P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3′ of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a ΔPodP3h2 mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in ΔPodP3h2 mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (  COL4A1, COL4A3, COL4A4, and COL4A5 are called collagen IV nephropathies. Alport syndrome and thin basement membrane nephropathy (TBMN) are 2 prominent examples of collagen IV nephropathies   have been described, most due to mutations in genes highly expressed in podocytes . One subs (FSGS) . The pres (FSGS) . Patients (FSGS) , 9.Collagen IV is the major structural GBM protein and has 6 different α-chains forming isomer-specific trimers  . CollageP3H2 gene can result in high myopia, lens subluxation, cataract formation, and a predisposition to retinal detachment (The enzyme prolyl 3-hydroxylase 2 (P3H2) is a posttranslational modifier and catalyzes 3′ hydroxylation (3Hyp) of proline residues of collagen IV. Proline is often found in position 2 or 3 of the Gly-Xaa-Yaa consensus repeats in the intermediate collagenous domain, and hydroxylation confers flexibility. P3H2 is localized in the endoplasmic reticulum–Golgi network and is highly expressed in tissues that have a rich basement membrane such as those of the kidney and eye . Mutatiotachment . The exatachment .P3H2, using an expression-based approach in a cohort of patients with albuminuria. The relevance of P3H2-mediated collagen modifications for GBM function were not known. We therefore explored the potential effects of P3H2 loss-of-function mutations on collagen IV synthesis and GBM homeostasis in vitro and in vivo. Our study shows that podocyte-specific P3h2-KO mice (referred to hereafter as ΔPodP3h2 or KO mice) mice initially have a TBMN phenotype that slowly progresses to a FSGS phenotype. These findings in mice were supported by data from 3 human kindreds with P3H2 loss-of-function mutations and ocular defects. In conclusion, we identify the what we believe to be the first nonstructural GBM modifier and prove its relevance for glomerular homeostasis.We identified what we believe to be a new albuminuria candidate gene, P3H2 gene was identified in a mouse and human glomerular expression database and highly rated in GO terms related to matrix biology. Subsequently, we identified a homozygous pathogenic nonsense mutation in a young female patient with steroid-resistant nephrotic syndrome (SRNS) and high myopia and cataracts. The P3H2 mutation at exon 4 caused a transition from cytosine (C) to thymidine (T) at position 1213, inducing the formation of a premature stop codon , while no other pathogenic variant of causative FSGS/SRNS genes could be detected  staining of the patient’s biopsy . FibrosiP3H2 mutations for urinary abnormalities. We analyzed the urine of 2 unrelated pedigrees. In the first family, there were 2 siblings carrying a homozygous P3H2 mutation in exon1 , inducing the formation of a premature stop codon. Both of these siblings had high myopia and cataract . Their ocular phenotype resembled that of a previously reported family from the same region with a different homozygous mutation in the gene (https://doi.org/10.1172/JCI147253DS1). These data from a total of 3 unrelated ancestries harboring different mutations highly suggest the modulating effects of P3H2 on the GBM in humans.Next, we analyzed patients with ocular abnormalities  related to cataract . Their uthe gene . UrinalyP3h2 mRNA was not detected in mouse embryonic tissue at E14.5 by ISH  mice to study the effect of P3H2 on GBM and GFB homeostasis. As indicated in P3h2 (fl/flP3h2) mice were generated by breeding tm1aWtsiP3h2 mice with FLPase mice. Subsequently, fl/flP3h2 mice were bred with WT/+hNphs2Cre mice to specifically delete the P3h2 gene in podocytes. fl/flP3h2 mice without Cre recombinase were used as WT controls in all experiments. We chose 3 time points  for further in-depth characterization of the ΔPodP3h2 mouse line. A lack of P3h2 expression in podocytes of the generated mouse line was proven by qPCR and immunofluorescence staining. In qPCR of sorted podocytes, the relative mRNA expression of P3h2 in WT podocytes was 75.7% ± 18.5% and 1.17% ± 0.48% in KO podocytes  analysis shows that until the age of 36 weeks, KO mice did not have albuminuria. At approximately 36 weeks, KO mice started to leak albumin 50.1 ± 12.7 mg/g (n = 8), whereas albumin in WT mice was 28.4 ± 16.4 mg/g (n = 8). Albuminuria further increased by 48 weeks, measuring 107.7 ± 10.3 mg/g (n = 8) in the KO mice  for WT mice and 58.9 ± 16.3 mg/dL (n = 8) for KO mice at 28 weeks. For 48-week-old mice, WT mice had 53.7 ± 9.9 ng/dL (n = 8) and KO mice had 67.4 ± 19.7 mg/dL (n = 8) serum urea  for WT mice and 777.2 ± 167.6 ng/mL (n = 8) for KO mice at 28 weeks. For 48-week-old mice, WT mice had 803.3 ± 144.5 ng/mL (n = 8) and KO mice had 867.4 ± 98.5 ng/mL (n = 8) cystatin C in serum. These values were within the normal range and were not significantly different between WT and KO mice at the age of 28 weeks and 48 weeks, respectively  in KO mouse urine and only 2 ± 1 cells per μL in WT mouse urine (n = 4) , while that of fl/flP3h2 mice was 154.1 ± 15.3 nm (n = 4) (P < 0.05). At 28 weeks, it was 149.9 ± 9.1 nm (n = 4) for ΔPodP3h2 and was 195.2 ± 7.5 nm for fl/flP3h2 mice (n = 4) (P < 0.001). At 48 weeks, the thickness of the GBM for ΔPodP3h2 mice was 387.5 ± 34 nm (n = 4), and for fl/flP3h2 mice, it was 230 ± 8.1 nm (n = 4) (P < 0.001) (n = 4) for KO mice and 310.8 ± 21.9 nm (n = 4) (P > 0.05) for WT mice. At 28 weeks, the mean foot process width was 499.9 ± 98.9 nm (n = 4) for KO mice and 317.7 ± 21.5 nm (n = 4) (P < 0.05) for WT mice. At 48 weeks, the mean values of the foot process width for KO mice were 409.9 ± 25.5 nm (n = 4) and for WT mice, 343.7 ± 19.2 nm (n = 4)   . Quantif < 0.01) .ΔPodP3h2 mice at 6 weeks and 28 weeks with PAS staining. At 48 weeks, glomerulosclerosis was observed and quantified in ΔPodP3h2 glomeruli, which showed further signs of podocyte injury. In fl/flP3h2 mice, the glomeruli appeared healthy at this time point (n = 5) and 10.4% ± 5.1% in WT glomeruli (n = 5) (P < 0.01). The number of glomeruli showing focal glomerulosclerosis was significantly higher in KO mice than in WT mice, confirming the FSGS phenotype in the KO mice . We used podometrics to calculate podocyte numbers, podocyte density, glomerular volume, and the average podocyte volume. Immunofluorescence staining showed reduced podocyte numbers (n = 6) podocytes and 87 (n = 6) podocytes per WT glomerulus  in KO mice and 36.7/μm3 (n = 6) in WT mice  for KO glomerular volumes and 2.95 × 105 μm3 (n = 6) for WT glomerular volumes  compared with 256 μm3 (n = 6) in WT podocytes  in KO mice than in WT mice (34.1% ± 10.4%)  and 5.7% ± 3.2% (n = 6) for WT mice  secrete more extracellular matrix (ECM), causing this increase in thickness of Bowman’s capsule at the beginning of FSGS development . To confP3H2-KO human immortalized cell lines were generated using CRISPR/Cas9 genome editing technology to evaluate ECM changes in vitro  signaling is one of them. This pathway regulates podocyte survival and growth. Its up- and downregulation can induce podocyte stress. Overactivity of mTORC1 signaling in podocytes has been observed in many kidney diseases and is thought of as a common hypertrophic stress pathway of podocytes . We founP3H2-KO immortalized podocyte cell lines. This finding might explain the thinner GBM observed in the KO mice. We observed a decrease not only in collagen IV, but also in collagen IV interactors . These findings might support a functional role of 3Hyp in protein-protein interaction. Indeed, a detailed analysis of collagen IV network formation using high-resolution expansion microscopy showed that the formation was unaffected in WT mice but highly disrupted in KO mice.Differential quantitative GBM proteome analysis showed that collagen IV subchains and other structural GBM proteins were significantly decreased in the KO mice . MoreoveThere are 2 explanations for the decreased abundance of collagen IV subchains in both the ECM and the GBM. First, there might be less secretion of collagen IV from podocytes to the extracellular space, since a lack of 3Hyp might affect its proper secretion. Second, collagen IV lacking 3Hyp within the GBM might have a shorter half-life than properly hydroxylated collagen IV. Lack of 3Hyp might make collagen IV less stable or might prevent proper cross-linking, which could induce its degradation by MMPs. MMPs were not increased in their abundance according to our proteomics data, but their activity could be increased. However, more detailed investigations are necessary to support these hypotheses.P3H2 mutation was different in our index patient compared with that in the other 2 families. Different mutations interfere with protein structure and affect the interaction, function, and activity of the mutated protein  cells from the UC Davis KOMP (Knock-Out Mouse Project) repository [P3h2tm1aWtsi (MGI: 4364924)]. B6.Cg-Tg(Pgk1-FLPo)10Sykr/J mice, obtained by the Central University Hospital Hamburg Eppendorf mouse facility from The Jackson Laboratory, were used to generate a conditional allele of P3h2. Floxed allele P3h2 (fl/flP3h2) mice were bred with Nphs2Cre+/+ (TghNphs2-Cre295Lbh) mice  to generate podocyte-specific P3h2–KO (ΔPodP3h2) mice.Mice were housed in a specific pathogen–free (SPF) facility with free access to chow and water and a 12-hour day/12-hour night cycle. Breeding and genotyping were done according to standard procedures. A conditional An expression-based candidate gene approach was applied to identify novel genes in a cohort of patients with albuminuria. A corresponding next-generation sequencing–based (NGS-based) panel for the parallel analysis of all previously described and potentially new causative genes was established. Candidate genes were selected using a comparative and sequential algorithm from genes heavily expressed in mouse podocytes. With the help of publicly accessible databases, the 750 most enriched transcripts in mice were tested for (a) their expression in human kidney cortex; (b) their expression in human glomerulus; (c) their occurrence in the murine podocyte proteome; (d) a significant regulation in human proteinuric diseases; or (e) a statistical overrepresentation in the respective GO term–based gene clusters. The most promising candidates were chosen according to their scores. The NGS technologies and comprehensive bioinformatics analyses utilized in this project are described in detail elsewhere , 27. CurPerformance of the wet-lab and bioinformatics processes was validated and controlled according to national and international guidelines reaching high sensitivity for single nucleotide variants (SNVs), indels, and CNVs using well-established reference samples as well as a large cohort of positive controls, especially for CNVs. For interpretation of the identified variants, we have developed our own published bioinformatic algorithms using a stepwise filtering