC induced ROS stress in E. coli cells were pretreated with antioxidant vitamin C (VC) (1 mg/mL) or N-acetyl-l-cysteine (NAC) (0.5 mg/mL) for 1 h prior to CFDC, CPM, or CAZ treatment, and the MIC was determined. Moreover, the MIC of CFDC against E. coli was significantly increased 3-fold, 10-fold, and 8-fold after pretreatment with DFO, VC, and NAC, respectively. However, the MIC values of CPM and CAZ increased by only 2-fold, 2-fold, and 4-fold after pretreatment with DFO, VC, and NAC, respectively  that inhibited ROS stress by interfering with electron transport in the respiratory chain. All NADH-quinone oxidoreductase gene-knockout E. coli strains showed less sensitivity to CFDC than the other two cephalosporins. In particular, the E. coli BW25113 ΔnuoJ strain produced 60-fold increases in the MIC of CFDC compared to that of the wild-type E. coli BW25113 strain  is considered one of the main sources for ROS in E. coli , the nuooduction . To eval3 strain . Taken tin vitro study of the mechanism of action of CFDC against E. coli was carried out by using a high-quality DIA-based quantitative proteomics approach.In recent years, multidrug-resistant infections caused by Gram-negative bacteria have become one of the core reasons for clinical anti-infective treatment failure. CFDC, the first clinically approved siderophore-antibiotic conjugate, has been a research hot spot due to its broad range of antibacterial activity against Gram-negative bacteria and the “Trojan horse strategy,” which was designed to bypass the Gram-negative bacterial cell wall. However, the antibacterial mechanism of CFDC is not well understood. Hence, the E. coli treated with CFDC. Bioinformatic analysis showed that energy synthesis; oxidation-reduction processes; NADH, FADH2, and NADPH biosynthesis; iron binding; penicillin binding; and cell motility were mainly induced by CFDC  and Fe2+ were overloaded in the CFDC-treated groups. The elevated Fe2+ could promote ROS production by Fenton reaction , and a reduction in ROS production contributed to E. tarda resistance to CAZ  in the respiratory chain promoted insensitivity of E. coli to CFDC far beyond the effects of CPM and CAZ. In particular, the E. coli BW25113 ΔnuoJ strain produced 60-fold increases in MIC to CFDC compared to the wild-type E. coli BW25113 strain  . The strains were grown overnight in Luria-Bertani (LB) medium at 37°C and then diluted 1:100 into fresh medium for reactivation.CFDC  was purchased from MedChemExpress and dissolved in sterile deionized water as a stock solution at 2 mg/mL. Wild-type 600) of ~0.05, and 500 μL of bacterial solution with drug at different final concentrations was added to each well. After incubating for 24 h at 37°C, the lowest concentration with no visible growth  was identified as the MIC  at 0 and 2 h after the addition of 0.4 μg/mL CFDC and then washed thrice with 1 M sterile precooled phosphate-buffered saline (PBS) (pH = 7.4). All samples were lysed using 8 M urea lysis buffer with intermittent sonication. The protein concentration was quantified using a BCA protein assay kit . The total proteins (200 μg) from each sample were reduced with 50 mM dithiothreitol (DTT) for 1 h at 37°C and then alkylated with 100 mM iodoacetamide (IAA) at room temperature for 30 min in the dark. Subsequently, proteins were digested with trypsin   for 14 h at 37°C. Peptides were dried with a cold-trap speed vacuum concentrator. The dried peptides were resuspended in deionized water containing 0.1% (vol/vol) formic acid. The pooled peptides collected from all samples of equivalent volume were preisolated into 6 fractions as previously described  in the Sequest HT (Proteome Discoverer v2.2) local server and Biognosys Spectronaut software Pulsar (DIA and data-dependent acquisition (DDA) MS analysis were used as previously described with some modification . Brieflye Pulsar . The DIAe Pulsar .P value was set as two-sided hypergeometric tests and adjusted via Bonferroni correction, and P < 0.05 was considered statistically significant. In KEGG pathway analysis, the ProteinRatio = the numbers of the DEPs in one KEGG pathway/the whole numbers of proteins in one KEGG pathway.The DEPs were analyzed by