Background: Almonertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is commonly used as a first-line treatment for non-small cell lung cancer (NSCLC) patients with EGFR T790M mutations. Rivaroxaban and apixaban are a selective, direct factor Xa inhibitor used to treat venous thromboembolism (VTE), which is a frequent complication of NSCLC. Rivaroxaban and apixaban are substrates of CYP3A4, P-gp and BCRP, whereas almonertinib is an inhibitor of P-gp and BCRP. Rivaroxaban or apixaban are often prescribed together with almonertinib in NSCLC patients, but clear information on pharmacokinetic drug interaction is lacking. Therefore, this study aimed to unravel the extent of interactions between almonertinib-rivaroxaban and almonertinib apixaban in rats, and whether the pharmacokinetic interaction can be mitigated by rivaroxaban and apixaban dose adjustment.Methods: Rats were divided into ten groups (n = 6) that received rivaroxaban (2 mg/kg) (group 1), apixaban (0.5 mg/kg) (group 2), almonertinib (15 mg/kg) , almonertinib with rivaroxaban (2 mg/kg) (group 5), almonertinib with rivaroxaban (1 mg/kg) (group 6), almonertinib with apixaban (0.5 mg/kg) (group 7), almonertinib with apixaban (0.25 mg/kg) (group 8), rivaroxaban (2 mg/kg) with almonertinib (group 9), apixaban (0.5 mg/kg) with almonertinib (group 10). The concentrations of drugs were determined by an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The levels of messenger RNA were determined using quantitative real-time polymerase chain reaction (qRT-PCR).Results and Discussion: The results indicate that almonertinib increased the Cmax and AUC0-t of 2 mg/kg rivaroxaban by 3.30 and 3.60-fold, 1 mg/kg rivaroxaban by 1.28 and 1.90-fold. Almonertinib increased the Cmax and AUC0-t of 0.5 mg/kg apixaban by 2.69 and 2.87-fold, 0.25 mg/kg apixaban by 2.19 and 2.06-fold. In addition, rivaroxaban also increased systemic exposure to almonertinib. The results of qRT-PCR showed that almonertinib reduced the expression of Cyp3a1 in liver and intestine, and Abcb1a, Abcg2 in intestine and kidney. The pharmacokinetic results suggest that it is important to take special care of the interactions of these drugs in clinical applications. Venous thromboembolisms (VTEs) are a common and clinically significant complication in cancer patients, contributing to their mortality and morbidity . Comparein vitro and in vivo studies indicated that the two drugs are substrates of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)  . Inductin (BCRP) . These cAlmonertinib is a novel, irreversible, and selective third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI)  that exeSeveral methods exist for determining the blood concentration of rivaroxaban and apixaban, but they each have limitations, such as a narrow range of calibration curves , a high 3  and Sciex Triple Quad 5,500 tandem triple quadrupole mass spectrometer . Chromatographic separation was performed using an XSelect HSS T3 column  at 40°C by gradient elution. The mobile phase consisted of water (A) and acetonitrile . The elution procedure was as follows: 0–1.0 min, 50% B; 1.0–1.5 min, 50%→98% B; 1.5–3.0 min, 98% B; 3.0–3.1 min, 98%→50% B; 3.1–4.1 min, 50% B. The flow rate was 0.35 mL min3, 437.3→145.0 for rivaroxaban, 460.3→443.4 for apixaban and 440.4→145.0 for rivaroxaban-d4 . The mixed calibration working solutions were acquired by diluting the stock solution with 50% acetonitrile-water to obtain final concentrations of 5, 20, 50, 100, 500, 1,000, and 2,000 ng/mL ; 50, 200, 500, 1,000,5,000, 10,000, and 20,000 ng/mL (rivaroxaban); 10, 50, 100, 500, 1,000, 5,000, and 10,000 ng/mL (apixaban). Quality control (QC) working solutions with concentrations of 10, 800, and 1,500 ng/mL , 100, 8,000, and 15,000 ng/mL (rivaroxaban) and 20, 4,000, and 8,000 ng/mL (apixaban) were prepared using the same method. The mixed IS working solution included 100 ng/mL of sorafenib-dThe calibration standards were prepared by spiking 5 µL of the mixed working solution with 45 µL of blank rat plasma. The final concentrations of the calibration curves were 0.5, 2, 5, 10, 50, 100, and 200 ng/mL for almonertinib, 5, 20, 50, 100, 500, 1,000, and 2,000 ng/mL for rivaroxaban and 1, 5, 10, 50, 100,500, and 1,000 ng/mL for apixaban. QC samples were obtained by the same method with the final concentrations of 1, 80, and 150 ng/mL for almonertinib, 10, 800 and 1,500 ng/mL for rivaroxaban and 2, 400 and 800 ng/mL for apixaban.Protein precipitation was used to process the plasma samples. 5 μL of mixed IS working solution, 50 µL of the plasma samples, and 150 µL of acetonitrile were vortex-mixed for 1.0 min. Then, the mixture was centrifuged at 13,000 × g for 10 min; 100 µL of 50% acetonitrile-water was added to 100 µL of supernatant, vortex-mixed, and centrifuged again. The supernatant was transferred to a 96-well plate for sample analysis.The method was validated according to the guidelines of the Bioanalytical Method Validation Guidance for Industry for the US Food and Drug Administration  and Chinese Pharmacopoeia (2020). The selectivity, calibration curve linearity, the lower limit of quantification (LLOQ), precision and accuracy, matrix effect, extraction recovery, and stability were assessed during the method validation course.The selectivity was determined by analyzing blank plasma spiked with a working solution at LLOQ and IS, blank plasma samples from six different batches of rats, and real plasma samples. In the absence of interference, the peak area of blank plasma should be less than 5% of the IS and 20% of the LLOQ within the retention time.2) least square linear regression method. The deviation between the back-calculated concentration values calculated from the calibration curve and the theoretical values were acceptable within ± 15%, whereas LLOQ should not surpass 20%.The calibration curves were evaluated at 0.5–200 ng/mL for almonertinib, 5–2,000 ng/mL for rivaroxaban and 1–1,000 ng/mL for apixaban, respectively. The linearity was determined by the peak area ratios of the analyte to the IS against nominal concentrations using the weighted , and the accuracy was expressed by relative error (RE) of six replicates of samples. The precision and accuracy of QC samples were accepted within ± 15%, and LLOQ samples within ± 20%.n = 6) with the analyte peak area in the corresponding pure solution. The extraction recovery was assessed by comparing the peak area of the analyte in extracted plasma samples at three concentrations of QC samples (n = 6) with the peak area of the analyte of a blank plasma extract spiked at the same concentration.The matrix effect was evaluated by comparing the analyte peak area in blank plasma samples at low, medium, and high concentrations of QC samples . The samples were verified to be stable if the deviation of the samples was not more than ±15% of the standard concentration.Healthy male Sprague-Dawley (SD) rats weighing 250 ± 20 g were purchased from Beijing Huafukang Biotechnology Co., Ltd. . The license number is SCXK (Beijing) 2019–0008. The study was approved and supervised by the Animal Ethics Committee of Hebei General Hospital  (No. 2023024). Adaptive feeding (sufficient food and water) was performed for 1 week before the experiment. All the rats were kept under suitable conditions . All rats were fasted for 12 h before starting the experiments while still given water.n = 6). Almonertinib was suspended in 0.5% methylcellulose (MC) with 1% DMSO, rivaroxaban was dissolved in 0.5% hydroxypropyl methylcellulose (HPMC), and apixaban was prepared in ultrapure water with 5% DMSO. Group 1 and 2 were treated with the control solvents of almonertinib for nine consecutive days, then with rivaroxaban 2 mg/kg (Group 1) or apixaban 0.5 mg/kg (Group 2) by gavage on the ninth day. Group 3 and 4 were treated with the control solvents of rivaroxaban (Group 3) or apixaban (Group 4) for five consecutive days, then with almonertinib 15 mg/kg by gavage on the fifth day. Group 5–8 received almonertinib 15 mg/kg for nine consecutive days, followed by rivaroxaban, 2 mg/kg (Group 5) or 1 mg/kg (Group 6); apixaban, 0.5 mg/kg (Group 7) or 0.25 mg/kg (Group 8) by gavage on the ninth day. Group 9 and 10 were treated with rivaroxaban 2 mg/kg (Group 9) or apixaban 0.5 mg/kg (Group 10) for five consecutive days, then with almonertinib 15 mg/kg via gavage on the fifth day. Approximately 0.1 mL of blood was collected in a heparinized centrifuge tube via the orbital venous plexus at the following time points: 0, 0.17, 0.34, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h for rivaroxaban; 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h for almonertinib; and 0, 0.08, 0.17, 0.25, 0.34, 0.5, 0.75, 1, 3, 5, 7, 10, 12, and 24 h for apixaban. Blood samples were centrifuged at 3,500 g for 10 min, and then the supernatant was gathered and stored in a −80°C refrigerator. After, rat intestine, kidney and liver tissues were harvested for molecular analysis on the ninth or fifth day after treatment with the corresponding drugs for each group. The tissues were placed immediately at −80°C.Sixty healthy rats were randomly divided into ten groups  was employed to quantify the purity and concentration of the total RNA based on the ratio of the absorbance between 260 and 280 nm. RNA samples of 1 µg were transformed to complementary DNA (cDNA) using the FastKing RT Kit. The real-time PCR assays were accomplished using a two-step amplification method based on the recommendations in a SLAN-96S Real-Time PCR system . NADPH was used as an internal control, and the PCR cycling criteria were as follows: 95°C for 15 min, then 40 cycles of 95°C for 10, and 60°C for 32 s. The sequences of primers are shown in max, Tmax, t1/2, CLz/F, Vz/F, and MRT. Statistical comparisons were conducted using a analysis of variance, t-test or nonparametric rank-sum test depending on the data type; p-value <0.05 was deemed statistically significant.DAS 2.1.1 Software  was applied to calculate pharmacokinetic parameters using non-compartmental analysis. SPSS 25.0 statistical software  was applied to statistically analyze the main pharmacokinetic parameters, including AUC, C3 was selected as the IS, which had excellent stability and eliminated the influence of the endogenous matrix. The protein precipitation method has the merits of simplicity, reduced environmental pollution and low cost, compared to liquid-liquid extraction. Acetonitrile was chosen as the protein precipitant.A UPLC-MS/MS method with high sensitivity and wonderful reproducibility was developed and used to evaluate the pharmacokinetic interactions of almonertinib with rivaroxaban or apixaban in rats. Acetonitrile with 0.1% formic acid was selected as the mobile phase because it showed greater elution capacity than methanol. The addition of 0.1% formic acid to the organic phase B obtained superior peak profiles and minimal background noise. Finally, water (A) and acetonitrile (containing 0.1% formic acid) (B) were chosen as the mobile phase. In general, isotope-labeled internal standards could eliminate mistakes caused by matrix interference and differential ionization properties of the analytes. The internal standard applied to detect almonertinib was a deuterated IS in previous studies , but deu4, almonertinib, sorafenib-d3, and apixaban in different plasma samples. These include blank plasma(I), blank plasma containing the target analytes at LLOQ and internal standard IS (II), and real plasma samples from rats after oral rivaroxaban, apixaban or almonertinib (III). No significant endogenous substance interferences were detected. The retention times for rivaroxaban, rivaroxaban-d4, almonertinib, sorafenib-d3, apixaban were 1.23, 1.23, 0.85, 2.63 and 1.30 min, respectively.Calibration curves were generated using linear regression analysis to concentration ranges of 0.5–200 ng/mL for almonertinib, 5–2,000 ng/mL for rivaroxaban, and,1–1,000 ng/mL for apixaban respectively. The typical calibration curves were Y = 0.373 X + 0.034(r > 0.999) for almonertinib, Y = 0.0651 X + 0.0466 (r = 0.999) for apixaban, and Y = 0.0414 X - 0.0667 (r > 0.999) for rivaroxaban. The LLOQ values for almonertinib, rivaroxaban and apixaban were 0.5, 5, and 1 ng/mL, respectively.To evaluate accuracy and precision, six replicates of QC samples and LLOQ were analyzed at four concentrations, as shown in The matrix effects for almonertinib, apixaban and rivaroxaban ranged from 91% to 96%, from 100% to 108, and from 97% to 103%, respectively . The resStability data for almonertinib, apixaban and rivaroxaban in rat plasma under various storage and processing conditions are summarized in max, AUC0-t, and AUC0-∞ of rivaroxaban in group 5 by 3.30, 3.60, and 3.44-fold; rivaroxaban in group 6 by 2.19, 2.06, and 2.02-fold, respectively, compared to the group 1. Additionally, the CLz/F of rivaroxaban in group 5 and 6 were significantly decreased by 3.37-fold and 4.57-fold, respectively. The Vz/F and MRT were also decreased by different extent, and the Tmax of rivaroxaban in group 5 and 6 were also 1.40-fold (2.21 h) and 1.26-fold (2.08 h) longer than that of the control group (0.92 h). However, there were no statistically significant changes in the t1/2z between the groups.The mean plasma concentration-time curves of rivaroxaban after administration alone and in combination with multiple doses of almonertinib are shown in max, AUC0-t, and AUC0-∞ of apixaban in group 7 were significantly increased by 2.69, 2.87, and 2.82-fold; apixaban in group 8 were significantly increased by 2.19, 2.06, and 2.02-fold, respectively. Additionally, the CLz/F of apixaban in group 7 and 8 were significantly decreased by 3.00-fold and 5.29-fold, respectively. The Vz/F, MRT0-∞ and t1/2z were also decreased by different extent. However, there were no statistically significant changes in the Tmax and MRT0-t between the groups.The mean plasma concentration-time curves of apixaban after administration alone and following multiple doses of almonertinib are shown in max, AUC0-t, and AUC0-∞ of almonertinib was significantly increased by 1.26, 1.17 and 1.17-fold, respectively, when administered after multiple doses of rivaroxaban. Furthermore, the CLz/F of almonertinib decreased significantly by 81.1%. However, other pharmacokinetic parameters, including Tmax, t1/2z, Vz/F, MRT0-t, and MRT0-∞, showed no significant difference between group 3 and 9. In addition, all the pharmacokinetic parameters of almonertinib in group 10 showed no statistically significant difference compared with group 4.The mean plasma concentration-time curves for almonertinib (15 mg/kg) when administered alone and after multiple doses of rivaroxaban (2 mg/kg) or apixaban (0.5 mg/kg) are presented in To investigate the possible mechanism underlying the pharmacokinetic interactions between almonertinib and rivaroxaban or apixaban involving transporters and metabolic enzymes, we assessed the mRNA expression of CYP3A4 (Cyp3a1) in liver and intestine, and P-gp (Abcb1a), BCRP (Abcg2) in intestine and kidney of rats. The results, as shown in H. perforatum extracts reduced rivaroxaban exposure and the pharmacodynamic effect. Another retrospective cohort study  after take orally for each of two drugs (Based on the above findings, the effects of lowering the dose on the blood concentration of rivaroxaban and apixaban were investigated in depth. Our study found that after a half dose reduction of rivaroxaban, multiple doses of almonertinib increased the Cwo drugs , and useThis study has several limitations. First, many of the mechanisms mentioned in the article are mostly inferred and require further study. Second, animal models with lung cancer or thromboembolism were not used to examine pharmacokinetic interactions. Third, studies have shown that CYP2J2 has a higher catalytic efficiency than CYP3A4, but our study did not investigate the effect of almonertinib on CYP2J2. Fourth, species differences exist between rats and humans exist, and the drug interactions we observed in rats may not necessarily occur in humans. Therefore, further studies in clinical settings are needed to confirm whether similar interactions occur in humans.In this study, we developed and validated a rapid method for the simultaneous determination of almonertinib, rivaroxaban and apixaban in rats. The method was successfully applied to investigate the pharmacokinetic interaction between the three drugs. Based on the experiments we speculate that almonertinib significantly increased systemic exposure to rivaroxaban and apixaban by inhibiting CYP3A4, BCRP and P-gp. Similarly, co-administration of almonertinib with multiple doses of rivaroxaban increased the bioavailability of almonertinib. The pharmacokinetic results suggest that active surveillance for adverse drug reactions should be conducted when the drugs are combined in clinical practice. However, further verification is necessary through clinical trials.


TPS9 and TPS12, were isolated from Aquilaria sinensis (Lour.) Gilg, and their functions were examined in Escherichia coli BL21(DE3), with farnesyl pyrophosphate (FPP) and geranyl pyrophosphate (GPP) as the substrate of the corresponding enzyme activities. They were both identified as a multiproduct enzymes. After incubation with FPP, TPS9 liberated β-farnesene and cis-sesquisabinene hydrate as main products, with cedrol and another unidentified sesquiterpene as minor products. TPS12 catalyzes the formation of β-farnesene, nerolidol, γ-eudesmol, and hinesol. After incubation with GPP, TPS9 generated citronellol and geraniol as main products, with seven minor products. TPS12 converted GPP into four monoterpenes, with citral as the main product, and three minor products. Both TPS9 and TPS12 showed much higher expression in the two major tissues emitting floral volatiles: flowers and agarwood. Further, RT-PCR analysis showed TPS9 and TPS12 are typical genes mainly expressed during later stages of stress response, which is better known than that of chromone derivatives. This study will advance our understanding of agarwood formation and provide a solid theoretical foundation for clarifying its mechanism in A. sinensis.Sesquiterpenes are characteristic components and important quality criterions for agarwood. Although sesquiterpenes are well-known to be biosynthesized by sesquiterpene synthases (TPSs), to date, only a few TPS genes involved in agarwood formation have been reported. Here, two new TPS genes, namely,  Aquilaria and Gyrinops species of the Thymelaeaceae family upon stimulation of the tree by various stress factors, including mechanical wounding, microbial infection, cutting, lightning strikes, and chemical wounding (Aquilaria species have been included in the IUCN Red List (A. sinensis (Lour.) Gilg. is the main plant resource for producing agarwood in China, with the species mainly distributed in South China  chromones are the main active constituents in agarwood . Furthervia two different routes, namely, either through the mevalonic acid (MVA) pathway , heat stress, salicylic acid (SA), ABA (abscisic acid), mannitol, H2O2 and NaCl, to further investigate the mechanism of agarwood formation. TPS9 and TPS12 showed very low identity with heterologously produced sesquiterpene synthases, and their expression profiles after Agar-Wit treatment and catalysis were very different from ASS1, a sesquiterpene synthase previously identified . Different tissues were collected from two-year-old trees, and materials for transcription sequencing were obtained from seven-year-old trees treated using the whole-tree agarwood-inducing technique (Agar-Wit)  . All colhttp://www.expasy.org) was used to calculate physical and chemical parameters of TPS9 and TPS12, such as molecular weight (MW), theoretical isoelectric point (pI), stability, and instability index. Conserved motifs of TPS9 and TPS12 were predicted using Multiple Expectation Maximisation for Motif Elicitation (MEME) with the default parameters. The SWISS-MODEL was used to analyse the molecular modelling of TPS9 and TPS12 proteins. Trans-membrane domains were predicted using TMHMM 2.0 and TMpred software. DNAMAN was used for multiple alignment analysis of the TPS9 and TPS12 amino acid sequences, ExPASy and three softwares, including SCLpredT, Predotar, and Wolf Psort, were used to predict the physiological and biochemical properties and subcellular localisation of TPS9 and TPS12. Phylogenetic analysis was performed using MEGA5 using the neighbour-joining method with 1,000 bootstrap replicates.The Expasy Proteomics Server  on a Light Cycler® 480II . The PCR cycling conditions were the same as has described by TPS9 and TPS12 were calculated using the 2−ΔΔCT method . A total RNA of 1 µg was reverse-transcribed to cDNA using the PrimeScript™ RT Reagent Kit  according to the protocol described by the manufacturer. The cDNA was stored at 20 °C for qRT–PCR analysis and gene cloning. PCR amplifications were performed using SYBRT method .TPS9 and TPS12 with restriction enzyme sites were amplified using primers F1 (TCTACACCAGCACTTGCCCTCTAC) and R1 (TACAACTCCTTCACTG CTTCCTGC), F2 (GCATTTCGCTGCTGTTTC), and R2 (AATGGATTTGAGGTGGGTC). The resultant fragments of TPS9 and TPS12 were digested with restriction enzymes XhoI and BamHI and inserted into the expression vectors pET21a and pET28a, respectively, which were also digested with the same restriction enzymes. The ligation product was transformed into E. coli DH5 α competent cells and spread on Luria-Bertani (LB) medium containing 100 µg/mL ampicillin and 50 µg/mL kanamycin. Positive clones were extracted and confirmed using digestion and sequencing. All bacterial strains were stored in the −80 °C refrigerator in the laboratory.Full-length sequences of E. coli BL21(DE3) competent cells. Empty vectors pET-2a and pET-28a were transformed into E. coli