process conducted by an experienced team of scientists and supported by various bioinformatics decision tools. Sequence variants of interest were verified by Sanger sequencing if the NGS results failed internal validation guidelines. If other family members were available, segregation of sequence variants with the disease was further assessed.P3H2 gene had one of the highest GO term values in the analysis. A young female patient with SRNS and histologically diagnosed with FSGS along with early cataract development and high myopia presented with a homozygous P3H2 mutation and did not harbor any other pathogenic variant in known causative FSGS and SRNS genes.The t test and a Mann-Whitney U test were used to test for significance between the experimental and control groups. When 3 or more groups were assessed, 1-way ANOVA with Tukey’s multiple-comparison post hoc test was used. A P value of less than 0.05 was set as the significance level. A P value of less than 0.01 was considered very significant, and P values of less than 0.001 and less than 0.0001 were considered highly significant.The data in the diagrams of the results section are shown in different types of plots. All statistical analysis were performed and plots were prepared using GraphPad Prism, version 8.4.0 (GraphPad Software). The data are presented as the mean with SD or the median with the IQR. An unpaired, 2-tailed Student’s Guide for the Care and Use of Laboratory Animals , as well as the German law for the welfare of animals. Animal procedures were approved by the Regierungspräsidium Freiburg (G16-122) and BGV Hansestadt Hamburg (Ü 004/2018). All human samples in this study were obtained with written informed consent accompanying the patients’ samples. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki and were approved by the IRBs of the State Key Laboratory of Medical Genetics (SKLMG) in Changsha, Hunan, China, and the King Faisal Specialist Hospital and Research Center in Riyadh, Saudi Arabia (All animal experiments were conducted according to the NIH’s i Arabia , 18.P3H2 human patient and urine data. HA, CB, TBH, and FG were responsible for conceptualization, interpretation of data, writing and editing the manuscript. TBH and FG supervised the study.All authors read and revised the manuscript before submission. HA, CK, S Lu, S Liu, DK, OK, GW, JMD, MM, TW, and FG performed experiments. OK performed all TEM observations and analysis. MM, CK, and HS performed differential quantitative mass spectrometry–based proteome analysis, data interpretation, and statistical analysis of the proteomics data and provided expertise. DK and VGP assisted with the expansion microscopy, morphometrics, and immunofluorescence analyses. S Lu and S Liu helped with the FACS analysis. KA and KB provided the patient biopsy tissue. MG, TW, and CB performed genetic analysis for the detection of novel genes. PT, ZMH, SMA, and AOK provided


Small-molecule protein kinase inhibitors are used for the treatment of cancer, but off-target effects hinder their clinical use. Especially off-target activation of the pregnane X receptor (PXR) has to be considered, as it not only governs drug metabolism and elimination, but also can promote tumor growth and cancer drug resistance. Consequently, PXR antagonism has been proposed for improving cancer drug therapy. Here we aimed to identify small-molecule kinase inhibitors of the Tübingen Kinase Inhibitor Collection (TüKIC) compound library that would act also as PXR antagonists. By a combination of in silico screen and confirmatory cellular reporter gene assays, we identified four novel PXR antagonists and a structurally related agonist with a common phenylaminobenzosuberone scaffold. Further characterization using biochemical ligand binding and cellular protein interaction assays classified the novel compounds as mixed competitive/noncompetitive, passive antagonists, which bind PXR directly and disrupt its interaction with coregulatory proteins. Expression analysis of prototypical PXR target genes ABCB1 and CYP3A4 in LS174T colorectal cancer cells and HepaRG hepatocytes revealed novel antagonists as selective receptor modulators, which showed gene- and tissue-specific effects. These results demonstrate the possibility of dual PXR and protein kinase inhibitors, which might represent added value in cancer therapy. Signaling cascades of protein kinases regulate multiple functions in cells, such as cell proliferation and survival. Their dysregulation frequently results in loss of apoptotic cell death and uncontrolled cell growth, which are among the hallmarks of cancer . Thus, iEven if small-molecule kinase inhibitors have been conceived as molecularly targeted drugs, it is now increasingly recognized that they demonstrate pronounced off-target effects, which not only account for adverse drug effects but also may contribute to the desired activity . A speciThe objective of this study was to identify novel PXR antagonists among kinase inhibitors. To this end, using an in silico approach we screened the in-house Tübingen Kinase Inhibitor Collection (TüKIC) compound library, which contains 8500 small-molecule kinase inhibitors, to identify PXR binding compounds. Candidate ligands were subjected to experimental confirmation by PXR-dependent reporter gene assays in agonist and antagonist modes. Overall, this screening strategy resulted in the identification of four novel inhibitors and one strong activator with a common phenylaminobenzosuberone scaffold. Comprehensive characterization using different biochemical assays, assessing direct ligand binding, and cellular assays, addressing the interaction of PXR with coregulatory proteins, as well as gene expression analyses in colorectal cancer cells and differentiated hepatocytes, identified the novel inhibitors as passive mixed competitive/noncompetitive antagonists of PXR, which elicit gene- and tissue-specific modulation of PXR target gene expression.3 were purchased from Sigma-Aldrich . CITCO was provided by ENZO Life Sciences . Rifampicin was purchased from Merck Chemicals . SR12813 and T0901317 were obtained from Tocris Bioscience . Minimum essential medium (MEM), Dulbecco‘s modified Eagle‘s medium (DMEM), William’s E medium, and Trypsin-EDTA solution were purchased from Thermo Fischer Scientific . L-glutamine, nonessential amino acids, sodium pyruvate, and penicillin–streptomycin mixture were provided by Biozym . Fetal bovine serum (FBS) was obtained from Biowest . Oligonucleotide primers were provided by Biomers . TaqMan probes were purchased from Applied Biosystems .DMSO and 1α,25-dihydroxy vitamin D2 has been reported earlier  was diluted with 150 mM NaCl to a final volume of 25 µL. Similarly, 0.6 µL JetPEI reagent was diluted with 150 mM NaCl to 25 µL. The diluted jetPEI was added to the diluted DNA mixture and incubated at room temperature for 15 min. In parallel, H-P cells were trypsinized and counted, and the cell number was adjusted to 40,000 cells in 200 µL, per well. The transfection mixture was added to the cell suspension and pipetted into a 96-well plate . After overnight incubation, cells were treated with chemicals for 24 h before cell lysis with 50 µL of passive lysis buffer (Promega). Firefly and Renilla luciferase activities were measured from 10 µL of sample with 150 µL of firefly luciferase assay solution  and 100 3Tk (as reporter for VDR), 0.01 µg pMetLuc2control, and 0.03 µg either CAR1, CAR3, VDR, or PXR(S208W/S247W/C284W) expression plasmids. In addition, 0.03 µg RXRα expression plasmid was added to CAR3 transfections. Metridia luciferase activity was measured from 10 µL of medium supernatant after adding 100 µL Renilla luciferase assay solution.For testing nuclear receptor selectivity and LBP-filled PXR mutant activation, transient batch transfection was conducted as above, but with HepG2 cells and the following plasmids, with amounts per well of 0.23–0.27 µg pGL4-CYP3A4(-7830Δ7208-364) or pGL3(DR3)In mammalian two-hybrid corepressor/coactivator interaction assays, per well 0.24 µg pGL4-G5, 0.01 µg pGL4.75[hRluc/CMV], 0.03 µg expression plasmid encoding VP16-AD/PXR LBD fusion, and 0.03 µg expression plasmids encoding GAL4-DBD/SRC1-RID or GAL4-DBD/SMRT-RID fusions, respectively, were used. For the SMRT corepressor interaction assay, additionally 0.015 µg of RXRα expression plasmid was included.For the mammalian two-hybrid PXR ligand-binding domain assembly assay, 0.24 µg pGL4-G5, 0.01 µg pMetLuc2control, and 0.03 µg each of expression plasmids encoding GAL4-DBD/PXR(132–188) and VP16-AD/PXR(189–434) fusion proteins were used per well. Otherwise, batch transfection was performed as above with HepG2 cells.The limited proteolytic digestion assay was performed as described previously . A total100 and 109 were tested with 7 different concentrations, ranging from 0.01 µM to 10 µM, for IC50 determination. Compound binding was calculated as % inhibition of the binding of the radioactively labeled PXR ligand [3H]SR12813 to recombinant human PXR, as described previously [The assay was performed by Eurofins Cerep . Compounds eviously .Total RNA was prepared from LS174T and HepaRG cells using the NucleoSpin RNA kit . RNA integrity was analyzed by formaldehyde-agarose gel electrophoresis. First strand cDNA was synthesized as described earlier . Relativ100 and 109 were profiled for inhibition of kinase activity at concentrations of 1 µM and 10 µM against a panel of 335 wild-type protein kinases with single measurements. The analysis was performed by ProQinase .Compounds t-test between two groups. Multiple comparisons were performed using ordinary or repeated measures one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test. Comparisons to a hypothetical value were conducted with one sample t-test corrected by the method of Bonferroni. Statistical analyses were performed with GraphPad Prism 9.3.1 .In quantitative real-time PCR analysis, if the standard deviation of Ct values in technical triplicates exceeded 0.2, the outlier was omitted. In the cell viability experiments, if the coefficient of variation of technical triplicates exceeded 20%, the outlier was omitted. Data are presented as mean ± SD of at least three independent experiments, each performed in technical triplicates, if not specified otherwise in the respective figure legend. In bar charts, individual experiments are illustrated as dots. In scatter plots, mean and individual samples are shown. Statistical significances were determined using unpaired or paired 73 and 100 demonstrated 60–70% inhibition of rifampicin-mediated induction, while only weakly activating the CYP3A4 reporter on their own, at about 10% of rifampicin activity  and 2.8 µM (95% CI 2.1–3.8 µM), respectively. Thereby these compounds demonstrated stronger inhibition of rifampicin-induced PXR activation compared to 2 and 12, which displayed IC50 values of 11.2 µM (95% CI 7.7–17.2 µM) and 33.7 µM (95% CI 20.2–61.6 µM), respectively (12 only) could not be used, IC50 values were calculated from concentration–response analyses, which did not result in a lower plateau, thereby limiting the significance of the absolute values. Furthermore, 73 and 100 activated PXR only weakly, with maximal effects (Emax) of about 2-fold, but with low EC50 values of 0.41 µM (95% CI 0.18–0.87 µM) and 0.15 µM (95% CI 0.04–0.45 µM), respectively  and by the large confidence interval with 12 . Interestingly, we identified also a structurally related strong activator, 109 (50 of 44 µM (95% CI 20–??? µM)  C. Due to12 has an inverted amide linking the R2 and is showing the highest IC50-value. The length of the R2 seems also important for PXR inhibition, as the shorter phenyl containing 109 does not inhibit PXR. Of note, 2, which also has a phenyl group in its R2, has an additional sterically large trifluoromethyl substituent in its phenyl ring. Furthermore, compounds with the highest inhibitory activity display a heavier substituent than H in their R1. Based on the lowest energy conformations in water , while in 12, with an H-atom in this position, this conformation is not preferred (11%)  .73 and 100, their effects on the concentration–response curve of rifampicin were assessed. 