The retrograde flow of endometrial tissues deposited into the peritoneal cavity occurs in women during menstruation. Classically (M1) or alternatively (M2) activated macrophages partake in the removal of regurgitated menstrual tissue. The failure of macrophage egress from the peritoneal cavity through the mesothelium leads to chronic inflammation in endometriosis. To study the migration differences of macrophage phenotypes across mesothelial cells, an in vitro model of macrophage egress across a peritoneal mesothelial cell monolayer membrane was developed. M1 macrophages were more sessile, emigrating 2.9-fold less than M2 macrophages. The M1 macrophages displayed a pro-inflammatory cytokine signature, including IL-1α, IL-1β, TNF-α, TNF-β, and IL-12p70. Mass spectrometry sphingolipidomics revealed decreased levels of ceramide-1-phosphate (C1P), an inducer of migration in M1 macrophages, which correlated with its poor migration behavior. C1P is generated by ceramide kinase (CERK) from ceramide, and blocking C1P synthesis via the action of NVP231, a specific CERK chemical inhibitor, prohibited the emigration of M1 and M2 macrophages up to 6.7-fold. Incubation with exogenously added C1P rescued this effect. These results suggest that M1 macrophages are less mobile and have higher retention in the peritoneum due to lower C1P levels, which contributes to an altered peritoneal environment in endometriosis by generating a predominant pro-inflammatory cytokine environment. The peritoneal cavity is covered by the peritoneum, comprising a single layer of flat, simple, stratified squamous epithelial cells, the mesothelium, which sits on a basal laminar. The microvilli on the surface of mesothelial cells secrete a protective barrier, called the glycocalyx, which is composed of glycosaminoglycans (GAGs) and provides a non-adhesive and protective surface for intracoelomic organs and tissues ,2. WithiEndometriosis is a common chronic estrogen-dependent disease that affects 6–12% of women, mainly during their reproductive years, and is characterized by the presence of ectopic endometrial implants primarily in the pelvic peritoneum, ovaries, and rectovaginal septum . RetrogrSphingolipid metabolism is dysregulated in endometriosis. Various types of sphingolipids, including glucosylceramide (GlcCer) and ceramide (Cer), were found to be accumulated in the serum and PF of endometriosis patients . Of thesIn this study, we aimed to ascertain the differences in the migratory properties of macrophage phenotypes across mesothelial cells in vitro and the correlation with C1P levels. Several in vitro models include 2D and more complex 3D cultures of endometrial stromal and peritoneal mesothelial cells ,25,26. HTo evaluate macrophage retention in the peritoneum, we established an in vitro model of MeT-5A peritoneal mesothelial cells to mimic the peritoneal membrane A. At fult-test, p < 0.01; p < 0.005; p < 0.005; Using the developed peritoneum in vitro migration model, the innate migratory rate of polarized macrophages was assessed and compared. Monocytes were polarized to the M0, M1, and M2 macrophages. To recapitulate the egress of macrophages out of the peritoneum and into the lymphatic system, polarized macrophages were added to the upper transwell chamber ; allowed to emigrate to the lower chamber  over 20 h; and counted. After 20 h, a larger proportion of M1 macrophages were retained in the upper chamber compared to M2. When normalized to unactivated M0 macrophages, the M1:M0 ratio was significantly (2.9-fold) lower than the M2:M0 ratio  and cell culture reagents from Life Technologies , unless otherwise stated.g and quantified by staining with Trypan Blue (Sigma) and counting using a hemocytometer. Cells were seeded at 0.2 × 106 cells/mL and grown to 80–100% confluence (0.8 × 106 cells/mL to 1 × 106 cells/mL). Cells were passaged every four to seven days. Cell viability was determined by staining with Trypan Blue and counting using a hemocytometer. THP-1 cells at passages 31–33 were used for experiments.Human monocyte THP-1 cells  were cultured in RPMI medium  supplemented with 10% heat-inactivated fetal bovine serum ; 1% penicillin-streptomycin ; and 1% amphotericin B , henceforth referred to as R10 medium. The FBS was heat-inactivated by being kept in a 56 °C water bath for 30 min. During passage, cells were pelleted at 300× 2. Cells were grown to 70–80% confluence in a 37 °C humidified incubator at 5% CO2 and passaged every three to four days. Cells were trypsinized and pelleted at 300× g. Cell viability was assessed by staining with Trypan Blue and counting using a hemocytometer. Passage 13 and 14 cells were used for experiments.Transformed mesothelial cells  were grown in Medium 199 supplemented with 1.5 g/L sodium bicarbonate, 10% FBS, 1% PS, 3.3 nM epidermal growth factor , 400 nM hydrocortisone, 870 nM zinc-free bovine insulin, 20 mM HEPES, 3.87 µg/L selenious acid, and trace elements of B liquid , henceforth referred to as mesothelial medium. MeT-5A were seeded in T-25 or T-75 flasks  at a density of 6000 cells/cm6 THP-1 were seeded onto 6-well plates (Nunc) in 1.5 mL of R10 containing 25 nM PMA. After 48 h of incubation, the media were replaced with R0 (RPMI 1640 + 1% PS + 1% ampB) for 72 h. Subsequently, M0 cells were polarized to M1 and M2 with the respective cytokines in R2 (RPMI 1640 + 2% HI FBS + 1% PS + 1% ampB). M1 polarization: 20 ng/mL IFN-γ (R&D Systems) and 10 ng/mL LPS; M2 polarization: 40 ng/mL IL-4 (R&D Systems) and 40 ng/mL IL-13 (R&D Systems); M0 polarization: R2 alone was added. All polarization experiments were incubated for 48 h.THP-1 monocytes were differentiated to unactivated M0 macrophages and subsequently polarized to M1 and M2 as described by others, with some modifications ,53. Brie5 MeT-5A were seeded into each transwell insert in 200 µL of mesothelial medium (1.5 × 105 cells/cm2). Care was taken to ensure that the lower chambers were left dry to prevent the premature migration of MeT-5A cells prior to the experiment. Cells were incubated for 24 h to form a confluent monolayer. A transwell insert was fixed after 24 h of seeding of MeT-5A with ice-cold methanol for 15 min. The inner membrane was stained with 100 µL crystal violet for 1 h. This served as a control to visualize and confirm that the mesothelial cells had grown to confluence and formed a monolayer.Sterile 6.5 mm transwell inserts with 5.0 μm or 8.0 μm pore polycarbonate membranes (Corning Transwell) were coated with 50 µL of 0.01% rat tail collagen type I (Corning) in 0.02 M acetic acid and incubated for 30 min at 37 °C. The coated membranes were washed twice with 200 µL 1 × PBS, and inserts were allowed to dry prior to use. Then, 0.5 × 105 macrophage cells in 200 µL migration media were added to each insert. M0, M1, M2 cells or 200 µL migration medium alone (blank) were added to the transwells, respectively. To the lower chamber, 700 µL of migration media was added. The transwells were incubated for 20 h to allow migration. After 20 h, the inner transwell membrane was washed in 1 × PBS, and the migrated cells were fixed in ice-cold methanol for 15 min. The cells were stained with Giemsa for 1 h then washed in milliQ water. Subsequently, the transwell membranes were cut out using a scalpel and mounted onto microscope slides using DPX mounting media and coverslips (VWR). Images at 20× magnification were acquired using an Olympus CKX41 microscope and Olympus cellSens imaging software (version 2.3). Migrated cells were counted using the Photoshop Portable counting tool. The total migrated cells per image were obtained and corrected for the basal migrated MeT-5A cells using the blank. The average number of M0, M1, or M2 macrophages migrated per image was obtained by averaging the replicates.Polarized macrophage cells were harvested by scraping and resuspended in migration media (50% M199 + 50% R2). Immediately prior to macrophage seeding, mesothelial media were removed from the MeT-5A monolayer in the transwell inserts. Then, 1 × 105 macrophage cells in 200 µL migration media were added to the inserts . Cells were treated with 0, 200, 500, and 1000 nM of NVP-231 , a potent and specific inhibitor of ceramide kinase (CERK) . Immunob inserts A. M0, M1After polarization, macrophage culture supernatants were collected and stored at −80 °C. Eight replicates each of the M0, M1, and M2 phenotypes were collected. The undiluted samples were randomized and assayed in duplicates for 45 cytokines, chemokines, and growth factors as previously described  16,54].,54.16,5418 reversed-phase LC  was used to separate reconstituted lipids at 400 µL/min before entering the mass spectrometer. The dwell times were set at 10–50 ms per MRM transition. The MS parameters were as follows: gas temperature 250 °C, gas flow 10 L/min, sheath gas temperature 350 °C, sheath gas flow 11 L/min, capillary voltage 5000 V, and nozzle temperature 1500 V. The column thermostat and autosampler temperatures were maintained at 40 °C and 6 °C, respectively. The mobile phase consisted of 5 mM ammonium acetate in water (mobile phase A) and 5 mM ammonium acetate in methanol (mobile phase B). Sphingolipids were eluted using linear gradients from 60 to 100% B for 2 min, maintained at 100% B for 7 min, eluted by a linear gradient to 5% B for 2 min, held for another 4 min, and finally returned to 60% B for 2 min for the next injection. Deuterated internal standards corresponding to each sphingolipid class were verified for their absence and used for quantification against a nine-point calibration curve . Data acquisition and processing were performed using MassHunter software . Sphingolipid quantification was performed using Agilent Quantitative software (version B.05). Only sphingolipids that had >100 intensity counts (approximately three times the limit of detection of our LC-MS instrument), and were present in >50% of the samples were retained for further analysis.Positive-ionization-mode liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in multiple reaction monitoring (MRM) mode using Triple Quadrupole 6460 with a Jet Stream  was applied for the relative quantification of sphingolipids as previously described, with some modifications ,55. Briet-test was used to make comparisons. The data analyzed were expressed as mean ± standard error, and p-values < 0.05 were considered significant.For the secretome analysis, cytokines were analyzed by partial least squares regression (PLSR) using the nonlinear iterative partial least squares (NIPALS) algorithm (Unscrambler X version 10.1) after the normalization of data by performing log2 transformation. This was carried out to determine if there were specific cytokine signatures (cytokine being the x variable) that could explain the macrophage phenotype (outcome variable). Full cross-validation was applied in PLSR to increase the model performance by leaving out one sample at a time from the calibration dataset, and the model was calibrated on the remaining data points. Under cross-validation, a number of submodels were created based on all the samples that were not kept out in the cross-validation segment. For every submodel, a set of model parameters, including β-regression coefficients, was calculated. The β values of the regression coefficients associated with each x variable (cytokine) provided an indication of how strongly each x variable (cytokine) contributed to the macrophage phenotype (outcome variable). Additionally, for the concentration of each cytokine or sphingolipid, Student’s two-tailed, two-sample equal-variance Macrophages play a key role in the pathogenesis of peritoneal endometriosis. M1 macrophages, which secrete an increased pro-inflammatory cytokine milieu, demonstrated a lower innate migration rate across the mesothelium in vitro due to their lower C1P levels, plausibly leading to the development of chronic peritoneal inflammation in endometriosis. Thus, upregulating the CERK metabolic pathways and/or controlling macrophages to skew towards M2 phenotypes are potentially novel pharmacologic approaches for treating peritoneal endometriosis.


