Reportedly, Saro_0803 undergoes resveratrol-mediated dissociation from the nov1 promotor and distinguishes resveratrol from its precursors, p-coumaric acid and trans-cinnamic acid, enabling the transcriptional factor to serve as a biosensor component for regulating resveratrol biosynthesis. However, little is known about the molecular mechanisms underlying the Saro_0803 interactions with either the nov1 promotor gene or resveratrol, which undermines the potential for Saro_0803 to be further modified for improved biosynthetic performance and other applications. Here, we report the discovery of the 22 bp A/T-rich Saro_0803 binding site near the −10 box of the nov1 promotor (named nov1p22bp). As validated by molecular docking-guided mutagenesis and binding affinity assays, the Saro_0803 binding of its target DNA sequence relies on charge-predominating interactions between several typical positively charged residues and nucleic acid. Furthermore, we semi-quantified the influence of resveratrol presence on Saro_0803–nov1p22bp interaction and identified a bilateral hydrophobic pocket within Saro_0803 comprising four aromatic residues that are crucial to maintaining the resveratrol binding capability of the transcriptional factor. Our data are beneficial to understanding saro_0803′s structural and functional properties, and could provide theoretical clues for future adaptations of this transcriptional factor.Saro_0803 is a transcriptional factor modulating the transcription of the stilbene-degrading enzyme gene  Novosphingobium aromaticivorans DSM 12444 and found to be encoded by the gene saro_0803 that is located reversely on the upstream of gene nov1 -labeled Saro_0803, the protein is expressed and purified as described in 15NH4Cl for [15N]-labeling, and prepared in an NMR buffer containing 20 mM MES pH 6.5, and 200 or 600 mM NaCl, 5% (v/v) D2O. One-dimentional [1H] and two-dimensional [1H-15N]-HSQC NMR spectra were acquired with Bruker Avance III spectrometers equipped with cryoprobes, with the proton resonance frequencies of 600 MHz, 700 MHz, and 850 MHz at 298 K. The pulse programs within the standard pulse library  were used for NMR data collection. The NMR data were processed with Bruker Topspin 4.1.3 and NMRPipe -HSQC NMR spectra were obtained using a Bruker Avance III 600 MHz spectrometer at 298 K. The protein Saro_0803 samples were prepared in an NMR buffer containing 20 mM MES pH 6.5, 200 mM NaCl, and 5% (v/v) D2O. The Saro_0803 sample was then temporarily transferred from the NMR tube into the sample tube containing dry DNA powder for equimolar titration. The protein sample of Saro_080321-157C39S was prepared in an NMR buffer containing 20 mM MES pH 6.5, 600 mM NaCl, and 5% (v/v) D2O, and we temporarily transferred the sample from the NMR tube into the sample tube containing resveratrol (CAS No. 501-36-0) for titration. Seven 2D [1H-15N]-HSQC spectra with protein: resveratrol ratios of 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:8, and 1:16 were recorded to observe the composite chemical shift perturbations ΔH-N upon ligand binding [To verify the interaction between Saro_0803 and the core DNA sequence  binding .Single-stranded DNA oligonucleotides were purchased from Tsingke biological technology Co., Ltd.  for in vitro assays. Double-stranded DNAs were prepared by annealing complementary oligonucleotides in buffer  to 95 °C and slowly cooling them to room temperature. Detailed sequences are listed in ® TGX™ Precast Gel  and separated in Tris/Glycine buffer using 100 V/50 min on ice. The gel was soaked in TAE buffer with SYBR® Safe stain , shaken for 15 min for staining, and photographed using the Bio-Rad Molecular Imager® Gel DocTM XRS system .DNA binding was assayed using Electrophoretic Mobility Shift Assays (EMSA) with a constant 5.0 µM DNA concentration and increased protein concentrations. Saro_0803 and the mutants were incubated with DNA at 37 °C for 30 min to allow complex formation. Subsequently, complexes were loaded onto 4–20% Mini-PROTEAN. The titration buffer was made of 50 mM Tris-HCl pH 8.0, 200 mM NaCl; 5% DMSO), with 20 µM protein in the cell, and 200 µM