BL21(DE3) cells as negative controls. Positive clones were selected and inoculated into LB medium containing 100 µg/ml ampicillin or 50 µg/ml kanamycin. The expression of pET21a-TPS9 and pET28a-TPS12 was induced under different IPTG concentrations (0.2 or 0.5 mM), incubation times , and temperatures (16 or 36 °C) to determine the optimal induction conditions. Then, positive clones of pET21a-TPS9 and pET28a-TPS12 were inoculated into LB medium containing 100 µg/mL ampicillin or 50 µg/mL kanamycin on a large scale after OD600 reached 0.6−0.8. Bacterial cells were induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 6 h at 36 °C and proteins were obtained by ultrasonic decomposition for 1 h. The solution was centrifuged for 20 min at 5,000 rpm and 4 °C, and the supernatant was discarded. The target protein was detected using 10 µL of the sample for separation using 10% SDS-PAGE.Recombinant plasmids pET21a-TPS9 and pET28a-TPS12 were transformed into Proteins were separated by 10% SDS-PAGE and transferred onto 0.45 µm polyvinylidene fluoride membranes . The membranes were blocked with TBST buffer  Tween 20) containing 5% fat-free milk powder and incubated at 4 °C for 4 h. The membrane containing the two expression vectors, TPS9-pET21a and TPS12-pET28a, with His-tag, was incubated overnight at 4 °C with anti-His antibody diluted at 1:2,000 , washed twice with TBST at 4 °C, and incubated with a secondary antibody conjugated to alkaline phosphatase diluted at 1:8,000 .E. coli cells with positive clones of pET21a-TPS9 or pET28a-TPS12 were inoculated into 200 ml LB medium until OD600 reached 0.6−0.8. To increase the protein expression level, a longer incubation time was needed, and thus, bacterial cells were induced with 0.5 mM isopropyl IPTG for 12 h at 37 °C. The bacterial solution was concentrated to 10 ml by centrifuging at 4 °C, followed by ultrasonic decomposition for 1 h. The solution was placed into 20 ml sample bottles with 60 µM FPP  or 60 µM GPP  and kept for 60 min in a water bath at 30 °C. According to the protocol by the manufacturer, vapour from the bottle was extracted with a solid phase micro extraction (SPME) fibre  for 90 min in a water bath at 75 °C, following injection of the sample into the gas chromatograph.−1. Samples were injected in the splitless mode at 250 °C. Thehe temperature operating conditions were as follows: 30 °C for 3 min, then increasing to 200 °C at a rate of 4 °C min −1, and maintaining that temperature for 5 min  gas chromatograph equipped with an Agilent DB-5MS capillary column  and a Varian 600 mass spectrometer with an ion-trap detector in full scan mode under electron impact ionisation (70 eV), as described by or 5 min . 1 µL C77–C40) under the same operating conditions. Kovats retention indices was calculated as tx is the retention time of the compound to be measured, tn is the retention time of the alkane that elutes prior to the compound, tn+1 is the retention time of the next eluting alkane, and n is the carbon number for the preceding alkane. Further identification was made by comparing their mass spectra with these stored in NIST 11 and with mass spectra from the literature (Sesquiterpenes were identified by comparing their Kovats retention indices (RI) with those from the literature , and comterature . We did TPS9 and TPS12 attracted our attention because transcriptomic studies of A. sinensis showed high expression levels of these synthases at the later stages of the plant response to external stimuli, suggesting a potential biological function in agarwood accumulation. Based on the sequences obtained from genome sequencing, we used PCR technique with specific primers to clone two sesquiterpene synthase genes, named TPS9 and TPS912. The isolated cDNAs have open reading frames (ORFs) of 1,383 and 1,632 base pairs, encoding predicted proteins of 460 and 543 amino acid residues, respectively. Aspartate-rich motifs, including the Rx8W, DDxxD, and NSE/DTE motifs, which are conserved in TPSs, were also found to be present in the amino acid sequences of TPS9 and TPS12 . The results showed that TPS12 did not express any protein. We then changed pET-21a to the pET-28a expression vector, and the TPS12-encoded protein was expressed in E. coli BL21 (DE3). SDS-PAGE analysis of crude extracts from transformed E. coli BL21 (DE3) showed the expected molecular mass with excessive amounts of polypeptides. As shown in TPS9 and TPS12 are eukaryotic genes.To obtain TPS9 and TPS12, full-length in vivo using total protein.Analysis by SDS-PAGE showed that most recombinant pET21a-TPS9 and pET28a-TPS12 were inclusion body proteins with no activity. Additionally, Western blot analysis showed a small amount of protein present in the supernatant. As functional enzymes are able to show catalytic activity even when present in small quantities, we attempted to detect such catalytic function E. coli BL21 (DE3) was examined by GC-MS, which indicated that the two enzymes are able to produce sesquiterpenes. Based on mass spectrum analysis using an equipped database, unknown sesquiterpenes, unidentified sesquiterpene RI 1404 (Rt = 12.642 min), β-farnesene (Rt = 12.711 min), cis-sesquisabinene hydrate (Rt = 15.837 min), and cedrol (Rt = 16.066 min) were detected by GC–MS in TPS9; the products contained major amounts of cis-sesquisabinene hydrate (34.61%), β-farnesene (31.79%), and smaller amounts of unidentified sesquiterpene RI 1404 (19.04%) and cedrol (14.56%). Incubation of TPS12 with FPP resulted in the formation of four sesquiterpenes, β-farnesene (41.11%), nerolidol (31.07%), γ-eudesmol (1.77%), and hinesol (26.05%), at retention times of 12.713, 14.842, 16.875, and 17.493 min, respectively , limonene (1.1%), 3-carene (1.8%), unidentified monoterpene RI 1005 (1.34%), citronellol (39.73%), geraniol (49.73%) and citral (3.93%), with retention times of 5.712, 6.323, 6.436, 6.605, 9.328, 9.739, 9.927 min, respectively. Four monoterpenes, β-myrcene (3.39%), unidentified monoterpene RI 1006 (2.38%), unidentified monoterpene RI 1089 (2.00%), citral (92.30%), with retention times of 5.724, 6.616, 7.936, 9.769 min, respectively. The same products of TPS9 and TPS12 were β-myrcene, and citral. Citronellol and geraniol were the principal monoterpene products of TPS9, but they were not detectable as products of TPS12. Among them, it is reported that myrcene and β-pinene also the common products generated by various sesquiterpenes synthase with GPP as substrate (δ-selinene synthase and γ-humulene synthases in Abies grandis (32), and it is also generated by At5g23960 TPS from Arabidopsis. Pinene, myrcene, limonene and cital are detected in the essential oil of Tunisian Conyza bonariensis   . In contwounding . Both TP21 month . The relTPS9 and TPS12 would respond to various abiotic stress, we analysed well-grown A. sinensis calli treated with heat, 100 µM MeJA, 100 µM SA, 100 µM ABA, 400 mM mannitol, 50 mM H2O2 and 300 mM NaCl at various times, and then used qRT-PCR to study the resulting transcript levels of TPS9 and TPS12. As shown in TPS9 and TPS12 were induced by all additional stresses, and they showed different expression under the stresses. TPS9 seems to be more sensitive to heat and MeJA treatment while TPS12 induces dramatically after MeJA and NaCl treatment.To examine whether TPS9 and TPS12 were positively induced by all three treatments but TPS12 was more sensitive to MeJA than TPS9, and significant induction (approximately 60-fold) of TPS12 was observed in response to stress at 24 h after treatment, after which there was a remarkable increase  at 7 d after treatment, with the trend still continuing. Meanwhile, the expression level of TPS9 transcripts increased by approximately 40-fold at 6 h after treatment and the induced transcription level was maintained as the treatment continued. As for heat treatment, it increased the transcript levels of both TPS9 and TPS12 but the changes were not as significant as those observed under MeJA treatment. The expression of TPS9 and TPS12 both increased first, until levels peaked at 80- and 20-fold, respectively, at 36 h after treatment, and then decreased. Under salicylic acid (SA) treatment, the relative expression of TPS9 showed an up-down-up pattern and peaked at 24 h with an expression level approximately four times higher than that recorded prior to treatment. Meanwhile, the expression level of TPS12 remained stable during 72 h, increasing significantly, approximately five-fold, after nine days; then, the expression increased to a maximum level, approximately 80-fold greater than that observed for controls, at 13 days after treatment. ABA plays a central role in responses to biotic and abiotic stresses  induced the expression of sesquiterpene synthase and the production of sesquiterpenes , and MeJstresses . Under At stress  and it hargonium , cassavaargonium , soybeanargonium . The TPSTPS9 and TPS12 are induced at all treated stresses, implying they might play a crucial role in protecting plants from environment abiotic stresses. Furthermore, under all stresses treatment, TPS9 and TPS12 were typical genes that are induced in the later stages of the plant response to stress conditions.These results suggest that As agarwood formation occurs occasionally and takes a long time in natural environments, and given that the demand for agarwood is continuously on the rise, naturally, at present, such demand already exceeds market supply. In response to this situation, various artificial methods have been tested to induce agarwood formation, including cutting , chemicaBdTPS gene is expressed in various tissues of Brachypodium distachyon, including roots, stems, leaves, and spikes  and β-farnesene (31.79%) as major products, and smaller amounts of unidentified sesquiterpene RI 1404 (19.04%) and cedrol (14.56%). cis-sesquisabinene has not been previously detected in A. sinensis. Although cedrol has been found in the essential oil from A. sinensis and A. crassna , nerolidol (31.07%), hinesol (26.05%), and traces of γ-eudesmol (1.77%), among which nerolidol is a characteristic sesquiterpene from agarwood , all belonging to the TPSa subfamily. Their products vary to a large extent with only one common product, β-farnesene  TPS9. (B) TPS12Click here for additional data file.10.7717/peerj.15818/supp-2Supplemental Information 2Conserved motifs identified with the MEME search tool. (A) Motifs are represented by boxes. Different colors boxes represent motif 1–4, respectively and the size of box indicates the length of motifs. (B) The structure diagram of motif 1–4.Click here for additional data file.10.7717/peerj.15818/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15818/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.15818/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.15818/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.15818/supp-7Supplemental Information 7Click here for additional data file.


