C9ORF72 gene is the main genetic cause of ALS/FTD (C9ALS/FTD). The repeat expansion leads to reduced expression of the C9orf72 protein. How C9orf72 haploinsufficiency contributes to disease has not been resolved. Here we identify the synapsin family of synaptic vesicle proteins, the most abundant group of synaptic phosphoproteins, as novel interactors of C9orf72 at synapses and show that C9orf72 plays a cell-autonomous role in the regulation of excitatory synapses. We mapped the interaction of C9orf72 and synapsin to the N-terminal longin domain of C9orf72 and the conserved C domain of synapsin, and show interaction of the endogenous proteins in synapses. Functionally, C9orf72 deficiency reduced the number of excitatory synapses and decreased synapsin levels at remaining synapses in vitro in hippocampal neuron cultures and in vivo in the hippocampal mossy fibre system of C9orf72 knockout mice. Consistent with synaptic dysfunction, electrophysiological recordings identified impaired excitatory neurotransmission and network function in hippocampal neuron cultures with reduced C9orf72 expression, which correlated with a severe depletion of synaptic vesicles from excitatory synapses in the hippocampus of C9orf72 knockout mice. Finally, neuropathological analysis of post-mortem sections of C9ALS/FTD patient hippocampus with C9orf72 haploinsufficiency revealed a marked reduction in synapsin, indicating that disruption of the interaction between C9orf72 and synapsin may contribute to ALS/FTD pathobiology. Thus, our data show that C9orf72 plays a cell-autonomous role in the regulation of neurotransmission at excitatory synapses by interaction with synapsin and modulation of synaptic vesicle pools, and identify a novel role for C9orf72 haploinsufficiency in synaptic dysfunction in C9ALS/FTD.Dysfunction and degeneration of synapses is a common feature of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). A GGGGCC hexanucleotide repeat expansion in the The online version contains supplementary material available at 10.1007/s00401-022-02470-z. C9orf72 gene is the most common genetic cause of ALS/FTD (C9ALS/FTD) . Patch-clamp recordings were performed at 12–13DIV as described by [2, 10 Na-HEPES, 10 Na-PiCreatine, 2 Mg2-ATP, and 0.3 Na3-GTP, pH 7.3, 300 mOsm. For mEPSC recordings, cells were bathed in an extracellular recording solution comprising (in mM): 152 NaCl, 2.8 KCl, 10 HEPES, 2 CaCl2, 1.5 MgCl2, 10 glucose, pH 7.3, 320–330 mOsm in the presence of TTX (300 nM), strychnine (20 µM) and PTX (50 µM). Recordings made at − 70 mV , low-pass filtered online at 2 kHz, digitized at 10 kHz and recorded to a computer using WinEDR V2 7.6 Electrophysiology Data Recorder . Synaptic data was analysed as described previously [Before electrophysiological recordings were carried out, hippocampal neurons at 11–12DIV were cultured overnight in 90% SGG [comprising, in mM; 114 NaCl, 5.292 KCl, 1 MgClribed by . Brieflyeviously . BrieflyFor extracellular multi-electrode array (MEA) electrophysiology, hippocampal neurons were cultured on glass arrays with 59 channels per array . Data were recorded from cultures bathed in 90% SGG/10% MEM medium using a MEA recording platform (Multi Channel Systems) at a sampling rate of 20 kHz. Data were filtered offline with a 2nd order Butterworth filter with a cut-off frequency of 200 Hz. Data was analysed using the Multi Channel Systems software. For each MEA, a minimum of 10 active channels per array were selected for further analysis. Spikes were identified as activity 5 times the standard deviation of the baseline. Individual burst detection parameters: maximum interspike interval (ISI) to start burst, 0.5 s; maximum ISI to end burst, 0.5 s; maximum beginning ISI, 0.1 s; minimum interburst interval (IBI), 0.8 s; minimum burst duration, 0.05 s; and minimum number of spikes in a burst, 3. Synchronised network bursts throughout the array were determined using the software that detected defined individual bursts on each array that began within 0.2 s of each other. Data were determined to be parametric or non-parametric before performing statistical analysis.C9orf72 KO mice were generated using the Cre-LoxP system. A floxed mouse line for the conditional deletion of C9orf72 exons 3–4, coding for the two C9orf72 ATG start codons, was generated in a mixed C57BL/6 and 129Sv strain . These floxed mice were cross-bred with CRE Deleter C57BL/6J mice constitutively expressing the Cre recombinase under the cytomegalovirus (CMV) promoter  to generate a constitutive knockout model for the C9orf72 gene (C9orf72-KO). The resulting heterozygous mice were interbred to obtain both homozygous C9orf72-KO mice and age-matched wild type (WT) controls.All mice were maintained on the C57BL/6J genetic background and back crossed at least 6 generations. Excision of exons 3 and 4 was confirmed by qualitative PCR  on DNA isolated from tail biopsies. RT-qPCR and immunoblots of brain tissue confirmed the absence of C9orf72 mRNA and protein in the brain of C9orf72-KO mice, respectively .Genotyping for C9orf72 was performed through Polymerase Chain Reaction (PCR) using genomic DNA extracted from tail biopsies collected from the mice after weaning, within twenty-one days of age. Caudal biopsies were completely digested using the Maxwell® 16 System (Promega), according to the manufacturer’s instructions. The amplification solution was composed of 1X Buffer 3 (Roche), 250 µM dNTPs (Promega), 0.5 µM forward primer (5′-GCCCTCCCCTTCCTGTTTTGTTCT-3′) , 0.5 µM reverse primer (5′-AGACGGCAACTCCTGTGAGCATAGTTG-3′) (Metabion), 2.6 U Expand Long Template Polymerase  and 20 ng of genomic DNA in a final volume of 10 µl. The MJ Research PTC-200 Thermocycler (Bio-Rad) carried out an amplification programme. Cycling conditions were as follows: 94 °C for 2 min to activate polymerase, followed by 15 cycles of 94 °C for 30 s (DNA denaturation), primer annealing at 65 °C for 30 s and amplification at 68 °C for 7 min. Per cycle, 20 s were added to the amplification step. Final elongation was 8 min at 68 °C. The amplification products are one or two bands of the following size : C9orf72 homozygous wildtype: 3 Kb; C9orf72 heterozygous knockout: 3 Kb and 1 Kb; C9orf72 homozygous knockout: 1 Kb.Absence of C9orf72 transcript was confirmed by RT-qPCR . For this RNA was extracted from brain using Trizol (Invitrogen) and purified with PureLink RNA columns . cDNA was produced using the High Capacity cDNA Reverse Transcription Kit (Life Technologies) and RT-qPCR was performed using the Taq Man Gene expression assay  on cDNA triplicates using Universal PCR master mix (Life Technologies) and specific probes for the C9orf72 gene  and reference gene β-actin . Data were analysed comparatively to control via the ∆∆Ct method . AbsenceProcedures involving animals and their care that were conducted at the Mario Negri Institute for Pharmacological Research IRCCS, Milan, Italy adhered to the Mario Negri Institute for Pharmacological Research IRCCS institutional guidelines, that comply with national  and Mario Negri Institutional regulations and Policies providing internal authorisation for persons conducting animal experiments , the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/UE). All animals were housed under specific pathogen-free (SPF) conditions at a temperature of 22 ± 1 °C, a relative humidity of 55 ± 10% and 12-h light/dark cycle, 5 per cage. Food (standard pellets) and water were supplied ad libitum.Procedures involving animals performed at the University of Sheffield, Sheffield, UK, were conducted according to the Animal (Scientific Procedures) Act 1986, under a Project Licence reviewed and approved by the University of Sheffield Ethical Review Sub-Committee, and the UK Animal Procedures Committee . The UK Home Office code of practice for the housing and care of animals used in scientific procedures was followed. Mice were bred and housed in an SPF environment using a 12 h light/dark cycle, and a standardised room temperature of 21 ˚C. Mice were fed 2018 rodent diet  and provided with water ad libitum.12-week-old mice were deeply anaesthetized with ketamine hydrochloride (150 mg/kg) and medetomidine (2 mg/kg) followed by intracardiac perfusion with PBS 0.1 M (pH 7.4) followed by perfusion with 4% paraformaldehyde  in PBS 0.1 M. Brains were removed, post-fixed overnight in PBS 0.1 M with 4% PFA followed by dehydration in PBS with 30% sucrose and embedded in cryostat medium OCT . Brains were frozen in N-pentane at − 45 °C for 3 min, stored at − 80 °C before 30 µm coronal sections were cut on a cryostat. Free floating sections were blocked and permeabilized in PBS 0.01 M with 10% goat serum and 0.2% Triton and incubated overnight at 4 °C with primary antibodies in antibody diluent (PBS 0.01 M with 1% goat serum and 0.2% Triton) before being washed with PBS 0.01 M and incubated for 1 h at room temperature with secondary antibodies in PBS 0.01 M with 1% goat serum. After washing with PBS 0.01 M, sections were mounted on glass slides with coverslips using FluorSaveTM . Z-stacks of images were acquired on an Olympus IX81 microscope with a confocal scan unit . Z-stacks of co-stained sections were acquired using the sequential scanning mode on an A1 Nikon confocal running NIS Elements at 40X magnification. For nuclear staining, sections were stained with Hoechst 33258  and images were acquired using an Olympus virtual slide system VS110 at 40 × magnification with VS-ASW software (Olympus) for image processing.C9orf72 status) is provided in Table ® DAB EqV Peroxidase (HRP) Substrate, Vector Laboratories Inc., Burlingame, CA, USA) with antibodies to Syn1, SV2 or C9orf72. Stained slides were digitised using the Hamamatsu NanoZoomer XP, visualized by NDP.view2 digital pathology software (Hamamatsu Photonics). Regions of interest from CA3 and CA4 areas were selected for digital image analysis by a qualified neuropathologist (JRH). Analysis was performed blinded to the genotype.Immunohistochemistry staining of human brain samples was carried out on formalin-fixed, paraffin-embedded blocks of hippocampus and middle frontal gyrus of ALS/FTD patients with hexanucleotide expansions of the C9orf72 gene, non-C9orf72 related FTD patients as well as neurologically healthy controls. These post-mortem tissues