73 induced a parallel dextral shift of the rifampicin concentration–response curve with corresponding increases in the EC50 values of rifampicin. Without compound 73, EC50 of rifampicin was 1.8 µM (95% CI 0.76–4.5 µM), which increased in the presence of 1 µM or 3 µM of compound 73 to 2.9 µM (95% CI 1.0–9.1 µM) or 6.4 µM (95% CI 2.5–19.6 µM), respectively. With 10 µM of compound 73, rifampicin EC50 was further elevated to 7.4 µM (95% CI 2.0–47.7 µM), and reduction of Emax was pronounced. Compound 100 also caused a dextral shift of the rifampicin concentration–response curve with corresponding increases in the EC50 values of rifampicin. EC50 value of rifampicin raised from 2.0 µM (95% CI 1.6–2.6 µM) in the absence of 100 to 4.8 µM (95% CI 3.4–7.0 µM) with 1 µM of 100, and further to 9.3 µM (95% CI 5.4–17.0 µM) with 3 µM of 100  . Similarly, PXR‘s interaction with SRC1 was induced by the activating compound 109  C. Releasound 109 D. In conpressors , while 173 and 100, as well as of the activator 109, to the PXR LBD, the respective in vitro limited proteolytic digestion assay was applied [Ligands of nuclear receptors physically interact with the respective ligand binding domain (LBD). To confirm the binding of the antagonists  applied . The ass applied . Figure  applied .100 and 109 was further analyzed by competitive radioligand binding assays. 100 and 109 inhibited the binding of 3H-labeled SR12813 to PXR with IC50 of 4.5 µM (95% CI 1.9–15.4 µM) and 2.9 µM (95% CI 1.9–4.7 µM), respectively. We also investigated the binding of compound 100 by microsecond timescale molecular dynamics (MD) simulations [100 in the ligand binding pocket (LBP) of PXR based on the simulation data. In conclusion, the results of the limited proteolytic digestion assay and competitive radioligand binding assay, supported by MD simulations, indicate a physical interaction of the novel compounds with PXR and thus confirm ligand binding.To corroborate these results, the binding of compounds ulations . Figure 73 and 100 and agonist 109 induced the assembly of the PXR LBD. PXR antagonism can occur either by competitive binding into the PXR ligand binding pocket (LBP), which has been demonstrated with SPA70 [100 did not affect constitutive activity of the PXR mutant, 73 still suppressed the respective activity by 40% . These results indicate that the novel PXR antagonists, and here especially 100, demonstrate only minor inhibitory effects on CAR and VDR.To assess the selectivity of the novel PXR ligands, we determined their effects on CAR- and VDR-mediated transactivation. CAR and VDR belong to the same NR1I group of nuclear receptors as PXR and share 37–45% sequence similarity in their LBD . Both 731 by 30% A. CompouO by 46% B and redO by 46% C, while 73 and 100, and these compounds also suppressed its rifampicin-mediated induction , supporting the observed biological results [Passive antagonism is suggested by the fact that the novel antagonists abolished the rifampicin-induced interaction of PXR with coactivator SRC1, as well as impairing the constitutive interaction of PXR with corepressor SMRT. Except SPA70, for which recruitment of SMRT was shown , previou and 100 ,46. Mole results .73 and 100 demonstrated gene-specific effects in LS174T colorectal cancer cells, not inducing on their own the expression of ABCB1, but inducing CYP3A4 expression to a similar extent as rifampicin. Furthermore, both compounds antagonized exclusively the rifampicin-mediated induction of ABCB1. The strong induction of CYP3A4 and absence of antagonism of the rifampicin-dependent activation is surprising, as the compounds were identified by their inhibition of rifampicin-mediated CYP3A4 enhancer/promoter activation. However, in contrast to the transfected reporter gene construct, the regulatory region of the endogenous gene resides in chromatin. Differences in the promoter context between genes, resulting in altered interaction with or altered activity of coregulators, have been suggested for the explanation of gene-specific effects of nuclear receptor ligands [100 was shown to inhibit PKA at 10 µM, it is conceivable that inhibition of the PXR-inhibitory kinase activity of PKA may participate in CYP3A4 induction, especially as it was shown that the activation of PKA resulted in repression of CYP3A4 expression [100 might result from divergent coactivator versus corepressor levels, varying PXR levels, or different activities of coactivators or PXR due to modulation of kinase signaling by the compound. These potential cellular variables have been shown previously to determine the activity of selective modulators of nuclear receptors [The novel antagonists  ligands . Alternapression . Even ineceptors .100, belong to the MAPK/ERK pathway. Hitherto and in conventional 2D cell culture, the MAPK/ERK pathway has not been associated with regulation of hepatic cytochrome P450 expression, as its inhibition by dominant-negative MEK1 did not induce CYP3A4 in HepG2 cells [100 might result in inhibition of this pathway, followed by release of PXR inhibition and consequently CYP3A4 and CYP2B6 induction. In this case, the induction of CYP3A4 and CYP2B6 by 100 would not indicate PXR agonism but indirect activation through MAPK/ERK pathway inhibition. Further research is required to distinguish between these possibilities.With BRAF, RAF1, p38β, and MAPKAPK3, four of the five kinases, which were strongly inhibited by G2 cells . HoweverG2 cells . By siRNG2 cells . If diffAdditional inhibition of PXR by protein kinase inhibitors may provide benefit for cancer therapy in several ways. First, many kinase inhibitors are metabolized by cytochrome P450 enzymes and transported by MDR1/P-glycoprotein ,64,65,66In conclusion, we have identified and characterized novel selective receptor modulators of PXR from an in-house kinase inhibitor compound library. Their common phenylaminobenzosuberone scaffold represents a previously unknown PXR ligand structure and may be used as a starting point for the synthesis of the suggested dual PXR and protein kinase inhibitors.


Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3′-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 ( Ribosomes are ribonucleoprotein complexes that carry out protein synthesis through the translation of genetic information into polypeptides. The ribosome consists of a large subunit (LSU), which contains the catalytic peptidyl transferase center (PTC) and the small subunit where messenger RNA (mRNA) is decoded. Nucleotides in the PTC critical for peptide bond formation are universally or highly conserved, and mutation of these nucleotides often results in severe growth defects or lethal phenotypes . HoweverE. coli ribosome, the interaction between the A-site tRNA 3′-CCA end and the A loop is formed in part by base pairing between universally conserved nucleotides C75 in the tRNA and G2553 in 23S rRNA (E. coli numbering)  docks in the A site of the ribosome. For proper tRNA substrate positioning to occur, the tRNA anticodon must base pair with the mRNA in the small subunit decoding site while the tRNA 3′-CCA nucleotides interact with the A loop, an RNA hairpin in the ribosomal RNA (rRNA) of the large subunit . Proper mbering) .O-methylation of U2552 is a conserved post-transcriptional modification that enables the proper folding of the A loop and ensures residues in the apical loop can interact with the A-site tRNA . Using archaeal sequences as a guide, we show that U to C mutations at positions 2554 and 2555 in 23S rRNA of the E. coli ribosome have a stabilizing effect on the 50S ribosomal subunit. This work identifies rare sequence variation in ribosomal RNA that likely leads to thermostability in the archaeal ribosome and describes how the specific stabilization of A loop structure can lead to global ribosome stabilization. These insights into how thermophilic organisms can stabilize their ribosomes provide a foundation for engineering ribosomes with increased mutational robustness and evolvability.Interestingly, positions 2554 and 2555 are sometimes found as cytidines in Archaea ,13. It icytidine  and 2′-Ohylation , and thahttps://doi.org/10.5281/zenodo.7465829) to identify relevant positions, and correlate data for average optimal living temperature from the TEMPURA database , which harbors a pCsacB plasmid containing the rrnB operon, the KanR gene, and the sucrose counter-selectable sacB marker, was used to examine E. coli growth supported by mutant ribosomes. Replacement of the pCsacB plasmid with the modified pLK35 plasmid was achieved as described with adaptations . 200 μl of each dilution was then pipetted into wells of a sterile 96-well plate. The OD600 was measured every 15 min using a Spark Plate Reader (Tecan) at 37°C with constant shaking. Doubling times were calculated by fitting the initial region of exponential growth to a Malthusian growth model in Prism.Liquid cultures of each transformant were grown overnight and then were diluted to OD 0.01 in LB media containing 100 μg/ml ampicillin and 25 μg/ml spectinomycin (SQ171) or 100 μg/ml ampicillin and 500 μM IPTG . Semiquantitative PCR was used to determine the ratio of tagged 23S rRNA to WT 23S rRNA as previously described . Brieflyq cells were transformed with the corresponding pLK35 plasmid carrying a tac promoter, MS2-tagged 23S rRNA, and the desired mutations. Overnight cultures of the transformants were diluted 1:100 into 2–3 l of LB broth containing 100 μg/ml ampicillin and grown at 37°C. Upon growth to OD600 = 0.6, rRNA transcription was induced with 0.5 mM IPTG and the cultures were grown for 3 h at 37°C. Cells were then pelleted, resuspended in 50–100 ml buffer A , and lysed by sonication. The lysate was then clarified by centrifugation at 14,000 rpm  for 45 min in a F14-14 × 50cy rotor (ThermoFisher). Clarified lysate was then loaded onto a sucrose cushion containing 24 ml of buffer B  with 0.5 M sucrose and 17 ml of buffer C  with 0.7 M sucrose in Ti-45 tubes (Beckman-Coulter). The tubes were then placed in a Ti-45 rotor and spun at 27 000 rpm (57 000 × g) for 16 h at 4°C. The next day, ribosome pellets were resuspended in dissociation buffer (buffer C with 1 mM MgCl2).Crude ribosomes were isolated as previously described with several adaptations . NEB Exp2). Crude tagged ribosomes (∼100–200 mg) were diluted to 15 mg/ml in buffer A-1, which dissociates 70S ribosomes into 30S and 50S subunits and decreases WT ribosome contamination. The diluted crude ribosomes were loaded slowly onto the MBP-trap column, which was then washed with 5 CV of buffer A-1 followed by 5 CV of buffer A-250 (buffer A with 250 mM NH4Cl and 1 mM MgCl2). Ribosomes were eluted with a 10 CV gradient of buffer A-1 containing 0–10 mM maltose. Fractions containing tagged ribosomes were concentrated in 100 kDa cut off spin filters (Millipore) and washed with buffer A-1. 