Hp1 family, causes reduced expression of H3K9 methyltransferases, and that this subsequently leads to the exit of embryonic stem cells (ESCs) from pluripotency and a reciprocal gain of lineage-specific characteristics. Importantly, the phenotypes of Hp1-null ESCs can be rescued by ectopic expression of Setdb1, Nanog, and Oct4. Furthermore, Setdb1 ablation results in loss of ESC identity, which is accompanied by a reduction in the expression of Hp1 genes. Together, our data support a model in which the safeguarding of ESC identity involves the cooperation between the H3K9 methylation writers and their readers.Histone H3 lysine 9 (H3K9) methylation, as a hallmark of heterochromatin, has a central role in cell lineage and fate determination. Although evidence of a cooperation between H3K9 methylation writers and their readers has started to emerge, their actual interplay remains elusive. Here, we show that loss of H3K9 methylation readers, the   •Hp1 family function redundantly to maintain ESC identityMembers of the •Hp1-null ESCs can be rescued by ectopic expression of Setdb1The phenotypes of •Functional crosstalks exist between the H3K9 methylation writers and HP1 in ESCs HP1 and H3K9 methylation, as hallmarks of heterochromatin, have a central role in cell lineage and fate determination, yet the underlying mechanisms remain elusive. Lixia et al. report that the safeguarding of ESC identity involves the crosstalk between the H3K9 methylation writers and their reader HP1. The covalent modification of histone tails is thought to play essential roles in chromatin organization and transcriptional regulation of eukaryotic genes . HistoneHeterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein that is involved in heterochromatin-mediated gene silencing in many organisms . MammaliSuv39h1/2 knockout mice show no developmental defects, double null mice are characterized by perinatal lethality associated with genome instability  and exhibit gross morphological abnormalities across all developing cell lineages  survival in vitro is compromised  to eliminate all HP1 activity  cells that failed to maintain their pluripotent state, as determined by the reduced expression of pluripotency markers at both the mRNA and protein levels  (Hp1 achieved with a tamoxifen inducible Cre (4 days after tamoxifen treatment)  D and S1Eeatment) . Additiosfection E. Therefparalogs J and S1CHp1 individual and compound mutants . Complete loss of all three Hp1 paralogs in ESCs resulted in substantial changes in gene expression   terms related to pattern specification process, cell fate commitment and tissue morphogenesis. Genes down-regulated in Hp1α/β/γΔ/Δ cells were related to regulation of axon guidance and cell junction organization, as well as regulation of cell morphogenesis  analysis in  mutants , and S3.paralogs A and 2B.d genes) , furtherogenesis C.Figure Hp1α/β/γΔ/Δ cells also showed a strong over-representation of embryonic germ layer lineage genes, including key endoderm markers , markers of ecto-mesoderm . and meiosis- and germline-specific genes . In contrast, expression levels of trophectoderm genes (Ets2 and Cyp11a1), either decreased or remained unaffected in the mutant , Nr5a2, Nanog, Sox2, and Dppa5a  family members we examined . To this end, ESCs were induced to differentiate upon the removal of both leukemia inhibitory factor (LIF) and feeder cells and growing in suspension culture  , and to a lesser extent, mesoderm (T and Flk1).To explore the  culture A. As sho Nestin) D. Strikiin vivo differentiation potential of Hp1-mutant ESCs, these cells were injected subcutaneously into immune-deficient mice. Histological analysis revealed that, while Hp1 single mutants produced teratomas containing all three germ-layer lineages, double mutants developed teratomas containing derivative of all three germ layers, but with an over-representation of the endoderm structures   A–5C, terd NANOG) D, sugges in vivo A–5D.FiguSetdb1, we engineered ESCs by inserting loxP sites in introns between exons 10 and 14 of the Setdb1 gene, which was designed to remove exons 11–13 after Cre-mediated recombination to inactivate the gene. Setdb1F/F displayed normal SETDB1 protein levels and formed colonies and teratomas that were phenotypically identical to wild-type ESCs  Setdb1 (Hp1 deficiency in ESCs (To determine to what extent the expression levels of H3K9 methyltransferases are needed to produce the observed defects in sferases B. Introd in ESCs B–6D. The/Δ cells D and S5Cm layers E. Intere) Setdb1 , suggest in ESCs D–S5F. ToNanog as a direct target of SETDB1 and OCT4, and revealed significant binding of SETDB1, OCT4, and NANOG to the promoter regions of Hp1β and Hp1γ .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jinzhong Qin . ESCs were separated from MEF feeder cells by trypsinization, followed by incubation on gelatin-coated dishes for 30 min. Non-adherent cells consisting mainly of ESCs were used for further experiments. The culture medium of HEK293FT and MEFs was DMEM added with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin. MEFs were derived from 13.5 dpc embryos using standard procedures (2.ESCs were routinely cultured on mitotically inactivated MEF feeder layers in gelatin-coated dishes with DMEM (15% fetal bovine serum [Gibco], 100 U/mL penicillin-streptomycin [Thermo Fisher], 1,000 U/mL LIF, 2 mM ocedures . All cel∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.Unless stated otherwise, all data were presented as mean values ± SD or mean values ± SEM for experiments conducted with at least three independent experiments. GraphPad Prism 5 was used to analyze data and perform statistical tests and generate p values using two-tailed the Student t test for comparisons between two datasets, and p values of less than 0.05 were considered to be statistically significant. Statistical significance was presented in figures in the following manner: Conceptualization, J.Q.; methodology, L.D., H.L., L.Z., J.W., C.W., and S.L.; formal analysis, L.D., J.W.,C.W., L.X., and J.Q.; mechanistic investigations, L.D., H.L., B.W., Y.S., C.W., Y.X., S.L., X.L., and J.Q.; writing – original draft, L.D. and J.Q.; and writing – review and editing, L.D. and J.Q.; funding aAcquisition, J.Q.; supervision, J.Q.


The monodispersed magnetic core (18 nm diameter) with its mesoporous silica shell (93 nm diameter) exhibited a notable superparamagnetic behavior useful for magnetic bioseparation. The hydrodynamic diameter, deduced from dynamic light scattering analysis, of the nanoparticle increased from 100 to 800 nm when the aqueous buffer changed from pH 10.0–3.0. A size polydispersion occurred from pH 7.0–3.0. In parallel, the value of the extinction cross section increased according to a negative power law of the UV wavelength. This was mainly due to light scattering by mesoporous silica, whereas the absorbance cross section remained very low in the 230–400 nm domain. The three types of resveratrol-grafted magnetic nanoparticles exhibited similar scattering properties, but their absorbance spectrum was consistent with the presence of trans-resveratrol. Their functionalization increased their negative zeta potential when pH increased from 3.0 to 10.0. The mesoporous nanoparticles were monodispersed in alkaline conditions, where their anionic surface strongly repulsed each other but aggregated progressively under van der Waals forces and hydrogen bonding when negative zeta potential decreased. The characterized results of nanoparticle behavior in aqueous solution provide critical insight for further study of nanoparticles with proteins in biological environment.In the framework of a protein–ligand-fishing strategy to identify proteins that bind to The online version contains supplementary material available at 10.1186/s11671-023-03805-9. Produced by plant species, it is considered with an increasing interest in many biological fields related to biocontrol −: m/z calc. 1014.4514. MALDI-TOF [M + Na] m/z 992.4.1H-NMR of 2: δ ppm : 9.65 , 7.76 , 7.64–7.56 , 7.18–7.08 , 6.80 , 6.41 , 3.76 , 3.05 , 1.53 , 1.16 , 0.59 . 13C-NMR: δ ppm  158.2 (C-5), 154.3 (C-3), 154.2 (C=O), 150.7 (C-4′), 138.9 (C-1′), 133.6 (C-1), 128.1 (C-β), 127.7 (C-5′), 127.3 (C-3′), 122.0 (C-α), 115.8 (C-2′), 115.6 (C-6′), 110.4 (C-2), 110.2 (C-6), 108.4 (C-4), 57.7 (Si–O–CH2–CH3), 43.2 (NH–CH2–CH2–CH2–Si), 23.5 (NH–CH2–CH2–CH2–Si), 18.1 (Si–O–CH2–CH3), 7.2 (NH–CH2–CH2–CH2–Si). HRMS (ESI) for C34H54N2O11Si2 [M–H]−: m/z calc. 721.3196. MALDI-TOF [M + Na + H]+ m/z 745.3.1H-NMR of 3: δ ppm : 9.24 , 7.76 , 7.57 , 7.09–7.02 , 6.44 , 6.17 , 3.77 , 3.05 , 1.53 , 1.18 , 0.59 . 13C-NMR: δ ppm  159.2 , 154.9 (C=O), 151.0 (C-4′), 139.4 (C-1′), 134.5 (C-1), 129.3 (C-β), 127.8 , 127.6 (C-α), 122.5 , 105.3 , 102.9 (C-4), 58.4 (Si–O–CH2–CH3), 43.8 (NH–CH2–CH2–CH2–Si), 23.5 (NH–CH2–CH2–CH2–Si), 18.8 (Si–O–CH2–CH3), 7.8 (NH–CH2–CH2–CH2–Si). HRMS (ESI) for C24H33NO7Si [M–H]−: m/z calc. 474.1947. MALDI-TOF [M + Na + H]+ m/z 498.2CS) nanoparticles, were added with 1 mL toluene . Three other flasks contained 150 mg of 1, 2, and 3, respectively, in 1 mL toluene. The 1 mL suspensions of CS nanoparticles were added to the latter flasks along with 60 µL of ultrapure water (18.5 MΩ) then stirred and heated up to 70 °C for 15 h. The functionalized CS1, CS2, and CS3 nanoparticles were then collected by washing with ethanol and drying under vacuum for 3–4 h.Three flasks, each containing 100 mg of core/shell  with 10 mm quartz cuvettes , and a Spectrum Two FT-IR spectrometer  in transmittance mode with KBr disks.The successful grafting of dH) of the nanoparticles was studied at 25 °C by dynamic light scattering (DLS) using a Zetasizer Nano-ZS , adapted to assess particle sizes between 5 and 1000 nm. The instrument was equipped with a 633-nm diode laser and a DTS1070 cell. DLS was analyzed at the back scattering angle of 173°. In this configuration, the contributions of rotational diffusion effects in the observed autocorrelation profiles can be neglected and the translational diffusion coefficient, D, can be assessed [The distribution of hydrodynamic diameter . The zeta potential is referred to as the electrostatic charge at the splining plane boundary separating ions within the diffuse layer which moves with the nanoparticles, and ions that remain with the bulk water, corresponding to the Stern shell. Zeta potential is related to the surface electrostatic charge of nanoparticles .For extinction cross section determination, all nanoparticles were initially suspended in absolute ethanol and were then diluted at the final suspension concentration of 50 ppm (50 µg/mL) in aqueous tris(hydroxymethyl)aminomethane (TRIS) buffer (50 mM), previously adjusted to pH 3.0, 5.0, 7.0, and pH 10.0 with HCl 37% and HCl 0.1 N. Extinction was recorded by EnSpire microplate reader  at 1 nm step in front face configuration using 96 wells black microplates  set up at 27 °C.As shown in Fig. These nanoparticles were inspected using TEM imaging Fig. B–C. TEM –3 m3/kg and 0.048 × 10–3 m3/kg, respectively.To understand the magnetic properties of these particles, the hysteresis loop and FC-ZFC curves of the two particles (few mg of samples) were measured at room temperature using a SQUID magnetometer (Quantum Design MPMS-XL). Figure Ti) of around 250 K and a well-defined maximum in the ZFC curves [Tb of 213 and 215 for core and CS (core/shell) nanoparticles, respectively. However, the maximum of ZFC curve for core/shell NPs is very broad suggesting non-negligible dipole–dipole magnetic interactions [Thermal magnetization behaviors in the zero field (ZFC)—field cooled (FC) were performed from 4 to 300 K, under an applied magnetic field of 20 Oe Fig. C curves  at the sractions  among irractions .Tb of the core/shell particles (− 58 ℃) meets very well the requirements of bio-applications at positive temperatures, i.e., much higher than Ti = 250 K, by ensuring that the CS particles will be in a superparamagnetic state and will therefore not aggregate, while retaining magnetization. The combination of Fe2O3 core and nanostructured Silica not only leads to interesting properties of nanocomposite system, but is also a good