Cytoscape software (version 3.8.3) with various plug-ins as previously reported . The Clum/z, the resolution was 60,000, automatic gain control (AGC) target was 4e5, and MS2 acquisition used the target ms2 module to monitor the target m/z list  with a resolution of 30,000, isolation window of 1.6 Da, 5e4 AGC target and maximum injection time of 120 ms, and HCD collision energy of 35. The PRM raw data were loaded into Protein Discoverer 2.2  to perform peptide identification, and the pdResults file containing peptide spectra was read by Skyline 20.1.0  per candidate protein were selected for PRM quantification. The chromatographic conditions were the same as those in the DIA experiment. The parameters of Orbitrap Fusion Lumos mass spectrometry were set as follows: MS1 scan range was 350 to 1,550 e 20.1.0 . With a The motility was monitored on semisolid agarose (0.5%) with 0, 0.4, and 0.8 μg/mL CFDC in accordance with our previous work , 55. SinE. coli BW25113 cells were grown in LB medium to an OD600 of ~0.6, treated with or without CFDC, CPM, CAZ, or 20 μM H2O2 (positive control) for 2 h and then washed three times with 1 M sterile precooled PBS. DCFH-DA was added to each well at a 1:200 ratio and incubated for 30 min at 37°C in the dark. After washing thrice with 1 M sterile precooled PBS, the levels of ROS in bacterial cells were detected at fluorescence excitation/emission wavelengths of 485 ± 20 nm/525 ± 20 nm and then normalized to the corresponding sample protein concentration.Intracellular ROS were probed using DCFH-DA  and measured using a BioTek Synergy 2 microplate reader according to the kit's instructions . Briefly, +/NADH assay kit with the WST-8 method  according to the manufacturer’s instructions. Briefly, E. coli BW25113 was grown in LB medium to an OD600 of ~0.6, cell pellets were harvested after treatment with 0, 0.4, or 0.8 μg/mL CFDC for 2 h, and then bacterial cells were lysed with 200 μL precooled lysis buffer. The lysed cell suspension was incubated at 60°C for 30 min, and then 20 μL supernatant with 90 μL alcohol dehydrogenase was added to a 96-well plate. The plate was incubated at 37°C for 10 min protected from the light. Finally, 10 μL chromogenic solution was added to the mixture, and the plate was incubated at 37°C for 30 min. A standard curve was generated and measured as the samples. NADH content was measured at 450 nm via a BioTek Synergy 2 microplate reader and then normalized to the sample protein concentration.The NADH content was measured using an NAD2O2 activity was measured using the Amplex Red hydrogen peroxide/peroxidase assay kit . Briefly, E. coli BW25113 was grown in LB medium to an OD600 of ~0.6, treated with 0, 0.6, or 0.8 μg/mL CFDC, 0.8 μg/mL CFDC +0.2/+0.4/+0.8 mg/mL DFO, or 20 μM H2O2 (positive control) for 2 h and then washed thrice with 1 M sterile PBS at room temperature. Immediately thereafter, all samples were prepared according to the manufacturer’s instructions. The H2O2 activity was measured using a BioTek Synergy 2 microplate reader with excitation at 530 ± 25 nm and fluorescence emission monitoring at 590 ± 35 nm and then normalized to the corresponding sample protein concentration.The HE. coli BW25113 cells were grown in LB medium to an OD600 of ~0.6, and cell pellets were harvested after treatment with 0, 0.4, or 0.8 μg/mL CFDC for 2 h. The iron concentration was measured by using an Iron assay kit  according to the manufacturer’s protocol. Briefly, bacterial cells were suspended in 1 M sterile PBS and homogenized using multiple freeze and thaw cycles, and 30 μL supernatant sample and 70 μL iron assay buffer were added to a 96-well plate. To measure total iron Fe2+ or total iron, 5 μL iron assay buffer or 5 μL iron reducer was added to each well. All samples were incubated at 25°C for 30 min protected from the light. Then, 100 μL iron probe was added to the mixture at 25°C for 60 min and protected from light. Thereafter, the absorbance was detected at 593 nm using a BioTek Synergy 2 microplate reader and then normalized to the corresponding sample protein concentration.t tests or one-way analysis of variance (ANOVA) test with Dunnett's correction and expressed as means ± the standard deviations (SD). Statistical analysis was conducted using Prism 8.0 . Results were considered significant at a P value of <0.05.Data were analyzed using two-tailed, unpaired Student PXD031624.The raw data and search results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier


CaF2: Ce, Gd, Nd NPs are small (15–20 nm), of uniform shape and size distribution, and show good biocompatibility and low immunogenicity in vitro. In addition, CaF2: Ce, Gd, Nd NPs possess excellent optical properties. CaF2: Ce, Gd, Nd NPs produce downconversion emissions in the second near-infrared window  under 808 nm excitation, with a strong emission peak at 1056 nm. Excitation in the first near- infrared window  has the advantage of deeper tissue penetration power and reduced autofluorescence, compared to visible light. Thus, CaF2: Ce, Gd, Nd NPs are ideally suited for in vivo fluorescence imaging. In addition, the presence of Gd3+ makes the NPs intrinsically monitorable by magnetic resonance imaging (MRI). Moreover, next to fluorescence and MR imaging, our results show that CaF2: Ce, Gd, Nd NPs can be used as imaging probes for photoacoustic imaging (PAI) in vitro. Therefore, due to their biocompatibility and suitability as multimodal imaging probes, CaF2: Ce, Gd, Nd NPs exhibit great potential as a traceable imaging agent in biomedical applications.Here, we describe the synthesis of a novel type of rare-earth-doped nanoparticles (NPs) for multimodal imaging, by combining the rare-earth elements Ce, Gd and Nd in a crystalline host lattice consisting of CaF Imaging holds a crucial role in the diagnosis of a variety of diseases such as cancer. Early-stage disease diagnosis is important to maximize treatment effects, and to personalize treatments based on the patient’s individual variability and medical profile. Molecular imaging techniques provide comprehensive anatomical, physiological and functional information on disease detection and the monitoring of treatment responses. The most commonly used diagnostic imaging methods during the past few decades in the medical field include MRI, X-ray computed tomography (CT) , positron structure and are widely used in the development of optical probes [3+-doped nanoprobes are downconversion NPs (DCNPs) ranging from NIR-I to NIR-II. They can emit at 890, 1060 and 1340 nm under 808 nm excitation and therefore can be used for near-infrared two-zone imaging [3+, Nd3+-doped NPs have a larger quantum yield than upconversion NPs (UCNPs), and the absorption cross-section of Nd3+ at 808 nm is larger than the absorption cross-section of Yb3+ at 980 nm, which leads to little absorption by water molecules at 808 nm when using Nd3+-doped nanoprobes. Therefore, compared to Yb3+- doped UCNPs, the overheating effect of biological tissues can be more effectively avoided in bioimaging [3+, Nd3+-doped NPs are undoubtedly an optical imaging probe with great potential that can be used for biosensing, biological process monitoring and imaging-guided therapy [Lanthanides display attractive photophysical properties, such as large anti-Stokes shift, narrow band and multiple emission and long-life luminescence due to their unique 4fl probes ,25,26,27 imaging ,29. Of noimaging . Because therapy .3+-doped nanomaterials is to choose a suitable host matrix material. CaF2 is a center-symmetric fluorite crystal with low phonon energy and a wide band gap (E g = 12.1 eV). It is optically transparent in the wavelength range from ultraviolet to visible light and near infrared, so it is widely used in various types of optical equipment [2 crystals strong optical activity in the visible and near-infrared regions [2 can be used as an optical matrix material for Nd3+-doped nanomaterials.Another key to obtaining high-quality Ndquipment ,35,36,37 regions ,39. Thus3+ is not much different from that of Ca2+, trivalent Nd ions replace the crystal sites of divalent Ca ions when they are doped into the CaF2 lattice, requiring more F− for charge compensation, but they do not cause obvious crystal changes. However, when the Nd3+ doping concentration reaches a certain limit, Nd3+ aggregates and some energy cross-relaxation occurs, which triggers the cluster effect