Secondary osteoporosis is triggered mostly by glucocorticoid (GC) therapy. Dexamethasone (DEX) was reported to inhibit osteogenic differentiation in zebrafish larvae and MC3T3-E1 cells in prior research. In this research, we primarily examined the protective impacts of epimedin C on the osteogenic inhibition impact of MC3T3-E1 cells and zebrafish larvae mediated by DEX. The findings illustrated no apparent toxicity for MC3T3-E1 cells after administering epimedin C at increasing dosages from 1 to 60 μM and no remarkable proliferation in MC3T3-E1 cells treated using DEX. In MC3T3-E1 cells that had been treated using DEX, we discovered that epimedin C enhanced alkaline phosphatase activities and mineralization. Epimedin C could substantially enhance the protein expression of osterix (OSX), Runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALPL) in MC3T3-E1 cells subjected to DEX treatment. Additionally, epimedin C stimulated PI3K and AKT signaling pathways in MC3T3-E1 cells that had been treated using DEX. Furthermore, in a zebrafish larvae model, epimedin C was shown to enhance bone mineralization in DEX-mediated bone impairment. We also found that epimedin C enhanced ALPL activity and mineralization in MC3T3-E1 cells treated using DEX, which may be reversed by PI3K inhibitor (LY294002). LY294002 can also reverse the protective impact of epimedin C on DEX-mediated bone impairment in zebrafish larval. These findings suggested that epimedin C alleviated the suppressive impact of DEX on the osteogenesis of zebrafish larval and MC3T3-E1 cells via triggering the PI3K and AKT signaling pathways. Epimedin C has significant potential in the development of innovative drugs for the treatment of glucocorticoid-mediated osteoporosis. Studies pathway . This il pathway . Hence, 2.MC3T3-E1 cells were grown in α-MEM  combined with 10 percent (v/v) fetal bovine serum (FBS)  solution, followed by incubation at 37°C in humid air that contained 5 percent COAfter dissolving Epimedin C in 100 mM dimethyl sulfoxide (DMSO) at a temperature of -20°C, we subsequently diluted it using α-MEM to the designed experimental concentration.3 cells/well). Thereafter, we concurrently cultured the cells with 10, 20, 40, 60, 80, and 100 μM epimedin C for 3 days, sequentially, to assess the toxicity of epimedin C in MC3T3-E1 cells. To evaluate the proliferation of epimedin C in DEX-mediated viability, the cells belonging to the experimental group were concurrently cultured in 10, 20, and 40 μM epimedin C correspondingly for 3 days. After the experiment, CCK-8 (10 μL) and complete medium (90 μL) were added, and cultured for 2 h in a CO2 incubator. The microplate reader  set the wavelength of 450 nm to detect the absorbance. The digested cells were seeded in 24-well plates, and BeyoClick™ EdU-594  was used to evaluate cell proliferation after 24 h. First, diluted EdU (10 µM) was added to the cell culture medium and incubated for 2 h. Cells were fixed with 4% paraformaldehyde for half an hour and treated with 0.5% Triton X-100 for 10 min. After washing with 3% BSA, cells were stained with Click Additive Solution for 30 min at room temperature. After washing with 3% BSA, cells were incubated with Hoechst 33342 stain for 10 min. Finally, the percentage of EdU+ cells at five light fields/well was observed and counted using a fluorescence microscope (Olympus Corporation).The viability of the cells was evaluated utilizing Cells Counting Kit-8 (CCK-8) tests . We then cultured MC3T3-E1 cells in 96-well plates  to conduct ALP staining.5 cells/well) using an osteogenic induction medium . Cells belonging to the experimental group were subsequently subjected to 10, and 20 μM epimedin C treatment, correspondingly, and 10 μM DEX for 21 days. This was followed by fixing of the MC3T3-E1 cells with cell fixative for 20 min, rinsing twice in PBS, and staining for 30 min utilizing alizarin red S. Alizarin red-stained mineralization nodes were photographed using an inverted microscope. The mineralization nodes stained with alizarin red S were isolated using 10 percent cetylpyridinium chloride , and the absorbance was quantitatively detected at 562 nm wavelength.Alizarin red staining was utilized to assess the mineralization nodes of MC3T3-E1 cells. The next step involved the induction of MC3T3-E1 cells , Anti-ALPL , Phospho-Akt (Ser473) , anti-RUNX2 , Akt (pan) (C67E7) , Phospho-PI3K p85 alpha (Tyr607) , PI3 kinase P110 alpha Antibody  and anti-β-actin antibody . Incubation of the PVDF was done at 4°C throughout the night with diluted antibodies. We subjected the PVDF to 1 h of incubation with goat anti-mouse IgG H&L (HRP)  or with goat anti-rabbit IgG H&L (HRP)  at 25°C. Chemiluminescence analysis using a gel imaging system  after incubating proteins with ECL luminescent solution.4, and 0.33 mmol/L CaCl2. Epimedin C and dexamethasone to appropriate concentrations and dissolved them into the culture water of zebrafish larvae for administration. This was followed by staining of the zebrafish larvae on nine dpf using alizarin red S and calcein staining. Firstly, zebrafish larvae were immobilized for 2 h in 4 percent polyformaldehyde and then dried for 30 min in 50 percent ethanol. The zebrafish larvae were once again bleached for 30 min using 1 percent H2O2 and 1 percent KOH. The zebrafish larvae were then subjected to staining for 6 h with 0.01 percent alizarin red staining in 0.5 percent KOH. Subsequently, the zebrafish larvae were decolorized for a duration of 6–8 h in differing concentrations of 0.5 percent KOH and glycerin . The lateral and ventral views of zebrafish were photographed with the aid of a stereomicroscope. For calcein staining, the zebrafish on the nine dpf were anesthetized with anesthesia, then 0.2% calcein solution was added for 15 min, rinsed thrice using water, and photographed with an upright fluorescence microscope. We anesthetized the zebrafish TG (ola.sp7:nlsGFP) treated with anesthesia, and placed them under an upright fluorescence microscope for fluorescence photography. Image-Pro Plus  was utilized to examine the mineralized region and integrated optical density (IOD) of skull alizarin red staining.Shanghai FishBio Co., Ltd. provided wild-type zebrafish larvae (AB strain) and zebrafish TG (ola.sp7:nlsGFP). Culturing of AB strain larvae was done within isothermal settings at 28.5°C in a medium that contained 10 ppm methylene blue, 5 mmol/L NaCl, 0.17 mmol/L KCl, 0.16 mmol/L MgSOp < 0.05 were interpreted as having statistical significance.The data were reported as mean ± standard deviations (SD). SPSS 17.0 was utilized to analyze statistical data. A one-way analysis of variance (ANOVA) was utilized for comparing several groups and statistical results with p < 0.01), while epimedin C (10 and 20 μM) had no effect on the inhibition of cell proliferation   and epimedin C . The findings of ALP staining and ALP activity illustrated that adding DEX considerably suppressed the function of ALP in MC3T3-E1 cells. Contrastingly, the inhibited ALP activity was significantly alleviated when epimedin C was added ( < 0.01) .p < 0.01)  and epimedin C  for 21 days. The mineralization nodes were visualized using Alizarin red staining. We found that DEX (10 μM) inhibited mineralization nodes of MC3T3-E1 cells in contrast with OM. Nevertheless, the inhibited effect was significantly alleviated when epimedin C was added ( < 0.01) .In order to explore the impacts of epimedin C on osteogenesis-related proteins, we extracted protein for 7 days. The findings demonstrated that the expression levels of OSX, RUNX2, and ALPL protein were substantially reduced in the DEX group, whereas the epimedin C group with 10 and 20 μM reduced the inhibitory impacts of DEX and considerably elevated the RUNX2, OSX, and ALPL protein expression levels in contrast with DEX group .The PI3K-AKT signaling pathway is critical for osteogenic differentiation. In DEX-treated MC3T3-E1 cells, we performed western blotting to quantify the proportions of AKT, phosphorylated AKT (p-AKT), PI3K, and phosphorylated PI3K (p-PI3K). In contrast with OM, DEX suppressed PI3K and AKT phosphorylation as illustrated by Western blotting. In contrast, epimedin C promoted the phosphorylation of PI3K and AKT in MC3T3-E1 cells subjected to DEX treatment on day 3 .p < 0.01). However, the use of LY294002 significantly inhibited ALP activity and mineralized nodules in MC3T3-E1 cells (p < 0.01) , DEX, and epimedin C. Epimedin C (20 μM) considerably promoted the ALP function and mineralization nodes in MC3T3-E1 cells that had been treated using DEX in contrast with the DEX group ( < 0.01) .Mineralization of the bones is an essential marker of bone development. The development of zebrafish bones was studied utilizing alizarin red S and calcein staining. The extent of mineralization of the skull can be assessed by performing alizarin red S and calcein with area staining. The DEX group’s bone mineralization region of the skull was clearly suppressed, according to the findings. In zebrafish, the administration of epimedin C (10 and 20 μM) ameliorated the impacts of DEX on the reduction in the mineralization area of bones in the skull . Bone map < 0.01). In DEX-mediated reduction of skull mineralization in Zebrafish, however, the introduction of LY294002 greatly suppressed the IOD and mineralization area of the skull elevated by epimedin C (p < 0.01) , DEX, and epimedin C. The results illustrated that epimedin C (20 μM) considerably increased the integrated optical density (IOD) and mineralization area of skull, compared with the DEX group ( < 0.01) .Glucocorticoid-induced osteoporosis (GIOP) is the most frequent secondary contributor to osteoporosis, resulting in severe morbidity. For the treatment of osteoporosis, postmenopausal patients with osteoporosis should choose estrogen therapy, however, it is not advised to utilize it for an extended period of time . BisphosAlkaline phosphatase is a well-known indicator of early bone formation and performs an instrumental function in bone growth . Cell mi(−/−) mice exhibit a craniofacial skeletal phenotype with severely diminished bone mineralization (RUNX2, a constituent of the Runx family of transcriptional factors, is essential for osteoblast differentiation, bone production, and chondrocyte maturation . Osterixlization . This relization . Therefoosteoblasts in vivo and in vitro via the PI3K/Akt signaling pathway (In osteoporosis, the PI3K/Akt signaling cascade is triggered, which performs a fundamental function in the osteoclast’s hyperactivation as well as the differentiation and maturation of osteoblasts . Astraga pathway . DEX supThe zebrafish is a feasible model organism that has been used in scientific research with great success. The zebrafish larvae are highly transparent, making it possible to readily observe their bone morphology. As a result, zebrafish might serve as an excellent model organism for investigating bone growth . The mosThe current research demonstrated the involvement of epimedin C in alleviating the antagonistic activity on osteogenic differentiation mediated by DEX via the mechanism of triggering the PI3K/AKT signaling pathway. Overall, our findings illustrated that epimedin C may be a promising option for development as a novel treatment approach for GIOP.