DNA or 2 mM resveratrol in the syringe at 25 °C. Seventeen repeats of 2.2-µL injections were performed at intervals of 120 s. The titration data were analyzed with the software Origin 9.0 and fitted using a one-site binding model.Experiments with expressed Saro_0803 and related mutants were performed using a MicroCal iTC200 instrument 21-157, and DNA nov1p22bp samples at respective 100 µM concentrations were analyzed via SEC as the control. Experimental groups include co-incubated 200 µL protein-DNA samples at 100 µM + 100 µM and 100 µM + 150 µM, respectively. The elution was collected for analysis.Analytical size exclusion chromatography (SEC) was performed utilizing Superdex 200 increase 10/300 GL column  equilibrated with a buffer . Saro_0803, truncated Saro_0803nov1p22bp, the predicted structure of Saro_0803 was retrieved from AlphaFold2  [nov1p22bp fragment was generated via Avogadro 1.2.0 [http://hdock.phys.hust.edu.cn/, accessed on 22 February 2022) [nov1p22bp complexes were ranked by the iterative knowledge-based scoring function ITScore-PP. The complexes with lower energy scores were selected and visualized using PyMOLTM 2.5.4 .Before performing the molecular docking of Saro_0803 with ry 2022) , and thero 1.2.0 . Then, try 2022) ,28. The TM, Nicoyalife, Kitchener, Canada). Once the NTA sensor chip was activated, His-tagged Saro_0803 was captured with the sensor chip to achieve a stable baseline. Diluted resveratrol with different concentrations was loaded to flow slowly over the sensor chip for 240 s, allowing protein–ligand interactions to occur. Lastly, the running buffer was allowed to flow for 360 s to collect the dissociation data. The flow rate of all solutions over the sensor chip was set to 20 µL/min at 25 °C. The binding kinetic parameters were obtained by fitting the signal response vs. time curve to a one-to-one binding model using TraceDrawer  software.The binding kinetics of purified His-tagged Saro_0803 to the resveratrol was measured by surface plasmon resonance , between which the nov1 promoter possesses canonical −10 and −35 boxes [E. coli MarR of up to 37.0%, and the relative conservation of secondary structural elements of MarR families  slightly larger than 15 kDa, consistent with its theoretical MW of around 18.75 kDa  possesses a length of 68 bp and includes an inverted repeat, GCAATA------TATTGC, overlapping with the predicted −10 box  sequential fragments were designed and investigated for the interaction of the purified Saro_0803 and the 58 bp nov1 promoter fragment by an electrophoretic mobility shift assay (EMSA) (nov1 promoter (nov1p22bp). Further, we tested two neighboring DNA fragments against the nov1p22bp sequence via EMSA to feature their Saro_0803 binding abilities  . Roughlyy (EMSA) b. Hence,promoter a. Then, promoter c,d. Thesbilities , where nov1p22bp .nov1 promoter, we first carried out EMSA experiments between nov1p22bp and increased concentrations of Saro_0803. Additionally, the semi-quantitative results showed that 5.0 μM Saro_0803 binds to more than half of the nov1p22bp at an equimolar ratio of nov1p22bp   b. In addov1p22bp c. Althounov1 promoter. As the molecular docking approach is widely applied for studying biologically relevant molecular interactions, we conducted free docking between Saro_0803 and nov1p22bp via HDOCK to characterize their interaction pattern [nov1p22bp, while residues such as R108 and K109 located in the interloop between β1 and β2 are distributed near the minor groove and disposed to the grooves at both sides of nov1p22bp, which is positively charged in the residues and induce specific interactions with its cognate DNA sequence , further strengthening the credibility of our docking model  and K109 mutants (K109A and K109E) were not detectable  to keep Saro_0803 in a compatible environment. We then found the binding affinity of Saro_0803 to resveratrol to be Kd ~79.8 µM by SPR and ~35.7 µM by ITC, indicating micromolar binding  is a stilbenoid and consists of two phenol-hydroxyl-bearing benzene groups linked by a vinylene group ,32,33. Cveratrol e.nov1p22bp (nov1 promotor in the future.We next sought to validate