were donated to the Sheffield Brain Tissue Bank (SBTB) with the consent of the next of kin. The SBTB Management Board gave ethical approval for this study under the provision to act as a Research Tissue Bank as approved by the Scotland A Research Ethics Committee . Human sample information  for 30 min. Samples were dehydrated through an ascending series of ethanol and propylene oxide, before embedding in Durcupan resin. During dehydration, sections were treated with uranyl acetate (1% in 70% ethanol) for 40 min. Durcupan resin was polymerised for 48 h in an oven set to 56 °C. Smaller regions of interest containing the CA3 region of hippocampus were identified and then cut from Durcupan-embedded sections before being mounted on Durcupan blocks and cutting ultra-thin sections (~ 70 nm) on an ultracut microtome (Leica) with an Ultra 45 Diamond Knife . Sections were collected on formvar-coated slot grids and stained with lead citrate before imaging on a JEOL transmission electron microscope equipped with a Gatan digital camera .12-week-old mice were deeply anaesthetized with ketamine hydrochloride (150 mg/kg) and medetomidine (2 mg/kg) followed by intracardiac perfusion with PBS 0.1 M followed by 2.5% glutaraldehyde  and 4% PFA in PBS 0.1 M. Brains were removed, post-fixed overnight in 2.5% glutaraldehyde and 4% PFA in PBS 0.1 M and washed 3–4 times for 1 h at 4 °C. A ~ 1 mmhttp://imagej.nih.gov/ij/) [Image analysis was performed using ImageJ/Fiji (gov/ij/)  and Cellgov/ij/) .http://imagej.nih.gov/ij/).PLA fluorescence intensities in EGFP-positive HEK293 cells were measured and the corrected total cell fluorescence  was calculated as integrated density—(cell area × mean background fluorescence) using Fiji . Synaptophysin staining accounts for variability in neuron culture density across coverslips.Synapse densities and properties of pre- and postsynaptic staining in 4-colour confocal images of cultured hippocampal neurons were analysed using an automated analysis pipeline in CellProfiler-4.1.3 . BrieflyTo quantify immune staining of brain sections of C9orf72-WT and heterozygous and homozygous C9orf72-KO mice, confocal imaging stacks were analysed using Fiji and CellProfiler. Stacks were background subtracted and Z-projected (Sum of slices). For single stain images Fig. , regions2) of the dentate gyrus using Fiji.On images of nuclear staining of the entire hippocampus, ROIs of the dentate gyrus were outlined manually and analysed blinded to the genotype to calculate the nuclei density .t test, or one sample t and Wilcoxon tests, according to the data structure and distribution. Multiple comparisons following ANOVA were performed using "Tukey's multiple comparisons test". Sample sizes and further details can be found in the figure legends.Calculations and statistical analysis were performed using Excel  and Prism 9 software. Statistical significance between experimental groups was determined by one-way analyses of variance (ANOVA), paired or unpaired two-tailed SYN1, SYN2 and SYN3, and alternative splicing generates a and b isoforms of Syn1 and Syn2 and isoforms a-f of Syn3 [To identify novel interactors of C9orf72, we screened a human full-length cDNA yeast two-hybrid (Y2H) library containing approximately 15,000 ORF clones using the long isoform of human C9orf72 (C9orf72L)  as bait. of Syn3 . Synapsi of Syn3 .To confirm the interaction between C9orf72 and synapsin, we first performed co-immunoprecipitation assays of transfected proteins in HEK293 cells. In agreement with the Y2H interaction, V5-tagged Syn3a (V5-Syn3a) co-immunoprecipitated with Myc-tagged C9orf72L (Myc-C9orf72L) from V5-Syn3a and Myc-C9orf72L co-transfected cells but not from cells transfected with Myc-C9orf72L or V5-Syn3a alone Fig. a. To detC9orf72 is known to form a complex with SMCR8 . Since tWe next investigated the C9orf72/synapsin interaction in its cellular context using proximity ligation assays (PLA). Similar to conventional immunofluorescence, PLA uses pairs of primary antibodies that selectively recognise putative interacting proteins in fixed cells but the secondary antibodies used are linked to complementary oligonucleotide sequences (PLA probes) that generate a fluorescent ligation product when the two probes are in close proximity (< 40 nm); each signal corresponds to an interacting protein pair . Using aGiven the presynaptic localisation of C9orf72 and synapsin , 70 thesTogether these data identify synapsin family proteins as novel binding partners of C9orf72 at synapses.upstream DENN) and a C-terminal DENN domain; C9orf72S encompasses only the N-terminal longin domain  protein family. C9orf72L contains an N-terminal longin domain , but differ at their C-terminus . The intThus, synapsin interacts via its conserved C domain with the N-terminal longin domain of C9orf72.Dysfunction of synapses is a common feature in many neurodegenerative diseases, including C9ALS/FTD , 56, 57.We first examined the effect of C9orf72 knockdown on excitatory synapses by co-staining of presynaptic Syn1 or Syn3 with the excitatory postsynaptic markers Homer or PSD95, respectively , and 22.78 ± 3.311%  reduction in postsynapses, respectively  and homozygous C9orf72-KO mice using antibodies to Syn1, Syn3 or the non-synapsin family presynaptic vesicle marker SV2. Staining for Syn1 and Syn3 was particularly prominent in the hippocampal mossy fibre system, namely the hilus area of the dentate gyrus CA4) and the CA3/CA2 region  by co-occurrence of the pre- and postsynaptic markers. C9orf72 again significantly reduced the number of Syn1 and Syn3-positive excitatory synapses in both C9orf72-HET and C9orf72-KO mice compared to the C9orf72-WT controls  and strychnine (glycine receptor blocker), to isolate glutamatergic miniature excitatory postsynaptic currents (mEPSCs). mEPSC recordings in miRNA-C9 treated neurons revealed a substantial decrease in the frequency of mEPSC events by approximately 58% and a corresponding increase in the cumulative probability of the inter-mEPSC event interval compared to miRNA-NTC treated neurons . To investigate functional synaptic properties, we measured miniature postsynaptic currents. Because our data indicated a specific effect of C9orf72 haploinsufficiency on excitatory but not on inhibitory synapses  become temporally organised into synchronized concentrated bursts. Synchronous bursts of network activity in these cultures rely on viable synaptic neurotransmission and network connectivity. Given the fact that we observed a substantially reduced frequency of synaptic mEPSC events in C9orf72-haploinsufficient neurons, we reasoned that this would impact upon neuron culture network activity.To explore this, we cultured hippocampal neurons on multi-electrode arrays (MEA) that permit the detection of culture wide network activity and excitability. Network activity was measured on 12-13DIV hippocampal neurons in which C9orf72 expression was knocked down by treatment with lentiviral miRNA-C9  and assessed it according to a defined set of criteria .We found that knockdown of C9orf72 increased the interval time between bursts  more than threefold, from 7.3 ± 2.1 s in miRNA-NTC to 22.6 ± 5.1 s in miRNA-C9-transduced neurons Fig. d, e. C9oC9orf72 haploinsufficiency is hypothesised to contribute to the disease phenotype in C9ALS/FTD, however the underlying mechanisms are unclear. Despite recent reports showing that C9orf72 is present in synapses , 84, theSynapsins are key proteins involved in the maintenance of the reserve pool of synaptic vesicles at the presynaptic terminal. Accordingly, perturbation or knockout of synapsins causes a dramatic reduction in synaptic vesicles in the reserve pool, particularly in excitatory synapses , 62, 73 Despite clear evidence that synapsins maintain the reserve pool, the underlying mechanisms remain unclear. On one hand, synapsins have been proposed to anchor synaptic vesicles to each other by inter-vesicle dimerisation of membrane-associated synapsins. Alternatively, there is evidence that synapsins create a liquid phase that traps synaptic vesicles (Reviewed in ). We shoSYN1, 2, and 3 did not affect the pool of docked vesicles at the active zone, suggesting that C9orf72 may regulate additional aspects of synaptic vesicle dynamics leading to reductions in the pool of docked synaptic vesicles. Rab3a is important for replenishment of docked synaptic vesicles in response to stimulation [In contrast to our observations in C9orf72-KO mice, triple-knockout of mulation . It is wmulation , and Rabmulation . Thus, imulation , similarmulation , 27, ourIn agreement with our data revealing a role for C9orf72 in the regulation of synaptic vesicle pools, downregulation of SV2 and a reduced rate of synaptic vesicle cycling has been observed at the neuromuscular junction in a zebrafish C9orf72 loss of function model . ReductiHow C9orf72 haploinsufficiency is involved in C9ALS/FTD is not yet clear. The data presented here show that C9orf72 haploinsufficiency per se can cause neuronal damage independent of gain-of-function mechanisms. Synaptic dysfunction and loss are a recurring theme in ALS/FTD, including in non-C9orf72 related familial and sporadic forms of the disease, and appear to precede neurodegeneration , 57, 71.UNC13A, which encodes the presynaptic protein Munc13-1, that have been identified as risk factors for ALS/FTD [It is worth nothing that polymorphisms in  ALS/FTD , 59 caus ALS/FTD . Munc13- ALS/FTD . Consequ ALS/FTD . Thus, aOur study demonstrates that C9orf72 directly interacts with the synapsin family of presynaptic vesicle proteins, impacting upon their levels in synapses and disturbing their role in modulating vesicle trafficking and neuronal function. These data support previous observations that excitatory synaptic dysfunction increases disease risk in ALS/FTD, and demonstrate for the first time that C9orf72 haploinsufficiency significantly contributes to altered synaptic density, regulation and function in C9ALS/FTD.Finally, our study reveals a potentially deleterious neurological effect of reducing C9orf72 protein levels, which should be taken into consideration when designing anti-sense oligonucleotide therapeutics targeting C9orf72.Supplementary file1 (PDF 7158 kb)Below is the link to the electronic supplementary material.