Mest) is an imprinted gene that is transcribed from the paternal allele. It is a marker of adipose tissue expansion; however, it is uncertain whether Mest expression promotes or suppresses adipogenic differentiation. To elucidate the effects of Mest expression on adipogenic differentiation, we transfected an expression vector or siRNA for mouse Mest into 3T3-L1 mouse preadipocyte cell line.The mesoderm-specific transcript (Mest overexpression decreased lipid accumulation. Conversely, gene silencing of Mest increased the accumulation of lipid droplets in adipocytes. These results demonstrate that Mest negatively regulates adipocyte differentiation. Further, Mest induced trans-differentiation of 3T3-L1 cells into hepatocytes, and its overexpression induced the expression of hepatocyte marker genes, including albumin and α-fetoprotein. In the presence of dexamethasone, the forced expression of the Mest caused morphological changes in 3T3-L1 cells. Cells were flat and polygonal shapes, with an increased accumulation of intracellular glycogen and other features that are typical of hepatocytes. Therefore, Mest inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inducing hepatocyte trans-differentiation.In differentiated 3T3-L1 adipocytes, The online version contains supplementary material available at 10.1186/s13104-022-06051-x. Mest) is an imprinted gene that is transcribed from the paternal allele and is expressed in the embryonic and extraembryonic mesoderm [The mesoderm-specific transcript  were seeded onto 60-mm dishes one day prior to transfection in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Calf serum (CS) . The cells were treated with a complex of Lipofectamine 3000  and 5 µg of plasmid DNA. After 24 h of incubation, the cells were trypsinized and seeded into 100-mm dishes; then, neomycin-resistant cells were selected with 1 mg/mL G418. The 3T3-L1 preadipocytes, mock-transfected cells, and Mest expression vector-transfected 3T3-L1 cells, designated 3T3-L1-Mest, were maintained in DMEM supplemented with 10% CS at 37 °C in the presence of 5% CO2.We employed the pcDNA3.1 plasmid vector containing both the nce gene . The 3T3The total cellular RNA was isolated using RNAiso Plus . The RNA from each sample was reverse-transcribed using a High-Capacity cDNA RT kit . PCR was performed within a linear range of amplification using the primer sets indicated in Additional file g for 15 min at 4 °C, aliquots of the supernatants were treated with 25 mM mercaptoethanol, boiled at 100 °C for 2 min, and 20 μg of protein from each sample was resolved using a sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in a 10% gel. The proteins were transferred from the gel to a polyvinylidene difluoride membrane in a basic transfer buffer  using an electroblotter. Proteins were visualized by immunostaining with a primary antibody  or a rabbit anti-β-actin IgG  ), a secondary antibody (either a horseradish peroxidase (HRP)-conjugated anti-goat IgG  or HRP-conjugated anti-goat IgG antibody (Abcam Ltd.)), and a chemiluminescent substrate  using an LAS-4000 mini image analyzer. Prestained protein markers  were used as the standard molecular mass proteins.The cells were digested using mammalian protein extraction reagent , which contained a protein inhibitor cocktail  After centrifugation at 204004 cells/cm2 and precultured for two days. Adipogenic differentiation of preadipocytes was induced on day 0 by replacing the original medium with DMEM containing 10% fetal bovine serum (FBS) (Sigma-Aldrich) supplemented with an adipogenic cocktail , and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich). After two days (day 2), the culture medium was changed to DMEM containing 10% FBS supplemented with 1 µg/ml insulin, and the cells were cultured for two more days. On day 4, the medium was replaced with DMEM containing 10% FBS, subsequently the cells were cultured until day 8. For trans-differentiation into hepatocytes, the cells were treated with 1 µM DEX in DMEM containing 10% FBS for four days, and then the medium was replaced and cells were treated DMEM containing 10% FBS for four more days.The cells were seeded at a density of 3 × 10Lipid accumulation was evaluated by measuring Oil Red O retention. The cells were fixed with 4% paraformaldehyde  and stained with 3 mg/mL Oil Red O (Sigma-Aldrich) in 60% 2-propanol. To quantify intracellular lipid accumulation, the absorbance of Oil Red O was measured at 520 nm using a microplate spectrophotometer.Mest mRNA and a RNAi negative control  at a final concentration of 20 nM using Lipofectamine RNAi MAX . The sequences of the siRNAs and negative control RNAs are shown in Additional file We transfected the cells with stealth siRNAs targeting mouse The cells were fixed in 4% paraformaldehyde. After rinsing in distilled water, the samples were oxidized in 0.5% periodic acid  for 10 min and then reacted with Schiff’s reagent  for 30 min. Cells were rinsed again, this time in tap water for 5 min. Samples were counterstained with Mayer’s hematoxylin for 1 min and rinsed with distilled water.http://imagej.nih.gov/ij).The cells were trypsinized and resuspended in DMEM + 10% CS. The cell suspension was then applied to a cell counter plate. Images were obtained using the TrueChrome II . The surface area of the cells in the images was calculated using the software functions of the adjusted threshold and analyzed using ImageJ  was used for the statistical analysis. Data sets were compared for significant differences by one-way analysis of variance using the Dunnett’s test or the paired Student’s Mest expression levels between 3T3-L1 cells, 3T3-L1-Mest, and mock-transfected cells. While the expression level of Mest in mock-transfected cells was similar to that of the parent cells, Mest mRNA was significantly expressed in the 3T3-L1-Mest cells  and α-fetoprotein (AFP), in 3T3-L1 mock and Mest cells. Increased expression of ALB mRNA and weak expression of AFP mRNA were observed in 3T3-L1-Mest cells  are multipotent cells, that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, and adipocytes , 14. Thesylation . We hypo. demonstrated that the Mest is a marker of adipose cell size in mice [Mest were positively correlated with adipocyte size in mouse adipose tissue [Mest overexpression in 3T3-L1 preadipocytes promotes adipocyte differentiation [Mest gene was induced, differentiation was suppressed rather than promoted  and (B) Original images of agarose gel electrophoresis from Figure 1 A.(C) and (D) Original images of western immunoblot data from Figure l B. M, marker; P, parent cells.Additional file 4: Figure S2. (A) and (B) Original images of agarose gel electrophoresis from Figure 2A. (C) and (D) Original images of western immunoblot data from Figure 2B. M, marker; P, parent cells. M, marker; NS, nonspecific bands.Additional file 5: Figure S3. (A), (B), (C) and (D) Original images of agarose gel electrophoresis from Figure 3B. M, marker.


Additionally, the BNTP and BDBC molecules also showed the HOMO–LUMO orbital gaps of 5.96 eV and 6.06 eV, respectively. Furthermore, the computation of UV-visible spectra of the titled compounds indicated a limited and/or no absorption above the 400 nm region, directing a good transparency and NLO property trade-off for both synthesized compounds that may play a significant contribution in the future for optoelectronic technologies.In the present study crystalline unsymmetrical diarylidene ketone derivatives BNTP and BDBC have been prepared by two sequential acid catalyzed aldol condensation reactions in a one pot manner. The crystal structures of both compounds were confirmed by single crystal X-ray diffraction analysis which revealed the presence of H-bonding interactions of type C–H⋯O, along with weak C–H⋯π and weak π⋯π stacking interactions that are involved in the crystal stabilization of both organic compounds. Hirshfeld surface analysis is carried out for the broad investigation of the intermolecular interactions in both compounds. The quantum chemical investigation was performed on the optimized molecular geometries of BNTP and BDBC to calculate optical and nonlinear optical (NLO) properties. The density functional theory (DFT) study showed that the third-order NLO polarizabilities of compounds BNTP and BDBC are found to be 226.45 × 10 In the present study crystalline unsymmetrical diarylidene ketone derivatives BNTP and BDBC have been prepared by two sequential acid catalyzed aldol condensation reactions in a one pot manner. Moreover, human health is also facing risks on account of the frequent use and misuse of anti-infectious medication causing the emergence of multidrug resistant species. Thus, constant efforts are required to develop new therapeutic drugs with effective potential against the resistant microbes. In this regard, curcumin (derived from rhizome) is considered to be one of most important classes of natural products with numerous biological and chemical applications.1 Traditionally, curcuminoids  have been used as herbal supplements and as spices in the south Asia reign. Beside this, the powdered form of turmeric has find vast applications in medicines,2 cosmetics, food flavoring, and fabric dying etc.3 Symmetrical and unsymmetrical curcuminoids are pharmaceutically active compounds,4 exhibiting various medicinal uses such as anti-parasitic,5 antibacterial,6 antioxidant,7 antimitotic,8 antimalarial,9 antitumor,10 neuro-protective,11 antimicrobial,12 antifungal,13 anti-inflammatory,14 anti-tubercular14 anti-fertility,15 anti-viral,16 anti-tubulin17 and anticancer.18The increasing global population and climate change has a meaningful effect on social health. The increase in pollution, greenhouse gases, deforestation, global warming, floods et al.,19 reported the conjugates of fullerene-curcumin for biological and photovoltaic applications and the reported conjugates have displayed HOMO/LUMO energy levels comparable to perovskite solar cells. Curcuminoid derivatives as borondifluoride complexes has also reported for optical and photovoltaic behavior as donor–acceptor–donor motifs.20 Saeed et al.,21 have reported the nonlinear optical properties of dihydropyridone curcumin derivatives through determination of nonlinear refractive index. The study of NLO aspects has captivated the interest of the scientific community  as materials with appreciable NLO capabilities have found numerous applications in the scientific arena,22 especially in the fields of biophysics, medicine, solid physics, atomic science, material science and chemical dynamics are worth mentioning.23 It is important to reference that organic materials have found considerable appreciation in the scientific community because of strong NLO capabilities accompanied with substantial advantages over the inorganic counterparts due to their low toxic nature, little cost, and simplicity in their synthetic process.24 Density functional theory  is commonly applied as an effective tool to analyze the NLO