and reduces the luminous efficiency of the nanomaterials [3+, Gd3+, Y3+, Yb3+) can effectively destroy the formation of Nd-Nd clusters, thereby improving the quantum efficiency of the material [2: Nd co-doped Ce3+ was 10.4 times higher than that of CaF2: Nd [3+ a common T1 contrast agent in MR bioimaging [3+, Gd3+ and Nd3+ co-doping CaF2 as the material in our strategy to construct an imaging mode combining NIR-II imaging with complementary MRI and PAI.Since the radius of Ndaterials ,41. In omaterial ,43,44,45material . Wang etCaF2: Nd . On the oimaging ,49. Ther2 as the matrix, co-doped with the three lanthanide elements—Ce, Gd and Nd. The resulting CaF2: Ce, Gd, Nd NPs had a small particle size, were stable in the presence of serum and showed excellent luminous intensity at 1056 nm. In vitro cell culture experiments demonstrated that CaF2: Ce, Gd, Nd NPs did not induce cellular cytotoxicity and were readily taken up by human breast cancer cells. In addition, the NPs showed low immunogenicity when cultured with antigen-presenting cells, and thus the NPs can be used as a potential imaging probe in vivo. Importantly, CaF2: Ce, Gd, Nd NPs are multimodal and can be detected by NIR-II/PA/MR imaging and therefore provide significant advantages in disease diagnosis. In summary, CaF2: Ce, Gd, Nd NPs represent a novel optical material that can be widely used in the field of medical imaging.In this study, we used a hydrothermal method to synthesize a citric acid-terminated nanomaterial with CaF3·6H2O, 99.9%), calcium chloride dihydrate , cerium (III) chloride heptahydrate , potassium citrate tribasic monohydrate (HOC(COOK)(CH2COOK)2·H2O, ≥99.0%), gadolinium (III) chloride hexahydrate , ammonium fluoride . Biolegend  supplied the anti-CD40-APC. Thermo Fisher Scientific  provided fetal calf serum (FCS), 4′,6-Diamidino-2-Phenylindole (DAPI), dulbecco’s Modified Eagle’s Medium (DMEM) and CD86-FITC. Promega  offered cell titer 96 AQueous MTS Reagent Powder. PeproTech  supplied lipopolysaccharide (LPS). Bioline  delivered agarose. Abcam  supplied the phalloidin- iFluor 488 Reagent. All of water used in the experiments was ultrapure deionized water.The following chemicals were acquired from Sigma-Aldrich : neodymium (III) chloride hexahydrate  were dissolved in 7 mL water and stirred for 10 minutes (min) until fully dissolved. Then, potassium citrate solution was added dropwise to the solution and stirred for 30 min. After that, NH4F solution was added, and the solution was stirred evenly. The final solution was transferred to a 50 mL Teflon bottle  held in a stainless-steel autoclave, put in an oven  and maintained at 180 °C for 10 hours (h). Finally, the obtained sample was centrifuged at 2.4× g for 20 min and washed three times with water followed by ethanol (99%). The samples were dried in a freeze-dryer .As previously described , we synt2: Nd NPs, CaF2: Ce, Nd NPs, CaF2: Ce, Gd NPs was similar to that of CaF2: Ce, Gd, Nd NPs. The doped concentration of Nd3+ was maintained at 0.02, and Ce3+ and Gd3+ were maintained at 0.15.The synthesis of CaF2: Ce, Gd, Nd NPs was performed by a Panalytical X’pert PRO  operating at a tube voltage of 40 kV and a tube current of 40 mA. The diffraction patterns were acquired using Cu Kα radiation (λ = 1.5405 Å) at a scanning rate of 6.0°/min in the 2θ range of 10° ≤ 2θ ≤ 70°.X-ray diffraction (XRD) analysis of the CaF2: Ce, Gd, Nd NPs solution (1 mg/mL) using a Malvern ZetaSizer 2000 . The analysis software applications were Zetasizer Software (Version 7.13) and GraphPad Prism 8.The hydrodynamic size and Zeta potential were obtained from CaF2: Ce, Gd, Nd NPs powder. The spectra were captured in the IRSpirit-TOAPC0027956 mode with a wavenumber range of 600–4000 cm−1 and a resolution of 4 cm−1. IR Pilot and Origin 8.5 were used for analysis.IRSpirit FTIR spectrophotometer  was used to measure the Fourier transform infrared (FTIR) spectra of CaF2: Ce, Gd, Nd NPs were examined using a Tecnai 12 Twin transmission electron microscope (TEM)  outfitted with a OneView Camera Model 1095  under 120 kV voltage. TEM samples were prepared by pipetting CaF2: Ce, Gd, Nd NPs aqueous solution (1 mg/mL) onto the surface of the copper grid.The