the above binding properties experimentally and, accordingly, applied alanine mutation to the residues F30, F43, F47, and W61 before ITC and EMSA measurements for mutant–resveratrol binding. It was found that compared to the wild-type Saro_0803, the binding affinity for the four mutants to resveratrol significantly decreased or reduced to an undetectable level . Furtherov1p22bp . Taken tSaro_0803, a putative member of the MarR family transcriptional factors, displayed distinctive potential as a valuable tool in synthetic biology. Reported as a gating sensor, Saro_0803 was incorporated into biosynthetic cell units to detect the concentration fluctuation of stilbenoids such as resveratrol and regulate their synthesis via a negative-feedback route . Howevernov1p22bp, which spans the sequence from −35 to −10 boxes of the nov1 promoter, with sequential specificity and affinity at the micromolar level. This result might aid the future explanation of the binding mode variety and the niche-dependent functional discrepancies among the MarR family TFs. In our docking model of a Saro_0803–nov1p22bp complex, the charge interactions between arginines/lysines and DNA backbone phosphate groups played a significant part in the binding interface. Differently, residues R108/R′108 were observed to stick to the nucleoside part of the minor groove. Nevertheless, to validate this speculation, the details of the binding dynamics of the Saro_0803–nov1p22bp complex have been under intensive investigation.In this study, we described a model where the MarR-like TF Saro_0803 binds a 22 bp cognate sequence p-coumaric acid and trans-cinnamic acid as substrates and regulates the expression of the stilbene-degrading enzyme NOV1 [Organisms, typically plants, produce molecules with antibiotic activities to encounter bacterial infection and damage, while certain bacteria co-evolve countermeasures to neutralize or consume the antibiotics. Particularly, Saro_0803 recognizes resveratrol and activates the NOV1 transcription to counteract high levels of resveratrol, which is different from certain MarR family regulators that employ a thiol group-mediated sensor mechanism to regulate bacterial stress responses ,35,36,37


Therefore, it is urgent to discover new potential targets. ERCC6L is an essential protein involved in chromosome separation during cell mitosis. However, the effect of ERCC6L on the tumorigenesis and progression of breast cancer is unclear.Breast cancer (BC) is the leading cause of morbidity and the second leading cause of death among female malignant tumors. Although available drugs have been approved for the corresponding breast cancer subtypes (ER-positive, HER22/M checkpoint signalling pathway. Additionally, we demonstrated that there is an interaction between ERCC6L and KIF4A, both of which are closely related factors in mitosis and are involved in the malignant progression of breast cancer.Here, we found that ERCC6L was highly expressed in breast cancer, especially in TNBC, which was closely related to poor outcomes of patients. An ERCC6L conditional knockout mouse model was first established in this study, and the results confirmed that ERCC6L was required for the development of the mammary gland and the tumorigenesis and progression of mammary gland cancers. In in vitro cell culture, ERCC6L acted as a tumor promoter in the malignant progression of breast cancer cells. Overexpression of ERCC6L promoted cell proliferation, migration and invasion, while knockdown of ERCC6L caused the opposite results. Mechanistically, ERCC6L accelerated the cell cycle by regulating the GWe first demonstrated that ERCC6L deficiency can significantly inhibit the occurrence and development of mammary gland tumors. ERCC6L was found to accelerate the cell cycle by regulating the p53/p21/CDK1/Cyclin B and PLK/CDC25C/CDK1/Cyclin B signalling pathways, thereby promoting the malignant progression of breast cancer cell lines. There was a direct interaction between KIF4A and ERCC6L, and both are closely associated with mitosis and contribute to growth and metastasis of breast tumor. To sum up, our results suggest that ERCC6L may be