characteristics of organic materials.Furthermore, Castro In this scenario, here we are presenting our findings regarding the acid catalyzed one-pot synthesis of unsymmetrical diarylidene cycloalkanones and its single crystal exploration accompanied with DFT exploration .via glass delivery tube.Standard chemicals were purchased and used without further purification from the reputed chemical suppliers like Sigma-Aldrich, Acros Chemicals, Macklin, and TCI. The reaction progress was observed by using TLC plates (pre-coated silica gel G-25-UV254). The dry HCl used as catalyst was generated by the reaction of sodium chloride and concentrated sulfuric acid and passed through reaction mixture H-pyran-4-one and benzaldehyde were taken (1 : 1) in a two necked round bottom flask (50 mL) using chloroform as solvent (20 mL). Dry HCl gas was passed through this reaction mixture till its color turns into red. Stirring was sustained for 4 to 6 hours. After accomplishment of intermediate 1in situ (monitered by TLC), 1 equivalent of 4-nitrobenzaldehyde was added and the reaction mixture was further stirred for 5 hours.25,26 After completion (TLC indication), the solvent of the reaction mixture was evaporated by rotary evaporator and the residue was diluted using ethyl acetate. The extraction was performed with NaHSO3 solution and the organic layer was dried with anhydrous Na2SO4. The crude product was purified using column chromatography. Finally, the recrystallization in ethanol gave the titled compound BNTP in 68% yield , 1 equivalent of 2,3-dimethoxy benzaldehyde was added and the reaction mixture was further stirred for 5 hours. After completion (TLC indication), the solvent of the reaction mixture was evaporated by rotary evaporator and the residue was diluted using ethyl acetate. The extraction was performed with NaHSO3 solution and the organic layer was dried with anhydrous Na2SO4. The crude product was purified using column chromatography. Finally, the recrystallization in ethanol gave the titled compound BDBC in 63% yield  in a two necked round bottom flask (50 mL). Dry HCl gas was passed through this reaction mixture till its color turns into red. Stirring was sustained for 4 to 6 hours.3% yield .27 and SHELXL 2019/228 software were used for structure solution and refinement, respectively. ORTEP-III,29 PLATON30 and Mercury version 4.031 were used for the graphical representations of single crystal XRD results. All the non-hydrogen atoms were refined by using anisotropic displacement parameters. The isotropic displacement parameters were assigned to H-atoms. The hydrogen atoms were refined by using riding model. The details of SC-XRD analysis are given in 1H and 13C NMR spectra of BNTP and BDBC are shown in Fig. S1 and S2,The unsymmetrical diarylidene ketone derivatives, BNTP and BDBC have been prepared by the two sequential acid catalyzed aldol condensation reactions in a one pot manner. The single crystal XRD data of both compounds was collected on Bruker Kappa Apex-II CCD diffractometer with molybdenum x-rays source. The SHELXT-20141H NMR  δ 8.34–8.27 , 7.87 , 7.51–7.43 , 7.36 , 4.98 , 4.93 . 13C NMR  δ 185.0, 147.7, 141.0, 137.6, 136.2, 134.4, 133.2, 132.5, 130.8, 130.6, 129.8, 128.8, 123.9, 68.7, 68.3. FT-IR (cm−1):  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.ngrt" format="PBM" resolution="300" x-size="9" y-size="11" xml:space="preserve">000110010001001100000000000000000000011000010001100110000100100000100100000100100000111000000010000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.454545pt" height="16.000000pt" viewBox="0 0 13.454545 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M240 840 l0 -40 -40 0 -40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 80 0 80 0 0 -40 0 -40 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 -80 0 -80 0 0 40 0 40 -80 0 -80 0 0 -40z M80 520 l0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -160 0 -160 40 0 40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 120 0 120 40 0 40 0 0 80 0 80 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -160 0 -160 -80 0 -80 0 0 160 0 160 -40 0 -40 0 0 40 0 40 -80 0 -80 0 0 -40z"/></g></svg> 3053  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C–H), 2833, 2924 , 1670 (CO), 1612 (CC), 1492 (–CH2), 1444 (–NO2) 1265 (C–O).1H NMR  δ 7.96 , 7.60 , 7.47–7.41 , 7.41–7.35 , 7.18 , 7.11 , 6.96 , 3.92–3.87 , 3.10–3.02 . 13C NMR  δ 196.3, 153.0, 149.3, 138.4, 137.5, 136.3, 135.9, 133.7, 130.7, 130.1, 129.3, 128.8, 128.3, 123.8, 121.6, 113.6, 61.5, 55.9, 26.6. FT-IR (cm-1):  3053 (C–H), 2833, 2924 , 1670 (CO), 1612 (CC), 1492 (–CH2), 1265 (C–O).H-pyran-4-one ring D (C8–C12/O3/O4) is puckered with puckering parameters (Q = 0.5328 (15) Å, θ = 59.51 (16)°, φ = 2.9 (2)°) and adopts a conformation very similar to envelope conformation. The dihedral angles D/A and D/B are 25.4 (4)° and 32.2 (4)°, respectively. The molecules of BNTP are interlinked in the form of dimers through C–H⋯O bonding to complete R22(12) loop, one of the O-atom of nitro group acts as H-bond acceptor for meta-positioned CH of phenyl ring attached with nitro group  is not planar with group A. The atoms of group C are at the distance of −0.0139 (4) and 0.0150 (3) Å, respectively from plane of group A whereas atoms of group D are at the distance of −1.1773 and 0.0631 Å, respectively from plane of group A. The cyclopentanone ring E (C11–C14/O3) is planar with r.m.s deviation of 0.0508 Å with dihedral angles E/A and E/B are 27.3 (7)° and 17.2 (1)°, respectively. The ring E is puckered with Pseudorotation parameters (P = 311.7 (6) Å, θ = 11.2(1)° for reference bond C10–C11) and adopts an exact envelope conformation on C14 atom. The molecules of BDBC are interlinked in the form of dimers through C–H⋯O bonding to complete R22 (14) loop, the carbonyl O-atom of acts as H-bond acceptor for ortho-positioned CH of unsubstituted phenyl ring , shape index, curvedness etc. We are going to explore the information about the single crystals provided by Hirshfeld surface over dnorm and shape index. Red, blue and white colour are the main feather of the HS plotted over dnorm. Red and blue spots represent short and long contacts, respectively.38–41 White spots represent the contacts for which the center to center distance between the atoms is equal to the sum of the van der Waals radii of the atoms involved. 42 Such sort of regions are present in  for BNTP whereas the major E_tot energies  for BDBC. The electrostatic energy is mostly attractive for the pair of molecules but it may be repulsive for a pair of molecule. In the present case, the electrostatic energy is positive for two pair of molecules  connected by  in BNTP whereas for BDBC, the electrostatic energy is positive only for a single pair of molecules (0.8 kJ mol−1) connected by . The calculations inferred that the dispersion energy is the major contributor of the total attractive energy contribution for both compounds. In order to understand the topology of the single crystals, the energy frameworks are constructed in terms of joining the centroids of the interacting molecules by the cylinders are displayed in 52 The radius of the cylinder is proportion to the strength of the interaction. The cut off energy is set to be 5 kJ mol−1 and size of the cylinder is set to be 80 and cluster of molecules within 1 × 1 × 1 unit cell is used for generating the energy frameworks. For both compounds, the contribution of the repulsive energy is greater than the contribution of the electrostatic energy in defining the total energy as the radius of cylinders for repulsive energy framework is greater than the radius of the cylinder for electrostatic energy framework is used for the calculations. A cluster of molecules within 3.8 Å of the reference molecule is generated for the calculations of the interaction energy that is the sum of four kind of energies .for BNTP  and for for BNTP . The scaframework.53–55 It is assumed that all the atoms are spherically symmetric while calculating voids. The void volume is 149.02 Å3 for BNTP and 273.41 Å3 for BDBC. The volume occupied by the voids is 9.72% in BNTP and 8.36% in BDBC. As the voids occupy very small space in both compounds so it indicates that there is no large cavity in the crystal packing of both compounds and both compounds are expected to have good mechanical properties M06-2X method with same basis set 6-311G*. The linear and nonlinear optical polarizabilities were calculated using Polar keyword. Our earlier pure computational studies shows more details58–60 to calculate nonlinear (NLO) responses of organic molecules using Kohn–Sham perturbation method through Gaussian 16.61 It is possible to determine the average third-order nonlinear polarizability (γ), isotropic (<α>) and anisotropic linear polarizability (Δα), and dipole moment μ, of the molecules using the following formulas.All quantum chemical calculations were achieved through the Gaussian 16 program.αiso) can be calculated by following equations:The average isotropic polarizability (α) and average static second hyper polarizability, these can be calculated by following equations:For anisotropy of polarizability  greater than BNTP, while the anisotropic polarizability values of BNTP and BDBC have only a small difference of 1.08 × 10−24 esu. Among the individual components, αzz shows maximum value of linear polarizability in both BNTP and BDBC molecules as 79.35 × 10−24 esu and 73.03 × 10−24 esu, respectively, which shows that polarization and ICT occurred mainly along z-axis. The linear isotropic polarizability αiso and anisotropic Δα for BNTP and BDBC are about similar, which indicates direction effects are negligible during induced polarization.The most basic electric response qualities are linear polarizability, and the calculation of these values has provided an excellent foundation for evaluating the accuracy of electronic state calculations with various theoretical models in the field of quantum chemistry.