size and morphology of CaF2: Ce, Gd, Nd NPs were mounted on scanning electron microscopy (SEM) specimen stubs. Then, an Apreo S LoVac SEM  equipped with an UltraDry energy-dispersive X-ray spectroscopy (EDS) detector  was utilized to analyze the sample. The measurement conditions were: 1500× magnification with 30 kV and 51 nA.CaF2: Ce, Gd, Nd NPs (4.51 mg).A Quantum Design Versalab physical property measurement system with VSM option  was employed to quantify the vibrating-sample magnetometry (VSM) of CaF2:Ce, Gd, Nd NPs powder was recorded using Fluorolog®-3 with FluoEssenceTM  equipped with a diode laser as the excitation light source, and the emission spectra of CaF2: Nd NPs (powder) and CaF2: Ce, Nd NPs (powder) were recorded using an Edinburgh FLS920 fluorescence spectrometer  with an 808 nm NIR diode laser (300 mW). The absorption spectra of CaF2: Ce, Gd, Nd NPs (10 mg/mL) and CaF2: Ce, Gd NPs (10 mg/mL) were obtained by SpectraMax® iD3 Multi-Mode Microplate Reader . Analysis tool was SoftMax Pro® 7 Software.The emission spectra of CaF2: Ce, Gd, Nd NPs in physiologically relevant buffers, we dissolved CaF2: Ce, Gd, Nd NPs at a concentration of 0.2 mg/mL in 50% FCS solution. The samples were kept in a shaker at 37 °C, and we used a Malvern ZetaSizer 2000  to measure the size and zeta potential at 0 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d and 7 d. Data analysis used Zetasizer Software (Version 7.13) and GraphPad Prism 8.To assess the stability of CaF2+/Mg2+-free PBS was used to dilute the blood 50 times. To extract red blood cells, the blood dilutions were centrifuged at 0.9× g for 15 min at 4 °C. The supernatant was removed, and two groups were chosen randomly as negative and positive controls. The negative control group was resuspended with 500 μL of saline, whereas the positive control group was resuspended with 500 μL of 1% Triton X-100 (v/v). For the experimental group, blood cells were resuspended with CaF2: Ce, Gd, Nd NPs isotonic saline at five different concentrations . The samples were then incubated at 37 °C for 4 h before being centrifuged at 0.9× g and 4 °C for 15 min to obtain the supernatant. The absorbance values were measured at 540 nm by an enzyme marker SpectraMax . The concentration of cytosolic hemoglobin in each sample was assessed according to the hemoglobin concentration standard curve.Briefly, a total of 100 μL of fresh blood was collected from the tail vein of BALB/c mice using a vacuum blood collection tube. CaThe hemolysis ratio was calculated using the hemolysis (HL%) equation follows:2: Ce, Gd, Nd NPs to cells was assessed by MTS assay using two different cell lines. Briefly, 4T1 cells (1 × 104) were seeded in 96 wells and incubated for 24 h at 37 °C. The cells were then treated for 24, 48 and 72 h with varied concentrations of CaF2: Ce, Gd, Nd NPs (0–2000 µg/mL). Meanwhile, a 96-well plate was inoculated with 1 × 105 peripheral blood mononuclear cells (PBMCs)/well, kept in a 37 °C incubator and then incubated with various CaF2: Ce, Gd, Nd NPs concentrations (0–125 µg/mL) for 24 h, 48 h and 72 h. Afterwards, the medium was removed and 100 µL of fresh medium and 20 μL of MTS reagent were added to each well as directed by the manufacturer’s instructions. The cells were then cultured for 1.5 h at 37 °C in an incubator. The absorbance (OD) was measured using Microplate reader  at 490 nm. The ratio to the untreated control group was used to assess cell viability. Data are presented as mean ± SD.The toxicity of CaF2: Ce, Gd, Nd NPs in 4T1 cells was determined by confocal microscopy. 