50S ribosomal subunits were quantified using the approximation of 1 A260 = 36 nM and were stored at −80°C.The MBP-MS2 fusion protein was purified as previously described . 10 mg oE. coli strain MRE600 were diluted 1:100 into 2–3 l of LB broth and grown to an OD600 = 0.6. Cells were then pelleted and lysed by sonication. Ribosomes were pelleted and resuspended as described above for crude ribosome preparations. After resuspension in dissociation buffer, the 30S and 50S subunits were separated on a 15–35% (w/v) sucrose gradient in dissociation buffer on a SW-32 rotor (Beckman-Coulter) spun at 28 000 rpm (97 000 × g) for 16 h. Fractions containing the 50S and 30S subunits were collected separately, washed with dissociation buffer, and concentrated in a 100 kDa cut off spin filter. To increase purity, concentrated subunits were separately run on second 15–35% sucrose gradients and appropriate fractions were washed with dissociation buffer and concentrated.Overnight cultures of 600 = 0.4, cells were induced with 1 mM IPTG for 3 h. Cells were then pelleted at 4000 × g for 15 min and resuspended in ∼20 ml 11S lysis buffer . Cells were lysed by sonication and the lysate was clarified by centrifugation at 18,000 rpm  for 30 min . Supernatant was then applied to a 1 ml HisTrap column (Cytiva). The column was washed with 10 CV of 11S lysis buffer and eluted with a 20 CV linear gradient form 20 to 500 mM imidazole in lysis buffer. Protein fractions were dialyzed against 11S lysis buffer without imidazole overnight at 4°C. The protein was then concentrated and stored at −80°C.A plasmid encoding 8xHis-tagged 11S was transformed into BL21 (DE3) Rosetta2 pLysS cells . An overnight culture was diluted (1:100) into LB media with 100 μg/ml ampicillin and grown at 37°C. When the culture reached OD2. This mixture was then incubated at the desired temperature for 30 min on a ProFlex (ThermoFisher) PCR cycler with a heated lid. After incubation, tubes containing the 50S subunits were placed on the bench and allowed to cool slowly to room temperature for 30 min. The cooling method influenced the return of mutant ribosome activity. Slow cooling at room temperature returned higher levels of activity as compared to snap-cooling on ice for the CC mutant ribosome (in vitro translation mixture was assembled using a ΔRibosome PURExpress kit (NEB). The mixture was assembled with the following: 3.2 μl solution A (NEB), 1 μl factor mix (NEB), 250 nM pre-incubated 50S ribosomal subunits, 500 nM WT untagged 30S ribosomal subunits, 1U/μl Murine RNAse inhibitor (NEB), 100 nM 11S NanoLuc protein . Ribosome activity was calculated by determining the slope of the initial linear region of each in vitro translation reaction. Each reported activity is the average from three separate HiBit reactions.50S ribosomal subunits were diluted to 1.4 μM in buffer A with a final concentration of 10 mM MgClribosome . It is p protein , 1:50 , 2 μl factor mix (NEB), 250 nM pre-incubated 50S subunit, 500 nM WT untagged 30S subunit, 1U/μl Murine RNAse inhibitor (NEB), and 10 ng/μl of a plasmid encoding a T7 promoter followed by the nanoluciferase gene . This reaction was then incubated at 37°C for 1 h. After incubation, 5 μl from the reaction was mixed with 30 μl of 11S buffer  and a 1:50 dilution of Nano-Glo substrate (Promega). This mixture was placed in a 384-well plate and luminescence was measured in a Spark Plate Reader (Tecan). Triplicate readings were measured and averaged for each in vitro translation reaction.An 2 to a final volume of 20 μl. This mixture was then incubated at the desired temperature for 30 min on a ProFlex (ThermoFisher) PCR cycler with a heated lid and placed on the bench for 30 min to cool slowly to room temperature. For 70S formation assays, 10 pmol 50S subunit and 20 pmol WT untagged 30S subunit were diluted in buffer A with 15 mM MgCl2 to a final volume of 20 μl. These samples were then incubated at 37°C for 30 min. Samples were loaded onto a 15–40% sucrose gradient in buffer A with 10 or 15 mM MgCl2. Gradients were then placed in a SW-41 Ti rotor (Beckman-Coulter) and centrifuged at 29 000 rpm (100 000 × g) for 13 h. An ISCO gradient fractionation system was used to measure A254 traces.For heat treated 50S subunits, 10 pmol 50S subunit was diluted in buffer A with 10 mM MgClxHyPO4 pH 6.5. Low ionic buffer conditions were used to shift the melting temperature to a value where both the initial and final baselines could be more accurately modeled. CD experiments were conducted in a 1-mm path-length cuvette with an Aviv 410 CD spectropolarimeter. For thermal melting experiments, CD at 262 nm was recorded in 2°C intervals from 5°C to 95°C. The temperature was moved to the target value, and the system was allowed to equilibrate for one minute before a measurement was taken. Thermal melting data was fit to a two-state model with sloping baselines . The protein was concentrated and stored at −80°C.A plasmid encoding 6xHis-tagged methionyl-tRNA synthetase (MetRS) was transformed into BL21 (DE3) Codon + RIL cells (Agilent). Overnight cultures were diluted (1:100) into ZYM-5052 autoinducing media  and growfMet, with a C1G mutation to increase transcription yield and deletion of the 3′-terminal adenosine (C1G -A), was amplified via PCR, yielding a double stranded DNA template. tRNAfMet was in vitro transcribed with transcription buffer , 2.5 mM NTPs, 1 U/μl murine RNase inhibitor (NEB), 0.5 U/μl T7 polymerase (NEB) and 0.5 U/ml YIPP (NEB). In vitro transcription reactions were incubated for 16 h at 37°C and then treated with 45 U/ml of RQ1 DNase (Promega) at 37°C for 30 min. Reactions were ethanol precipitated and then resolved on a 12% denaturing polyacrylamide gel. Gel slices containing the tRNA were excised, crushed and soaked in 300 mM sodium acetate pH 5.2 overnight at 4°C. The RNA was then ethanol precipitated, resuspended in water, and stored at −80°C.A single stranded DNA template encoding a T7 promoter and tRNAfMet lacking the 3′-terminal adenosine (A76) was then treated with the tRNA nucleotidyl transferase from Archeoglobus fulgidus (Af) and 3′-NH2-ATP (nATP) (Axxora) in the following conditions: 100 mM glycine pH 9.0, 10 mM MgCl2, 2 μM nucleotidyl transferase, 2 μM tRNA(-A), 0.5 mM nATP, 1 mM DTT and 2 U/ml YIPP (NEB) (Af tRNA nucleotidyl transferase was purified with the same method and buffers as the MBP-MS2 fusion protein (tRNAPP (NEB) . Af tRNA protein . The reaE. coli methionyl-tRNA synthetase (MetRS) was used to charge 3′-amino tRNAfMet in the following conditions: charging buffer , 10 μM tRNAfMet-NH2, 10 mM methionine, 1 μM MetRS, 10 mM ATP, 1 U/μl murine RNase inhibitor. The reaction was incubated at 37°C for 30 min. Charged tRNA was phenol-chloroform extracted, ethanol precipitated, resuspended in water, and stored at −80°C.2 at 37°C for 45 min. The ribosome mixture was then loaded onto a 15–40% (w/v) sucrose gradient in buffer C with 10 mM MgCl2. Gradients were centrifuged at 28 000 rpm (97 000 × g) for 16 h in a SW-32 rotor (Beckman-Coulter). An ISCO gradient fraction system was used to isolate the 70S fraction  mutant 50S subunit and 0.6 mg of WT untagged 30S subunit were incubated in buffer C with 10 mM MgClfraction .fMet, 5 μM mRNA, and 100 nM ribosomes in buffer AC . Paromomycin at a concentration of 100 μM was used to ensure nonenzymatic A-site tRNA binding. The complex was incubated at 37°C for 30 min and then held at 4°C. The mRNA sequence was 5′-GUAUAAGGAGGUAAAAUGAUGUAACUA-3′ (IDT). Met codons are underlined. This mRNA sequence placed Met-tRNAfMet in both the A and P sites. Cryo-EM grids were prepared and samples were frozen as previously described (Ribosome–tRNA–mRNA complexes were prepared as previously described  with modescribed .Å) in RELION 3.1 . Ra. Ra35). LION 3.1  and motiLION 3.1 . The CTFLION 3.1 . The estLION 3.1  with 10 LION 3.1  from PDBLION 3.1 . ClassesLION 3.1 . The sepLION 3.1 . Upon clLION 3.1  and BayeLION 3.1  in RELIOLION 3.1  was usedLION 3.1  45). Th. ThÅ) inLION 3.1  and manuE. coli numbering) , even among thermophiles, suggesting that this change may play a role in ribosome stability  Figure . Both po) Figure . Class N) Figure . We find) Figure . Organistability ,49. HoweE. coli ribosome. We first assessed whether mutations in the A loop influence bacterial growth. E. coli strain SQ171, which does not contain genomically encoded rRNA and requires a plasmid containing an rRNA operon  is translated. At 37°C, both the CU and CC ribosomes produce similar levels of active nLuc protein compared to WT ribosomes  to a global resolution of 2.2 Å .,42.syn cd in RNA . In the 4 Figure . No new E. coli ribosome structure, where all A-site tRNA classes had density for the CCA-end  luciferase complementation assay, in which a truncated and catalytically inactive form of nLuc, termed 11S, is complemented by an y Figure  31). Th. Thin viy Figure , which iy Figure .E. coli protein components of an in vitro translation reaction are not thermostable  thermal melting experiments, the RNA constructs demonstrated cooperative and reversible unfolding , all of which contained 2′-nfolding . The WT,nfolding  in low infolding . FurtherE. coli has a less stable AU base pair at this position. As thermophilic organisms have increased rRNA GC content  Figure . This GC content , we hypo similar .T. thermophilus A-loop sequences, we designed two mutant E. coli 50S ribosomal subunits to test for global stability. The first incorporates the A-loop sequence from T. thermophilus (Tt A loop), which swaps the order of the A and U at base pair 1 and changes base pair 2 to a GC base pair. These changes are predicted to stabilize the secondary structure of the A loop hairpin  decreased tRNA binding to the ribosome , EMDB-28218 (50S focus refinement map), EMDB-28229 (30S focus refinement map), and EMDB-28230 (70S map from A-site tRNA class 2). Sequences, temperature data, and scripts used in the sequence and phylogenetic analysis are available at gkac1273_Supplemental_FileClick here for additional data file.