γ is characteristic of two photon absorption phenomena. For second order hyperpolarizability, along with individual tensors, we have also calculated average third order polarizability(<γ>) that are shown in γ> of BNTP and BDBC compounds are considerately larger mounting to 226.45 × 10−36 esu and 238.72 × 10−36 esu, respectively, which indicate their relatively higher NLO response. The average second-order NLO polarizability of compound BDBC is larger than BNTP. It will be also interesting to make a comparative analysis of <γ> amplitudes of our synthesized compounds to some other standard and previously known molecules. For this purpose, we have also calculated the <γ> amplitude of para-nitroaniline (p-NA), which is a typical push–pull organic compound and has been used as a reference molecule in several investigations. The <γ> amplitude of p-NA was found to be  studied by Essid et al.;64 acyl thiourea BTCC derivative (27.30 × 10−36 esu at M06-2X/6-311G*) studied by Ashfaq et al.;65 donor–acceptor chalcone derivatives (79.31 × 10−36 esu at PBE0/6-311G**) studied by Muhammad et al.,60 and also better than calculated <γ> amplitudes for C60 fullerene and its different heteroatom doped molecules.66 This comparison indicates a semi-quantitative insight for the real-time potential of our indigenously synthesized molecules in NLO applications.Besides linear polarizability and second-order NLO polarizability, third-order NLO polarizability of BNTP and BDBC compounds have been also calculated at the same M06-2X/6-311G* levels of theory. The third order non-linear polarizability γ> amplitudes can be traced from wildly used three-state approximation67 by means of optical parameters as calculated by TD-DFT method and given in γzzzz in current calculations can be correlated with optical transition parameters. According to three-state approximation, a lower transition energy with larger oscillator strength and larger change in dipole moment between ground to excited states can cause a larger γzzzz amplitude. The γzzzz amplitudes of BNTP and BDBC compounds are found to be 892.88 × 10−36 esu and 549.19 × 10−36 esu, respectively. The larger amplitude of γzzzz for BNTP might be attributed to the lower energy transitions (S0 to S1 and S0 to S3) in BNTP as compared to BDBC compounds as shown in Regarding the origin of relatively larger NLO polarizabilities, it is seen that both entitled molecules illustrate a decent intramolecular charge transfer character. It is important to pen down that origin of <n and π orbitals of the ground state to the σ* and π* orbitals of higher excited states.68 The absorption spectrum of both molecules were obtained through time-dependent-DFT calculations as well as through experimental analysis as shown in n to π* and π to π* transitions, respectively, as perceived from The UV– isible spectrum offers useful information on the electronic structure, optical and photovoltaic properties of the molecule. The absorption peaks in the optical spectrum frequently involve the transfer of electrons from σ, 69–71 The way of interaction of the molecule with other is determined by these orbitals. The kinetic stability and chemical reactivity of a molecule can be determined using the energy gap between frontier orbitals. A molecule with a small frontier orbital gap is more polarizable and is often associated with low kinetic stability and strong chemical reactivity.72 The lower value between HOMO and LUMO energy gap in BNTP than BDBC  makes it less stable and more reactive. The LUMO orbital primarily act as electron acceptor while HOMO is the orbital that mostly act as an electron donor. The frontier molecular orbitals of BNTP and BDBC compounds are shown in The most important orbitals in the molecule are the lowest-lying unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), also known as frontier molecular orbitals (FMO). The FMOs play a crucial role in molecule interactions as well as molecule electronic spectra.The interaction of energy between electrons of the molecule produced from the electrical charge and a unit positive charge is used to describe the molecular electrostatic potential in the environment around the molecule. The molecular electrostatic potential (MEP) is related to electron density and is a useful tool for determining where electrophilic and nucleophilic assaults. It also enables to visualize the size, charge density, shape, and charge related properties of molecules. To find the reactive sites for nucleophilic and electrophilic attacks, MEP of BNTP and BDBC compounds were draw and shown in via the Aldol condensation reaction. The title compounds BDBC and BNTP are characterized by the single crystal x-rays diffraction and NMR analysis. Crystal packing of both compounds are stabilized by C–H⋯O, C–H⋯π and π⋯π stacking interactions. The intermolecular interactions are comprehensively explored by Hirshfeld surface analysis. In order to explore the topology of BDBC and BNTP, the interaction energy between molecular pairs are calculated and energy frameworks are constructed. Void analysis inferred that there is no large cavity in the crystal packing of both compounds. The quantum chemical computations were also successfully applied on the synthesized molecules. The geometries were optimized to the lowest energy ground states and compared with their counterpart experimental bonding parameters. The optimized geometries were further used for the calculation of optical and NLO properties. The average static third order nonlinear polarizability <γ> of BNTP and BDBC compounds are considerately larger mounting to 226.45 × 10−36 esu and 238.72 × 10−36 esu, respectively. A semi-quantitative evaluation illustrates that BNTP and BDBC compounds possess the <γ> amplitudes, which are approximately 28 and 30 times larger than that of p-NA, respectively, as calculated using same methodology. The non-zero amplitudes of third-order NLO polarizabilities indicate that entitled molecules possess decent potential for their future applications for optoelectronic and NLO applications.The crystalline unsymmetrical diarylidene alkanones are synthesized Akbar Ali: supervision, writing – original draft, data curation. Shabbir Muhammad: DFT study, Software, supervision, methodology. Zia Ud Din: data curation, writing – original draft preparation. Muhammad Ibrahim: statistical analysis, data curation, writing – original draft preparation. Abdullah G. Al-Sehemi: methodology, software conceptualization. Muhammad Ashfaq & Muhammad Nawaz Tahir: writing – reviewing and editing, resources of the SC-XRD part. Edson Rodrigues-Filho: supervision, resources. Dania Gull: methodology, writing – original draft preparation, Muhammad Suleman: reviewing, editing, UV and IR data curation.All authors agree to declare no conflict of interest regarding any content or publication of this work in the form of a manuscript.RA-013-D2RA07681K-s001RA-013-D2RA07681K-s002


N2 = N2/(N2+Ar). XPS measurements were done one month later. Oxides were detected on the top surface of the samples. 2p, 3d and 4f core level peaks were fitted in order to determine accurately the chemical composition of the nitride films. Al2p, Ti2p, Zr3d, Ta4f, and Hf4f reveal the formation of nitrides of all elements constituting the films. Atomic percentage of each element was calculated revealing an increase of nitrogen loading and decrease of the metallic fractions of the elements as RN2 grows from 5% to 50%. Nitridation behaviour of each element, as a function of the nitrogen flow rate, is investigated and presented.The data presented in this article are related to the published research of “Effect of nitrogen content on structural and mechanical properties of AlTiZrTaHf(-N) high entropy films deposited by reactive magnetron sputtering”. This database contains X-ray photoelectron spectroscopy (XPS) measurements, performed in order to determine the extents of nitrides formed in AlTiTaZrHf high entropy films. The latter were prepared by DC magnetron sputtering technique in reactive mode by adding the nitrogen to argon gas. The nitrogen flow rate is calculated by R A table of all parameters  of each fitting for each peak is provided. The data are accessible with the following link: https://figshare.com/s/a4d038f514016b19bd6b.Additional amazed data which correspond to the fitting of the peaks of all elements of XPS measurement are presented figshare sever. All these data are uploaded with excel files including then the fitting peaks of N1s, Al2p, Ti2p, Zr3d, Ta4h and Hf4f as a function of the nitrogen flow rate  and 460.0 eV (Ti2p1/2), respectively. The nitride TiN is formed when the nitrogen is introduced. The components of Ti-N are found at 456.6 eV (Ti-N(Ti2p3/2)) and 462.4 eV (Ti-N(Ti2p3/2)). Intermediate states were also found and correspond to Ti-O-N, its components are at 457.8 eV (Ti-O-N(Ti2p3/2)) and 463.5 eV (Ti-O-N(Ti2p1/2)) N2 increases from RN2 = 20% to 50%, Ti is totally transformed to nitride and no metallic peaks is observed . The peaks of Hf metal are fund around 14.6 eV and 16.3 eV for orbital 4f7/2 and 4f5/2 respectively. They disappear when RN2 increases from 20 to 50% and Hf-N nitrides are formed. The peaks of these nitrides are located at 15.9 eV for orbital 4f7/2 and 17.6 eV for orbital 4f5/2.Ta4f and Hf4f XPS spectra are plotted together and presented in the N2. The atomic percentage was estimated from the fitting of all XPS data according to RN2. Two metals Ta and Hf show a quick increasing when RN2 increases from 5 to 20% followed by a stable evolution as RN2 continuous to grow from 20 to 50%. However, Ti behaviour under nitridation effect displays a very slight or even quasi-stable evolution as RN2 increases. A part of Ti-N is oxidised but the quantity of oxynitride decreases when RN2 increases. The Al element shows a percentage varying between 58.9 and 70% and is the first nitride element compared to others.N2 = 5%), Hf-N and Ti-N are found at low extents compared to the other nitrides Al-N, Ta-N and Zr-N. However, when RN2 increases, Ti-N and Al-N exhibit a quasi-stable evolution, whereas the quantities of other nitrides increase to reach the saturation point followed by steady state.After introducing a small amount of nitrogen (RThe data are the authors' own original work, which have not been previously published elsewhere.Mohamed El Garah: Conceptualization, Investigation, Formal analysis, Validation, Writing – review & editing, Writing – original draft. Djallel Eddine Touaibia: Writing – review & editing. Sofiane Achache: Writing – review & editing. Alexandre Michau: Conceptualization, Funding acquisition. Elizaveta Sviridova: Investigation, Methodology, Data curation. Pavel S. Postnikov: Investigation, Data curation. Mohamed M. Chehimi: Investigation, Formal analysis, Writing – review & editing. Frederic Schuster: Conceptualization, Funding acquisition. Frederic Sanchette: Conceptualization, Funding acquisition, Writing – review & editing.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.