For the µ-opioid receptor (MOR), early experimental evidence from a single study suggested that G protein signaling mediates analgesia and sedation, whereas ß-arrestin signaling mediates respiratory depression and constipation. Then, receptor mutations were used to clarify which residues interact with ligands to selectively regulate signals in a ligand-specific manner. However, there is no systematic study on how to determine these residues and clarify the molecular mechanism of their influence on signal pathways. We have therefore used molecular docking to predict the amino acid sites that affect the binding of ligands and MOR. Then, the corresponding sites were mutated to determine the effect of the structural determinant of MOR on Gi/o protein and ß-arrestin pathways. The pharmacological and animal behavioral experiments in combination with molecular dynamics simulations were used to elucidate the molecular mechanism of key residues governing the signaling. Without affecting ligand binding to MOR, MORY7.43A attenuated the activation of both Gi/o protein and ß-arrestin signaling pathways stimulated by fentanyl, whereas it did not change these two pathways stimulated by morphine. Likewise, the activation peak time of extracellular regulated protein kinases was significantly prolonged at MORY7.43A compared with that at MORwildtype stimulated by fentanyl, but there was no difference stimulated by morphine. In addition, MORY7.43A significantly enhanced analgesia by fentanyl but not by morphine in the mice behavioral experiment. Furthermore, the molecular dynamics simulations showed that H6 moves toward the cellular membrane. H6 of the fentanyl–Y7.43A system moved outward more than that in the morphine–Y7.43A system. Y7.43 mutation disrupted hydrophobic interactions between W6.48 and Y7.43 in the fentanyl–Y7.43A system but not in the morphine–Y7.43A system. Our results have disclosed novel mechanisms of Y7.43 mutation affecting MOR signaling pathways. Y7.43 mutation reduced the activation of the Gi/o protein pathway and blocked the ß-arrestin2 recruitment, increased the H6 outward movement of MOR, and disrupted hydrophobic interactions. This may be responsible for the enhanced fentanyl analgesia. These findings are conducive to designing new drugs from the perspective of ligand and receptor binding, and Y7.43 is also expected to be a key site to structure optimization of synthesized compounds.G protein–coupled receptors can signal through both G proteins and  MOR signaling is regulated by the phosphorylation of intracellular C-terminal serine and threonine residues, which stabilizes the recruitment of ß-arrestin2, which leads to receptor internalization and additional kinase-driven signaling events. Upon the activation, there are two main downstream signaling pathways of MOR, namely, the G protein–dependent signaling pathway and the ß-arrestin2–mediated signaling pathway  . They aror (KOR) . MOR is  pathway , which a pathway . The disß-arrestin–dependent signaling pathway mediates respiratory depression . The hOPRM1 cDNA sequence was obtained from the NCBI website. The Vazyme® Fast Mutagenesis Kit V2  was used to introduce the Y7.43A site mutation to the MOR recombinant plasmid using the following primers: F 5′-CTCTAGGTgctACAAACAGCTGCCTCAACCCA-3′ and R 5′-GTTTGTagcACCTAGAGCAATGCAGAAGTGCC-3′. The nucleotide sequences of mutant MOR were confirmed through DNA sequencing and sequence alignment.To construct MOR and Y7.43A mutant plasmids, full-length hOPRM1 cDNA was subcloned into the Tag-liteß-arrestin2 were purchased from Cell Signaling Technology . Polyclonal rabbit MOR antibody was purchased from Sigma-Aldrich . GAPDH monoclonal antibody was purchased from Abcam . HRP-coupled secondary antimouse and antirabbit antibodies were obtained from Jackson ImmunoResearch .Antibodies recognizing phosphorylated p44/42 MAPK (p-ERK1/2), ERK1/2, and Morphine, 7,8-Didehydro-4,5-epoxy-17-methyl--morphinan-3,6-diol hydrochloride, item no. 20020201 was purchased from Qinghai Pharmaceutical Factory . Fentanyl, N-Phenyl-N-[1-(2-phenylethyl) piperidin-4-yl] propanamide 2-hydroxypropane-1,2,3-tricarboxylate (1:1) was synthesized by the Military Medical Research Institute . Forskolin was purchased from Sigma-Aldrich . Naltrindole hydrochloride was purchased from Tocris Bioscience .Homo sapiens active MOR was built by homology modeling with the X-ray structure of Mus musculus active MOR (PDB code: 5C1M), using Discovery Studio 3.5. Align Sequences module was utilized to align Homo sapiens MOR and murine MOR sequences. All water molecules in the X-ray structure were retained. The Ramachandran plot was used to evaluate the validity of the homology models.The Homo sapiens active MOR. Also, DAMGO was involved in docking as a reference agonist. The Induce Fit Docking module of the Schrödinger 3.5 software was used to dock them into the binding site of the active MOR. The two systems were subjected to Monte Carlo Multiple Minimum conformational searches using the OPLS_2005 force field, and the optimal conformations were chosen as binding conformations.The fentanyl and morphine were docked into the 3D structure of The MD simulations were conducted using Gromacs 5.1.2 package. We built six systems for MD simulations: 1) MOR without ligand (Apo); 2) fentanyl-bound active MOR; 3) morphine-bound active MOR; 4) Y7.43A mutant MOR without ligand (Apo-Y7.43A); 5) fentanyl-bound active Y7.43A mutant MOR; and 6) morphine-bound active Y7.43A mutant MOR. All systems were embedded into the hydrated, equilibrated palmitoyloleoylphosphatidylcholine bilayer using the CHARMM-GUI interface. Sodium and chloride ions were added to neutralize systems and adjusted NaCl concentration to approximately 0.15 mol/l. All systems were minimized and gradually equilibrated in an NPT ensemble at 310 K and 1 bar. The MD simulations were conducted for 100 ns. All analyses of MD trajectories were conducted using tools implemented in the GROMACS 5.1.2 package.® binding assay recommended protocol . HEK293T cells were cultured in a DMEM medium  containing 10% fetal bovine serum at 37°C with 5% CO2. They were grown on 60 mm dishes and transiently transfected with 4 μg WT/Y7.43A mutant MOR plasmid using Lipofectamine® 3,000  according to the manufacturer’s instructions. All cells were labeled in batch with 700 μl/dish SNAP-Lumi4-Tb and suspended in Tag-lite® labeling buffer (TLB), and then, cells were dispensed in a 384 well small volume white microplate  at a density of 5,000 cells/well.Homogeneous time-resolved fluorescence (HTRF) competitive binding assay was conducted according to the Tag-lited of the fluorescent ligand, which is a naltrexone derivative labeled with a red-emitting HTRF fluorescent probe , cells were incubated with a series of concentrations  of the fluorescent ligand in TLB. Nonspecific binding signal wells were incubated with 100 nM Naltrindole hydrochloride. For competition binding experiments, cells were added fentanyl or morphine  in competition against the 8 nM fluorescent ligand. All samples were mixed with a final volume of 20 μl and incubated at room temperature for 3 h. After incubation, HTRF signals were measured using Envision Multilabel Reader  after excitation at 337 nm at both 620 and 665 nm emission, and the HTRF signal was calculated as a two-wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)].To determine the saturation binding constant Kwildtype/MORY7.43A was performed using the GloSensor cAMP biosensor  according to the manufacturer’s protocol. In a word, HEK293T cells were grown on 60 mm dishes and transiently cotransfected with 2 μg MORwildtype/1.58 μg MORY7.43A plasmid and 1.4 μg pGloSensor-22 F cAMP plasmid . After 8 h of transient transfection, the cells were digested by trypsin and distributed evenly into the white 96-well poly-D-lysine-coated plate . After 24 h, cell media were replaced by 90 ml of fresh full DMEM with 2% v/v GloSensor cAMP Reagent  and incubated for 120 min at 37°C. Firefly luciferase activity was measured using Envision Multilabel Reader. After initial measurement of the baseline signal for 15 min, cells were stimulated with 10 μl of 10× drug solution of the MOR agonist fentanyl or morphine  for 10 min. Then, 1 μl of forskolin  solution was added for 15 min, and the real-time chemiluminescent signal was measured at once. The inhibition of cAMP production was calculated using the baseline signal, drug response signal, and forskolin signal. Then, sigmoidal dose–response curves with variable slope (three-parameter logistic regression) were plotted. Results are from three independent experiments.Measurement of ligand-induced cAMP production in HEK293T cells transiently expressing MORß-arrestin2 was monitored using a NanoBiT protein:protein interaction assay . MORwildtype and MORY7.43A were cloned into pBiT2.1-N [TK/SmBiT] vector and ß-arrestin2 into pBiT1.1-C [TK/LgBiT] vector. For the negative control, the MOR-SmBiT construct was substituted with the NanoBiT negative control vector . The positive control consisted of SmBiT-PRKACA and LgBiT-PRKAR2A vectors; 2.4 μg WT/Y7.43A-SmBiT together with 0.8 μg LgBiT-β-arrestin2 were used to transiently transfect HEK293T cells in 60 mm dish using Lipofectamine 3,000 according to the manufacturer’s protocol. After 8 h, the cells were digested by trypsin and distributed evenly into the white 96-well poly-D-lysine-coated plate. The next day, 30 min prior to the assay, the culture medium was substituted for 100 μl/well serum-free Opti-MEM medium . The nanoluciferase activity was measured using Envision Multilabel Reader. After the initial measurement of the background signal, 25 μl/well Nano-Glo® Live Cell Assay System  was added and cells were equilibrated and basal luciferase activity was measured for 15 min. Then, cells were stimulated with 10 μl of 13.5× drug solution of the MOR agonist fentanyl or morphine , and a chemiluminescent signal was measured for 15 min . The ß-arrestin2 recruitment was calculated using the baseline signal and the drug response signal. Then, sigmoidal dose–response curves with variable slope (three-parameter logistic regression) were plotted. Results are from three independent experiments.Coupling between MOR and ß-arrestin2 recruitment assay were seeded in six-well plates. The next day, RNA was extracted using TRIzol . cDNA was synthesized using the RevertAid first strand cDNA synthesis kit . Relative MORwildtype and MORY7.43A expressions were determined on 250 ng cDNA using SYBR Green quantitative polymerase chain reaction (PCR) reagents , normalized to GAPDH expression levels. The forward primer of MORwildtype/MORY7.43A was GCCCTTCCAGAGTGTGAATTAC, the reverse primer of MORwildtype/MORY7.43A was GTGCAGAGGGTGAATATGCTG, the forward primer of GAPDH was AGGTCATCCCAGAGCTGAACG, and the reverse primer of GAPDH was TCAGATGCCTGCTTCACCAC.The transfected samples for the wildtype and MORY7.43A plasmids into HEK 293T cells, the transfected cells were starved in serum-free DMEM medium for 2 h. Then, the cells were exposed to 10 μM fentanyl and morphine for an indicated time. Immediately following stimulation, all cells were lysed in an ice-cold RIPA lysis buffer  supplemented with protease inhibitor cocktail and phosphatase inhibitor . Protein samples were resolved by 10% SDS-PAGE and transferred onto PVDF membranes . After the membranes were blocked with 5% milk, they were incubated overnight with primary antibodies against p-ERK1/2, ERK1/2, MOR, and GAPDH at 4°C. The membranes were washed with TBST and incubated with the secondary antibody for 1 h at room