The synthesized composite (MCGO–HNPa) showed high stability, good water solubility and biocompatibility, expected magnetic targetability, and good photostability for PDT even in low concentrations. Our research reveals that MCGO nanomaterials can promote the production and release of singlet oxygen (ΦΔ = 62.9%) when compared with free HNPa. In addition, the in vitro cell uptake experiments suggested that the MCGO nanomaterials can accelerate the penetration of HNPa drugs into the tumor cell nucleus and that the drug release behavior is pH-sensitive. The MTT assay results against human hepatoma cell lines HepG-2 clearly show that the MCGO–HNPa composite can effectively result in cell damage and apoptotic cell death under light, and that the nanocomposite can improve the PDT antitumor effect of PS agents with negligible dark toxicity. Meanwhile, the research on the photoreaction mechanism reveals that Type I and Type II photodynamic reactions can occur simultaneously in this PDT process, and their relative contributions depend on the type and dose of the photosensitizer. Type II has a greater effect on PDT than Type I, especially for a higher HNPa photosensitizer dose. All the results reveal the promising application of the presented novel strategy.Photodynamic therapy (PDT) is an increasingly recognized alternative to treat various cancers in clinical practice. Most second-generation photosensitizers (PS) are hydrophobic and have poor targeting selectivity, which limit their efficacy for PDT. In this paper, graphene oxide (GO) coupled with magnetic Fe Prepared magnetic chitosan/graphene oxide composite loaded with a novel photosensitizer (MCGO–HNPa) showed excellent PDT activity against HepG-2 cells. Generally, an ideal PS should have several advantages such as good water solubility, deep penetration in tissues 11,12 Graphene, which has a large surface area due to its unique two-dimensional (2-D) structure, is a promising drug carrier.13–15 Graphene oxide (GO) exhibits favourable biocompatibility, low cytotoxicity and a local thermal effect, which make it a good candidate for a drug-loading/-delivery agent.16,17 Functional groups such as epoxy, hydroxyl, and carboxylic acid attached to GO sheets not only enable its good dispersion in physiological environments but also facilitate easy modification with biomolecules, thus improving the stability, water solubility, biocompatibility, or even targetability.18–20 Moreover, natural biodegradable polymers are viable polymeric materials that can be used to strengthen the biocompatibility of PS drugs for improving the value of therapeutic molecules.21 Among them, chitosan (CS) is an ideal polymer as a nanomaterial modifier for biological applications owing to it being non-toxic, hydrophilic, biocompatible, biodegradable and anti-bacterial.22–25 The chelation and cation properties of chitosan promote electrostatic interaction with negatively charged GO sheets. The solubility of chitosan in endosomal pH (5.3) of cancer cells and insolubility in physiological pH (7.4) prevents untimely release of encapsulated drug before reaching the target site.26Furthermore, to increase the water solubility of PS molecules and improve their delivery into cancer cells, various nanocarrier systems have been designed in recent years.27 Hence, drug delivery systems need to be primarily directed to tumor sites. Therefore, the development of an efficient delivery system has to focus on the ability to enhance the special target cellular uptake of antitumor drugs, resulting in intelligent and controlled release. Recently, embedding magnetic Fe3O4 nanoparticles into composites for constructing multifunction materials has been widely reported for targeted drug delivery, with the merits of magnetic separation and magnetic targeting as an external targeting strategy.28–30In addition, nonspecific accumulation in normal tissues of photosensitizer drugs taking a long time can lead to serious side effects and decrease the therapeutic efficacy.31–33 In this article, through a one-pot solvothermal method, we established a passive targeting system based on magnetic chitosan/graphene oxide (MCGO) composites that contain a biocompatible CS polymer and superparamagnetic iron oxide nanoparticles on the surface of GO, making them promising candidates for magnetic targeting.34 Furthermore, this text reports a simple structural modification of a chlorophyll-a degradation product, methyl pyropheophorbide-a (MPPa). A hydrophilic hydroxyl group and a carboxyl group were introduced into MPPa to increase its solubility in the human physiological environment, and the substitution at 131 site ring carbonyl groups with two cyano groups aimed to extend the absorption wavelength of the photosensitizer (λmax = 698 nm) to the near-infrared region. The obtained photosensitizer 3-[1-hydroxyethyl]-3-devinyl-131-β,β-dicyanomethylene-131-deoxopyropheophorbide-a (HNPa) was expected to be effective in cancer treatment due to the extended conjugate structure. The novel photosensitizer HNPa as an anti-tumor drug model was then loaded onto the surface of the MCGO nanohybrid via π–π stacking and hydrogen bonding.35 The composite MCGO–HNPa was comprehensively characterized by various methods. Cellular experiments in vitro were conducted to evaluate the potential of the novel photosensitizer HNPa as an anti-tumor drug in PDT and MCGO as an expected magnetic targeting delivery system that can transport anticancer drugs to tumor cells effectively.The assembly of various functional materials on the GO nanosheet surface remains a popular research topic.2.2.1.3), potassium permanganate (KMnO4), hydrogen peroxide solution , hydrochloric acid, ferric chloride hexahydrate , sodium acetate (NaOAc), chitosan (CS), dimethyl sulfoxide (DMSO) and 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. 1,3-Diphenylisobenzofuran (DPBF), sodium azide (SA) and d-mannitol (DM) were bought from Sinopharm Chemical Reagent . Dulbecco's modified eagle medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), and phosphate buffered saline (PBS) were purchased from Beijing Dingguo Biotechnology Co. . PBS used in other experiments was prepared by mixing stock solutions of NaH2PO4 and Na2HPO4. Ethanol, methanol, dichloromethane, triethylamine (TEA), hydrobromide/acetic acid , glacial acetic acid , concentrated sulfuric acid , and lithium hydroxide (LiOH) were analytical reagents. All the chemicals and reagents were of analytical grade and used without any purification. All the solvents were distilled and purified by standard procedures. Pure water was obtained from Milli-Q synthesis system .Methylene cyanide, natural graphite powder, sodium nitrate  as eluents in 47% (59 mg) yield as a dark blue powder.125 mg methyl pyropheophorbide-a (MPPa) was added into 10 mL 33% HBr/acetic acid solution with 4 mL 99% glacial acetic acid and stirred at 52 °C for 5 h. The acid and solvent were removed under reduced pressure. The resulting residue was dispersed in 20 mL distilled water and stirred at room temperature for 0.5 h. Then methyl alcohol (20 mL) and 2 M LiOH (5 mL) were added. The solution was refluxed for 2 h in a water bath under nitrogen atmosphere. 2 M acetic acid was added to adjust the pH to 6–7. 60 mL dichloromethane was added to the mixture, and it washed with saturated salt water (120 mL × 3), dried with anhydrous sodium sulfate and filtered. The organic solvent of the filtrate was evaporated in vacuum. The residue was chromatographed using CH2.2.2.2Cl2/MeOH  as eluents in 80% (80 mg) yield as an emerald green powder. 1H-NMR (MeOD) δ (ppm): 0.01 , 0.89 , 1.59 , 1.74 , 2.02–2.16, 2.25–2.65 , 2.16 , 3.19, 3.21, 3.31 , 3.51–3.64 , 4.07–4.10 , 4.21–4.37 , 4.84 , 5.28 , 6.34 , 8.31 (8.25), 8.46, 9.58 (9.54) ; 13C-NMR (MeOD) δ (ppm): 9.2, 12.0, 14.4, 17.3, 19.4, 23.7, 30.4, 30.7, 33.0, 48.1, 49.2, 55.1, 94.3, 100.2, 103.5, 105.0, 110.0, 116.0, 115.8, 116.3, 129.9, 131.3, 134.1, 135.3, 137.3, 138.9, 139.1, 144.8, 148.7, 151.8, 158.3, 161.3, 167.1, 173.3, 175.9, 179.5.100 mg HEPa was dissolved in 4 mL absolute ethanol, and 180 mg methylene cyanide and 195 μL triethylamine were added. The solution was refluxed for 5 h in an oil bath at 85 °C. The mixture was evaporated in vacuum to remove ethanol, the residue was extracted with dichloromethane and washed with saturated salt water several times, and dried with anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuum. The residue was chromatographed using CH2.2.3.36,37 The typical procedure was as follows: 1 g graphite and 0.5 g NaNO3 were added in a 250 mL three-necked round bottom flask, and 23 mL 98% H2SO4 was added to the mixture under 0 °C using an ice bath and was stirred evenly for 0.5 h below 2 °C. Subsequently, 3 g KMnO4 was added to the reaction system step-wise over 1 h, and the temperature of the mixture was kept below 15 °C. Then, it was stirred for 4 h in an oil bath at 35 °C. Afterwards, 138 mL deionized distilled water was slowly added into the solution, increasing the temperature to 95 °C instantly, while stirring for 0.5 h. Finally, 83 mL deionized distilled water and 67 mL 30% H2O2 were poured into the reaction system, and bright yellow suspensions were obtained. GO was separated by filtration, and the filter cake was washed with 40 mL 5% HCl and a large amount of deionized distilled water until the filtrate reached a pH = 7, and was dried for 12 h in vacuum. Tan slices of GO were obtained.Graphene oxide (GO) was prepared using purified natural graphite according to a modified Hummers method.2.2.4.3·6H2O was ultrasonically added to the mixed solution for 15 min. 0.37 g NaOAc was added to the above solution and it was stirred vigorously for 20 min. Subsequently, 0.1 g chitosan already dissolved in 5 mL (2%) acetic acid was added. The mixing solution formed was stirred for 30 min and then placed in a hydrothermal reactor at 185 °C for 6 h. Finally, the solution was cooled to room temperature, and the remaining deposit was washed thoroughly with ethanol and deionized distilled water and dried in a vacuum oven at 50 °C for 12 h.0.1 g GO was ultrasonically added to 70 mL ethylene glycol at 70 mL for 1 h using an ultrasonic cell mill to form a clear solution. Then, 0.16 g FeCl2.2.5.−1) by ultrasonic dispersion. 10 mg HNPa dissolved in a small amount of absolute ethanol was cautiously added dropwise. The mixture was ultrasonically treated for 6 h and stirred vigorously for 24 h at room temperature in the dark. The obtained MCGO–HNPa composite nanoparticles were separated by using an external magnet, and washed with absolute ethanol several times to remove unloaded photosensitizer and finally dried at room temperature for 24 h.The composite was prepared by direct π–π stacking and hydrogen bonding. Typically, magnetic chitosan/graphene oxide was dispersed in absolute ethanol to form a suspension . The Fourier transform infrared (FT-IR) spectra were recorded on a Vertex 80 FTIR spectrometer  using KBr disc samples in the absorption mode at a resolution of 4 cm−1 in the range of 4000–400 cm−1. UV-vis spectra were measured using a LAMBDA 25 spectrometer (PerkinElmer) at room temperature in a quartz cuvette with a path length of 1 cm. Fluorescence emission spectra was obtained using a fluorescence spectrometer Fluorolog-3 (Horiba Scientific) with a 150 W xenon lamp as the visible excitation source at an excitation wavelength of 430 nm. The surface charge of the samples in zeta potential and dynamic light scattering (DLS) were carried out using a NanoBrook ZetaPALS zeta potential analyzer (Brookhaven). For the MTT assay, a Biotek ELx800 absorbance microplate reader was used. Cell imaging was performed using a fluorescent inverted microscope .The microscopic size and shape of the composite were observed when a drop of the sample in absolute ethanol was carefully deposited on carbon-coated copper grids by a Tecnai G2 F200 S-TWIN transmission electron microscope (TEM)  operating at 200 kV. Magnetic characteristics were recorded at 300 K using a 7410 vibrating sample magnetometer (VSM) . Powder X-ray diffraction (XRD) patterns were recorded on a SIEMENS D5005 X-ray diffractometer with a Cu Kα radiation  from 10° to 80° and a rate of 3° min2.4.where mtotal HNPa is the total mass of initial drug HNPa for loading, mresidual HNPa is the mass of residual HNPa in the supernatant after being loaded, and mMCGO is the mass of MCGO