4T1 cells were seeded at 2 × 104/well on 13 mm3 circular coverslips in 24-well plates. After 24 h, 4T1 cells were co-incubated with 250 µg/mL of CaF2: Ce, Gd, Nd NPs medium at 37 °C for 1, 4, 24 and 48 h. Cells treated with medium without NPs served as a control group. At the end of the incubation time, the cells were washed 5 times with PBS to remove the unabsorbed NPs. At room temperature, the cells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, treated with 0.1% Triton PBS for 10 min and washed 3 times with PBS. Finally, cell nuclei were labeled with DAPI for 5 min. After washing with PBS, coverslips were sealed on slides with fluorCare sealer. The slides were imaged using a SP8 LIGHTNING Confocal Microscope  and analyzed by LAS X (Leica Application Suite X) software.Qualitative uptake of CaF2: Ce, Gd, Nd NPs on immune cells, we used flow cytometry to assess the expression of DC maturation/activation markers. Briefly, murine D1 DCs and 125 µg/mL CaF2: Ce, Gd, Nd NPs were co-cultured in a 96-well plate for 24 h in a 37 °C incubator. PBS/EDTA  was used to detach the cells, which were then washed with FACS buffer and stained with anti-CD40-APC  and anti-CD86-FITC  antibodies. The cells were washed and resuspended in 100 μL FACS buffer after 30 min. A LSR-II cytometer  was used to measure the samples, and FlowJo (version 10) was utilized to analyze the data.In order to evaluate the effect of CaF2: Ce, Gd, Nd NPs were suspended in ddH2O at 2 mg/mL, while the InGaAs camera was chilled to −20 °C at mid-gain setup. We obtained photos at various wavelengths using an 808 nm laser excitation at 50 mW/cm2. Images were recorded with the KIS NIR-II system.We employed an in vivo NIR-II optical imaging system  to evaluate the NIR-II imaging capabilities of our NPs. CaF2: Ce, Gd, Nd NP solution was injected into an 0.5% agarose gel. The experiment was carried out with the center transmit of 40 MHz and the axial resolution of 40 μm. Vevo LAB 5.5.0 was used to analyze the data.The PA and B-mode ultrasound images were acquired using Vevo LAZR-X , and a MX550D transducer was utilized. CaF2: Ce, Gd, Nd NPs were dissolved in 0.5% agarose solution, and the concentrations were 0 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL, 7.5 mg/mL and 10 mg/mL. The microwave oven was used to heat the solutions and create the gel sample. The measurement settings were as follows: 500/11 ms for the repetition time/echo time (TR/TE), 4 for the number of excitations (NEX), 128 × 128 for the matrix size (MTX), 40 × 40 mm2 for the field of view (FoV) and 1.20 mm for the slice thickness (SL). To investigate the MRI characteristics of NPs under biological conditions, we fixed C57BL/6J mouse cadavers with a tiny animal coil after subcutaneously injecting 100 μL of CaF2: Ce, Gd, Nd NPs (10 mg/mL) and took MR images before and after the injection. The measurement conditions were TR/TE = 10/2.8 ms, FoV = 40 × 40 mm2, matrix = 256 × 256.MRI studies were carried out using a 7T Bruker BioSpec , and ParaVision 360 (Version 2.0. pl.1) software was used to analyze attenuation images. CaFp < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.The statistical tests performed by GraphPad Prism software 8 . Statistical differences were considered significant at * 2 as the matrix, co-doped with the three lanthanide elements—Ce, Gd and Nd—was feasible, we adapted a previously published hydrothermal method to obtain Yb3+/Tm3+-doped MF2  colloids to synthesize CaF2: Ce, Gd, Nd. Briefly, Ca2+, Ce3+, Gd3+, Nd3+ were dissolved in water. Next, solutions of potassium citrate tribasic monohydrate and NH4F were added in turn to obtain the final solution. The resulting solution was heated in an oven  at 180 °C for 10 h to synthesize NPs. The obtained sample was centrifuged, washed and freeze-dried  have been successfully doped into CaF2.First, to confirm that the main matrix of the synthesized NPs consisted of CaFanalysis a. Througon peaks a. This i crystal . In addi crystal ,53. Thisperature b. There te bands c. On the, Nd NPs . In ordeanalysis d. The TE2: Ce, Gd, Nd NPs and CaF2: Ce, Gd NPs in aqueous solution at a concentration of 10 mg/mL  , resulting in a deeper penetration depth and smaller autofluorescence effect of the NPs [2: Ce, Gd, Nd NPs  b. The ex(300 mW) c. The NPectively ,59. The ficiency d. On the the NPs . Based o, Nd NPs e. Under 2: Ce, Gd, Nd NPs have good colloidal stability, the NPs were redispersed in NaCl containing 50% FCS, and then the particles were measured repeatedly (three times) using a Malvern ZetaSizer 2000  at different time periods to determine the particle size. The results showed that there was no significant change in the size and zeta potential of the NPs in 50% FCS  hemolysis was studied. To this end, murine peripheral blood cells were incubated at 37 °C for 4 h with