temperature. The immune complexes were detected using western blot detection reagents .After 24 h of transient transfection of MOROprm1tm1Kff/J  was obtained from The Jackson Laboratory. Mice were genotyped via PCR analysis of genomic tail-biopsy DNA using the following primers: 5′-GCCAGAGGCCACTTGTGTAG-3′, 5′-ATCTTCACCCTCTGCACCAT-3′, and 5′-TGCTGGGCTCAAGCTTTAAT-3’. JAX™C57BL/6J  mice from The Jackson Laboratory were used as wild-type control. Animals were housed four to six per cage under a 12 h light–dark cycle (lights on at 8 a.m.) and ventilated at a controlled temperature with ad libitum access to food and water. In hot plate experiments, we used female mice aged 8–16 weeks because the genitals of male mice touched the hot plate, which can produce an allergic reaction. Each animal was used only once for each dose and each drug tested. Studies were conducted in parallel such that age-matched mice received the same drug treatment at the same time. All experimental protocols were approved by and conducted in accordance with the Animal Care and Use Committee of the Beijing Institute of Pharmacology & Toxicology .MOR knockout mouse strain B6.129S2-wildtype/MORY7.43A was rAAV2-EF1α-EGFP-WPRE-hGH polyA, AAV-PHP.eB vector containing MORwildtype/MORY7.43A insert of mice.The AAV  that overexpressed MORThe heads of mice were firmly fixed by the thumb and index finger. A 25 µl tip microsyringe was used to slowly inject AAVs (5 µl) into the right cerebral lateral ventricle  .4, after which whole-brain dissections were performed. Brains were postfixed overnight in 4% paraformaldehyde in 0.1 M PO4 followed by incubation for a minimum of 48 h in 30% sucrose in 0.1 M PO4 ) after placement on a hot plate maintained at 55 ± 0.5°C . To avoid and Ki. For EC50 values of concentration–response curves, the best-fit line was generated following nonlinear regression analysis, as described in each section above. The results of the HTRF competitive binding assay, cAMP measurement assay, and ß-arrestin2 recruitment assay were evaluated via an unpaired two-tailed t-test. For the hot plate test experiment, data were analyzed using two-way ANOVA, followed by Bonferroni’s post hoc tests. In all the experiments, a value of p < 0.05 was considered significant and all calculations were performed using GraphPad Prism 8.0 software.Data are presented as mean ± SEM of at least three individual experiments. For HTRF competitive binding assay, concentration–response curves were fit to one-site binding models provided in GraphPad Prism software to determine KHomo sapiens active OR was obtained by homology modeling with the X-ray structure of Mus musculus active MOR (PDB code: 5C1M) and is shown in Homo sapiens MOR and murine MOR were 95.2% and 99.6%, respectively. In the Ramachandran plot, 96.7% amino acids were located in the most favored regions and 3.3% amino acids were located in additional allowed regions (The 3D model of  regions . The snaThe molecular docking diagram showed residues of MOR that interact with agonists. The most reasonable conformation of fentanyl in MOR was selected in our docking process based on the following criteria: 1) the lowest binding free energy and 2) the most frequently binding pose. The docking pose of fentanyl was subsequently optimized through MD simulation. It is consistent with the message-address concept: the protonated amine of the piperidine ring in fentanyl formed electrostatic interaction with the acidic D3.32 (message site), and the n-alkyl phenyl formed extensive hydrophobic contacts with transmembrane helices (TM) 2/3 (address site). The result was consistent with de Waal’s studies . Also, twildtype/MORY7.43A was estimated using a competitive TR-FRET (time-resolved fluorescence resonance energy transfer)–based assay  and (35.49 ± 2.97) nM, respectively, at MORwildtype and MORY7.43A. In addition, we determined the expression of MORwildtype and MORY7.43A. There was no statistical difference in both whole cell and cell membrane via western blot and fluorescence labeling, respectively  nM for MORY7.43A, which is much higher than that observed for MORwildtype (0.53 ± 0.15) nM. By contrast, the EC50 of morphine was (291.13 ± 181.44) nM for MORY7.43A, with no significant difference between the MORY7.43A and MORwildtype (119.94 ± 52.02) nM  nM . Next, wectively , indicatß-arrestin–dependent signaling pathway, we focused on the interaction between MOR and ß-arrestin2 using NanoBiT assay. The results showed that MORY7.43A markedly inhibited the ß-arrestin2 recruitment induced by fentanyl , leading to the 7.3 and 8.4 Å outward movement of H6 in the fentanyl–WT and fentanyl–Y7.43A systems, respectively. This result suggested that the Y7.43 site mutation slightly increased the outward migration of H6 compared with WT. In the morphine–receptor activated system, H6 moved outward 6.3 Å after site mutation, which was less than 8.9 Å in the WT system (intracellular view). Besides, in the fentanyl–WT and fentanyl–Y7.43A systems, we also found the degree of I3.40 flipping was consistent compared to the apo state, while it varied in the two activated states of morphine binding with a receptor. In summary, the binding of fentanyl with MORY7.43A broke up hydrophobic interactions and rearranged the conserved P5.50-I3.40-F6.44 triad. The rearrangement of the conserved P5.50-I3.40-F6.44 triad induced the outward movement of H6, which ultimately may lead to a differential impact on signal pathways by agonists stimulating the receptor.The whole simulation process lasted for 100 ns, and all the systems remained stable . In the with MOR . As showY7.43A to characterize the binding affinities and functional activities of fentanyl and morphine. The conventional hot plate test was used to determine the analgesic effect in vivo when the Y7.43 site was mutated. It showed that MORY7.43A reduced the activation of Gi/o protein–dependent signaling, blocked ß-arrestin2 recruitment, and altered the kinetics of ERK1/2 phosphorylation induced by fentanyl. That is, MORY7.43A delayed the occurrence of maximum ERK1/2 phosphorylation induced by fentanyl, compared with the MORwildtype group. It is worth noting that we observed that MORY7.43A mice exhibited significantly greater analgesic potency of fentanyl than MORwildtype. Moreover, compared with WT, Y7.43 site mutation broke up the hydrophobic interactions and promoted the outward movement of H6 in MOR stimulated by fentanyl but not morphine. Taken together, we show for the first time that Y7.43 site mutation reduced Gi/o protein signaling and blocked ß-arrestin2 recruitment, which may engage the enhanced analgesia induced by fentanyl.In our studies, we first predicted the key amino acid residues playing an important role in agonists binding to the receptor by molecular docking. We then constructed MORi/o protein signaling and ß-arrestin2 recruitment. In general, it is necessary to measure the affinity of MOR and ligands in determining the functional activity of MOR. In our research, we used HTRF competitive binding assay to determine the affinity of drugs in binding with MOR. d value was obtained from this curve. 50 of drugs was obtained from this curve. The Ki value was calculated as the Cheng–Prusoff equation: i value directly reflects the affinity of drugs binding with MOR as shown in Y7.43A and MORwildtype. The relative affinity of fentanyl at MORY7.43A was lower than that of MORwildtype. The pKi value of fentanyl was 91% of MORwildtype pKi. In combination with the practical application of Cisbio’s protocol, if the Ki value difference between changes is less than four times, it is usually considered that there is no difference in affinity. Therefore, we believe that the Y7.43 mutation almost did not change the affinity of the drugs in binding with MOR. On this basis, we monitored the activity of signaling pathways downstream of MOR.Functional activity at MOR was characterized using cell-based assays designed to measure GY7.43A did not change both cAMP and ß-arrestin2 signaling response by morphine, as it was similar to MORwildtype. In complete contrast, decreased cAMP activity and no ß-arrestin2 activity were discovered at MORY7.43A by fentanyl.Recent research has clarified how and where a ligand should bind with a receptor to cause adequate signaling activation. Y7.43 of MOR TM7 forms a ligand-binding pocket with W7.35 as highlighted by recent receptor crystallography data  and prev5.50-I3.40-F6.44 triad rearrangement, which were mentioned in the result section. The first index is a three to seven lock, which was formed by the hydrogen bond between D3.32 and Y7.43, and plays a key role in the activation of MOR ) . The biassay2)) . Bias fassay2)) . HoweverY7.43, we might improve the chance of successfully designing new analgesics with fewer side effects and stronger analgesic ability.In summary, we indeed first elucidate the novel mechanisms of Y7.43 mutation affecting MOR signaling pathways. Further, functional selectivity in fentanyl and morphine can engage different mechanisms to induce analgesia. Through a better understanding of the molecular mechanisms of ligands specific binding with MOR, it is anticipated that we can bridge the knowledge gap on how chemical information encoded within the receptor regions’ controlled-effector coupling is decoded into the effect mediated by signaling pathways through the specific interactions it makes within the receptor binding site. Thus, by rationally designing novel compounds to interact predictably with MOR


Coriolopsis gallica. Decolorization was optimized in a 1 mL reaction mixture using the response surface methodology (RSM) to test the influence of five variables, i.e., laccase-like activity, dye concentration, redox mediator (HBT) concentration, pH, and temperature, on dye decolorization. Statistical tests were used to determine regression coefficients and the quality of the models used, as well as significant factors and/or factor interactions. Maximum decolorization was achieved at 120 min (82 ± 0.6%) with the optimized protocol, i.e., laccase-like activity at 0.5 U mL−1, dye at 25 mg L−1, HBT at 4.5 mM, pH at 4.2 and temperature at 55 °C. The model proved significant (ANOVA test with p < 0.001): coefficient of determination (R²) was 89.78%, adjusted coefficient of determination (R²A) was 87.85%, and root mean square error (RMSE) was 10.48%. The reaction conditions yielding maximum decolorization were tested in a larger volume of 500 mL reaction mixture. Under these conditions, the decolorization rate reached 77.6 ± 0.4%, which was in good agreement with the value found on the 1 mL scale. RB5 decolorization was further evaluated using the UV-visible spectra of the treated and untreated dyes.The textile industry generates huge volumes of colored wastewater that require multiple treatments to remove persistent toxic and carcinogenic dyes. Here we studied the decolorization of a recalcitrant azo dye, Reactive Black 5, using laccase-like active cell-free supernatant from  Water is essential for all forms of life, making it the most important resource on Earth. However, water decline and depletion is an escalating problem driven by the increasing global population ,2. This −1 of dyes)  for laccase production. Coriolopsis gallica strain CLBE55 was deposited at the culture collection “Centre International de Ressources Microbiennes” (CIRM) under accession number BRFM 3473.This study used C. gallica were performed on PDA media that contained 39 g of dehydrated media (Accumix) suspended in 1000 mL of distilled water sterilized by autoclaving at 120 °C for 30 min. Liquid pre-cultures were performed in 25 mL of Malt Extract medium  containing 30 g Malt Extract per L at pH 5.5 sterilized by autoclaving at 120 °C for 30 min. The pre-cultures were inoculated with 3 agar plugs (6 mm in diameter) cut from the growing edge of a plate stock culture and incubated at 30 °C for 3 days at 160 rpm. Mycelia from these three-day precultures were then partially ground down using glass beads (0.6 mm). The mycelial mixture obtained was used to inoculate 500-mL Erlenmeyer flasks containing 100 mL of M7 medium [−1) glucose 10, peptone 5, yeast extract 1, ammonium tartrate 2, KH2PO4 1, MgSO4, 7H2O 0.5, KCl 0.5, and 1 mL of trace element solution. Composition of the trace-element solution was (in g L−1): B4O7Na2, 10H2O 0.1, CuSO4, 5H2O 0.01, FeSO4, 7H2O 0.05, MnSO4, 7H2O 0.01, ZnSO4, 7H2O 0.07, (NH4)6Mo7O24, 4H2O 0.01. pH was adjusted to 5.5, and cultures were incubated in a rotary shaker for 7 days at 30 °C and 160 rpm. On day 3 of incubation, 300 µM CuSO4 was added as a laccase inducer.Solid cultures of 7 medium ,63. The C. gallica was filtered on a Miracloth membrane  and centrifuged at 30 °C for 5 min at 10,000× g prior to use. The laccase-like activity of the cell-free supernatant was assayed by monitoring the oxidation of 5 mM 2,6-dimethoxyphenol (DMP) in 50 mM citrate buffer, pH 5  in the presence of 50 µL of supernatant. The assay was carried out at 30 °C for 1 min. One unit of DMP-oxidizing activity was defined as the amount of enzyme oxidizing 1 µmol of substrate per minute.The supernatant of a 7-day culture of C. gallica. The chemical properties of the RB5 dye are reported in An experimental design was used to optimize the enzymatic decolorization of the recalcitrant azo dye Reactive Black 5 (RB5)  by using the culture supernatant of 1), initial dye concentration (x2), HBT concentration (x3), pH (x4), and temperature (x5). The center point tested the linearity of the experimental points , medium (coded 0), and high (coded +1)  . Table 3i is initial absorbance of the dye at the maximum wavelength before incubation with the enzyme, and At is dye absorbance after 2 h of incubation. All experiments were done in a total volume of 1 mL reaction mixture in 50 mM citrate buffer.Decolorization was measured every 30 min over a 2-h incubation period by tracking the decrease in absorbance of the RB5 dye (598 nm). Percentage decolorization was calculated using the following formula (Equation (2)):0, βi, βij and βii are the model’s intercepts, linear, interactions and quadratic coefficients, respectively, xi is level-coded factor variable, and k is 5 in our experimental setup.The best condition for RB5 decolorization was determined using RSM based on a Box–Behnken design with influential variables obtained previously via the Plackett–Burman design. A total of 46 runs  were pert-test were used to identify the level of significance of both the fitted model and the factors and their interactions. The probability (p < 0.05) and high F-values (Fisher’s test) demonstrated that the model and factors were significant. The quality of the model was analyzed using a coefficient of determination (R²), adjusted coefficient of determination (R² adj), and root mean square error (RMSE).Minitab 16 Statistical Software  was used for the experimental designs and statistical analysis. Factor coefficients were determined using the least-squares method. Analysis of variance (ANOVA) and a Student’s −1 RB5 dye, 4.5 mM HBT, and 0.5 U mL−1 crude C. gallica laccase. The reaction mixture was incubated at 55 °C for two hours, and 1-mL samples were taken every 30 min over the 2-h incubation period. Percentage rates of decolorization were calculated following Equation (2).The optimal conditions determined for RB5 decolorization in a reaction volume of 1 mL were then tested on a larger scale (500 mL). Experiments were performed in duplicate in 1 L Erlenmeyer flasks containing 500 mL of the following reaction mixture: 50 mM citrate buffer pH 4.2, 25 mg LThe UV-visible spectrum of RB5 was analyzed to visualize its color and absorbance peak shift. Decolorization was carried out for 24 h in reference conditions described in C. gallica was active and stable at acidic pH (4–7) for 24 h of incubation retaining from 40 to 80% of its initial activity and the same high temperature (up to 50–55 °C) as tested here [Preliminary experiments were performed to fix the levels of factors. With a view to potential scale-up for use in textile industry applications, reaction time was set at 2 h. Note that we ran tests at several reaction times (>2 h) and found no change in the final decolorization of the dye. In addition, a previous study had demonstrated that the main laccase of ted here  and thatted here . Based otfactor > tstudent (p = 0.05) = 2.024; p < 0.05) and that enzyme concentration was the most effective variable (p < 0.001) compared to pH (p < 0.001), initial dye concentration (p < 0.001) and HBT concentration which was the least effective variable (p < 0.01). Temperature was found to be a non-significant variable but was kept for the second step of the RSM-based optimization protocol for two reasons: (1) its p-value (0.073) was almost ruled in as significant ; (2) temperature was found to be insignificant as the responses at extreme points  were very close to each other, but the center point was very significantly different to these values (p < 0.001).Statistical results obtained from Minitab 16 gave the model coefficient, and the fitted form of the equation was then represented as follows (Equation (4)):1), HBT (x3) concentrations, and temperature (x5) were positive-signed whereas dye concentration (x2) and pH (x4) were negative-signed. Moreover, the coefficient of determination (R²), the adjusted coefficient of determination (R²(adj)), and the root mean square error (RMSE) were 85.62%, 83.35%, and 12.5% (units of RB5 decolorization), respectively, thus demonstrating the goodness-of-fit of the adopted model (Equation (4)).A positive-signed main effect of a factor indicates that using higher levels gave the most effective response whereas a negative-signed main effect indicates that using lower levels was more effective. Thus, according to Equation (4), laccase-like activity (xp < 0.001), indicated a second-order model was able to fit decolorization as a function of all factors. We consequently applied a second response-surface experimental design for the same variables in our protocol optimization step.C. gallica. The Box–Behnken design also statistically predicted the same 5 variables as previously tested by the Plackett–Burman design. Response surface methodology based on a Box–Behnken design was performed to optimize  the conditions of RB5 decolorization by crude laccase obtained from The second-order polynomial equation, indicating the main effects, interactions among variables, and the quadratic effect with coefficients obtained from statistical analysis are represented as follows (Equation (5)):1), initial dye concentration (x2), and pH (x4) were the most effective variables with the highest coefficients in a linear regression with positive (x1) and negative  effects. Moreover, as illustrated in p-values were less than 0.001 and 0.01 for linear regression (factors without their interactions), thus making all factors highly significant. Regarding the quadratic effect of factors, some of them were statistically non-significant with p-values over 0.05, such as initial dye concentration (p = 0.465) and temperature (p = 0.089), whereas the rest of the quadratic-term factors  had a significant influence (p < 0.001). Regarding the interactions between the five variables, 5 among the 10 were significant. The highest p-values found were for pH × temperature (p = 0.592) and dye × HBT concentration (p = 0.561). pH × laccase-like activity and pH × HBT concentration were the most significant and valuable interactions ) and had a significant influence on RB5 decolorization (p < 0.001). Moreover, the initial dye concentration × pH interaction was found to be significant with a positive-signed effect and a p-value < 0.01.According to Equation (5), laccase-like activity  to 125 mg L−1 (level +1) decreased RB5 decolorization from 80% to 30%. Increasing HBT concentration to 2.5 mM  improved RB5 decolorization compared to the condition with 0.5 mM of HBT. Contour plots for the laccase-like activity× temperature interaction (Iso-responses plots (contour curves) were analyzed to visualize the interaction between factors. eraction B showed eraction C showed eraction D and pH eraction E reachederaction F.−1—high level (+1)), dye concentration (25 mg L−1—low level (−1)), HBT concentration (4.5 mM—high level (+1)), pH (4.2—close to level 0 (−0.203)), and temperature (55 °C—high level (+1)). The best values for each variable to obtain maximum decolorization as predicted by the Box–Behnken design are highlighted in red  obtained via the Box–Behnken design was tested in a total reaction volume of 500 mL to test the conditions in larger volumes. The optimized condition-set  accordingly a decrease in the peak absorbance of RB5 at 598 nm. Note that there was no decrease in absorbance when RB5 was incubated with HBT but without enzyme.Spectrum analysis showed that the maximum absorbance peak of RB5 was at 598 nm, which is in the visible-light region . MoreoveC. gallica to decolorize RB5 could therefore be an efficient solution to minimize RB5 concentrations in wastewater. The experimental design successfully performed here used a screening plan that came with five factors and indicated that 4 of the 5 factor variables tested were significant . Moreover, for the fifth variable, which was temperature, we choose to rule it as significant as it was very close to the p = 0.05 limit (p = 0.073). Daâssi et al. (2012) [T. trogii. However, whereas the Pareto chart  and Reactive Red 239 (RR239), was unaffected by the enzyme concentration of a commercial laccase from Aspergillus sp. [C. gallica is 3.0  [Trametes sp. was found to be sensitive to small pH changes [P. eryngii F032, the optimum pH occurred at 3, affording a maximum of 94.56% dye removal, whereas an increase in pH up to 10 decreased RB5 decolorization down to just 25.9% [T. versicolor and Yarrowia lipolytica NBRC 1658, increasing pH values increased the RB5 decolorization rates [Textile manufacturing encompasses a complex cluster of various different technologies and machinery  that ult. (2012)  found thto chart  indicatellus sp. . Furthert p = 0.0. Daâssi llus sp. . For ins. (2016) ) but the. (2016) . Our ana changes . When usst 25.9% . Howeveron rates ,78.