for loading.The amount of HNPa loaded was calculated as follows: first, the standard absorption curve of HNPa was determined by UV-vis spectroscopy at the wavelength of 698 nm from a series of HNPa solutions in different concentrations of ethanol for quantitative analysis. During the MCGO–HNPa fabrication process, the HNPa-containing supernatant was collected and the residual amount of HNPa was determined by measuring the absorbance at 698 nm using UV-vis spectroscopy relative to the above calibration curve recorded under identical conditions, allowing the drug-loading efficiency to be estimated. The drug-loading capacity was calculated according to the following formula:in vitro from the synthesized nanocarrier MCGO–HNPa was studied at the physiological temperature of 37 °C and pH of 5.3 , 7.4  and 8.9. Briefly, 3 mg of the MCGO–HNPa composite was sealed in a dialysis membrane tube immersed in 10 mL PBS solution with a pH of 5.3, 7.4 or 8.9, followed by placing it in a water bath maintained at 37 °C. An aliquot containing about 2.5 mL drug release medium was withdrawn after 1 h, 3 h, 6 h, and 12 h, and thereafter every 12 h until 72 h. The amount of HNPa in the PBS solution was quantified using UV-vis absorption spectroscopy using the same method as described above. After each measurement, the aliquot was poured back into the release system. Given that the measurement time was very short, while the predetermined time interval of drug release was significantly large, the influence of the returned aliquot on drug release during the measurement time was expected to be insignificant. All the drug release experiments were repeated at least three times.The drug release profile 2.5.1O2 trapping reagent, in DMF solution. In a typical experiment, 3 mL DPBF (6 × 10−5 mol L−1) in DMF solution containing 20 μL free HNPa (2.8 × 10−5 mol L−1) or MCGO–HNPa with the same concentration of the free photosensitizer as the HNPa solutions in DMF was placed in a sealed quartz cuvette. The solution was irradiated by a 10 J cm−2 NIR Nd:YAG laser diode at λ = 700 nm ± 10 nm every 10 s for 120 s. Each decrease in absorbance caused by photobleaching of DPBF was measured with an ultraviolet-visible spectrophotometer at 415 nm. The 1O2 quantum yield was calculated using the following equation:ΦΔ is the 1O2 quantum yield, IαT is the total amount of light absorbed by photosensitizers, AT is the corresponding absorbance at irradiation wavelength under a specific illumination time t, and κ is a slope fit by the first-order linear curve plotted against −ln([DPBF]t/[DPBF]0) as a function of irradiation time t, where [DPBF]0 and [DPBF]t represent the UV-vis absorbance of DPBF at 415 nm before and after irradiation time t, respectively. S and R represent the sample and reference compound, respectively. The 1O2 quantum yields of HNPa and MCGO–HNPa in DMF were calculated using methylene blue (ΦΔ = 49.1%) as a standard.Singlet oxygen production was observed using 1,3-diphenylisobenzofuran (DPBF), a sensitive 2.6.−5 M, 2.8 × 10−5 M, and 5.6 × 10−5 M. Then irradiation with a 10 J cm−2 NIR Nd:YAG laser diode at λ = 700 nm ± 10 nm was performed every 10 min for 60 min. The maximum absorption at 703 nm was recorded using an ultraviolet visible spectrophotometer after irradiation time of 0 min, 10 min, 20 min, 30 min, 40 min, 50 min and 60 min. All experiments were repeated three times and carried out at room temperature and in the dark.The photostability of the composite was studied as follows: MCGO–HNPa was dissolved in PBS and transferred to a sealed quartz cuvette at three different concentrations of 1.4 × 102.7.−1 penicillin–100 μg mL−1 streptomycin, Life Technologies, USA) in a 95% humidified atmospheric incubator with 5% CO2 at 37 °C.The human hepatocellular carcinoma cell line (HepG-2) was obtained from Harbin Engineering University, Harbin, China. Cells were cultured with Dulbecco's modified Eagle's medium  containing 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic (100 μg mL2.8.H-tetrazolium bromide (MTT) was used to check the cell viability; first, HepG-2 cells were seeded in two 96-well plates at 2 × 105 cells per well in 100 μL DMEM and incubated at 37 °C in 5% CO2 for 24 h. Then, 100 μL DMEM containing free HNPa or MCGO–HNPa with a series of equivalent concentrations of HNPa  was administered to the cells in the experimental groups and allowed to uptake for 4 h, while the control group was given only 100 μL DMEM without the photosensitizer or the composite. Then cells were fed for 20 h after exposure to calibrated visible light for 10 min . Then, MTT solution in DMEM  was added to each well; after the 4 h incubation with the MTT, the media were removed and 150 μL of dimethyl sulfoxide (DMSO) was added to solubilize the produced formazan crystals. The cell toxicity efficacy was measured with a Biotek ELx800 absorbance microplate reader at a wavelength of 490 nm and then calculated by the following equation:A490(sample) is the average absorbance values of the wells treated with the same concentration of HNPa or MCGO–HNPa and A490(control) is the average absorbance values of the wells treated under the same conditions without the photosensitizer. Data presented are averaged results of six experiments.The 3--2,5-diphenyl-22.9.5 cells per well and incubated at 37 °C for 24 h. Then the free HNPa or MCGO–HNPa composite was added in the same concentration as the free HNPa photosensitizer  to the wells for 0.5 h, 1 h, and 3 h. Afterwards, the cells were washed with 1 mL PBS three times and subsequently fixed with 1 mL of glutaraldehyde aqueous solution (2.5%) for 10 min at 37 °C. Then the glutaraldehyde aqueous solution was removed and the cells were rinsed with PBS three times, and stained with 1 mL of a 1 μg mL−1 DAPI nuclear probe for 10 min. Finally, the DAPI dye was removed by rinsing with 1 mL PBS three times, and the cells were placed on a glass slide after removing the coverslip for fluorescence imaging by FIM.Cell uptake studies were performed using HepG-2 cells, a human hepatocellular carcinoma cell. To investigate the uptake of MCGO–HNPa composite by HepG-2 cells, the cellular uptake was observed by a Leica DM IL IED fluorescent inverted microscope (FIM) . HepG-2 cells were plated into 6-well plates in DMEM at a density of 2 × 102.10.5 cells per well in 100 μL DMEM and incubated at 37 °C in 5% CO2 for 24 h. Then 100 μL DMEM containing free HNPa or MCGO–HNPa with a series of equivalent concentrations of HNPa  was administered to the cells in the experimental groups and allowed to uptake for 4 h, followed by exposure to calibrated visible light for 10 min . After irradiation, the cells were fed for an additional 20 h. In addition, the dark toxicity control group conditions were identical to the experimental group conditions but without irradiation. Analysis of cell cytotoxicity using the MTT assay was conducted as described above.The cell culture condition, cell culture medium volume and the cell number used for 96-well plates were similar to that used for the MTT assay. To investigate the cytotoxicity of the MCGO loaded with the anti-tumor drug HNPa towards tumour cells, HepG-2 cells were seeded in two 96-well plates at 2 × 102.11.5 cells per well in 100 μL DMEM and incubated at 37 °C in 5% CO2 for 24 h. Then the MCGO–HNPa with equivalent concentration of HNPa  was added for 4 h incubation, and subsequently irradiated for 10 min. Then the cell morphological changes in the bright field were observed by DAPI fluorescence staining after 3 h, 6 h, 9 h, 12 h and 24 h. Briefly, HepG-2 cells were incubated with MCGO–HNPa with an equivalent concentration of HNPa  and irradiated for 10 min and incubated for 4 h. After removing the culture medium, it was fixed with 1 mL of glutaraldehyde aqueous solution (2.5%) for 10 min, and then 1 mL fluorescence staining DAPI (1 μg mL−1) solution was added to the cells in each well covered with a coverslip. Then morphological variation was observed by FIM. The results were compared with those of normal HepG-2 cells.Cell morphological changes after PDT were analysed by DAPI fluorescence staining, which was used to label the nucleus. Typically, HepG-2 cells were seeded in 6-well plates at a density of 2 × 102.12.d-mannitol (DM), an effective scavenger for specific hydroxyl radicals, were used to perform the experiments to quench the ROS generated from a photodynamic reaction. Briefly, the test was divided into four groups: (1) MCGO–HNPa–PDT groups, different concentrations of MCGO–HNPa and irradiation; (2) MCGO–HNPa–PDT–SA groups, different concentrations of MCGO–HNPa with SA  and irradiation; (3) MCGO–HNPa–PDT–DM groups, different concentrations of MCGO–HNPa with DM  and irradiation; (4) blank group, without MCGO–HNPa and no irradiation. That is, for the MCGO–HNPa–PDT groups, HepG-2 cells were seeded in 6-well plates at a density of 2 × 105 cells per well in 100 μL DMEM and incubated for 24 h as described above. Then the cells were incubated with different concentrations of MCGO–HNPa for 4 h and irradiated for 10 min. But, for MCGO–HNPa–PDT–SA and MCGO–HNPa–PDT–DM groups, the plate was similarly treated but SA  and DM  were added into the culture medium, respectively. The final HNPa equivalent concentrations of MCGO–HNPa were 0.5, 1.5, 2, 2.5, 3 and 5 μg mL−1. The cells were further cultured for an additional 24 h as described above. Then DMEM containing MCGO–HNPa was removed and the cells were washed with 1 mL PBS three times. Cell viability was determined by MTT assay.During PDT, reactive oxygen species (ROS), such as oxygen centered radicals including hydroxyl radicals (HR), superoxide anions and hydrogen peroxide (Type I reaction mechanism of PDT) and singlet oxygen (Type II reaction mechanism of PDT) play important roles. In order to visualize the photochemical mechanism of PDT, sodium azide (SA), a quenching agent for singlet oxygen, and 2.13.t-test using the SPSS 19.0 for Windows (SPSS Inc.). p < 0.05 was considered to indicate statistical significance.All experiments were performed in triplicate and the data and figures were given as mean ± standard error. Statistical analysis comparisons between two groups were determined by Student's 3.3.1.9 The second step of the chemical reaction retained the hydrophilic group on the basis of compound 2, and at the same time, the introduction of two strong electron-withdrawing cyano groups in 131-bit extended the conjugated system. The desired product 3-[1-hydroxyethyl]-3-devinyl-131-β,β-dicyanomethylene-131-deoxopyropheophorbide-a (HNPa) (m/z 600.7094) was obtained in 80% yield. The structure of HNPa was confirmed by 1H-NMR spectra, 13C-NMR spectra, UV-vis spectra and fluorescence spectra.The chlorophyll-a based photosensitizer HNPa was prepared as shown in 3O4 nanoparticles and biocompatible chitosan (CS) (MCGO) was prepared by a one-pot solvothermal method. The obtained MCGO nanomaterials had high stability, good water solubility and biocompatibility, and expected magnetic targetability for potential use as a drug carrier. Then MCGO was used as a nanocarrier to load the new photosensitizer HNPa (λmax = 698 nm) via hydrogen bonding interactions and π–π stacking by ultrasound and vigorous stirring.The preparation of the magnetic chitosan/graphene oxide composite loaded with the novel photosensitizer HNPa for enhanced photodynamic therapy is also illustrated in 3O4 spheres were decorated and anchored uniformly in interlayers of GO and many small chitosan molecules and HNPa as the photosensitizer was successfully assembled on the surface of GO layers with a high density.The obtained MCGO–HNPa was characterized by various methods. The morphology of GO in TEM  showed a2, respectively, increasing potential value of GO surface, respectively. After loading with HNPa, MCGO–HNPa showed a zeta potential of −8.81 ± 2.02 mV possibly due to more negative groups of the photosensitizer HNPa. Based on the above investigation, it was clear that HNPa was successfully loaded on the MCGO nanoparticles. Meanwhile, the hydrodynamic diameters of MCGO–HNPa were measured using dynamic light scattering (DLS) shown in Zeta potentials of GO, MCGO and MCGO–HNPa in ethanol solutions of pH = 7.0 were further investigated. As shown in ε = 29 975 M−1 cm−1) with a high intensity. An ideal photosensitizer should have near-infrared absorption spectra at long wavelengths, which allows deeper tissue