candidate for applications in biomedicine because both are biocompatible in biological systems due to their low toxicity and biodegradability.The low Based on the works of Wagner & Fisher  and Leontrans-resveratrol molecules was inspected by observing the appearance of the C=O group at 1700 cm−1 and the disappearance of –N=C=O group at 2270 cm−1 in the FT-IR transmittance spectra propyl isocyanate to obtain compound 3. The structures of three main products propyl isocyanate were found on the 1H-NMR spectra at 1–4 ppm and 13C-NMR spectra at 7–60 ppm with the proper ratio for 3, 2, and 1 substituted groups.In the 2 and 3 were tested to pinpoint the –OH conjugated position. The signal of –OH group appearing at 9.65 ppm on the proton spectrum of compound 3 proved that the two hydroxyl groups remained on the A ring of trans-resveratrol. Moreover, HMBC spectra showed the coupling of C-4′ with H-3′ and H-5′ and the drop of the chemical shift of C-4′ from 157.4 ppm to 151.1 ppm revealed the substitution of –OH at C-4′ position of compound 3. The structure of compound 2 was elucidated by the key couplings between protons and carbons on the HMBC spectrum: (1) –OH (9.65 ppm) with C-4, and C-6 proved the presence free of hydroxyl group on the A ring; (2) H-4 (6.41 ppm), H-2 and H-6 with C-3 and C-5 show that one hydroxyl group of A ring is substituted and makes the imbalance of A ring; therefore, C-3 and C-5 have the different chemical shift, 154.3 ppm, and 158.2 ppm, respectively; (3) H-3′ and H-5′ with C-4′ on the B ring prove the –OH at C-4′ is modified with the chemical shift decrease of C-4′ from 157.4 ppm in the trans-resveratrol structure to 150.7 ppm of compound 2.Precisely, the two-dimension NMR spectra of compounds 1) corresponds to 3′,4,5′-tri((3-(triethoxysilyl)propyl)carbamate)-trans-stilbene, compound (2) is 5′-hydroxy-4-((3-triethoxysilyl) propyl)carbamate)-3′-bis((3-(triethoxysilyl)propyl)carbamate)-trans-stilbene, and finally, compound (3) is 3′,5′-dihydroxy-4-((3-(triethoxysilyl)propyl)carbamate)-trans-stilbene.In conclusion, NMR characterization showed that compound  and 1709 cm−1 (CS3) corresponded to C=O bonds of in frameworks of 1, 2, 3. These findings were consistent with a successful covalent coating of core/shell nanoparticles surfaces with trans-resveratrol. The slight differences between the wavenumber of the maximum absorption of the C=O band in the IR spectra CS1, CS2, and CS3 and between the maximum wavelengths on the UV–Vis spectra could reflect conformation changes of silane-resveratrol derivatives when they were coated with a silica shell and/or the position of silane that was grafted. We observed that absorption maxima of CS1, CS2, and CS3 were between 280 and 340 nm. We observe that they are centered near the 304 nm corresponding to the main optical electronic HOMO–LUMO transition from Π → Π* of trans-resveratrol in water, assessed by theoretical DFT calculation [In parallel, we compared the FT-IR transmittance spectra of ca shell , respectculation .vitro protein interaction studies. The correlation coefficient increases with nanoparticle size or upon particle aggregation, and which is associated with a slower diffusion speed of particles. For CS, the nearly single exponential decay of the autocorrelation function obtained by DLS suggests a narrow distribution of CS sizes in the suspension. Figure CS, CS1, CS2 and CS3 are monodisperse at pH 10.0. Resveratrol grafting onto CS has a negligible effect on the hydrodynamic radius  at this alkaline pH value. At pH 10.0, nanoparticles dispersed in absolute ethanol had the same size distribution as in TRIS buffer, which evidenced their monodispersed colloidal behavior  of around 200 nm or 300 nm diameter, respectively, whereas the non-reacting CS1 remains as a single nanoparticle of 95–100 nm diameter. Similarly, CS2 has 2 silane groups and may form covalent dimers. The DLS analysis of CS1 and CS2 suspended in alkaline TRIS buffer (pH 10.0) revealed that dimers and trimers may exist only in very negligible proportions. CS1, CS2, and CS3 populations can thus be considered as a population of single nanoparticles after their functionalization.DLS measurements were carried out to examine the colloidal stability of nanoparticles within the 3.0–10.0 pH range generally used for in dH distribution of CS shifted drastically and widened up to 800 nm, evidencing an aggregation process during acidification. The highest dH value was reached for the most acidic condition  is preferentially grafted to silanized resveratrol, which resveratrol hydroxyl(s) remain(s) free for CS2 and CS3. Additional investigations would be necessary to fully explain the observed pH-control of the surface charge density.As shown in Fig. CS3, which contained the resveratrol-silane derivative with two free hydroxyls on one of its two phenolic groups and was also deprotonated at pH 10.0. In neutral and acidic conditions, the low zeta-potential values corresponded to those of nanoparticle aggregates of an average dH up to 825 nm diameter.The most negative value was obtained for A(λ)) spectra of CS, CS1, CS2, and CS3 have been recorded both in clear bottom microplates using the EnSpire plate reader in absorbance mode ) spectra of CS conformed to an exponential decrease of the wavelength for all the TRIS buffer pH values tested, fitting very well to the Rayleigh–Gans–Debye model [Extinction cross section  increased with pH value for a wavelength between 240 and 500 nm , with three covalent Si–O links with the bulk silica, are located at a far distance from neighboring H-donors or H-acceptors, which prevents hydrogen bonding. Q3 are the most acidic silanols (pKa around 4.8) and therefore become the first deprotonated sites at pH 5.0. They are followed by hydrogen-bonded vicinal (Q1) and germinal (Q2) silanols, which have an intermediary acidity (8.5 < pKa < 9.3) and are progressively deprotonated between pH 7.0 and pH 10.0. Finally, inner silanols (Q4) are the least acidic ones (pKa > 11.0). They thus remained always protonated in the pH range of our experiments (3.0 and 10.0) [Mesoporous silica exhibits four types of silanol groups, which can be separately studied by both NMR , 45 and nd 10.0)  Fig. 6)3), with CS remained very reduced at pH 3.0, and therefore, the electrical double-layer repulsion of counter-ions had a very weak strength. Then, attractive forces between nanoparticles such as van der Waals dispersion forces became sufficient for spontaneous nanoparticle aggregation [π–π stacking forces. They may explain differences in aggregation processes (reaction-limited and diffusion-limited cluster aggregation) and in morphology , resulting in contrasted dH distributions between CS1, CS2, and CS3. In the case of CS3, the presence of the resorcinol group at their surface may also create a specific affinity site for the silica surface of the neighboring nanoparticles [Consequently, as observed on Fig. regation . Dependiarticles . Indeed,articles .2 and Q3 silanols became deprotonated, the surface charge density became high and the long-range electrostatic repulsion of counter-ions became strong enough to stabilize the water suspensions in a monodispersed state, even at high ionic strength. This pH-dependent charge density of CS was observed in the case of many metal oxides: α-quartz and α-cristobalite [2O3 [2O3 [2) [The distribution of the more acidic silanol groups on the mesoporous silica surface may be possibly heterogeneous, at the origin of the puzzle of more negative areas and more positive areas as pH increased. Electrostatic forces increased with the deprotonation of the surface silanols and may participate in repulsive forces between nanoparticles. Finally, when the majority of Qtobalite , α-alumitobalite , smectittobalite , illite tobalite , magnetiite [2O3 , maghemi2O3 [2O3 , titaniu[2O3 [2) , zinc ox[2O3 [2) .CS net charge with the increase of the pH value. The surface charge of metal oxides originates from four major mechanisms [4+ by cations of lower valence  creates structural charges which are pH-independent; (2) under-coordinated Si4+ of silica layer edges hydrate into silanols in an attempt to complete their coordination sphere. Usually, siloxanes of surface layers are potentially partially oxidized in silanol when they experienced a rich O2 medium. Protonations/deprotonations of interfacial silanols generate charges depending on the aqueous pH buffer; (3) some charges also originate from additional lattice imperfections and impurities; (4) finally, interfacial complexation of protons or other ions also occurs within the inner-sphere or the outer-sphere complexes.Interestingly, the zeta potential of coated nanoparticles became more negative than the chanisms : (1) theTo assess colloidal stability of our nanoparticles CS, CS1, CS2, and CS3, we added in Additional file describing step by step the DLVO equations that we apply, including the magnetic aggregative energy, and its extended XDLVO form , at 3660 and 3620 cm−1 , at 3740 cm−1 , and at 1110, 1050, 1085 cm−1 (siloxane) [29Si could reveal if the number of silanols and their Q2–Q3–Q4 proportions are modified after the three functionalizations , resulting in an average pKa shift. Finally, potentiometric acidity titration may also highlight surface charge density of functionalized nanoparticles.In a future work, using small amounts of nanoparticles, vibrational infrared spectroscopy could be used to assess silanol proportions. Indeed, changes in their proportion should induced changes in absorption bands at 3747 cmiloxane) . Using mtrans-resveratrol derivatives and their aggregation behavior in an aqueous environment. Thanks to their superparamagnetic property at room temperature, MNPs coupled with trans-resveratrol are expected to be good candidates for protein–ligand fishing and immunoassay design. Three trans-resveratrol derivatives were synthesized, purified and their structures were identified before their grafting on the surface of MNPs. This was an essential step for further understanding the interactions of these MNPs with protein. Since cis-resveratrol exhibits a maximum absorbance at 280–285 nm [trans-resveratrol was not isomerized into cis-resveratrol during the preparation process. The colloidal behavior/aggregation of these nanoparticles in aqueous solution is mainly governed by pH. They are monodispersed at pH 10.0 and tend to aggregate at neutral and acidic pH, due to the decrease of the surface charge density , resulting in lower electrostatic repulsion forces. The aggregation process results in the decrease of the extinction cross section, mainly due to the decrease of the light scattering cross section. A negligible difference in aggregation behavior was observed between uncoated and coated nanoparticles. The understanding of the aggregation behavior of nanoparticles in aqueous solution at different pH is important for their uses in quantitative applications.This work reports the preparation of novel magnetic nanoparticles grafted with 0–285 nm , we deduMonodisperse MNPs are expected for protein–ligand fishing and immunoassay design. Consequently, we may hypothesis that the proposed functionalizations have to be improved for these applications. Fluorescence binding analysis of these MNPs with CpLIP2 lipase was carried out in acidic, neutral and basic pH by Nguyen , and revAdditional file 1: Table S1. The parameters of the Rayleigh-Gans-Debye model. Table S2. Parameters. Fig. S1. Transmission spectra of silanized trans-resveratrol. Fig. S2. Structure of silanized trans-resveratrol. Fig. S3. Size distribution of nanoparticles. Fig. S4. Distribution of zeta potential of nanoparticles at different pH. Figs. S5 to S26. NMR spectra of compound 1, 2, and 3. Fig. S27. Mass spectroscopy of compound 1, 2, and 3. Figs. S28 to S30. MALDI-TOF spectra of compounds 1 to 3. Fig. S31. Interaction energy of two spherical nanoparticles in aqueous TRIS buffer calculated using DLVO and XDLVO models. Fig. S32. Potential energy of interaction of two spherical nanoparticles. Fig. S33. Energy barrier of colloidal suspension of nanoparticles. Fig. S34. Surface charge density of silica nanoparticles.