−1 negatively affected RB5 decolorization by Peniophora sp. CBMAI 1063, possibly due to the inhibitory effect of the dye on laccase-like enzymes at high concentrations [T. trogii and laccase from Trametes sp. strain CLBE55 at up to 1 mM, [The Box–Behnken experimental design and RSM then served as a suitable approach to facilitate the process modeling and determine the optimized conditions for improving the decolorization rate. The contour plots indicated that increasing the initial dye concentration has a negative effect on RB5 decolorization. Our results are consistent with data from El Bouraie and El Din (2016)  and Khantrations ,83. To itrations . Analysitrations , Murugestrations  and Neiftrations  all obsetrations . HBT alrto 1 mM, ,87.C. gallica laccase, which showed a correlation between the increase in temperature and laccase activity in the range of 20–60 °C [Aspergillus oryzae, Wang et al. (2011) [Pleurotus sajor-caju, where increasing the temperature led to high levels of dye decolorization, with a maximum found at 35–40 °C. However, a study using cauliflower (Brassica oleracea) bud peroxidase to remove dyes  found that the decolorization rate was maximal at 40 °C and decreased at temperatures above 70 °C, probably due to the thermal denaturation of the enzyme [Increasing the temperature parameter improved dye decolorization up to 55 °C. This result is in good agreement with the optimum temperature profile of the 20–60 °C . Using t. (2011)  reported. (2011)  reached e enzyme . −1, 25 mg L−1, 4.5 mM, 4.2, and 55 °C, respectively, yielding 82 ± 0.6% color removal after 2 h of incubation. This result was in the same range for the decolorization of Tubantin direct dye bath for pH (4) and enzyme concentration (0.6 U mL−1 of laccase-active cell-free supernatant from Trametes sp.) leading to 88% decolorization [−1 of Tubantin bath dye, 1 mM HBT, 30 °C, and 20 h [Trichoderma asperellum was also reported to decolorize 90% of 50 mg L−1 RB5 within 24 h in the presence of an HBT mediator [−1 of RB5 within 24 h using purified NADH-dichlorophenol indophenol (NADH-DCIP) reductase and lignin peroxidase (LiP) obtained from Sterigmatomyces halophilus SSA-1575 [The optimized conditions obtained here using RSM for laccase-like activity, dye concentration, HBT concentration, pH, and temperature were 0.5 U mLrization . Howeverand 20 h . The lacSSA-1575 . In concBjerkandera adusta removed 95% of the azo dye RB5 (0.2 g L−1) in a 5 L aerated stirred tank bioreactor within 20 days. Pavko (2011) [T. versicolor was capable of decolorizing 97% of the synthetic dye Orange G in a 1.5 L bioreactor within 20 h. In a 3 L-volume bioreactor, Pseudomonas putida SKG-1 strain was able to decolorize 98% of the reactive orange 4 dye within 60 h [Tests were run at a larger scale (500 mL reaction volume) to increase the total quantity of dyes with the best condition-set and achieved 77.6 ± 0.4% decolorization within 2 h and compared to controlled bioreactor processes. Mohorčič et al. (2004)  reportedo (2011)  reportedhin 60 h . This shC. gallica, demonstrating that the laccase mediator system efficiently decolorizes RB5. Our results confirmed those of Tavares et al. (2008) [The UV-visible spectrum of RB5, in the range of 200 to 800 nm, indicated peak absorbance of RB5 at 598 nm together with the presence of benzene and naphthalene ring signatures at 254 and 310 nm, respectively, ,95. Spec. (2008)  who showC. gallica was active on RB5 dye using the mediator HBT, and that an enzymatic treatment of 2 h was sufficient to achieve up to 82 ± 0.6% decolorization in a lab-scale reaction volume of 500 mL Further scale-up experiments are required to demonstrate that this treatment can be made amenable to pilot scale and to future industrial applications. This work provides proof-of-concept for the biodegradation of textile-industry dyes as a promising alternative route to physicochemical treatments. Further experiments could usefully measure the biochemical oxygen demand (BOD) and chemical oxygen demand (COD) of the treated dye solution to evaluate the toxicity of the residual dye. Processes using cheaper and eco-friendly alternative plant-origin mediators should be tested in an effort to engineer a more sustainable system.The statistical design-of-experiments approach employed here showed that 4 factors among the 5 studied were key factors for the degradation of the RB5 dye. Among all these parameters, the main factor was the concentration of laccase activity used in the process. This is a critical point, as the price of the enzyme dictates the economic viability of the process. The second most critical factor was pH, probably due to the biochemical properties of the enzymes (pH optimum and pH stability). However, the present study confirmed that the laccase cell-free supernatant of


This study aims to clarify the mechanistic action of long non-coding RNA (lncRNA) SNHG12 in the development of renal cell carcinoma (RCC), which may be associated with promoter methylation modification by KMT2B and the regulation of the E2F1/CEP55 axis.TCGA and GEO databases were used to predict the involvement of SNHG12 in RCC. Knockdown of SNHG12/E2F1/CEP55 was performed. Next, SNHG12 expression and other mRNAs were quantified by RT-qPCR. Subsequently, CCK-8 was used to detect cell proliferation. Wound healing assay and Transwell assay were used to detect cell migration and invasion, respectively. The in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) was explored by matrigel-based capillary-like tube formation assay. ChIP assay was used to detect H3K4me3 in SNHG12 promoter region. The binding of E2F1 to CEP55 promoter region was analyzed with ChIP and dual luciferase reporter assays. RIP assay was used to detect the binding of SNHG12 to E2F1. Finally, the effect of SNHG12 on the tumor formation and angiogenesis of RCC was assessed in nude mouse xenograft model.SNHG12 was highly expressed in RCC tissues and cells, and it was related to the poor prognosis of RCC patients. SNHG12 knockdown significantly inhibited RCC cell proliferation, migration, and invasion and HUVEC angiogenesis. KMT2B up-regulated SNHG12 expression through modifying H3K4me3 in its promoter region. In addition, SNHG12 promoted CEP55 expression by recruiting the transcription factor E2F1. Knockdown of SNHG12 blocked E2F1 recruitment and down-regulated the expression of CEP55, thereby inhibiting tumor formation and angiogenesis in nude mice.The evidence provided by our study highlighted the involvement of KMT2B in up-regulation of lncRNA as well as the transcription of CEP55, resulting in the promotion of angiogenesis and growth of RCC.The online version contains supplementary material available at 10.1186/s12935-022-02607-w. Renal cell carcinoma (RCC) is the main pathological type of kidney cancer, accounting for 70% to 90% . EpidemiLong non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 nucleotides. It is involved in the multi-level regulation of gene expression and its abnormal expression and mutation are usually closely related to tumorigenesis and metastasis –8. In adIn this study, we explored the role of SNHG12 promoter methylation in RCC development through series loss- and gain-of-function experiments. Our findings may provide evidence for identifying new molecular targets for the diagnosis and treatment of RCC.https://xena.ucsc.edu/). The GSE71963 dataset was obtained from GEO database (https://www.ncbi.nlm.nih.gov/gds). In total, we obtained 535 RCC tissue samples and 72 normal tissue samples from TCGA, as well as 32 RCC samples and 16 normal tissues samples from GSE71963 dataset. The R language “limma” package (http://www.bioconductor.org/packages/ release/bioc/html/limma.html) was used to screen differentially expressed lncRNAs, with |logFC|> 1 and p < 0.05. Kaplan–Meier survival analysis was performed using the R software “survival” package , with p < 0.05. ENCORI database (http://starbase.sysu.edu.cn/index.php) was used for correlation analysis. LncMAP database (http://bio-bigdata.hrbmu.edu.cn/LncMAP/) was used to predict the transcription factors of SNHG12 regulating centrosome protein 55 (CEP55) in RCC. The UCSC database (http://genome-asia.ucsc.edu/) was used to analyze the epigenetic modification of the SNHG12 promoter region.RNA sequencing data and corresponding clinical data of RCC tissue and normal tissue samples were downloaded from TCGA database through UCSC Xena  at Mianyang Central Hospital from January 2017 to January 2019. All patients were confirmed to have RCC by surgery and pathological analysis. The inclusion criteria: 1) Patients with pathologically confirmed RCC cases; 2) Patient did not receive any chemotherapy, radiotherapy, or other antitumor treatment before operation; 3) Patients had complete clinical data. The exclusion criteria: 1) Patients without pathological confirmation; 2) Patients with recurrence and distant metastasis after treatment; 3) Patients with a history of mental illness; 4) Patients with dysfunction of the heart, liver, pancreas, and other important organs; 5) Patients with respiratory and circulatory diseases; 6) Patients with non-RCC tumors. Tissue samples were frozen in liquid nitrogen and stored at − 80 °C. Among the included patients, 33 cases at stage I-II, 13 cases at stage III-IV, 28 cases at stage G1-2, and 18 cases at stage G3; 11 cases with lymph node metastasis and 35 without lymph node metastasis. The current study was approved by the Ethics Review Committee of Mianyang Central Hospital (P2020030) and performed in strict accordance with the Human umbilical vein endothelial cells (HUVECs), purchased from Zhong Qiao Xin Zhou Biotechnology , were cultured in 500 mL endothelial cell basal medium containing 25 mL fetal bovine serum, 5 mL endothelial cell growth factor and 5 mL penicillin/streptomycin solution. RCC cell lines A498, 786-O, Caki-1, and 769-P, and human normal kidney cell line HK-2 purchased from ATCC  were cultured in modified Eagle’s medium  containing 10.0% FBS (Gibco) and 1.0% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin).Cells were transfected with plasmids of sh-SNHG12 (short hairpin RNA-SNHG12), oe-SNHG12 (SNHG12 overexpression), sh-KMT2B, oe-E2F1, oe-SNHG12 + sh-E2F1, sh-E2F1 + oe-CEP55, using the Lipofectamine 2000 reagent . Corresponding negative control (NC) groups were set up. The plasmids were all constructed by GenePharma Co., Ltd. , and the plasmid concentration was 50 ng/mL. The shRNA sequences are shown in Additional file 5 cells/mL (100 μL/well). After routine culture overnight, the cells were treated according to CCK-8 kit , and the cell viability was detected at 24 h, 48 h, and 72 h. The OD450 was detected with a microplate reader.After 48 h of transfection, cells were seeded into 96-well plates at a density of 1.0 × 105 cells/mL and cultured for 24 h. Next, a 200 μL sterile pipette tip was used to make a scratch on the cells perpendicular to the horizontal line on the back. Cells continued to culture in the renewed serum-free medium. At 0 and 48 h, the scratch distance was measured and recorded under an optical microscope , and images were acquired under an inverted microscope. Image J software was used to determine the width of the scratches in each well. The relative distance of cell migration to scratch area was measured, and the actual migration distance was calculated according to the scratch area distance of cells.Cells were seeded in a 6-well plate at 5 × 105 cells/mL . In the lower chamber, 700 μL of pre-cooled DMEM medium containing 10% FBS was added. The chamber was then incubated in a 37 °C, 5% CO2 saturated humidity incubator. After 24 h, the cells in the lower chamber were fixed with methanol, and stained with 0.1% crystal violet. Invaded cells were photographed and counted using randomly selected 5 visual fields in each chamber under an inverted microscope. Cell migration experiments were conducted using the same procedures except for ECM gel.The Transwell upper chamber  was pre-coated with ECM gel (Sigma-Aldrich). After starving culture for 24 h, the cells were added to the upper chamber at 2.5 × 10RCC cells were transfected as above described. After 48 h, the cell supernatant was collected. The tumor-conditioned medium was prepared according to the ratio of 4:5:1 (tumor supernatant: DMEM medium: FBS). HUVECs were seeded into a 96-well plate pre-coated with Matrigel and incubated with tumor-conditioned medium for 8 h. Finally, 4 fields of view were randomly selected from each well and the tube