penetration and decreased nonspecific lesions. For instance, 630 nm light penetrates less than 0.5 cm and 700 nm light reaches a depth of no more than 0.8 cm.38 Compared with the approved photosensitizer Photofrin , HNPa has a stronger absorbance at the longest possible wavelength, which makes it a potential photosensitizer for enhanced PDT.39 The MCGO nanohybrid properties before and after loading with HNPa are further confirmed by UV-vis spectra as shown in UV-vis absorption spectra are shown in −1 correspond to C–O–C stretching vibrations, C–OH stretching, and C–C stretching mode of the sp2 carbon skeletal network, respectively, while the peaks located at 1743 and 3416 cm−1 correspond to C–O stretching vibrations of the –COOH groups, and O–H stretching vibration. There are two characteristic absorbance bands centered at 1615 and 1597 cm−1 owing to the C–O stretching vibration of –NHCO– and the N–H bending of –NH2, respectively, which proves that –NH2 groups on the chitosan chains react with the –COOH groups of GO and therefore are converted to –NHCO– graft points. In addition, the peak at 596 cm−1 in the MCGO spectrum is the characteristic Fe–O peak of Fe3O4. These observations confirm that magnetic Fe3O4 and CS are successfully grafted on GO. For HNPa, the peak at 2215 cm−1 reveals the existence of stretching vibrations of C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N, while the peak at 2270 cm−1 of the MCGO–HNPa is indicative of CN stretching vibration of HNPa, which suggests the loading of HNPa on MCGO.The FTIR patterns of CS, HNPa, MCGO and MCGO–HNPa are shown in In the fluorescence experiment, the emission spectrum of HNPa shows an intense peak at 703 nm at an excitation wavelength of 430 nm, which is compared with the MCGO–HNPa sample containing the same amount of HNPa. The emission intensity of MCGO–HNPa in 29 The Raman spectrum of MCGO in −1, respectively. After loading with HNPa, the Raman spectra of MCGO–HNPa also shows the characteristic peaks of the GO in MCGO and significantly background upward drift owing to the effect of photosensitizer fluorescence coverage; the decreased intensity in D bands may be due to the ultrasonic stirring reaction process weakening the defect density of MCGO. These results further support the wrapping of HNPa on MCGO.Raman spectroscopy can be used to characterize the lamellar structure and oxidation degree observed by the ratio of the strength of the D peak to the G peak of graphene oxide.−1 at 300 K, while that of MCGO–HNPa is about 21.6 emu g−1, which further indicates the superparamagnetic nature of MCGO and MCGO–HNPa after drug loading.Photos of MCGO–HNPa in aqueous solutions without and with a magnet clearly demonstrate its excellent magnetic properties . The mag3O4, MCGO and MCGO–HNPa are shown in 3O4  observed in the samples, marked by their indices , indicating the existence of superparamagnetic Fe3O4 nanoparticles that can be used for the magnetic targeting of PDT. In addition, the XRD patterns of MCGO and MCGO–HNPa show a broad peak from 15° to 30°, which is generally considered to be the diffraction peak of the amorphous structure of chitosan, suggesting that CS is successfully loaded on the surface of MCGO. The decrease in peak intensity of MCGO–HNPa is due to the fact that the content of Fe3O4 in the nanocomposite is low and that the crystal structure is destroyed after high drug loading. The above analysis clearly shows the superparamagnetic nature of the composite and sheds light on the application of MCGO–HNPa in magnetically targeted PDT.XRD patterns of pure Fe3.2.via a simple sonication mixture method by π–π stacking and hydrophobic interactions. As shown in y = 0.0499x + 0.0147 (R2 = 0.9992). The unbound drug was removed by centrifugation and the loading efficiency of HNPa on MCGO was calculated by measuring the concentration of unbound drugs using UV-vis spectra. The HNPa loading capacities of the MCGO is as high as 57.6% when the initial concentration of the HNPa solution is 1 mg mL−1.HNPa as an anti-tumor photosensitizer drug is loaded on the surface of the multi-functionalized GO (MCGO) + in the solution competes with the hydrogen bond-forming group and weakens the above outlined hydrogen-bonding interaction, which may lead to a greater release of HNPa. Hence, the amount of released HNPa from MCGO is much higher under the acidic conditions than under neutral conditions. The interaction between the MCGO sheet and HNPa is due to the π–π stacking as the loading of HNPa on MCGO is still high. In summary, in view of the different releasing behaviors of HNPa on MCGO under different pH environments, this multi-functionalized MCGO can be used as a good candidate material for intelligent drug release.The HNPa release from the MCGO–HNPa composite at a temperature of 37 °C in the phosphate buffer solution  is given in 3.3.1O2) is the vital reactive agent of photosensitizer-induced photodynamic therapy (PDT). Thus, in this experiment, in order to measure the production of 1O2, 1,3-diphenylisobenzofuran (DPBF), a sensitive 1O2 trapping reagent, was utilized to check singlet oxygen production in PDT.9 The remarkable reduction of DPBF absorbance at 415 nm with increasing irradiation time  owing to the quenching reaction with 1O2 indicated that HNPa could photo-produce 1O2 efficiently. As shown in ΦΔ) of HNPa was calculated as 42.6%, while that of MCGO–HNPa with an equivalent concentration of HNPa was 62.9%, using methylene blue as the reference compound (ΦΔ = 49.1%). It is clear that MCGO nanomaterials can promote the production and release of singlet oxygen from HNPa to some extent. Compared with HNPa, MCGO–HNPa nanoparticles increased the ΦΔ to as high as 62.9%. The result indicated the MCGO–HNPa nanoparticles could enhance photodynamic therapy because the high generation of singlet oxygen would cause excellent cell toxicity, as confirmed by our MTT assay (shown later).Singlet oxygen  of the MCGO–HNPa composite in PBS with irradiation  every 10 min for 60 min by measuring the UV-vis absorption of the composite at 703 nm after different irradiation times. The photobleaching percentage plots are shown in −5 M. However, under concentrations of 2.8 × 10−5 M and 5.6 × 10−5 M, the MCGO–HNPa composite was not photobleached at all. After irradiation for 20 min, 5.6 × 10−5 M of the MCGO–HNPa composite began to be photobleached. After irradiation for 60 min, 1.4 × 10−5 M of the MCGO–HNPa composite was photobleached by about 15%; yet, at concentrations of 2.8 × 10−5 M and 5.6 × 10−5 M, the photobleaching percentage was about 8% and 8.8%, respectively. This result may be attributed to the concentration of the photosensitizer. According to research, under the same conditions, photobleaching will slow down with an increase in photosensitizer concentration.40 All these results indicated that the MCGO–HNPa composite had good photostability for PDT even in lower concentrations.Photobleaching is the phenomenon accompanying the consumption of photosensitizers during the photodynamic reaction. For the target tissue to be destroyed, photobleaching has a negative effect on reducing the production of reactive oxygen species (ROS) in photodynamic therapy (PDT). The slower the rate of bleaching and the lower the amount of bleaching, the better is the photodynamic therapy. Therefore, optimizing the photobleaching intensity of the photosensitizer is of great significance for the research and application of PDT. In this section, we studied the photobleaching of three different concentrations (1.4 × 103.5.−1) at 37 °C for 0.5 h, 1 h and 3 h. In order to visualize intracellular uptake, 4′,6-diamidino-2-phenylindole (DAPI), a widely used nuclear staining agent showing blue fluorescence, was utilized to observe the cell nucleus. Cell imaging was performed with a fluorescence inverted microscope. As shown in The efficient internalization by the cells is crucial for nanohybrids to achieve a better therapeutic efficiency. To examine the cellular delivery of HNPa by the MCGO–HNPa composite in PDT, we compared the cellular uptake behaviors of MCGO–HNPa composite with that of free HNPa. In this study, HepG-2 cells were incubated with free HNPa or MCGO–HNPa with an equivalent concentration of HNPa  of the MCGO–HNPa composite was lower than that of free HNPa (2.013 ± 0.138 μg mL−1) by exposure to calibrated visible light for 10 min , suggesting that the MCGO–HNPa composite could enhance photodynamic therapy efficiency to a certain extent, which may be attributed to the higher 1O2 quantum yield of the MCGO–HNPa composite reported previously. Meanwhile, as shown in The 3.7.−1) after PDT for 3 h, 6 h, 9 h, 12 h and 24 h gradually became rounder and their shading degree decreased significantly. It showed that the tumor cells were damaged due to necrosis and apoptosis with increasing PDT time. After PDT for 3 h, the cells began to become brighter and rounder. After PDT for 12 h, most of the cells were destroyed and died. After PDT for 24 h, almost all the cells were dead, and the number of cells in the field of vision reduced because of the decreased cell adhesive ability, resulting in partially dead cells detached from the wall of the culture dish. Based on the above results, the morphological variation of cells clearly indicated that the MCGO–HNPa composite under light could effectively result in cell damage and apoptotic cell death in HepG-2 cells, which was in good agreement with results of the photodynamic activity of MCGO–HNPa.In order to study the specific process of photosensitizer action on cells after PDT, the cell morphological changes of HepG-2 cells in the bright field were analyzed by a fluorescent inverted microscope (FIM). The control group (0 h) cells without treatment with the MCGO–HNPa composite presented a spindle-shaped normal cell morphology. However, it was clear that the HepG-2 cells incubated with the MCGO–HNPa composite (2.5 μg mL3.8.1O2) (Type II photoreactions) to tissue oxygen through energy transfers. These ROS show very strong activity to induce cell death and necrosis of tumor components. In order to visualize the photochemical processes mechanism of PDT, corresponding ROS of Type I and Type II generated from the photodynamic reaction were quenched using specific quenching agent DM  and SA (a singlet oxygen quencher), respectively. As shown in 1O2 generated from the MCGO–HNPa composite in the HepG-2 cells after PDT has been quenched effectively. In addition, the cell viability of the MCGO–HNPa–PDT–SA group was much higher than that of the MCGO–HNPa–PDT–DM group with increasing concentration of HNPa, suggesting that the production of 1O2 is more than that of the hydroxyl radical. In conclusion, Type I and Type II photodynamic reactions can occur simultaneously in this PDT, and their relative contributions depend on the type and dose of photosensitizer. Type II has a greater effect on PDT than Type I, especially in a higher photosensitizer dose.PDT involves the use of a three-component system comprising a photosensitizer, light of specific wavelength and molecular oxygen, resulting in the formation of reactive oxygen species (ROS) including singlet oxygen and free oxygen radicals that are directly responsible for the death of cancer cells. The reaction mechanism can be of two types. Photoactive drug photosensitizer, which is administered to cancer cells followed by appropriate wavelength light activation, can be excited form the ground state to the excited triplet state T to produce free oxygen radicals (type I photoreactions) such as hydroxyl radicals (HR), superoxide anions and hydrogen peroxide through electron transfer from a substrate molecule or singlet oxygen  (62.9%) and efficient cell toxicity against human hepatocellular carcinoma cell line (HepG-2) (IC50 = 1.504 ± 0.522 μg mL−1) by irradiation for 10 min . Furthermore, cellular uptake experiments suggested that the MCGO nanoparticles could accelerate the penetration of HNPa drugs into tumor cell nuclei. In addition, the photoreaction mechanism investigations showed that Type I and Type II photodynamic reactions could occur simultaneously in this PDT and Type II had a greater effect on PDT than Type I, especially in a higher photosensitizer dose. All the results showed that the prepared MCGO–HNPa composite had great potential application for PDT.In summary, the magnetic FeThere are no conflicts to declare.