The present work, based on high-performance liquid chromatography (HPLC), highlights the frequency and levels of AFM1 in coalho and mozzarella cheese samples (n = 28) from the main cheese-processing plants in Araripe Sertão and Agreste in the state of Pernambuco, Brazil. Of the evaluated cheeses, 14 samples were artisanal cheeses and the remaining 14 were industrial (manufactured) cheeses. All samples (100%) had detectable levels of AFM1, with concentrations ranging from 0.026 to 0.132 µg/kg. Higher levels (p < 0.05) of AFM1 were observed in artisanal mozzarella cheeses, but none of the cheese samples exceed the maximum permissible limits (MPLs) of 2.5 µg/kg established for AFM1 in cheese in Brazil and 0.25 µg/kg in the European countries by the European Union (EU). The high incidence of low levels of AFM1 found in the evaluated cheeses underscores the need for stringent control measures to prevent this mycotoxin in milk used for cheese production in the study area, with the aim of protecting public health and reducing significant economic losses for producers.Cheese is one of the most susceptible dairy foods to accumulating aflatoxins due to their high affinity to caseins. The consumption of cheese contaminated with high levels of aflatoxin M Aspergillus, mainly A. flavus, A. parasiticus, and A. nomius, during growth on food and feed products [1 (AFB1), G1 (AFG1), B2 (AFB2), and G2 (AFG2) [1, the compound is biochemically converted in the animal’s liver into the hydroxylated aflatoxin M1 (AFM1), which is excreted in the milk and other biological fluids [1 in milk and milk-derived products is a major concern for public health, as the AFB1 and AFM1 metabolites were classified as group 1 (human carcinogens) by the International Agency for Research on Cancer (IARC) [Aflatoxins are mycotoxins produced as secondary metabolites by species of the fungal genus products . These f2 (AFG2) . All the2 (AFG2) . When anl fluids . The occr (IARC) ,6,7,8. 1 (AFM1) binds strongly to casein in milk, resulting in greater levels in dairy products that are high in protein, such as cheeses. Moreover, traditional processes used in milk product manufacture, such pasteurization or sterilization, do not destroy AFM1 [. Previous reports have shown that AFM1 levels are approximately 4 and 5 times higher in soft and hard cheeses, respectively [1, depending on the moisture content of the product [1 in cheeses from the states of São Paulo, Amazonas, and Santa Catarina in Brazil [1 level in cheeses from the state of Pernambuco, Brazilian.Aflatoxin Mroy AFM1 . Previouectively ,10. Soft product , as shown Brazil ,12,13. A1 frequencies and levels include variations in cheese manufacturing practices, changes in milk contamination, cheese ripening conditions, geographical and seasonal changes, and the analytical methods used [1, Brazil has adopted regulatory limits for this mycotoxin, with maximum permissible limits (MPL) of 0.5 µg/L for fluid milk and 2.5 µg/kg for cheese [1 in milk products have been monitored [1 concentrations in milk and its derived products is an essential aspect of food safety efforts, aimed at protecting consumers’ health protection [1 in artisanal and industrial cheeses produced in the main dairy plants in the state of Pernambuco, Brazil.The main factors affecting the AFMods used . Given tr cheese . The occonitored  using seonitored , high-peonitored , and HPLonitored . In thisotection . The aim1 in different cheeses from the main production plants in the Araripe Sertão and Agreste regions of Pernambuco state in Brazil. The occurrence and levels of AFM1 were determined by HPLC, as given in 1 occurrence in all analyzed cheese samples . In addition, the levels of AFM1 were assessed, with artisanal mozzarella cheese  demonstrating levels ranging from 0.026 µg/kg to 0.093 µg/kg, and the manufactured mozzarella cheese  indicating levels ranging from 0.037 to 0.132 µg/kg. Similarly, artisanal coalho cheese  exhibited AFM1 levels ranging from 0.035 µg/kg to 0.045 µg/kg, whereas the manufactured coalho cheese  revealed AFM1 levels ranging from 0.035 µg/kg to 0.046 µg/kg. Throughout the analyzed cheese samples, the mean level of AFM1 for 25% of the artisanal mozzarella cheeses (n = 7) was 0.07 ± 0.02 μg/kg, which was significantly higher (p < 0.05) than the remaining artisanal or manufactured coalho cheeses.This study is the first to evaluate the incidence and levels of AFM1 in cheese samples were further assessed by distributing them into quartiles  cheese revealed AFM1 levels at a minimum of 0.035 µg/kg and a maximum of 0.045 µg/kg, with a median of 0.04 µg/kg. In turn, the manufactured cheese (MC) revealed AFM1 levels ranging from 0.035 µg/kg to 0.047 µg/kg, with a median of 0.041 µg/kg. However, the MC had greater variability in the data compared to the AC  cheese represented AFMo the AC .1 in cheeses collected from production plants in Araripe Sertão and Agreste in the state of Pernambuco in Brazil  with varying levels  to another food  during fabrication, even under strict processing and handling procedures. When the resulting cheese products contain high levels of AFM1, they can pose a risk to human health. To reduce this risk exposure and increase awareness in the food industry, the occurrence and levels of AFM1 are evaluated in various milk products, comprising different cheeses produced in Brazil.This study, based on HPLC, evaluated the incidence and levels of AFMn Brazil . The resg levels . None of.5 µg/kg  or the E1 in different types of cheeses produced in various regions of Brazil. The investigation of AFM1 in this study is in line with previous data demonstrating the incidence of AFM1 in different types of cheeses produced not only in Brazil, but also in other countries , around 59,543.14 tons of cheeses were produced in Brazil in 2020 . The couountries . 1 in Minas cheese, mozzarella, and cheddar, which were sold in Campinas, São Paulo, Brazil. Unfortunately, their pioneering study did not distinguish AFM1 in the tested cheese samples, as the evaluation was conducted using thin layer chromatography [1 in cheese products with varying levels. One of these studies, using HPLC, investigated three types of cheeses, namely Minas frescal (n = 7), canastra (n = 18), and Minas standard (n = 50). The results showed that the entire samples (n = 75) contained AFM1, with levels ranging from 0.03 to 0.18 μg/kg in Minas frescal , 0.02 to 1.7 μg/kg in canastra , and 0.02 to 6.92 μg/kg in Minas standard  cheese [1 was highest in the Minas standard cheese (0.62 μg/kg), followed by canasta (0.36 μg/kg), and frescal cheese (0.08 μg/kg), as given in 1 at levels ranging from 0.142 to 0.118 μg/kg and 0.118 to 0.054 μg/kg, respectively [1 (0.142–0.118 μg/kg) compared to Minas standard cheese (0.118–0.054 μg/kg) (n = 20), Minas frescal (n = 30), and Minas standard (n = 8) cheese, a higher occurrence of AFM1 was found in 67.2% of the cheese samples, with the levels ranging from 0.01 to 0.304 μg/kg [n = 23) produced in Brazil, incorporating prato (n = 9) and parmesan ralado (n = 14), were analyzed and showed higher occurrence of AFM1. The levels of AFM1 in prato cheese  and parmesan ralado cheese  were found to be 0.02–0.54 μg/kg and 0.04–0.30 μg/kg [n = 30), 60% (n = 18) showed AFM1 positivity at levels ranging from 50 to 690 μg/kg [n = 40/88) of parmesan cheese was found to contain AFM1, with levels ranging from 0.02 to 0.66 μg/kg [In accordance with tography . Further) cheese . In thisectively . This st4 μg/kg) . Amongst04 μg/kg . Likewis30 μg/kg . Overall90 μg/kg . These p90 μg/kg . In cont66 μg/kg . n = 75/111) of the locally fabricated or imported white and yellow cheese types, comprising Halloumi, Naboulsi, Feta, Baladi, and Akkawi, were contaminated with high occurrence of AFM1 at levels ranging from 0.00561 to 0.315 μg/kg [1 tremendously exceeded the EU permissible level of 0.25 μg/kg [n = 194/360) of the locally produced Lighvan, Koozeh, Siahmazgi, Khiki Talesh, and Lactic cheeses were contaminated with higher levels of AFM1 at 0.0505–0.3087 μg/kg [n = 17/30) of Oaxaca cheeses tested contained AFM1 at an average level of 1.7 μg/kg [1 compared to Oaxaca cheeses fabricated in Mexico City [n = 39/46) of various types of cheeses had higher occurrence of AFM1, with a mean level of 0.1977 μg/kg [n = 82/82) of the cottage cheese samples tested positive for AFM1, with levels recorded at 5.58 ± 0.08 μg/kg [n = 42/60) of the cheeses produced in Serbia were found to be contaminated with AFM1 at levels exceeding 0.25 μg/kg [n = 10/25) from Turkey indicated the incidence of AFM1 at concentrations ranging from 0.00246 to 0.035 μg/kg (mean: 0.01714 ± 0.0042 μg/kg) [1 was detected in 29% (n = 8) of the Minas frescal cheese manufactured in São Paulo State, at levels ranged from 0.113 to 0.092 μg/kg [1 in Minas frescal cheese marketed in the northeast region of São Paulo, Brazil [Following additional research, a study conducted in Lebanon  found th87 μg/kg . A study.7 μg/kg . In addiico City . Additio08 μg/kg . In a re25 μg/kg . The ana2 μg/kg) . Similar92 μg/kg . However, Brazil . The inc, Brazil ,39.1 in cheese depend on the type of cheese, the amount of water eliminated, the curd temperature, the pH of the saturated brine, and the duration of cheese pressing. Reports suggest that AFM1 levels may be higher in hard cheese compared to soft cheese, due the higher amount of the milk proteins, mainly casein, to which AFM1 has a greater binding affinity [1 in a range of milk and milk-derived products, containing several cheese samples (n = 57) [1 at a level of 0.695 µg/kg [n = 58), incorporating Minas frescal light, Minas frescal, Minas standard, and other type of cheese, found that 67.24% of the cheeses were contaminated with AFM1 (0.01–0.304 µg/kg), as given in The results of the coalho and mozzarella cheeses can be attributed to variations in manufacturing methods and physicochemical characteristics of each product, as described in previous studies ,41. The affinity . A study(n = 57) . The stu95 µg/kg . Moreove1 compared to other studies conducted on almost same cheese samples in northern Brazil [1 in artisanal and manufactured mozzarella cheeses ranged from 0.026 to 0.093 μg/kg and from 0.037 to 0.132 μg/kg, respectively, while artisanal and manufactured coalho cheeses showed 0.035–0.045 μg/kg and 0.035–0.046 μg/kg, which were comparatively lower than those detected in northern Brazil. In the northern region, especially the state of Amazonas in Brazil, 25 cheese samples  were analyzed, and none of the samples exceeded the Brazilian MPL of 2.5 μg/kg for AFM1 [1 was analyzed in Serrano artisanal cheeses at four different maturation periods , and it was observed that only four samples had AFM1 levels of 0.505, 0.875, 0.093, and 1.03 μg/kg [1 levels below the Brazilian MPL of 2.5 μg/kg, some exceeded the EU MPL of 0.25 μg/kg. Although seasonal factors were not considered in the current study, the incidence and level of AFM1 in cheese samples analyzed can also be influenced by different time periods of cheese manufacture. The artisanal and coalho or mozzarella cheeses produced between March and May 2022 (Brazilian autumn) give AFM1 levels ranging from 0.026 to 0.132 µg/kg  in the occurrence and level of AFM1 in traditional cheese produced in the summer and winter seasons [1, further studies have also been performed. For example, in a study of traditionally produced Egyptian cheeses over two years (2016–2017), the occurrence and concentration of AFM1 in karish Egyptian cheese was found to range from 1.11 to 0.632 µg/kg [1 at 1.34 µg/kg (2016) and 0.855 µg/kg (2017) only in the winter, compared to the other three seasons [It is important to note that the impact of seasonality on AFMescribed . In thiscotoxins . This de seasons . In rega32 µg/kg . However seasons . 