length was quantified under a phase-contrast microscope.g for 30 s. The sample was subsequently eluted with 40 μL of Elution solution by centrifugation at 12,000 g for 30 s and with 10 μL of Elution solution. RT-qPCR was used to detect the expression of SNHG12, with GAPDH as the cytoplasmic marker and U6 as the nuclear marker.Nuclear and cytoplasmic fractions of cells were isolated using the PARIS kit . RCC cells were trypsinized and centrifuged at 500 g and 4 °C for 5 min. The pellet was rinsed with PBS, added with 500 μL Cell Fractionation Buffer, gently dispersed, left to stand on ice for 5–10 min, and centrifuged at 500 g and 4 °C for 5 min. Next, the supernatant (cytoplasm) was transferred into a new 2 mL sterile enzyme-free tube, and centrifuged at 500 g and 4 °C for 5 min. The pellet (nucleus) was mixed with 500 μL Cell Fractionation Buffer, 500 μL of 2 × Lysis/binding solution at room temperature, pre-cooled 500 μL Cell Disruption Buffer, and 500 μL of anhydrous ethanol. The adsorption column was put into a collection tube, which was added with 700 μL of reaction solution each time, and centrifuged at 12,000 786-O cells transfected with sh-NC or sh-KMT2B were fixed with 1.0% formaldehyde and incubated for 10 min to generate DNA–protein crosslinks, which were then halted by glycine. Next, the cells were subjected to ultrasonic treatment to obtain the DNA fragments. For ChIP, the supernatant was incubated with the rabbit anti-IgG antibody , anti-H3K4me3 antibody , and anti-E2F1 antibody   at 4 °C overnight. The endogenous DNA–protein complexes were precipitated with Protein Agarose/Sepharose . The DNA fragments were extracted with phenol/chloroform. RT-qPCR was used to detect the enrichment of H3K4me3 in the SNHG12 promoter and E2F1 in the CEP55 promoter.RIP kit (Millipore) was used to detect the binding of SNHG12 and E2F1 protein. The 786-O cells were lysed with RIPA buffer  containing protease inhibitor for 5 min. A portion of cell lysates were taken out as Input, and another was incubated with antibodies against E2F1  and NC IgG  for immunoprecipitation. The antibody-magnetic bead complexes, which were prepared by incubating magnetic beads with the above antibodies, were incubated with the supernatant at 4 °C overnight. After that, the samples were digested with proteinase K and RNA was extracted for subsequent RT-qPCR detection.http://jaspar.genereg.net). After that, the pGL3-CEP55-WT and pGL3-CEP55-MUT plasmids were constructed with wild type (WT) and mutant type (MUT) binding sequences of E2F1, respectively. Then, these plasmids were co-transfected with oe-NC and oe-E2F1 into the HEK-293 T cells (ATCC). After 48 h, the Dual-Luciferase Reporter Assay System kit (Promega) and the TD-20/20 Luminometer were used to detect the luciferase activity.The potential binding site of E2F1 in the promoter region of CEP55 was analyzed through the JASPAR website . The total RNA of cells and tissues as well as nucleus and cytoplasm of RCC cells was extracted by Trizol (Invitrogen). RT-qPCR was conducted as reported previously . The priTotal protein of tissues or cells was extracted with RIPA . Protein concentration was determined using BCA kit (ThermoFisher Scientific). After separation by polyacrylamide gel electrophoresis, 50 μg protein was transferred to PVDF membranes and blocked with 5% BSA for 1 h. The detailed experimental method was similar to a previous report .7 cells/mL (0.1 mL) stably transfected with sh-SNHG12 + oe-E2F1, sh-E2F1 + oe-CEP55, and corresponding NCs (n = 6 for mice upon each treatment). The tumor volume was measured weekly. The tumor volume was calculated by length × width2 × 0.5. Five weeks after inoculation, mice were euthanized by intraperitoneal injection of overdose pentobarbital sodium (100 mg/kg). The tumors were dissected, and weighed, with the tumor length and width measured. The tumor tissue was frozen in liquid nitrogen and stored at − 80 °C for subsequent experiments. Animal experiments were approved by the Animal Ethics Committee of Mianyang Central Hospital (P2020030). All efforts were made to minimize animal suffering.BALB/c female nude mice  were housed in the SPF laboratory at 18–22 °C and 50–60% humidity under a 12-h light/dark cycle. The health of the mice was observed before the experiment. The mice were randomly grouped and inoculated with 786-O cell suspension containing 2 × 10Paraffin sections of tumor tissues were dewaxed, hydrated with gradient alcohol and antigen retrieved in water bath. Next, the sections were blocked with normal goat serum blocking solution  and incubated with anti-VEGF antibody  at 4 °C overnight. After that, sections were incubated with goat anti-rabbit IgG secondary antibody , developed with DAB, and counterstained with hematoxylin. The results were evaluated by two experienced pathologists in a blinded manner. The positive rate of VEGF was calculated as the ratio of positive cells to total number of cells. Specific experiment procedures were similar to previous reports , 27.p < 0.05 indicates that the difference was statistically significant.All data were analyzed using SPSS 21.0 software, and expressed as Mean ± SD. Statistical analysis for data included paired t-test for paired data comparison between two groups, unpaired t-test for unpaired data comparison between two groups, one-way ANOVA with Tukey’s test for data comparison between multiple groups, two-way ANOVA for comparison of OD values at different time points or Bonferroni-corrected repeated measures ANOVA for comparison of tumor data at different time points. Following intersection analysis of the differentially expressed lncRNAs in the RCC tissue samples in TCGA database and the GSE71963 dataset, 5 highly expressed lncRNAs were identified in RCC tissue samples  showed up-regulated SNHG12 expression, with the 786-O cells showing the highest SNHG12 expression (Fig. Collectively, the above findings suggested the abundant SNHG12 expression in RCC tissues and cell lines.SNHG12 was knocked down by three shRNA sequences in 786-O cells, RT-qPCR results showed that the three shRNA sequences successfully reduced the expression of SNHG12, with sh-SNHG12-1 presenting the highest knockdown efficiency (Fig. Moreover, CCK-8, wound healing assay and Transwell assay results showed that SNHG12 knockdown inhibited RCC cell proliferation, migration and invasion (Fig. Thus, SNHG12 knockdown can repress the proliferation, migration, and invasion of RCC cells and HUVEC angiogenesis.As shown in Fig. KMT2B mRNA and protein expression was increased in RCC tissues compared with normal adjacent tissues Fig. D. ComparThe aforementioned data supported that KMT2B could increase SNHG12 expression through the H3K4me3 modification of the SNHG12 promoter.In RCC, SNHG12 expression was positively correlated with CEP55 expression in RCC samples Fig. A. AnalysTo further verify this hypothesis, we showed that SNHG12 was mainly located in the nucleus Fig. D. RIP reFinally, in the 786-O cells, CEP55 expression was increased by SNHG12 overexpressing, but reduced by further silencing E2F1. When overexpressing SNHG12 and silencing E2F1 at the same time, CEP55 expression was reduced Fig. J.The above results indicated that the SNHG12 elevated CEP55 expression in RCC cells by recruiting E2F1 in RCC cells.Compared with oe-SNHG12 + sh-NC, E2F1 and CEP55 expression was decreased by oe-SNHG12 + sh-E2F1. Compared with sh-E2F1 + oe-NC, E2F1 expression remained not changed by sh-E2F1 + oe-CEP55 treatment, but CEP55 expression was elevated (Fig. In addition, compared with SNHG12 overexpression alone, further E2F1 silencing reduced cell proliferation, migration and invasion and capillary-like tube formation ability while CEP55 overexpression reversed the above effects of E2F1 silencing (Fig. Taken together, these results demonstrated that SNHG12 favored RCC cell malignant phenotypes and HUVEC angiogenesis by recruiting E2F1.As shown in Fig. CEP55 expression, while further E2F1 overexpression reversed this trend. Compared with silencing E2F1 alone, simultaneous silencing E2F1 and overexpressing CEP55 up-regulated CEP55 (Fig. Compared with NC, silencing SNHG12 reduced E2F1 and P55 Fig. D.Immunohistochemical detection showed that compared with NC, silencing SNHG12 decreased VEGF protein expression, which was reversed by further E2F1 overexpression. Compared with silencing E2F1 alone, simultaneous silencing E2F1 and overexpressing CEP55 increased VEGF expression Fig. E. MeanwhOverall, these data revealed that silencing of SNHG12 blocked its binding to E2F1 and downregulated CEP55 expression, ultimately inhibiting RCC growth and angiogenesis.RCC is a heterogeneous tumor that originates from the renal parenchyma and is one of the deadliest malignant tumors in the urinary system . So far,SNHG12 exerts a carcinogenic effect in a variety of cancers , 32–34. Here, in this study, the biological function and mechanism of SNHG12 in the occurrence and development of RCC were investigated. We first found that SNHG12 was highly expressed in RCC tissues and cells. In addition, the high expression of SNHG12 was related to the tumor grade and poor prognosis of RCC patients, and the expression of SNHG12 in the tissues of informed RCC patients was related to tumor grade, TNM staging, and lymph node metastasis. To further explore the effect of SNHG12 on the biological functions of RCC cells, we knocked down SNHG12 in 786-O cells and found that SNHG12 knockdown inhibited RCC cell proliferation, migration, and invasion and HUVEC angiogenesis. A meta-analysis showed that high SNHG12 expression in a variety of tumors reduced the overall survival rate and recurrence-free survival rate of tumor patients and that the high expression of SNHG12 suggested unfavorable clinicopathological results, including larger tumors, lymph node metastasis, distant metastasis, and later clinical staging .To further reveal the mechanism of high expression of SNHG12 in RCC, we searched the UCSC database and found that the promoter region of SNHG12 was enriched in H3K4me3. Next, we tested the enrichment of H3K4me3 in the promoter region of SNHG12 through ChIP and found that KMT2B up-regulated the expression of SNHG12 by mediating the modification of H3K4me3 in the promoter region of SNHG12. In addition, we predicted the possible binding sites of E2F1 in the CEP55 promoter region, and knocked down E2FF1 to explore the downstream factors of SNHG12 in RCC. We found that in RCC cells, SNHG12 overexpression promoted CEP55 expression by recruiting E2F1. Finally, we constructed a nude mouse xenograft tumor model. It was found that SNHG12 knockdown blocked the recruitment of E2F1 by SNHG12, and then down-regulated CEP55 expression, leading to the inhibition of RCC growth and angiogenesis. Our results confirmed that the SNHG12/E2F1/CEP55 axis affected RCC growth and angiogenesis.In summary, KMT2B can up-regulate SNHG12 expression through the modification of H3K4me3 in the SNHG12 promoter region, which in turn recruits the transcription factor E2F1, and ultimately promotes expression of CEP55, and the proliferation, migration, and invasion of RCC cells, as well as the angiogenesis of HUVECs Fig. . These aAdditional file 1: Table S1. shRNA sequences. Table S2. Primer sequences for RT-qPCR. Table S3. Relationship between SNHG12 expression and clinicopathological characteristics of RCC patients. Table S4. The transcription factors involved in the regulation of SNHG12 on CEP55 in RCC as predicted by LncMAP. Table S5. JASPAR analysis of the binding site of the E2F1 in the promoter region of CEP55.