Long intergenic non-protein coding RNA 1833 (LINC01833) exhibits elevated expression in the non-small cell lung cancer (NSCLC) tissues, while its molecular mechanism in NSCLC progression remains elusive. Herein, the proliferation, migration, invasion as well as apoptosis of NSCLC cells were assessed. The potential N6-methyladenosine (m6A) modification site was predicted by the m6aVar tool. RNA pulldown and m6A-specific immunoprecipitation assays were used to detect the interaction between LINC01833 and methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3). RNA pull-down together with mass spectrometry were performed to assess the binding relationship between LINC01833 and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) in NSCLC. Tumor xenograft mice model was established, and the tumor size and weight were measured. The results demonstrated that LINC01833 expression was elevated in NSCLC samples. Overexpression of LINC01833 promoted proliferative, migratory, and invasive abilities and inhibited HCC827 cell apoptosis. LINC01833 knockdown inhibited tumor growth in mice. LINC01833 is further demonstrated to be modulated by METTL3, which is highly expressed in NSCLC samples. In addition, RNA pulldown and m6A-specific immunoprecipitation assays indicated that LINC01833 might form a complex with HNRNPA2B1. In conclusion, m6A transferase METTL3-induced LINC01833 m6A methylation promotes NSCLC progression through modulating HNRNPA2B1 expression. Our findings indicated that LINC01833 might be a therapeutic target for NSCLC.  Since NLncRNAs have over 200 nucleotides in length and lack the frame of open reading, with little or without protein coding ability ,5. It haN6-methyladenosine (m6A) methylation widely exists in mammals and regulates gene expression after transcription without changing the base sequence . m6A metHeterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) is a protein that ubiquitously participates in RNA‐binding and pre‐RNA processing, regulating cancer cell metabolism, proliferation, migration, invasion, and apoptosis . HNRNPA2Based on bioinformatics analysis, there are m6A modification sites on LINC01833. We therefore hypothesized that METTL3 mediates m6A modification in LINC01833 to regulate NSCLC progression. Detailed functions of METTL3 and LINC01833 in NSCLC were further explored. Moreover, we provided an insight into the downstream molecular mechanism of LINC01833 in NSCLC.2.2.1http://m6avar.renlab.org/) was used to reveal the m6A modification sites on LINC01833. RNA binding proteins of LINC01833 were obtained from online tool Starbase (https://starbase.sysu.edu.cn/), which were listed based on the number of CLIP-seq experiments. HNRNPA2B1 was selected since it ranks the first.The online tool m6aVar  and were cultured in Roswell Park Memorial Institute (RPMI) 1640 basic medium supplemented with 10% fetal bovine serum (FBS) in a humidified with 5% CO2.3The cDNA of LINC01833 or METTL3 was synthesized and cloned into the expression vector pcDNA3.1 . shRNA plasmids were designed by GenePharma to knock down LINC01833 or METTL3. The pCDNA3.1 vectors and shRNA plasmids were transfected into NSCLC cells HCC827 using RNAiMAX following the manufacturer’s manual after the cell confluence reached 70–90%. Cells were collected at 48 h post-transfection.2.4−ΔΔCT method. The primers sequences are as follows: LINC01833-F, 5’-CATTACAGGCATCACCCAT-3’, LINC01833-R, 5’-GACAAACCCAGGTACCT-3’; HNRNPA2B1-F, 5’-CCAGGGACATTTACTCAGACCAAT-3’, HNRNPA2B1-R, 5’-GGACACCTACCTTTATCATTCCC-3’; U6-F, 5’-TACTTACCCAGGACCAGG-3’, U6-R, 5’-CCATTAGACCTGGACTG-3’; GAPDH-F, 5’-GGACCTGCATTACTCCGATCT-3’, GAPDH-R, 5’-CGGGCCATATTACATACT-3’.Total RNA was extracted from tissue samples or cells using TRIZOL regent and the concentrations of total RNA were analyzed by Nanodrop. Reverse transcription of total RNA was performed using the PrimeScript RT Reagent Kit (Takara). The quantitative polymerase chain reaction (qPCR) was performed by ABI7500 using the SYBR Premix Ex Taq (Takara). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The relative gene expression levels were calculated using the 22.5Proteins were isolated by RIPA buffer (Beyotime) and determined using a BCA protein kit (Beyotime). Thirty µg protein was loaded into a 10% sodium dodecyl sulfate polyacrylamide gel and then electrophoresed, transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk for 2 h at room temperature and incubated with primary antibodies against METTL3 and HNRNPA2B1 overnight at 4°C. Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated immunoglobulin G secondary antibody for 2 h at room temperature. Enhanced chemiluminescence reagent was applied to the membranes for visualization. Protein bands were analyzed by Quantity One software version 4.62 (Bio-Rad). Original Western blot images were provided in Supplementary material 2.2.62 incubator for 24 h and then were harvested, resuspended in a culture medium, and seeded into 96-well plates at a density of 2 × 103 cells/well. Cell proliferation was assessed by incubating cells with 10 µl of CCK-8 solution for 0, 24, 48, and 72 h at 37°C for 2 h. The optical density (OD) was measured at 450 nm using a Tecan microplate reader.The cells with different transfections were incubated at 37°C in a 5% CO2.7For cell apoptosis analysis, cells were harvested at 48 h after transfection. Propidium iodide (PI) and fluorescein isothiocyanate (FITC)-Annexin V were used to stain the cells in the dark. Cell apoptosis was measured by a flow cytometer . Original flow cytometry data were provided in Supplementary material 3.2.85 cells/ml. For cell migration assay, upper chamber was equipped with 8-µm porous membranes (BD Biosciences) and added with 100 µl of the suspension. Six hundred µl of complete culture medium was added to each lower chamber to induce migration. The non-migrated cells were removed with a cotton swab after incubation for 24 h at 37°C with 5% CO2 whereas the migrated cells were fixed with 4% (v/v) paraformaldehyde for 20 min and stained for 20 min with 0.1% crystal violet. The number of migrated cells was taken by an inverted microscope. For the cell invasion assay, chambers were precoated with Matrigel (BD Biosciences).Transfected cells were collected and suspended to a density of 1 × 102.9RIP assays were conducted using an EZ Magna RIP kit (Millipore) with the Protein A/G Agarose Beads (Santa Cruz) according to the manufacturer’s instructions. The cells were lysed, and the lysates were incubated with magnetic beads conjugated with the HNRNPA2B1-specific antibody or control IgG antibody for 36 h at 4°C. The beads were incubated with proteinase K after washing with wash buffer, the purified RNA was eluted and analyzed for the presence of LINC01833 4 by qRT-PCR.2.10.The m6A content in the total RNAs was measured using an m6A RNA methylation quantification kit . Two hundred ng RNAs were added into the assay wells. By detecting the absorbance at wavelength of 450 nm, the m6A content was quantified following the manufacturer’s protocol.2.11in vitro using T7 RNA polymerase and purified using RNA PURE Kit. The transcribed LINC01833 RNA was biotinylated with a Biotin RNA Labeling Mix. Biotinylated RNAs were incubated with streptavidin-conjugated magnetic beads at room temperature. Cell was lyzed, the beads were then washed, and the eluted proteins were examined by mass spectrometry and Western blot analysis.LINC01833 or its antisense RNA was transcribed 2.126 HCC827 cells stably expressing sh-LINC01833 or sh-NC were injected subcutaneously into the ventral side of male BALB/c nude mice in the according groups with five mice in each group. Five mice were sampled each time. Tumor size and weight were examined at 28 days after injection. The volume was calculated as follows: V (mm3) = 0.5 × D × d2 . The tumors were excised, weighed, and collected for IHC staining. The test results were repeated three times. All protocols were approved by the Committee on the Ethics of Animal Experiments of the Shanxi Provincial Cancer Hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.Male BALB/c nude mice (4 weeks old) were purchased from Beijing Vital River Company and fed in a specified pathogen-free environment. For the tumorigenicity studies, 3 × 102.13The tumor tissues were formalin-fixed, paraffin-embedded and cut into 3-μm sections. Next, the sections were deparaffinated with xylene and rehydration, and 10 mM citrate buffer (pH 6.0) was used for antigen retrieval for 20 min. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide. Next, samples were incubated with primary antibodies against METTL3 and HNRNPA2B1 overnight at 4°C and the biotinylated goat anti-mouse or goat anti-rabbit IgG secondary antibody (Dako) for 30 min. Next, the sections were incubated with streptavidin peroxidase for 30 min. The slides were stained with diaminobenzidine (DAB) and counterstained with hematoxylin, dehydrated by a graded ethanol series, and photographed.2.14The statistical analysis was performed using SPSS 21.0 software and data were presented as a mean ± standard deviation (SD). There are 3 technical repeats for all assays. An equivalent non-parametric test, the Kruskal–Wallis Test, was performed to test the assumption of normality. Homogeneity of variance was tested using Bartlett’s test. Comparisons between two or more groups were detected using Student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test. A P value of less than 0.05 was considered to be statistically significant.3.3.1The expression profile of LINC01833 and its functions in NSCLC were investigated. The results showed that LINC01833 expression was elevated in 30 NSCLC tissues . Additio3.2In order to know whether LINC01833 is modulated by m6A RNA methylation, the potential m6A modification site was predicted using m6aVar online tool. There was three potential m6A modification sites on LINC01833 sequence , suggest3.3The expression profile of LINC01833 and its functions in NSCLC were subsequently explored. METTL3 level in NSCLC tissues was detected with IHC as well as Western blot. METTL3 presented upregulation in NSCLC tissues and cell lines . METTL3 3.4To clarify the regulatory mechanism of LINC01833, the RNA binding proteins of LINC01833 were examined by Starbase. The results indicated that HNRNPA2B1 is a potential RBP of LINC01833 . Subsequ3.5in vivo.Role of LINC01833 was further investigated by knocking down LINC01833 in tumor xenograft mice model. The expression of LINC01833 was reduced in sh-LINC01833 group . The tum4.Multiple reports have indicated that lncRNAs exert crucial impacts on various kinds of cancer progression by regulating different cellular processes, including cycle, migration, apoptosis, and physiological changes . In the m6A modification at the post-transcriptional level in the eukaryotic genome exerts an important role in the pathophysiological process of multiple cancers ,19. Li eHNRNPA2B1 is an important RNA binding protein affecting the RNA splicing and transport and stability of its downstream target genes. HNRNPA2B1 exerts crucial impacts on lung cancers. Dai et al.  and Dowlin vivo studies. HNRNPA2B1 is an RNA binding protein and may bind with mRNAs to stabilize or destabilize their expression. We used the starbase tool to identify the downstream targets of HNRNPA2B1 and found 11,130 mRNAs. Proteins of 100 mRNAs among these 11,130 mRNAs were predicted as transcription factors for METTL3 . LINC01833 suppression-induced reduction of METTL3 may be associated with the 100 genes that were regulated by HNRNPA2B1 (positively regulated by LINC01833) and spontaneously modulated METTL3.Interestingly, it was found that the upstream modulator of LINC01833, METTL3, is suppressed by LINC01833 knockdown in m6A transferase METTL3-triggered LINC01833 m6A methylation facilitates NSCLC progression through modulating HNRNPA2B1, which provides a novel direction for the better understanding of the pathogenesis of NSCLC.Click here for additional data file.