used as a promising target for the treatment of BC.The online version contains supplementary material available at 10.1186/s13046-023-02806-x. According to the latest cancer statistics released by CA , the incidence rate of breast cancer ranks first among female malignant tumors  and has been on the rise since the mid-2000s . In addition, breast cancer is the second leading cause of cancer death in women  [Cell division is the process of dividing cells into two, which is of great significance for growth, tissue repair and regeneration, and asexual reproduction of single cells. It is a strictly regulated and highly ordered process that includes interphase and M phases, monitored by inherent cycle checkpoints within the cell to ensure the fidelity of cell replication . It was ERCC Exception Repair 6 Like (ERCC6L), belonging to the SWItch/Sucrose Non-Fermentable (SWI/SNF2) protein family, which is also named PLK1-interacting checkpoint helicase (PICH), is a DNA helicase closely related to cell mitosis . It is aSome studies reported that the deletion of ERCC6L could induce chromosomal abnormalities , embryonic lethality, DNA damage, p53 activation, and increased apoptosis , 13. TheIn this study, we first demonstrated the potential function of ERCC6L using an ERCC6L conditional knockout mouse model. Compared with wild-type mice, ERCC6L deletion delayed mammogenesis. By hybridizing ERCC6L conditional knockout mice with MMTV-PyMT mice (a mouse model in which specific expression of polyomavirus middle T antigen (PyMT) driven by the mouse mammary virus MMTV could rapidly facilitate multifocal mammary tumor formation), it was found that ERCC6L deficiency can significantly inhibit the occurrence and development of mammary gland tumors. Mechanistically, ERCC6L could accelerate the cell cycle by regulating the p53/p21/CDK1/Cyclin B and PLK/CDC25C/CDK1/Cyclin B signalling pathways, thereby promoting the malignant progression of breast cancer cell lines. Additionally, we also revealed the potential relationship between KIF4A and ERCC6L, that is, there was a direct interaction between KIF4A and ERCC6L, and both are closely associated with mitosis and contribute to tumor growth and metastasis. Taken together, our results suggest that ERCC6L plays a vital role in the occurrence and development of BC and might be a potential target for BC treatment.2 at 37 °C. The human BC and adjacent normal tissues used in this article were gifted by Dr. Bailin Zhang  and were approved by the Institutional Review Board (IRB).The BC cell lines ZR-75-1, MCF-7, T47D, BT474, HCC1937, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, and SKBR3 were purchased from American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640  with 10% foetal bovine serum (FBS) were used to culture MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, SKBR3 and BT474, HCC1937, T47D, ZR-75-1 cells. The serum was purchased from Procell Life Science & Technology . All cells were cultured in a humidified incubator with 5% COFor the construction of MCF-7 and T47D cell lines stably overexpressing ERCC6L, control and ERCC6L lentiviruses were used to infect both cell lines for 72 h, followed by selection with 3 µg/mL puromycin. For stable silencing of ERCC6L, MDA-MB-231 cells were infected with control, shERCC6L#1 and shERCC6L#2 lentiviruses. Subsequently, they were screened with 3 µg/mL puromycin. For transient overexpression of KIF4A, the human full-length KIF4A cDNA clone was transfected into 231 cell lines using Lipofectamine 3000 reagent in accordance with the manufacturer’s instructions.flox/flox and MMTV-Cre/Line D mice provided by Biocytogen Pharmaceuticals Co., Ltd. Mice were kept in the Laboratory Animal Center, Institute of Materia Medica, Chinese Academy of Medical Sciences under 12 h/d light, 22 ± 3 °C temperature and 50 ± 5% relative humidity. All experimental conditions and experiments performed on animals were in accordance with the guidelines of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences (Ethics number: 00008101).The ERCC6Lflox/wt Cre+/− mice were generated by crossing ERCC6Lflox/flox mice with MMTV-Cre