The COVID-19 pandemic significantly impacts the increase in plastic waste from food packaging, masks, gloves, and personal protective equipment (PPE), resulting in an environmental disaster, if collected, processed, transported, or disposed inappropriately. Plastic waste has a very long deterioration time in the environment (soil and water), cheap, and plentiful. Additionally, construction waste disposal is a process that transfers debris to a state that does lead to any sustainable or environmental problems. The core objective of this current research work is to provide safety and efficacy by partial substitution of both ultrafine demolition waste (UDW), incorporated with nanoplastic waste (NPW), for eco-white cement (E-WC) composition. E-WC is designed by partially substituted WC with UDW ; incorporated with NPW (1.0 and 3.0 wt.%); to adequately protect people and the environment over long periods. The context examines the high performance, physicomechanical properties and high durability of blends as presences of silica in UDW proposed a hydraulic filler material, plus; high surface area of NPW. The microstructure and workability are characterized by X-Ray Fluorescence (XRF), Scanning Electron Microscope (SEM), and Transmission Electron Microscope (TEM) measurements. The record results show greatly enhanced in the mechanical strength due to the combination of NPW and UDW (active silica). With the presence of NPW and UDW in WC matrix, the highest level of crystallization formed consequently a decrease in whiteness reflection (Ry) and total porosity. In summary, WC blend with NPW and UDW reflects better workability and energy saving qualities, which are economical and environmentally beneficial and may result in decreased construction budget and improve a long-term raw material sustainability. Plastic waste (PW) is present in the surrounding environment with less use for recycling or as a substitute for industrial raw materials. There are seven types of plastic, all of which are harmful except for the fifth type (BPA free). Modern waste management techniques reduce the rapid depletion of both resources, including raw and combustible materials. Solid waste has significant negative drawbacks to the ecosystem and living conditions , porosity, and microstructure.2 g−1. Figure In the current lab program, white cement (WC), ultrafine demolition debris waste (UDW), and nano-plastic-waste (NPW) represent the core materials. White cement  was purchased from Sinai White Cement Company (SWCo.), . Demolition debris waste was brought from Tourah area, . Crushing then milling until reach ultrafine size passes through 63-µm mesh, specific surface area is, e.g., Blaine 4199 cmxGx composites.Generally, two sets of works are proposed. WC was substituted with various portions, e.g.,  by wt. % UDW, in the presence of 1.0% and 3.0% NW by wt.% (fixed ratio), and all proposed sets are tabulated in Table xGxspecimens, whiteness reflection, compressive strength, setting time, and porosity variations were estimated. Elerpho French apparatus was used to detect the whiteness reflection (Ry) comply with DIN 5033 specs , using mercury intrusion data  dimensions, and then, cubes set for hydration in 95 ± 5% actual humidity (RH) at ambient laboratory temperature. One day later, the cast prisms were de-molded and hydrated directly in tap fresh water for up to 28 days of hydration. The ASTM stander was the guide for our practical work as detailed elsewhere . The morphology of the specimens was determined with , “an energy dissipation X-ray analyzer”. The transmission electron microscopy (TEM) instrument reported that the effective particle size for NPW is 56.93 and 81.14 nm. This indicated the NPW in nanosized powder, as seen in Fig. xGx composites, as shown in Table xGx composites pastes have the following order: M0 ˃ M1G1 ˃ M2G1 ˃ M3G1 ˃ M4G1 ˃ M5G1, as shown in Fig. Whiteness reflection (Ry) is one of the major indicators of white cement quality, so replacement will affect badly on WC reflection profile on the 3-axis  NORM . Hinter xGx blends—group I—has the following order: M0 ˂ M1G1 ˂ M2G1 ˂ M3G1 ˂ M4G1 ˂ M5T1, as shown in Fig. Increasing the consistency of the water extends the initial and final setting time of the white cement composites. NPW acts as an inert filler, while UDW is a positive hydraulic filler. The setting time for the MxGx-Group I paste composites. Clearly, notice that the morphology of WC reflects good surface microstructure under the SEM apparatus with an arranged Skelton structure. MxGx-Group I paste composite morphologies are very interesting during SEM operation, as the appearance of linkage fibrous proves the presence of C–S–H gel and C–A–H phase  and medium hydraulic filler (UDW). M1G1 paste shows beater CMS at early and late age of hydration, which may be attributed to the equal ratio from inert filler/medium hydraulic filler of 1:1% wt.%. Equal ratios make neutralization effect as high surface area of NPW and UDW fills the open pores of the WC microstructure, leading to solidification and hardness of M1G1 paste compared to the WC. Paste composites have the following order at an early age (3 days of hydration) in the CMS scale, e.g.: M1G1 (29 MPa) ˃ M0 (28 MPa) ˃ M2G1 (26 MPa) ˃ M3G1 (22 MPa) ˃ M4G1 (19 MPa) ˃ M5G1 (11 MPa)  is the key performance indicator for WC quality and workability. A low pore structure of WC reflects on CMS profile, as shown in Fig. −, Na, and K) ion penetration and reduces the alkali attack phenomena, e.g., corrosion, and this will lead to increase composites half-life time and cracking occurrence  and medium hydraulic filler (UDW). NPW 3.0% wt. % showed a decrement in CMS at early and late age of hydration, which may be attributed to the high ratio from inert filler and medium hydraulic filler. In addition, the high surface area of NPW and UDW fills the open pores of the WC microstructure, leading to solidification and hardness of MxGx-Group II paste composites but lower the control sample. Paste composites have the following order at an early age (3 days of hydration) the CMS scale, e.g., M0 (28 MPa) ˃ M1G1 (24 MPa) = M2G1 (24 MPa) ˃ M3G1 (19 MPa) ˃ M4G1 (12 MPa) ˃ M5G1 (8 MPa). At late age of hydration (curing for 28 days), both M4G1 and M5G1 pastes failed in the CMS test and recorded 51 and 42 MPa, respectively, which was attributed to the high ratio of substitution from hydraulic cementitious material. The addition of UDW produces extra Alite clinker, but at limited ratios, as mentioned earlier that UDW can reach 8.0% wt. % substitution while saving the hydraulic properties , porosity decreases sharply and recorded to less than 22.0%, e.g.: M0 (4.52%) ˃ M1G1 (6.87%) ˃ M2G1 (9.88%) ˃ M3G1 (11.37%) ˃ M4G1 (12.64%) ˃ M5G1 (15.24%). By default, decreasing composites permeability delays the alkali  ion penetration and reduces the alkali attack phenomena, e.g., corrosion, this will lead to increase composites half-life time and cracking occurrence . MIG1 (1.0% NPW + 1.0% UDW), (M2G1 (1.0% NPW + 5.0% UDW) and M1G2 (3.0% NPW + 1.0% UDW) showed better workability, whiteness reflection (Ry), and microstructure. In addition, NPW filled WC open pores enhance the physicomechanical features and reduce alkali leaching, improving sulfate attack properties. It recommend that until 3% of NPW can be applicable in the cement sector with additional physicomechanical features than the neat white cement.Solid-waste recycling is a major challenge nowadays. Reaching sustainability in raw material resources is the SDGs for industry, innovation, and infrastructure. The current practical investigation based on the 2050 vision is to reduce raw material consumption and CO


Bacillus strains , which are known for their ability to survive harsh environmental conditions, as AGP replacements in animal feed. Four of these Bacillus strains  showed antimicrobial activity against the pathogenic strains Clostridium perfringens, Escherichia coli and Staphylococcus aureus at 25 μg/mL, with BPR-16 and BPR-17 also able to inhibit Pseudomonas aeruginosa and Salmonella enterica at 100 μg/mL. Further chemical investigation of BPR-17 led to the identification of eight metabolites, namely C16, C15, C14 and C13 surfactin C (1–4), maculosin (5), maculosine 2 (6), genistein (7) and daidzein (8). Purified compounds (1–4) were able to inhibit all the tested pathogens with MIC values ranging from 6.25 to 50 μg/mL. Maculosin (5) and maculosine 2 (6) inhibited C. perfringens, E. coli and S. aureus with an MIC of 25 μg/mL while genistein (7) and daidzein (8) showed no activity. An animal trial involving feeding BPR-11, BPR-16 and BPR-17 to a laboratory poultry model led to an increase in animal growth, and a decrease in feed conversion ratio and mortality. The presence of surfactin C analogues (3–4) in the gut following feeding with probiotics was confirmed using an LC–MS analysis. The investigation of these Bacillus probiotics, their metabolites, their impacts on animal performance indicators and their presence in the gastrointestinal system illustrates that these probiotics are effective alternatives to AGPs.Antibiotic growth promoters (AGPs) suppress the growth of infectious pathogens. These pathogens negatively impact agricultural production worldwide and often cause health problems if left untreated. Here, we evaluate six  Antimicrobial growth promoters (AGPs) are currently utilised in the agricultural industry to improve livestock production, feed-energy conversion and to prevent the spread of infectious diseases ,2,3. TheBacillus  D23.0 +7.0 ; 1H NMR and 13C NMR data see 1H, COSY, HSQC and HMBC NMR spectra see C15 surfactin C (2): white powder; [α] D23.0 +7.8 ; 1H NMR and 13C NMR data see 1H, COSY, HSQC and HMBC NMR spectra see C14 surfactin C (3): white powder; [α] D23.0 +8.1 ; 1H NMR and 13C NMR data see 1H, COSY, HSQC and HMBC NMR spectra see C13 surfactin C (4): white solid; [α] D23.0 −20.1 ; 1H NMR and 13C NMR data see 1H, COSY, HSQC and HMBC NMR spectra see Maculosin (5): white solid; [α] D23.0 −16.0 ; 1H NMR and 13C NMR data see 1H, COSY, HSQC and HMBC NMR spectra see Maculosine 2 (6): white solid; [α] 1H NMR and 13C NMR data see 1H, COSY, HSQC and HMBC NMR spectra see Genistein (7): brown powder;  1H NMR and 13C NMR data see 1H, COSY, HSQC and HMBC NMR spectra see Daidzein (8): brown powder; n = 3).Bacteria were streaked on plates containing MHA . A colony was picked, added to MHB and incubated at 37 °C until a starting culture of 0.1 OD was obtained. To test for activity, bacterial broth (78 µL) was added to either the crude extracts  or the EtOAc extracts . The plate was then incubated at 37 °C overnight. A resazurin dye (25 µL) was then added into each well and incubated at 37 °C for 6 h, and the antimicrobial activity was quantified by the optical density at 525/580 nm on a plate reader. The serial dilutions were prepared with DMSO. Gentamicin (Sigma-Aldrich) was used as a positive control and DMSO was used as a negative control. Each concentration was tested in triplicate  and the assay was conducted as described for the extracts. The MIC was determined by the inhibition of visual growth of bacterium.n = 128 per experimental group). The feed-supplemented probiotic was added to the basal diet in the facility, and the diet was thoroughly mixed prior to the start of the experiment. The experimental treatments included a standard micro crumble wheat–soybean diet (control) and control +500 g/t probiotics (F1). The F1 probiotic added to the feed was supplied by Bioproton Pty Ltd.   on day 1–18, and 37.5 °C and 65% RH on day 18–21. The hatchlings were individually weighed and birds with similar weight (±2 g) were randomly assigned to one of two experimental groups (control and probiotic). Each experimental group had 16 replicate pens with eight birds in each pen  .The broiler chickens in each group were reared in floor pens (120 cm × 120 cm × 80 cm) with wood shavings as bedding and had ad libitum access to feed and water for 28 days. Throughout the trial period, the ROSS 308 Management Handbook was followed to meet the nutritional and environmental recommendations of the broiler, including temperature, relative humidity (RH) and lighting program . The temThe BWs of individual birds and the FIs of each replicate pen were recorded weekly. The ADG, ADFI and FCR ratio between feed intake over BW of birds per replicate were calculated. Animal mortality was recorded and the data was used to adjust subsequent measurements.2O (6 mL) and ethyl acetate (6 mL). Each suspension was vortexed for 5 min and centrifuged at 12,000× g for 10 min at 4 °C. Subsequently, the supernatant (400 µL) was removed, concentrated and redissolved in 100% methanol (30 µL). The samples were analysed by the LC–MS system with a gradient elution from 5% H2O/95% MeOH/0.1% formic acid to 100% MeOH/0.1% formic acid within 13 min, at a flow rate of 0.3 mL/min. Ions were extracted using both positive and negative modes.The caecum samples from the animal trial were stored at −80 °C and were analysed following a protocol previously described by Martias . The cae