The X-ray diffraction analyses revealed that this complex crystallizes in the monoclinic system with space group P21/n. The most remarkable structural feature of 1 is that it contains two types of 1D right-handed helical chains. The molecules are linked by intermolecular hydrogen bonds and π⋯π interactions, then a 3D supramolecular network was constructed. Moreover, the intermolecular interactions on the crystal packing of 1 have been further studied using Hirshfeld surface analysis and corresponding 2D fingerprint plots. Binding interaction of this complex with calf thymus DNA (CT-DNA) has been investigated using absorption and emission studies, viscosity experiments and circular dichroism studies. Complex 1 shows significant binding to the DNA. The results of fluorescence spectroscopy and UV absorption spectroscopy, CD spectroscopy and viscosity indicated that this complex interacted with CT-DNA in a groove binding mode where the binding constant was 1.3 ± 0.2 × 104 L mol−1. Our fluorimeteric study showed that the reaction between 1 and CT-DNA was exothermic . Antibacterial activities of the complex were screened by the disc diffusion method against three Gram-positive bacteria , and three Gram-negative bacteria . The results indicated that this complex demonstrated acceptable antibacterial activities.A new binuclear copper( ii) Schiff base complex was synthesized using a template method in which the tridentate N2O Schiff base ligand was derived from [1 + 2] condensation of 2,6-diformyl-4-methoxyphenol and p-methyl aniline.A new binuclear copper( Reviews have shown the significance of such complexes9 and structures with a single helix,10 double helices,11 and triple helices,12 whether ligand directed,13 spacer controlled,14 or anion induced,15 employing many different kinds of N, O, and/or S containing coordinating ligands. Such helical molecules have been designed and assembled by the selection of basic components such as the coordination geometry of metal ions, the binding site of donating atoms, and the length of spacers.16–18 Single stranded helical structures are the most commonly observed form compared to double and triple stranded structures, either with enantiopure or achiral ligands. The secondary structure of helical complexes is determined by weak metal–ligand covalent interactions, halide ions such as iodide and supramolecular interactions such as hydrogen bonding and π⋯π stacking.19–24 The design and synthesis of small synthetic systems that can recognize specific sites of DNA through formation of non-covalently associated complexes are important areas of much current interest.25 Such physical complexation, more often, may produce important pharmacological effects by interfering with the biological processes in which DNA/RNA takes part. Such investigations have also provided insights as to the mechanism of action for antitumor antibiotics.26 The metal complexes can bind to DNA by different connection modes, such as π⋯π interactions which happen when the ligand contains planar heterocyclic ring systems, or when there is groove binding in large molecules through hydrogen bonding and van der Waals interactions.27 Beside, metal complexes of Schiff-bases have been emerged for the development of efficient variety of science.28–32Helical structures retain a unique place in life since genetic information is encoded within helical DNA molecules. Much effort has been devoted in recent years to the design and fabrication of metal complexes exhibiting helical architectures, not only in modeling of biological systems, but also for applying them in the fields of supramolecular chemistry, asymmetric catalysis, and nonlinear optical material,ii) Schiff base complex, [Cu2L2−(NO3)2]·2CH3OH (1)  containing two types of 1D right-handed helical chains. We also examined the close intermolecular interactions between the molecules in the solid state of the crystal using Hirshfeld surface analysis. In addition, antibacterial activity of complex was investigated. Furthermore, DNA binding of 1 was studied using different physico-chemical methods. A list of abbreviations used in the text with explanations shows in Table S1.†In the present study, we reported crystal structure of helical binuclear copper nitrate hydrate, 4-methoxy phenol, hexamethylenetetramine, trifluoroacetic acid were purchased from commercial sources and used as received. 2,6-Diformyl-4-methoxy phenol was synthesized by the reported procedure.51 Commercially pure chemicals such as Tris–HCl , were provided and used without purification. Highly polymerized calf thymus-DNA (CT-DNA) was procured from Sigma Company. All the experiments of DNA interaction were carried out in Tris–HCl buffer solutions and double distilled water . The stock solution of CT-DNA was procured by dissolving approximately 1–2 mg of CT-DNA fibers in 2 mL Tris–HCl buffer and stored at 4 °C for 24 hours. The concentration of DNA in stock solution was expressed in monomer units, as determined by spectrophotometry at 260 nm using the extinction coefficient (εp) of 6600 M−1 cm−1. DNA solutions were employed after no more than 4 days.High purity 2.2.−1. The absorbance spectra were recorded for 1 using an HP spectrophotometer (Agilent 8453) equipped with a thermostated bath (Huber polysat cc1). The absorbance measurements were performed by keeping the complex concentration constant (2.5 × 10−5 M) while varying the DNA concentration from 0 to 8.0 × 10−5 M (ri = [DNA]/[complex] = 0.0–3.2). The spectra were recorded in the range of 200–500 nm. CD measurements were recorded on a JASCO (J-810) spectropolarimeter, keeping the concentration of DNA constant (5 × 10−5 M) while varying the complex concentration from 0 to 3.0 × 10−5 M (ri = [complex]/[DNA] = 0.0–0.6). Viscosity measurements were made using a viscosimeter (SCHOT AVS 450) preserved at 25 °C  0.5 °C in a constant temperature bath while the DNA concentration was fixed at 5 × 10−5 M and flow time was admeasured with a digital stopwatch. The data is reported as (η/η0)1/3versus ri (ri = [complex]/[DNA] = 0.0–1.0), where η0 is the viscosity of the DNA solution alone. All fluorescence measurements were performed on a JASCO spectrofluorimeter (FP6200) using a quartz cell of 1 cm path length by keeping the concentration of the complex constant (1 × 10−5 mol L−1) while varying the DNA concentration from 0 to 3.4 × 10−5 M at three different temperatures . The samples were excited at 260 nm and the emission spectra were recorded in the range of 300 to 480 nm. The quenching experiments were conducted by adding stoichiometric small aliquots of potassium iodide stock solution (0.1 mol L−1) to 1 and CT–DNA–Cu complex solutions, respectively. The fluorescence intensity was recorded and the quenching constants (ksv) were calculated.In order to characterize and assess the structure of 1, the following instruments were used: the C, H, N data were obtained by using a Costech ECS 4010 elemental analyzer. FT-IR spectra were recorded on Bruker Vertex 70 spectrometer in KBr pellets from 400–4000 cm2.3.33 and refined by full-matrix least-square technique on F2, using JANA 2006 (ref. Uiso(H) was set to 1.2Ueq. The molecular structure plots were prepared using the ORTEP III35 and Mercury.36 Crystallographic data and refinement parameters are summarized in Single crystal X-ray diffraction intensity data of 1 was performed at 120 K using Gemini four-circle diffractometer from Agilent Technologies, equipped with a mirror collimated Cu Kα radiation (1.54184 Å) and area detector Atlas. The structure was solved by direct methods using SUPERFLIP program006 ref.  program 2.4.37–40 HS and the associated 2D fingerprint plots (FP) have been generated using Crystal Explorer 3.0 program.41 When the structure input file in the CIF format was uploaded into the CrystalExplorer software, all bond lengths to hydrogen were automatically modified to typical standard neutron values42 . The 2D fingerprint plots were represented by using the standard 0.6–2.4 Å view with the de and di distance scales displayed on the graph axes, where de is the distance from the Hirshfeld surface to the nearest nucleus outside the surface and di is the corresponding distance to the nearest nucleus inside the surface. The function dnorm is a ratio that comprises the de, di and the van der Waals radii (rvdW) of the atom. The dnorm values were mapped onto the Hirshfeld surface using a red-blue-white colour scheme as follows: red regions with the negative values of dnorm represent the intermolecular contacts shorter than the sum of the van der Waals radii; blue regions with positive values of dnorm represent the intermolecular contacts longer than the sum of the van der Waals radii; white regions denote the distance of contacts exactly corresponding to the van der Waals separation with a dnorm values of zero.43 Furthermore, two colored properties namely, shape-index and curvedness surfaces can be specified based on the local curvature of the Hirshfeld surface, which indicates the interactions between neighboring molecules.44Hirshfeld Surface (HS) analysis has become an attractive and growing technique in description of crystal structures.2.5.Staphylococcus epidermidis ATCC 34384, Staphylococcus aureus ATCC 25923 Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumonia ATCC 70063 were obtained from Hacettepe University, Department of Medical Microbiology. Bacterial strains were cultured overnight at 37 °C in nutrient broth. During the survey, these stock cultures were stored in the dark at 4 °C.The cultures of 2.5.1−1 and sterilized by filtration through 0.45 μm millipore filters. Antimicrobial tests were then carried out by the disc diffusion method using 100 μL of suspension containing 108 CFU mL−1 bacteria, which was spread on a nutrient agar (NA) medium. The discs (6 mm in diameter) were impregnated with 25 μL of compound (200 μg per disc) at the concentration of 8 mg mL−1 and placed on the inoculated agar. DMSO impregnated discs were used as negative control. Sulfioxazole (300 μg per disk) were used as positive reference standards to determine the sensitivity of one strain/isolate in each microbial species tested. The inoculated plates were incubated at 37 °C for 24 h for bacterial strains isolates. The antimicrobial activity in the disc diffusion assay was examined by measuring the zone of inhibition against the test organisms. Each assay in this experiment was repeated twice.45,46For the investigation of the antibacterial activity, the synthesized complex, 1, was dissolved in dimethylsulfoxide (20% DMSO) to a final concentration of 10 mg mL2.5.2−1) to be tested, and then serial, two-fold dilutions were made in a concentration range from 15.625 to 2000 μg mL−1 in 10 mL sterile test tubes containing nutrient broth. The MIC values of compound against bacterial strains were determined based on a micro-well dilution method.47 The 96-well plates were prepared by dispensing 95 μL of nutrient broth and 5 μL of the inoculums into each well. 100 μL from the test compound initially prepared at the concentration of 2000 μg mL−1 was added into the first wells. Then, 100 μL from each of their serial dilutions was transferred into eight consecutive wells. The last well containing 195 μL of nutrient broth without compound, and 5 μL of the inoculums on each strip, was used as negative control. The final volume in each well was 200 μL. The contents of the wells were mixed and the micro plates were incubated at 37 °C for 24 h. The compound tested in this study was screened twice against each microorganism. The MIC was defined as the lowest concentration of the compounds to inhibit the growth of microorganisms.48The minimal inhibition concentration (MIC) values, except one, were also studied for the microorganisms sensitive to compound determined in the disc diffusion assay. The inocula of microorganisms were prepared from 12 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The test compound dissolved in dimethylsulfoxide (DMSO) was first diluted to the highest concentration  was added dropwise to a solution of Cu(NO3)2·2H2O  and 2,6-diformyl-4-methoxy phenol  in the same solvent (15 mL). The mixture of reaction was heated for 24 h. Then, the deep green solution was kept in air for slow evaporation. After about a week, dark green colored crystals appeared. Yield: 0.722 g (70%), anal. calc. for C46H42Cu2N6O10, 2(CH4O): C, 55.97; H, 4.89; N, 8.16. Found: C, 56.00; H, 4.90; N, 8.16%. ΛM: (1 × 10−3 M in DMF) 73.86 Ω−1 cm2 mol−1). IR : 3408 (O–H), 2831 (C–H), 1624, 1614  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N), 1584 (CC); UV-vis +, 814 [Cu2L2–OMe + 2H+]+, 747 [L2 + CH3OH]+A methanolic solution (10 mL) of 3.3.1.ii) salt, 2,6-diformyl-4-methoxyphenol and p-methyl aniline in methanol solvent at reflux conditions. The exact coordination sphere of 1 was determined by single-crystal X-ray crystallography. In FT-IR spectra, the peaks at 1614 and 1624 cm−1 is due to CN stretch.49 Conductivity measurements performed in DMF  73.86 Ω−1 cm2 mol−1) confirm the non-electrolyte nature of the complex.501 was stable in air at room temperature and soluble in common polar organic solvents such as ethanol, methanol, DMF, and DMSO. Moreover, the 1 was found to be stable in the solution phases used, as evidenced by the FT-IR spectra  ion.54The electronic spectrum of 1 was recorded in EtOH. The maximal