mice. Then, ERCC6Lflox/wt Cre+/− mice were hybridized with ERCC6Lflox/y Cre+/− mice and ERCC6Lwt/y Cre+/− mice to obtain ERCC6Lflox/flox Cre+/− mice and ERCC6Lwt/wt Cre+/− mice, respectively. At 6, 8 and 12 weeks, the fourth groin mammary glands of female mice were weighed, and a whole mount assay was performed.The flox-Cre recombination system was applied to generate mice with mammary epithelial cell-specific ERCC6L deletion. ERCC6LTo generate PyMT mice, we crossbred female ERCC6L mice of different genotypes with MMTV-PyMT mice. At 8 weeks, whole mount staining was performed on the fourth groin mammary gland of female mice to evaluate multifocal dysplasia. To assess palpable tumor onset, animals were palpated weekly. After the formation of palpable tumors, the body weight of mice was recorded, and tumor volume was measured using Vernier callipers within 42 days. At 16 weeks, the mice were dissected, and their body weight, the number of tumors per mouse, and the wet weight of each tumor were recorded. Tumor tissues, lungs, and spleens were soaked in 4% paraformaldehyde (PFC) for immunohistochemistry (IHC) and immunofluorescence (IF). The genotypes of all female mice were identified by specific PCR analysis of mouse tail genomic DNA. Primer information is listed in Supplementary Table 1.7/0.1 mL). After the tumor had grown (less than 1000 mm3), each mouse was dissected to measure the volume of the tumor. The calculation formula is V = 0.5xLxW2 mm3 .Female BALB/c-nu nude mice, purchased from Beijing Vital River Laboratory Animal Technology, were divided into three groups (n = 6) and separately injected with MDA-MB-231 shNC, MDA-MB-231 shERCC6L#1, and MDA-MB-231 shERCC6L#2 cells  for 1–2 days. They were then decolorized with 70% ethanol containing 2% hydrochloric acid for 2 h, immersed in gradient concentrations of ethanol (70-95%-100%) for 15 min, defatted with xylene for 20 min, and finally stored in methyl salicylate.The fourth inguinal mammary gland was removed, spread on a glass slide, and air-dried for 5–10 min. Fixation was performed with Carnoy’s solution  for 2–4 h. The slides were then immersed in gradient concentrations of ethanol (75%-50%-30%) for 15 min. After immersion in ddH24/48/72 h/96 h-OD0h)/OD0h. In terms of migration and invasion assays, the cells were counted and inoculated in 8 μm polycarbonate membranes in Costar 24-Transwell plates. Then, after being cultured for 3–4 h (after cell attachment), serum-free medium and 10% FBS medium were added to the upper and bottom chambers, respectively. After 19 h (for migration) and 22 h (for invasion), cells were fixed with 4% paraformaldehyde (PFA) and stained with 1% crystal violet. Subsequently, after cells in the upper chamber were wiped off, the chambers were placed under a microscope for observation. Note that in the invasion assay, the upper chambers were coated with Matrigel™ diluted in serum-free medium (1:7) before seeding the cells.Cell Counting Kit-8 (CCK-8) assays were carried out to check cell proliferation. The cells were first seeded in 96-well plates and then incubated in incubators for 0, 24, 48, 72 and 96 h. Finally, the plates were placed in SpectraMax M5, and the absorbance value of each well was measured at 450 nm for quantification. Relative cell proliferation rate (100%) =  and 1:10 (0.35% agar) and successively spread in 6-well plates to form the bottom and upper layers. The upper layer contained prepared cells (approximately 3000/well). Before being placed into the incubator, plates were first placed in a 4 °C refrigerator for 30 min. After being plated in an incubator for 2–3 weeks, cells were stained with MTT solution (5 mg/mL) and then counted. For the 3D Matrigel assay, the cell suspension and Matrigel were mixed in a 1:1 ratio and inoculated in 96-well plates. After two weeks, clones were observed and photographed.Cells were plated in 96-well plates. EdU reagent (5-Ethynyl-2’-deoxyuridine) (1:1000 dilution) was added to plates and incubated for 2 h. The cells were then fixed with 4% PFA for 30 min, washed with 2 mg/mL glycine solution and PBS 2–3 times, and then permeabilized with PBS with 0.5% Triton