different concentrations of CaF2: Ce, Gd, Nd NPs, and the hemolysis rate of the NPs was calculated by comparing the absorbance (λ = 540 nm) of the samples with the positive (1% Triton X-100) and negative  controls [2: Ce, Gd, Nd NPs have excellent blood compatibility and can be used for intravenous in vivo imaging.To evaluate the NPs’ biocompatibility with blood components, the effect of CaFcontrols  . Cell viability was significantly higher at a concentration of 2000 µg/mL (** p < 0.01). Values higher than 100% indicate that the NPs may have a positive effect on cell proliferation [2: Ce, Gd, Nd NPs contain citric acid groups on the surface, which may participate in the intracellular tricarboxylic acid cycle reaction, resulting in increased cell activity. At 48 h, cell viability was significantly reduced by 15% when the concentration of CaF2: Ce, Gd, Nd NPs was 1000 µg/mL compared to the control (* p<0.05). At 72 h, the doses of 500 and 250 µg/mL CaF2: Ce, Gd, Nd NPs showed a significant inhibitory trend on 4T1 cells . There was no difference between the cell survival rates when the concentration was < 125 µg/mL compared to the control group (p > 0.05). As cancer cell lines show biological differences compared to normal, healthy cells, we also tested the effect of CaF2: Ce, Gd, Nd NPs (0–125 µg/mL) on the viability of human peripheral blood mononuclear cells (PBMCs). As shown in 2: Ce, Gd, Nd NPs show great promise for biological applications, but further studies are needed to determine the optimal concentration and potential cytotoxic effects.To determine whether the NPs induce cellular toxicity, the effect of CaFTS assay . 4T1 is feration . Secondl2: Ce, Gd, Nd NPs by murine breast cancer cells. As demonstrated in 2: Ce, Gd, Nd NPs overlap with the excitation and emission spectra of commonly employed dyes in flow cytometry and microscopy applications, such as Alexa Fluor 568. Thus, we reasoned that our NPs might be inherently monitorable by confocal microscopy. In order to determine the NP uptake rate and intracellular localization of NPs in 4T1 cells, we performed a confocal analysis  at 37 °C for 1 h, 4 h, 24 h and 48 h, followed by co-labeling with DAPI (blue) to stain the nucleus and phalloidin (green) to stain the actin cytoskeleton. Visual inspection confirmed that CaF2: Ce, Gd, Nd NPs could be effectively visualized as distinct dots by confocal microscopy. After 1 h co-incubation, few NPs were found attached to the cell membrane. With increasing incubation time, more red fluorescent dots were found in the cytoplasm, indicating that more NPs entered the cells  encounter various activation stimuli, they will mature and increase the expression of costimulatory markers on their surface [2: Ce, Gd, Nd NPs for 24 h and assessed the expression of the DC costimulatory receptors CD86 and CD40 by flow cytometry. As positive control for DC maturation, we included D1 cells that were treated with LPS. After duplicate exclusion, the expression of CD86 and CD40 was analyzed on single D1 cells , 7% of the cells co-expressed CD86 and CD40  and an applied magnetic field of 1.5 T, we noticed that the NPs enhance their magnetic properties as the magnetic field increases, showing a typical paramagnetic behavior consistent with the magnetic characteristics of Gd ions , resulting in a bright image on the T1 weighted imaging (2: Ce, Gd, Nd NPs can be used as MRI contrast agents.Since Gd Gd ions a. The dins (2/kg ,67. In o imaging c,d. To b imaging e. These 3+, Gd3+ and Nd3+ into CaF2 crystals through a simple hydrothermal process, resulting in the synthesis of CaF2: Ce, Gd, Nd NPs suitable for multimodal imaging. The synthesized NPs were highly pure, and showed low toxicity, good biocompatibility and no immunogenicity. CaF2: Ce, Gd, Nd NPs themselves exhibited dual modes because Ce3+ and Nd3+ dopants contribute to NIR-II and PAI, and the presence of Gd3+ shows a high-contrast T1-enhancing effect for MRI. Therefore, CaF2: Ce, Gd, Nd NPs may be an informative NIR-II/PA/MR multimodal probe for clinical diagnosis. This research also laid the foundation for the use of CaF2: Ce, Gd, Nd NPs for biological imaging of cells and deep tissues.In summary, we doped Ce