absorption peaks at 270 nm and 454 nm arising from intraligand π–π* transitions and charge transfer transition, respectively.3.3.1/n and each asymmetric unit contains half of the crystallographically unique molecule (ii) center displays a six-coordinate distorted octahedral geometry with CuN2O4 configuration (2-O1 atoms and two imino nitrogen atoms (N1 and N2) from two deprotonated Schiff base ligands (L−). Moreover, the axial positions are occupied by the two bridging oxygen atoms from two nitrate anions (O3 and O4). In the CuN2O4 coordination sphere, the axial Cu–O contacts of μ2-NO3 bridged anions  Å) and Cu–O4, 2.504 (16) Å) are much longer than  Å and Cu–O1, 1.953 (2) Å), demonstrating the Jahn–Teller effect55 (ii) complex with nitrogen donor atoms.56–59The ORTEP diagram of 1 is shown in molecule . Each Cuguration , where teffect55 . The Cu–ii) complexes.60,61 The distance of dinuclear center is 3.027(5) Å. This long distance between two Cu(ii) centers suggests that there is no interaction between these two metal centers.61 We found two intermolecular C–H⋯O hydrogen bonds between the hydrogen atoms of aromatic rings of ligands and oxygen atoms of bridging nitrate anions as well as one intramolecular π⋯π stacking interaction between the aromatic rings of two ligands with a distance of 3.423 Å in the complex which, play an important role in stabilizing the structure. The O⋯H distances and C–H⋯O angles are 2.349 (17) Å, 154.06° for C13–H1⋯O3 and 2.31 (17) Å, 153.82° for C17–H1⋯O4, respectively. As shown in Fig. S2b,3− forms bifurcated hydrogen bonds with the hydrogen atoms attached to the imino and phenyl carbon atoms of Schiff base ligand, C7–H1⋯O5 and C6–H1⋯O5, respectively  Å, C–H⋯O (C4–H1⋯O1m 2.698(29) Å, C15–H1⋯O1m 2.519(26) Å, C21–H1⋯O1m 2.675(30) Å) and O–H⋯O hydrogen bonds (O1m–H1⋯O5 2.614(4) Å) to form 2D supramolecular structure 2] monomers are connected through C–H⋯O hydrogen bonds between the C–H group of a phenyl ring and O-methoxy group of Schiff base ligands (C22–H2⋯O2 2.59(17) Å) belonging to neighboring units in complex 1 (ac plane (b* axis) by the hydrogen bonding between the nitrogen atom of the coordinated nitrate group and C–H group of a phenyl ring Schiff base ligand (C20–H1⋯N3 2.596(2) Å)  Fig. S3.3.4.dnorm, shape index, and curvedness) of 1 are mapped over dnorm (ranging from −0.303 (red) to 1.370 (blue) Å), shape index (ranging from −1 to 1.0 Å), and curvedness (ranging from −4.0 to 0.4) Å. The molecular surfaces of 1 are shown as transparent to allow the visualization of the molecular moiety, around which they were calculated. The red spots on the dnorm surface of 1 indicate the intermolecular close contacts involved in the hydrogen bonds such as, C–H⋯O  and another one as an acceptor (de < di). The 2D fingerprint plots of 1 show that the main intermolecular interactions between its molecules are H⋯H, H⋯N, H⋯O, H⋯C, and C⋯C intermolecular interactions  area  and a spike at the top left (donor) area  of the 2D fingerprint plot. The H⋯C/C⋯H intermolecular interactions comprise 5.9% of the total Hirshfeld surface area which emerge as very short spike at the top left (H⋯C 6.2%) and bottom right (C⋯H 7.6%) of the 2D fingerprint plot. The H⋯H contacts appear in the largest region accompanied by the short and tenuous spike at the middle of fingerprint plot, which has the most significant contribution to the total Hirshfeld surfaces (53.7%). The 2D fingerprint plots of 1 show that the π⋯π (C⋯C) stacking interactions comprise 5.8% of the total Hirshfeld surface area. Furthermore, the presence of π⋯π (C⋯C) stacking interactions in the crystal packing of 1 are clearly confirmed by characteristic ‘bow-tie’ patterns in the shape index function . Indeed,The quantities of interactions with percentages are shown as a histogram in 3.5.in vitro for their antibacterial activity against three Gram-negative species  and three Gram-positive species  of bacterial strains by the disc diffusion and micro dilution methods. The antibacterial activities obtained by the two methods are given in The test compound was screened 62 The remarkable activity of such ligands may arise from existence of two hydroxyl groups in these ligands which may play an important role in antibacterial activity. However, Cu(ii) complex (1) in this paper has no hydroxyl group in its structure and has a lower antibacterial activity than similar Cu(ii) Schiff base complex in previous works.63,64The disc diffusion assay results demonstrate  that 1 h3.6.3.6.1.65,66 Absorption spectra of 1 in the absence and presence of DNA (pH 7.4) showed a shoulder in the 200–350 nm regions and a peak at range of 400–500 nm  and nickel(ii) containing the drug mesalamine that bound to DNA through groove mode.68,69Band 454 gently decreased with the increasing concentration of DNA and a blue shift was observed from 454 to 433 nm. Also, an isobestic point at 280 nm provided evidence for the new complex–DNA formation.Kb was determined from the spectral titration data using the following equation:εa, εf and εb correspond to Aobs/[compound], the extinction coefficient for the free complex and its entirely DNA-bound combination, respectively. In the plots of [DNA]/(εa _ εf) versus [DNA], Kb was obtained by the ratio of the slope to intercept  with DNA.70 This Kb value is similar to those reported for well-established groove binding agent ([Ni(phen)3]+2, 1.4 × 104 M−1),71 ([Ho(phen)2Cl3]·H2O (1.36 × 104 M−1),72 .73 It was detected that the binding mode between the complex and DNA was groove binding.In order to further illustrate the binding strength of 1 with CT-DNA, the intrinsic binding constant t Fig. S5. The bin3.6.2.i.e., viscosity and sedimentation) are considered to be one of the least ambiguous and most exacting tests of a binding mode in solution in the absence of crystallographic structural data. The sensitivity of viscosity measurement largely depends on the changes in the length of DNA that occur as a consequence of its different binding modes with guest molecules. Intercalating agents of ligand such as the classical intercalations are expected to elongate the double helix to incorporate the small molecules in between the bases leading to an increment in the viscosity of DNA.65 Reciprocally, a partial and/or non-classical intercalation of ligand could bend (or kink) the DNA helix, reducing its effective length and, simultaneously, its viscosity.74,75 The values of (η/η0)1/3 were depicted against [complex]/[DNA] that η and η0 are the relative viscosities of DNA in the presence and absence of 1, respectively  suggest the distortion in native conformation of B-DNA due to its interaction with the complex 1. These spectral variations show the presence of some C form DNA features in native conformation of DNA upon interaction. Moreover, reduction in 248 nm CD band is considered a key marker of C form of DNA.77,78 However, it seems that the perturbation in DNA conformation is limited to few base pairs because when complete B to C transition occurs, then the CD band at 248 nm shows a 66% decrease in its intensity. However, not much decrease was observed in the concerned band (40.1%). Therefore, there are possibilities for the formation of an intermediate form of DNA having features of both B and C conformation. Similar results have been observed in the case of cationic lipid and neutral lipid binding with DNA, which could be ascribed to a non-cooperative augment in DNA.79,80 Moreover, increase in winding angle (or decrease in propeller twist) causes widening in DNA groove that enables proper positioning of small molecules in the groove pocket.81The CD spectra of DNA with different values of the compound exhibit decreases in the positive peak and also decreases in the negative band, which clearly indicates a non-classical intercalation between the complex and DNA.3.6.4.−5 mol L−1) of 1 were titrated with increasing amounts of CT-DNA. The complex emits luminescence in Tris–HCl buffer with two maximums that appear at 357 and 408 nm. The fluorescence titrations spectra of 1 in the absence and presence of CT-DNA at 298 K are given in Fig. S7.2Cl2] complex that interacted with DNA by groove binding.71 This phenomenon of the quenching of luminescence of 1 by DNA may be ascribed to the photoelectron transfer from the guanine base of DNA to the excited levels of the complex.82,83Fixed amounts (2.5 × 103.6.4.1.84 Since in both cases the fluorescence intensity is related to the concentration of the quencher (DNA), the quenched fluorophore can be used as an indicator for the quenching agent.85 Fluorescence quenching is described by the Stern–Volmer equation . Of course, these assumptions should be modified by considering the point that quenching can occur through different mechanisms: the inner filter effect, collisional quenching, and binding-related changes in fluorescence. Thus emission needs to be corrected for inner filter effect by the following equation:Quenching is carried out by different mechanisms, which are usually classified as dynamic and static. Dynamic quenching refers to a process in which the fluorophore (here the complex) and the quencher (DNA) come into contact during the transient existence of the exited state, but static quenching refers to fluorophore–quencher complex formation. In general, dynamic and static quenching can be known by their differing dependence on temperature and excited-state lifetime. The dynamic quenching process increases with an increase in the temperature because upon this condition the compounds move faster causing more collision probability. As the formation of composite is responsible for the static quenching, an increase in the temperature diminishes the stability of the composite and causes the fluorescence quenching to descend.equation .2F0/F = Fcor and Fobs are the fluorescence intensities corrected and observed, respectively. Moreover, Aex and Aem are the absorption of the system at the excitation and the emission wavelength, respectively. Then the Fcor is used in ksv) decreased from (3.59 ± 0.02) ×103 M−1 to (2.70 ± 0.02)×103 M−1 for 1  for the complex formation between 1 and DNA was assayed using 86,87F is fluorescence intensity change, Kb is the binding constant, and ΔF0 is maximum fluorescence intensity change. The binding constant can be obtained by plotting 1/ΔF vs. 1/[Q] and then dividing intercept by slope.That 1 Fig. S8. It meanKb for 1 at room temperature is comparable to N,N-bis (3β-acetoxy-5α-cholest-6-yl-idene)hydrazine (4.7 × 103 M−1)88 and resistomycin (3.23 × 103 M−1)89 which binds to DNA in an groove binding mode. The plot of log Kbversus 1/T , entropy (ΔS) (G) change by the van't Hoff equation, (H) is independent of temperature over the range of applied temperatures.In the present study, the binding constants of 1 were obtained at various temperatures . The valT Fig. S9 allows topy (ΔS)  and freequation, , assuminH) and entropy changes (ΔS), the model of interaction between the complex and biomolecule like DNA can be concluded: (1) ΔH > 0 and ΔS > 0, hydrophobic forces; (2) ΔH < 0 and ΔS < 0, van der Waals interaction and hydrogen bonds; (3) ΔH < 0 and ΔS > 0, electrostatic interactions.89 It can be seen that the negative value of ΔG revealed the DNA interaction process is spontaneous; the positive ΔH and ΔS values indicated that hydrophobic forces play main roles in binding the 1 to DNA.4.ii) Schiff base complex, [Cu2 L2− (NO3)2]·2CH3OH (1) from reaction of 2,6-diformyl-4-methoxy phenol, p-methyl aniline and Cu(NO3)2·2H2O in methanol solvent. The complex was characterized by FT-IR, elemental analysis (CHN), UV-vis spectroscopy and molar conductivity measurement. Furthermore, structure of 1 was determined by single crystal X-ray analysis. This complex is neutral and found to have a distorted octahedral geometry with the six donor atoms (N2O4) of the ligand. The crystal structure and Hirshfeld surface analysis represent the presence of various intermolecular interactions such as H⋯H, H⋯N, H⋯O, H⋯C and C⋯C in assembling the molecules of 1 into a 3D supramolecular network. The 2D fingerprint plots of 1 show that the H⋯H interactions have the largest share of the total intermolecular interactions with contributions of 53.7%. Also, the shape index function accompanied by its 2D fingerprint plot indicate the presence of the π⋯π interactions (5.8%) in the crystal lattice of compound 1. Moreover, the existence of two 1D helical chains in 1 was observed, one along the b axis and other one along the perpendicular to ac plane (b* axis) via C–H⋯O and C–H⋯N hydrogen bonds. This complex exhibited moderate antibacterial activity against the three Gram-positive bacteria: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 23212, S. epidermidis ATCC 34384, and against the three Gram-negative bacteria: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumonia ATCC 70063. Finally, DNA binding of 1 was investigated using UV-vis spectroscopy, fluorescence quenching, CD spectrum and viscosity methods. Fluorescence quenching and UV-Vis spectroscopy results represent that 1 binds to CT-DNA with binding constant of 1.3 ± 0.2 × 104 L mol−1. The results of spectroscopic techniques suggested that 1 binds to CT-DNA through groove binding.The work described in this paper involved the synthesis and characterization a binuclear copper(There are no conflicts to declare.RA-012-D2RA00719C-s001RA-012-D2RA00719C-s002