X. After permeabilization, the prepared 1x Apollo staining solution and Hoechst 33,342 staining solution were added successively, and the cells were incubated in the dark at room temperature for 30 min. Finally, the cells were observed under a microscope.The cells were seeded in 60-mm medium dishes. After 48 h, cells were collected, washed with PBS, and fixed overnight with 70% ethanol at 4 °C. Then, the cells were washed again with PBS and incubated successively in PBS with RNase (20 µg/mL) and PBS with 50 µg/mL PI and 0.5% Triton X for 30 min at room temperature in the dark. Finally, the cell suspension was filtered through a 300-mesh screen, and its fluorescence was detected using Accuri C6. FlowJo v10 was used for data processing.2O2 and Triton X) for 30 min to reduce endogenous peroxidase activity. Subsequently, the sections were subjected to antigen repair and blocked using sodium citrate buffer (pH 6.0) and serum. ERCC6L, KIF4A, Ki67, F4/80, and CD31 (1:200 dilution) were added to the tissue sections for incubation at 4 °C overnight. The next day, sections were washed and incubated with secondary antibodies for 1–2 h at 37 °C. Diaminobenzidine (DAB), Mayer’s Haematoxylin Stain Solution and neutral balata were subsequently applied to stain, counterstain (negative controls were treated with rabbit serum) and seal slides. Finally, the stained sections were photographed under a Leica TCS SP8X confocal microscope. The staining density and positive rate of protein were analyzed by Image J (V1.8.0).Human BC tissue samples and animal tissues were prefixed and made into paraffin sections. After roasting, dewaxing, and hydration, the sections were infiltrated with preheated blocking permeabilizing solution  and ERCC6L (1:100) antibodies were added to the bottom of the dish, and the cells were incubated overnight at 4 ℃. Subsequently, the cells were stained with the corresponding fluorescent secondary antibodies for 1 h at room temperature (Alexa 488 for ERCC6L and Alexa 574 for KIF4A). Finally, the cells were further stained with DAPI for 30 min. A confocal microscope was used to acquire images.Total proteins in human BC tissue samples, animal tissues and cells were extracted by RIPA lysis buffer with protease and phosphatase inhibitors  and subsequently quantified by the BCA method. For immunoblotting, equal amounts of proteins were electrophoresed on 8-10% SDS‒PAGE gels and subsequently transferred to PVDF membranes. After blocked the membranes with 5% BSA or 5% defatted milk for 1 h at room temperature, the membranes were incubated with primary antibody overnight at 4 °C. After being washed with TBST, membranes were incubated with commensurable secondary antibodies. Finally, the membrane was cleaned again with TBST and then exposed in Tanon 5200  by dropping ECL regents on it. Data were analyzed by Image J V1.8.0. The antibodies used are shown in Supplementary Table 1.Cells seeded into 100 mm dishes were lysed using IP lysis buffer and centrifuged to obtain the supernatant. Part of the supernatant could be directly applied for Western blotting after denaturation. For Co-IP, the supernatant was mixed with protein A/G agarose beads and anti-DDK beads and then rotated overnight at 4 °C. Subsequently, the beads were washed, mixed with 2x SDS‒PAGE loading buffer, and heated at 95 °C for 5 min for Western blotting analysis.P values were used to evaluate the significance of the differences, which were calculated by unpaired two-tailed Student’s t test or one-/two-way ANOVA. There were statistically significant differences when P values < 0.05.The mean ± standard deviation (SD) was used to present the results. in silico assays were performed using the UALCAN , Oncomine (www.oncomine.org), and GEPIA (http://gepia.cancer-pku.cn/) databases. The results showed that ERCC6L was highly expressed in BC regardless of the BC subtype compared with the normal controls  and found that most BC cell lines had high expression levels of ERCC6L. It is worth mentioning that triple-negative BC cell lines had an overall higher expression level of ERCC6L  . Control cells infected with scramble shRNA lentiviruses were named 231 shNC. BC cell lines MCF-7 and T47D stably overexpressing ERCC6L protein were constructed by infection with pHBLV-ERCC6L lentivirus . Control cells were designated MCF7 NC and T47D NC. The effects of ERCC6L knockdown and overexpression were determined by Western blotting , ERCC6Lfl/wt Cre+/− (ERCC6L+/−) and ERCC6Lwt/wt Cre+/− (ERCC6L+/+)  Fig. B. The de/+) Fig. b.