1 levels at 0.07 μg/kg that do not exceed any of the described MPLs. Conversely, in a study of parmesan cheese commercially available in Rio de Janeiro, Brazil, overall samples showed AFM1 levels below the Brazilian MPL of 2.5 µg/kg, but 26.7% samples still exceeded the EU’s MPL of 0.25 µg/kg [n = 82) of locally produced and industrialized cheese contained AFM1 at levels of 5.58 ± 0.08 μg/kg, exceeding the limits set by Egypt, the EU, and Morocco, and 88% of the samples even exceeded the MPL settled by the United States [In the current study, none of the analyzed cheese samples exceeded the MPL of 2.5 µg/kg set by Brazil . This li25 µg/kg . A studyd States .1 compared to mozzarella, edam, cheddar, cream, and moshalal varieties [1 in halloumi and kashkaval cheeses to factors such as maturation time, manufacturing method , milk source, and time of production [In a study carried out in Qatar, halloumi and kashkaval cheeses were found to have significantly higher levels of AFMarieties . The autoduction . The varoduction ,43,44.1 was detected using HPLC in both artisanal and industrially produced cheeses in Pernambuco, Brazil. The levels of AFM1 were low and all the samples were found to be below the limit of 2.5 µg/kg established in Brazil and 0.25 µg/kg adopted in the EU. The study found significant differences between the levels of contamination of artisanal mozzarella-type cheese and manufactured coalho cheese. Based on these results, there is a need for strict quality control measure to further reduce the presence of this mycotoxin in the milk used for cheese manufacture in the studied area. Moreover, future studies should also evaluate the cheeses throughout different seasons to determine the incidence and level of AFM1, as seasonality can have a significant impact on cheese production in the same regions of Pernambuco, Brazil.According to the findings of this study, a high incidence of AFMIn this study, a non-probabilistic convenience sampling was employed, which accounted for approximately 70% of the producers in the study area. Participation in the research was voluntary and not all producers agreed to take part. The collection of the samples was performed for the first time in two main cheese-processing plants in the Agreste and Araripe Sertão regions of Pernambuco state, Brazil. A total of 28 samples of mozzarella- and coalho-type cheeses, 14 of which were artisanal and the remaining 14 were industrially produced, were collected between March and May 2022. Individual samples  were stored at 4 °C prior to transport to the laboratory for immediate analytical procedures.3OH, Dinâmica®, São Paulo, SP, Brazil), and 13 mL of ultra-pure water (Mili-Q). Individual mixtures after homogenization (1 min), shaking (10 min), and centrifugation (6000 rpm × 15 min) were filtered through membrane filters (0.22 µm) in glass tubes. At this stage, 20 mL of the individual extracts were diluted in 30 mL of ultra-pure water (Mili-Q) and submitted to re-centrifugation (6000 rpm × 15 min). Now the final filtrates were subjected to passing through the immunoaffinity columns  connected to the glass syringes and vacuum system (2–3 mL/min flow rate). The columns were washed with 20 mL ultra-pure water, and targeted AFM1 in the samples was eluted with 1 mL of methanol. Then, the samples were subjected to dryness on evaporation under the nitrogen flux (MultiVap-54), and the dried extracts were one-by-one reconstituted in a 1 mL solvent mixture of methanol/water . The analysis of the cheese samples was performed according to a previously in-house validated analytical method . Individ1 were carried out on a high-performance liquid chromatography (HPLC) system  equipped with 10 AXL fluorescence detectors (Excitation at 360 nm and emission above 440 nm). A Kinetex C18 column  4.6 × 150 mm, 2.6 µm particle size, and an in-line filter of 0.5 µm were used. The isocratic mobile phase consisted of methanol/water/acetonitrile  with a flow rate of 0.50 mL/min.The determinations of AFM1 standard  in acetonitrile (CH3CN) at the concentration ranges of 2.5, 5, 10, 20, 40 ng/mL. The limits of detection (LOD) and quantification (LOQ) were calculated based on the signal-to-noise ratio (S/N) of 3:1 and 10:1. The LOD and LOQ values for AFM1 in cheese samples under study were 0.017 and 0.055 µg/kg, respectively. All HPLC runs were carried out in triplicate and the data average values were expressed in the form of mean ± standard deviation.Calibration curves with five points were prepared by diluting AFMp < 0.05. All analyses were carried out using XLSTAT 2022 software (v.24.2.1300), with descriptive statistics and Microsoft Excel (v.14.0.4760.1000) also being used to summarize the data. Additionally, R version 4.0.5 software was used.The results obtained from the trial were subjected to one-way analysis of variance (ANOVA) to determine differences among the cheese samples and the Tukey 5% test was applied. The significance level was accepted at the probability


Compound 1 was also confirmed by single crystal XRD, showing a slightly distorted square planar geometry. The antibacterial results obtained via the agar-well diffusion method for compound 1 were maximum among the screen compounds. All the compounds have shown good to significant antibacterial results against the tested bacterial strains, Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus, except 2 against Klebsiella pneumonia. Similarly, the molecular docking study of compound 3 has shown the best affinity with binding energy scores of −8.6569, −6.5716, and −7.6966 kcal/mol against Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus, respectively. Compound 2 has exhibited the highest activity (3.67 µM), followed by compound 3 (4.57 µM), 1 (6.94 µM), and 4 (21.7 µM) against the DU145 human prostate cancer cell line using the sulforhodamine B (SRB) method as compared to cisplatin (>200 µM). The highest docking score was obtained for compounds 2  and 3 . Compound 2 shows that the Cl atom of the compound acts as a chain side acceptor for the DR5 receptor residue Asp B218 and the pyridine ring is involved in interaction with the Tyr A50 residue via arene-H, while Compound 3 interacts with the Asp B218 residue via the Cl atom. The physicochemical parameters determined by the SwissADME webserver revealed that no blood-brain barrier (BBB) permeation is predicted for all four compounds, while gastrointestinal absorption is low for compound 1 and high for the rest of the compounds (2–4). As concluding remarks based on the obtained in vitro biological results, the evaluated compounds after in vivo studies might be a good choice for future antibiotics and anticancer agents.One homoleptic ( Cancer is a devastating disease, yet many types can be entirely treated if discovered early on, and for many others, patients’ lives can be greatly prolonged. Surgery, radiation therapy, and chemotherapy  are threIn actuality, all anticancer medications also impact healthy cells, resulting in a variety of side effects, including nausea, vomiting, and exhaustion ,4. Since2+ produces both nonfunctional and bifunctional adducts.Many efforts have been made to clarify cisplatin’s biological targets since its discovery as an anticancer medication . Cisplat1H NMR was used to explore the mechanism of interaction between Pd(II) ions and tetracycline. Through the amide and hydroxyl groups at ring A, all three antibiotics create 1:1 compounds with Pd(II). Tetracycline’s Pd(II) complex was 16 times more efficient than tetracycline at slowing down the growth of E. coli HB101/pBR322, a bacterial strain that is resistant to tetracycline, and it was just as successful at stifling the growth of two E. coli susceptible bacterial strains. In the resistant strain, the palladium(II) combination with doxycycline increased its activity by a factor of two [−4 mol/L, several palladium (II) compounds showed broad-spectrum antibacterial action against a variety of human diseases [Khan et al. ,18,19 prr of two . At concdiseases ,22. Dithdiseases ,24. The diseases ,26.Palladium(II) and platinum(II) complexes have structural and thermodynamic similarities ,28,29,30The physical data of the reported compounds, including molecular formula, yield, melting point, and elemental analysis, is given in the −1 and 3040–3052 cm−1 was attributed to the sp3 hybridized aliphatic C-H and the sp2 hybridized aromatic C-H, respectively. The Raman peaks for Pd-Cl, Pd-P, and Pd-S bonds appeared at 298–308 cm−1, 210–221 cm−1 and 380–395 cm−1, respectively. The appearance of the formation of the Pd-S verifies the Pd dithiocarbamate bond formation. The peak at 1092–1097 cm−1 was assigned to the SCS group, while that at 1520–1530 cm−1 was ascribed to the CN moiety. The partial double bond character in the C-N bond for dithiocarbamate complexes was confirmed from the appearance of the peak in the range of 1520–1530 cm−1 as the single C-N bond appears at 1251–1361 cm−1 while the C=N shift value is reported at 1640–1690 cm−1 [−1 indicates the bidentate bonding mode of the dithiocarbamate ligand with palladium moiety; for monodentate bonding, two peaks are to be observed in this region if the dithiocarbamate [2.The FT-IR and Raman data of the prepared compounds are given in the 690 cm−1 . The prearbamate . Figure 1H NMR data is almost similar, with no conspicuous differentiation. The 13C NMR of the characteristic SCS fragment is observed in the range of 207.3–208.2 ppm and provides an important indication for the complex’s formation. A slight up field from the ligand due to the accumulation of electronic density over the carbon atom in SCS after the complexation with palladium metal was observed. The 31P NMR of the synthesized compounds is observed in the range of 20.3–27.5 ppm, and it also depicts a slight up field from the ligand as a result of the transfer of electron density from the ligand to the Pd2+ center. 