Inhibiting the sPLA2IIa enzyme with an effective molecule can reduce the inflammatory response and halt cancer progression. The present study evaluates quercitrin, a biflavonoid, for sPLA2IIa inhibition and anticancer activity. Quercitrin inhibited sPLA2IIa activity to a greater extent—at 86.24% ± 1.41 with an IC50 value of 8.77 μM ± 0.9. The nature of sPLA2IIa inhibition was evaluated by increasing calcium concentration from 2.5 to 15 µM and substrate from 20 to 120 nM, which did not alter the level of inhibition. Intrinsic fluorescence and far UV-CD studies confirmed the direct interaction of quercitrin with the sPLA2IIa enzyme. This significantly reduced the sPLA2IIa-induced hemolytic activity and mouse paw edema from 97.32% ± 1.23–16.91% ± 2.03 and 172.87% ± 1.9–118.41% ± 2.53, respectively. As an anticancer activity, quercitrin reduced PC-3 cell viability from 98.66% ± 2.51–18.3% ± 1.52 and significantly decreased the IL-6 level in a dose-dependent manner from 98.35% ± 2.2–37.12% ± 2.4. It increased the mean survival time (MST) of EAC-bearing Swiss albino mice from 30 to 35 days. It obeyed Lipinski’s rule of five, suggesting a druggable property. Thus, all the above experimental results were promising and encouraged further investigation into developing quercitrin as a therapeutic drug for both inflammatory diseases and cancers.Human phospholipase A  The inflammatory response mediates cell initiation, promotion, malignant conversion, invasion, and metastasis . Intrins2IIa enzyme is a crucial inflammatory enzyme that catalyzes the hydrolysis of membrane phospholipids into arachidonic acid and lysophosphotidate. The arachidonic acid is metabolized into proinflammatory mediators like prostaglandins, prostacyclins, thromboxanes and leukotrienes; the lysophosphotidate is converted into platelet activating factor (PAF) that continues to cause inflammation , atherosclerosis, cancers, and trauma , CM—Sephadex C-25, quercitrin, genistein, and lipopolysaccharide (#L2018) were sourced from Sigma-Aldrich. Mouse Anti-Human IL-6 (Cat No: 340527) was sourced from BD Biosciences. Six well cell culture plates were obtained from Biolite - Thermo. The PC3 cell line was obtained from the NCCS, Pune. Diphenyl picryl hydrazyl radical (DPPH), cell culture medium , FBS (#RM10432), and D-PBS (#TL1006) were procured from HiMedia Laboratories, Mumbai, India. Ethylenediaminetetraacetic acid (EDTA) and dimethyl sulfoxide (DMSO) were acquired from SRL (Sisco Research Laboratories), India. The solvents and chemicals utilized in the entire investigation were standard laboratory grade.The Institutional Human Ethical Committee of Mangalore University, India, approved the use of human blood (MU/IHEC/2018/1). Blood samples were collected after obtaining consent letters from healthy volunteers by giving prior information about the study.Swiss albino mice (20–25 g) were used in the neutralization of edema and anti-tumor studies. The animals were handled and supervised according to regulations made under Indian laboratory animal welfare legislation. The approval number of the Institutional Animal Ethical Committee is NGSMIPS/IAEC/NOV-2019/166 .2IIa was purified according to the method of The sPLA2IIa (1POE) structure was retrieved from the Protein Data Bank. The 3D structures of phytoconstituents were taken from the PubChem database. The molecular docking study was conducted using Autodock Vina. The drug molecule was flexible during the docking study, while the sPLA2IIa molecule was rigid. The chosen molecule had the lowest binding score with maximum binding affinity. After completing the docking runs, the interactions of different sPLA2IIa residues with inhibitors via hydrogen bonds, hydrophobic interactions, and electrostatic interactions were examined.The PLAThe ADME toxicity prediction was conducted using the ADMETSAR server, which included predictions of absorption, metabolism, distribution, excretion, and toxicity .2IIa enzyme activity was assessed according to the modified method of 2IIa inhibition was carried out with 0–18 µM concentrations of quercitrin. Genistein, a known sPLA2Iia inhibitor proven as an anti-inflammatory molecule .The nature of sPLA2IIa enzyme alone or with quercitrin was measured using the Horiba Jobin Yvon Fluorolog-3 spectrofluorometer. 2 ml of the reaction mixture in a 1 ml path length cuvette consisting of sPLA2IIa (20 μg/ml) with different concentrations of quercitrin (0.02–0.1 µM) used in the study. Fluorescence spectra were recorded between 300 and 380 nm and corrected empirically using the tryptophan standard (The fluorescence intensity of the sPLAstandard .2IIa (30 μg/ml) was recorded with and without an IC50 concentration of quercitrin using a Jasco J-810 spectropolarimeter between 200 and 240 nm. The response time was 2 sec and the bandwidth was 1 nm. A total of 10 scans were made into the final spectrum. The spectrum of the standard reaction mixture was subtracted to correct the spectra.The UV-CD spectra of sPLA2IIa enzyme at 37°C for 30 min and then 1 ml of the substrate was added and incubated at 37°C for 45 min. We then added 9 ml of ice-cold PBS to halt the reaction, which was then centrifuged at 1,500xg for 20 min. The hemolytic activity of the sPLA2IIa enzyme in terms of released hemoglobin was measured at 530 nm.The assay was carried out according to the method of 2IIa (5 µg) alone or with quercitrin (ranging from 0 to 18 µM) in a total volume of 20 µL was injected into the right hind footpad of the mice; 20 µL saline was injected into the respective left hind footpad as normal control. The animals were euthanized after 45 min by anesthesia . The limbs were amputated at the ankle joint and were weighed individually.The method of 2 incubator for 3 h. The media containing excess MTT was removed and formazan crystals were dissolved using 100 µL DMSO. A reading was taken at 570 nm for the purple color formazan solution and at 630 nm to subtract the background reading. The percentage viability of PC3 cells was calculated.The protocol of i.p.) 1×106 EAC cells. Group 1 received only buffers and served as vehicle control, and Group 2 served as disease control; Group 3 animals received quercitrin  and Group 4 served as the positive control. Animals were treated after 24 h of tumor induction and continued for the next 10 days. Experimental animals were observed for clinical signs and mortality. The weight of the animals was recorded and the percentage increase in mean survival time (% MST) was calculated.The anti-tumor activity of the quercitrin was evaluated using the Ehrlich ascites carcinoma (EAC)-bearing mice model . The queThe IL-6 level was measured by following the manufacturer’s instructions  for PE Mn = 3). The IC50 value was calculated by Graph Pad Prism version 9.0  and using one-way ANOVA , followed by Tukey’s test as a single post hoc test to compare the mean obtained from the experiment. The results are all shown with a mean standard error. If the p value is < 0.05, then statistical significance is assumed.The obtained test results were given as the mean standard deviation . AutoDock4 (v4.2.6) was used to analyze the binding pattern and energy values of molecules with human sPLA2IIa (1POE). Among the above molecules, quercitrin showed a greater energy value of −8.31 than standard genistein (−7.96) (5280459) interacts with Ala17, Tyr21, and His47 residues of the sPLA2IIa enzyme (H bond). Similarly, genistein (PubChem ID 5280961) interacts with His47 and Lys62 residues of the sPLA2IIa enzyme  . Quercita enzyme . Any ligin silico ADMET study was carried out to check the pharmacokinetics and pharmacodynamics properties of the inhibitors. The ligand physical descriptors and pharmaceutically essential properties were analyzed using the ADMETSAR server. The parameters used in ADME-Toxicity prediction are based on Lipinski’s rule of five; accordingly, the quercitrin showed 51.083% of human intestinal absorption −1.277 (log BB) of blood-brain barrier (BBB) permeability, whereas standard genistein showed 93.387% of human intestinal absorption −0.71 (log BB) of the blood-brain barrier. Both compounds did not show AMES toxicity. Thus, the compound quercitrin displayed drug-like qualities.The 2IIa inhibition in vitro; it better inhibited the sPLA2IIa enzyme in a concentration-dependent manner  and exhibited drug-like qualities. Hence, quercitrin was further employed for sPLAt manner . It inhi2IIa catalysis either by binding the substrate or chelating calcium ; it showed a constant sPLA2IIa inhibition of 48.79% ± 0.8 over all the ranges of calcium ; it showed a constant inhibition of 47.27% ± 1.0 over all the ranges of substrate concentrations  from 172.87% ± 1.9–118.41% ± 2.53 at 18 μM concentration  rise in body weight up to 46.6 ± 4.0 g compared to the normal control (G1) body weight which was 33.3 ± 2.8g, whereas the quercitrin treated group (G3) showed significantly reduced body weight, (p < 0.05) at 39.1 ± 3.7g, while the standard treated group (G4) showed 34.8 ± 2.8 g. In addition, G2 showed a viability of EAC cells of 56.4% ± 5.9, whereas the quercitrin treated group (G3) decreased to 48.51% ± 8.7; the standard treated group (G4) showed 50.97% ± 10.48 (p < 0.05) against EAC-induced tumors in mice.To better understand the anticancer effect of quercitrin, we established an EAC-induced mouse model. Treatment with quercitrin and 5-fluorouracil (standard) showed protective effects against EAC-induced mortality. Animals treated with quercitrin (G3) showed increased mean survival time (MST) to 35 days compared to disease/toxic control (G2), which showed 30 days, while the standard 5-fluorouracil treatment group (G4) showed a maximum MST of 40 days . The dis ± 10.48 . Along wThe elevated concentrations of IL-6 in the tumor microenvironment regulate the several signaling pathways of cancer, including apoptosis, proliferation, angiogenesis, invasiveness, and metastasis. IL-6 is known to activate numerous signaling cascades such as the JAK-STAT pathway, the p38 MAPK pathway, the ERK pathway, and the PI3-kinase pathway, reflecting the strong correlation between inflammation and cancer . The ele2IIa enzyme in vitro and neutralized sPLA2IIa-induced mouse paw edema, thus proving itself to be an effective anti-inflammatory molecule. Quercitrin significantly reduced the cell viability and inflammatory cytokine IL-6 in the PC3 cell line and increased the mean survival time in the EAC-bearing mice model. It displayed drug-like qualities by ADME toxicity analysis, which is mandatory for ensuring its use as a drug. Thus, quercitrin has shown promising anti-inflammatory and anticancer effects.An effective anti-inflammatory drug with anticancer activity is needed to treat cancers. Quercitrin inhibited inflammatory sPLA