1H, 13C, and 31P NMR spectra of complex 1.The NMR data of the ligands and their palladium complexes is given in the 1 single crystal XRD data was collected at 200 K using a STOE IPDS image plate detector using MoKα radiation, as depicted in Complex 1 in a monoclynic crystla system with the P21 space group is given in The ORTEP view of complex 2 > 3 > 4 > 1. The higher activity of the heteroleptic compounds 2, 3, and 4 containing different organophosphine ligands despite sharing a dithiocarbamate group is due to the electronegative nitrogen atom included in the organophosphine ligand, which can interact with the DNA strands of the carcinoma cells in a variety of ways’. This may be responsible for the greater activity of compound 2.1 is the most active, while complex 3 is the least active compound. The higher activity of complex 1 may be attributed to the fact that it has no dithiocarbamate ligand, while the other three complexes (2–4) have dithiocarbamate ligands, which can react with other active compounds inside the cell, so they may not have reached the target moiety.In order to make a crucial decision on a daily basis about which compound should be synthesized, tested, and promoted as a drug candidate, the study of pharmacokinetics has utmost importance. Not only the efficacy and toxicity but also other parameters such as absorption, distribution, metabolism, and excretion are thoroughly studied before the processing of a molecule to be marketed as medicine for the benefit of patients.SwissADME webserver. The physicochemical properties show that the complexes 1, 2, 3, and 4 have 10, 5, 7, and 5 rotatable bonds, respectively, as shown in o/wLogP (iLOGP) is zero for all four complexes, while o/wLogP (XLOGP3) is 9.49, 6.46, 7.54, and 7.56 for complexes 1, 2, 3, and 4, respectively. Poor solubility in water is predicted, which is in agreement with the experimental observation. No blood-brain barrier (BBB) permeation is predicted for all four complexes, while gastrointestinal (GI) absorption is low for complex 1 and high for the rest of the three complexes. No drug likeness for complexes 1, 3, and 4 and yes for complex 2 is predicted by the Lipinsky model, while the Veber model has predicted positive drug likeness for all four complexes. The medicinal chemistry parameter has predicted zero alerts for all four complexes by PAINS, one alert for complexes 1, 2, and 4, and two alerts for complex 3 by Brenk.The pharmacokinetic parameters of the synthesized complexes were studied by 3 have shown the highest binding affinity of −8.6569, −6.5716, and −7.6966 kcal/mol against E. coli, K. pneumonia, and S. aureus, respectively. The next highest binding energy is shown by compound 4 against all three bacterial strains, which is evident from the binding energy value given in 3 against the three bacterial proteins are given in His 203 and Lys 162 residues of E. coli. 3 interacts with the Asn B220 residues of K. pneumonia via arene–H.The compounds docking results with the selected antibacterial targets are provided in 1–4 with DR5 (1BU3) shows that the maximum binding affinity was observed for compounds 2  and then for compound 3 . The maximum in vitro antitumor activity was also observed for compound 2. The docking results reinforced the in vitro results. The interaction with the DR5 receptor of representative compounds 2 and 3 is given in 2 shows that the Cl atom of the compound acts as a chain side acceptor for the DR5 receptor residue Asp B218 while the pyridine ring is involved in interaction with the Tyr A50 residue via arene–H. Similarly, compound 3 interacts with the Asp B218 residue via the Cl atom.The interaction of the compounds 3)2PdCl2, where PR3 = diphenyl–2–pyridylphosphine, diphenyl–2–ethoxyphenyl phosphine, and diphenyl–p–tolylphosphine) was checked against the three bacteria receptors: E. coli (PDB_ID: 6G9S), K. pneumonia (PDB_ID: 4EXS), and S. aureus (PDB_ID: 5ZH8), as well as DR5 (1DU3).The interaction of the starting precursors  organophosphine precursor having a diphenyl–p–tolylphosphine moiety , followed by a diphenyl-2-pyridylphosphine moiety . Similarly, against K. pneumonia and S. aureus, the maximum binding affinity was observed for the Pd(II) organophosphine precursor having a diphenyl–2–ethoxyphenyl phosphine moiety , followed by a diphenyl–p–tolylphosphine moiety , respectively.Against p–tolylphosphine moiety . From the comparison of binding energy, we can say that the starting Pd(II) organophosphine precursors have low binding affinity as compared to their corresponding Pd dithiocarbamate complexes. The results are shown in The best binding affinity against DR5 (1DU3) was observed for the Pd(II) organophosphine precursor having a diphenyl–2–ethoxyphenyl phosphine moiety , followed by a diphenyl–2 was obtained from Alfa-Aesar, whilst organophosphines, diphenyl-n-propylphosphine, diphenyl–2–pyridylphosphine, diphenyl–p–tolylphosphine, diphenyl-2–ethoxyphenyl phosphine, potassium hydroxide, hydrochloric acid, methanol, and dichloromethane were acquired from Sigma–Aldrich and put to use directly without further purification.PdCl1H NMR (300.13 MHz): CDCl3 (7.26 from SiMe4). 13C NMR (75.47 MHz), internal standard TMS; 31P NMR (121.49 MHz): CDCl3. IR spectra were recorded on a Nicolet 6700 FT-IR instrument in the range of 400–4000 cm−1 and Raman spectra (±1 cm−1) were measured with an InVia Renishaw spectrometer, using argon ion (514.5 nm) and near infrared diode (785 nm) lasers. Wire 2.0 software was used for the Raman data acquisition and spectra manipulations. The elemental analyses were conducted on a LECO-183 CHNS analyzer. Melting points were measured on the Stuart SMP10 apparatus and are uncorrected. GraphPad Prism was applied for statistical analysis.NMR spectra were recorded on Mercury 200 MHz and Bruker 300 MHz spectrometers. The synthesized compounds were prepared in three steps. In step-1, dithiocarbamate was prepared; in step-2, palladium(II) organophosphine complexes were prepared; and in step-3, heteroleptic palladium complexes containing both organophosphine and dithiocarbamate ligands were prepared ,18.A carbon disulfide solution (30 mL) in dry methanol was added dropwise to dimethyl amine and potassium hydroxide dissolved in methanol (30 mL) in an equimolar ratio. The reaction mixture was stirred for 4 h at 0 °C . A white1) was prepared by reacting PdCl2 dissolved in methanol (30 mL) along with 4 drops of concentrated HCl with organophosphine dissolved in acetone (25 mL) in 1:2. The reaction mixture was reflexed and stirred for 3 h  organophosphine complex ( for 3 h . The pro2–4) were prepared by the reaction of potassium salt of dithiocarbamate dissolved in dichloromethane (25 mL) with palladium phosphine complex dissolved in dichloromethane (30 mL), Heteroleptic or mixed ligand palladium(II) complexes (2 was reacted with 0.20 mL (0.90 mmol) of diphenyl–n–propylphosphine. Yield: 79% (0.23 g). Melting point: 161–162 °C. Elemental analysis for complex 1 having a molecular formula C30H34Cl2P2Pd: % Calculated (found): H, 5.41 (5.39); C, 56.85 (56.71). FT-IR (cm−1): 2922, 2854 υ, 3052 υ. Raman (cm−1): 298 υ(Pd–Cl), 221 υ(Pd–P). 1H NMR (ppm): 0.96  CH3CH2CH2-, 1.49–1.55 (m) CH3CH2CH2-, 2.39–2.44  CH3CH2CH2-, 7.25–7.71 (m) Ar-H. 13C NMR (ppm): 14.7 (CH3CH2CH2-), 23.0 (CH3CH2CH2-), 28.9 (CH3CH2CH2-), 128.3, 128.4, 134.5, 134.6 (Ar-C). 31P NMR (ppm): 17.1.An amount of 0.08 g (0.45 mmol) of PdCl2 having a molecular formula C20H20ClN2PPdS2: % Calculated (Found) S, 12.21 (12.26); N, 5.33 (5.30); H, 3.84 (3.87); C, 45.72 (45.80). FT-IR (cm−1) analysis: 1096 υ(SCS), 1548 υCN, 2925, 2861, υ, 3056 υ. Raman (cm−1): 380 υ(Pd–S), 300 υ(Pd–Cl), 218 υ(Pd–P). 1H NMR (ppm): 2.80 (s), 3.58 (s) (CH3), 7.37–8.73 (m) (Ar-H). 13C NMR (ppm): 35.5, 38.6 (CH3), 125.0, 128.9, 130.0, 130.8, 131.3, 134.7, 134.6, 134.9, 149.3, 157.6 (Ar–C) 207.5 (SCS). 31P NMR (ppm): 27.5.A total of 0.05 g (0.41 mmol) of potassium salt of dimethyldithiocarbamate was reacted with 0.29 g (0.41 mmol) of Pd-phosphine complex. Yield: 78% (0.23 g). Melting point: 197–198 °C. Elemental analysis for complex 3 has a molecular formula. C22H23ClNOPPdS2: % Calculated (found) for: S, 11.57 (11.60); N, 2.53 (2.57); C, 47.66 (47.70). FT-IR (cm−1): 1097 υ(SCS), 1528 υ(CN), 2930, 2850 υ, 3052 υ. Raman (cm−1): 390 υ(Pd–S), 301 υ(Pd–Cl), 215 υ(Pd–P). 1H NMR (ppm): 3.14 (s), 3.26 (s) (CH3), 3.98 , 1.33 , 6.90–7.76 . 13C NMR (ppm): 14.3 (-CH2CH3), 65.2 (-CH2CH3), 120.1, 126.3, 128.2, 130.4, 131.7, 133.2, 164.0 (Ar-C), 208.2 (SCS). 31P NMR (ppm): 20.3.A total of 0.03 g (0.25 mmol) of potassium salt of dimethyldithiocarbamate was reacted with 0.19 g (0.25 mmol) of Pd-phosphine complex. Yield: 85% (0.17 g). Melting point: 223–224 °C. The elemental analysis for complex 4 having a molecular formula C22H23ClNPPdS: % Calculated (found) for: S, 11.91 (11.87); N, 2.60 (2.64); H, 4.31 (4.40); C, 49.08 (49.00). FT-IR (cm−1) analysis: 1092 υ(SCS); 1530 υ(CN) 2935, 2852, υ, 3040 υ. Raman (cm−1): 395 υ(Pd–S), 308 υ(Pd–Cl), 210 υ(Pd–P). 1H NMR (ppm): 3.09 (s), 3.27 (s) (CH3), 2.38 (s) (Ph–CH3), 7.43–7.92 (m) (Ar–H). 13C NMR (ppm): 36.4, 39.1 (CH3), 128.3, 129.5, 134.0, 136.5, 136.8, 137.1, 137.6, 141.7 (Ar–C), 22.5 (Ph–CH3), 207.3 (SCS). 31P NMR (ppm): 22.8.A total of 0.04 g (0.33 mmol) of potassium salt of dimethyldithiocarbamate was reacted with 0.24 g (0.33 mmol) of Pd-phosphine complex. Yield: 77% (0.19 g). Melting point: 216–217 °C. Elemental analysis for complex E. coli, K. pneumoniae, S. epidermidis, S. aureus, and B. subtilis [4–106 colony-forming units (CFU)/mL of bacterial inoculums were dispersed on top of nutritional agar. The tested compounds having a concentration of 2 mg/mL in DMSO were transferred to each well, and then the plates were incubated for 20 h at 37 °C, and finally the inhibition zone (in mm) was measured to determine the antibacterial activity. Streptomycin was used as the standard drug and DMSO as a positive control for the determination of growth inhibition, and the experiment was performed three times [The antimicrobial potential of the prepared compounds was evaluated using the agar-well diffusion method against five bacterial strains: two Gram-negative and three Gram-positive strains: subtilis . Using aee times .50. The test for growth inhibition was repeated three times [Using the technique described in the referred articles, the anticancer activity of the synthesized compounds was assessed against DU145 human prostate cancer (HTB-81) cells ,41,42. Cee times ,41,42.The attrition rate for clinical trials used to develop new drugs has increased to 90% during the last ten years. Over a five-year period, on average, 26.8 small molecules were approved as FDA drugs. Only 12 innovative small-molecule medicines were approved by the FDA in 2016—the fewest such approvals over the previous fifty years ,44. PharK. pneumoniae, S. aureus, and E. coli, as well as trial receptor DR5, for which the PDB files were obtained from the RCSB PDB homepage.The binding mode and affinity of a small molecule or ligand with a macromolecule, such as a protein or DNA, can be predicted using molecular docking. In the case of drug discovery, molecular docking is used to identify potential drug candidates that can bind to a target protein with high affinity and specificity. Here we have treated Palladium (II) complexes against three bacterial strains: Klebsiella pneumonia [Staphylococcus aureus [Escherichia coli [MOE-Dock software version 2015 was used to perform docking studies so as to identify the binding interactions of the screened compounds in the active site of three antibacterial targets, such as PDB_ID: 4EXS from neumonia , PDB_ID:s aureus ,49, and hia coli , as wellhia coli . Two con1 shows that the geometry around the palladium atom is square planar with slight distortion. The synthesized compounds have shown significant antibacterial activity against the selected targets. Compound 2 has shown the maximum antitumor activity against DU145 human prostate carcinoma cells. The lower binding energy or higher inhibition values indicate a stronger binding affinity of complex 3 with the target proteins. The molecular docking study of the evaluated compounds with DR5 (1BU3) shows the highest binding affinity for compound 2  and then for compound 3 . In silico studies have been performed by the SwissADME webserver, and the compounds generally have shown one or two violations of Lipinski’s rule of five.A total of four Pd(II) complexes, including one homoleptic and three heteroleptic, have been prepared quantitatively. The single crystal analysis of complex