+/+ mice were both higher than those of ERCC6L+/− and ERCC6L−/− mice aged 6 and 8 weeks. Furthermore, the numbers of TEBs and branches in 6- and 8-week-old ERCC6L+/− mice were also significantly greater than those in ERCC6L−/− mice. However, there was no difference in the mammary gland organ index among the three genotypes of mice , having a high ability of division and proliferation, are composed of cap cells, myoepithelial cells and luminal epithelial cells and are closely related to mammogenesis. When the mammary gland enters a period of rapid development, the duct can extend towards the mammary fat pad under the guidance of the TEB and then form secondary and tertiary branches . Therefoice Fig. I, which +/+ mice aged 6 and 8 weeks penetrated the lymph node (LN). However, at 6 weeks, the TEBs of most ERCC6L+/− mice were located near the LN, while those of most ERCC6L−/− mice remained at the initiation site of the ductal and even approached the LN at 8 weeks. Moreover, regardless of whether at 6 or 8 weeks, mammary fat pads of ERCC6L+/+ mice had longer ductal outgrowth than ERCC6L+/− and ERCC6L−/− mice  of 78.5 days (n = 19) and 68.5 days (n = 10), respectively, which were significantly higher than those of ERCC6L+/+ PyMT mice   Fig. b.+/+ PyMT mice was significantly faster than that in mice with the ERCC6L deletion, and there was also a statistically significant difference in the tumor growth rate between ERCC6L+/− PyMT (P = 0.002) and ERCC6L−/− PyMT mice (P = 0.0016) without a significant change in body weight , tumor burden, and individual tumor weight (the largest tumor weight) per mouse were counted. Compared with that in the ERCC6Loup Fig. E. As shoies Fig. G-I. Howeies Fig. J. All th+/+ PyMT group  , whose members are involved in the transport of membranous organelles, protein complexes, mRNA and other substances . Further80) Fig. a and was80) Fig. b. Interf80) Fig. c-d. ThesNonetheless, the role of ERCC6L and KIF4A in tumor progression remains unclear. To determine whether there is an interaction between KIF4A and ERCC6L, immunofluorescence assays and Co-IP were carried out. First, the confocal immunofluorescence assay confirmed the colocalization of KIF4A and ERCC6L Fig. e. Then, To further validate the regulatory role of KIF4A on ERCC6L and whether KIF4A exerts its effect on cell functions and downstream pathways through ERCC6L, we transfected the KIF4A plasmid into 231 shERCC6L and 231 shNC cells. The expression levels of KIF4A and ERCC6L in 231 cells are shown in Fig. BC, which is highly heterogeneous and can be divided into numerous subtypes, has received much attention due to its high incidence in women. Drug reactivity and patient prognosis also vary according to BC subtypes. However, there is still no cure for advanced metastatic breast cancer and TNBC. Thus, discovering new potential targets is of great significance for the development of targeted drugs and prognosis improvement of patients. In this study, ERCC6L was found to be highly expressed in BC patients, and its expression level was positively correlated with the malignant grade of the tumor and the poor prognosis of patients, which to some extent suggested that ERCC6L played an essential role in the malignant progression of BC. Moreover, a higher expression level of ERCC6L was discovered in TNBC patient tissues and cell lines than in the tissues and cell lines of patients with other subtypes, indicating the potential value of ERCC6L in TNBC. Cell experiments in vitro were also performed and found that ERCC6L could facilitate the malignant progression of BC cells by promoting cell proliferation, migration and invasion. Knocking down ERCC6L significantly inhibited tumor growth in nude mice. In summary, using both in vitro cell culture and in vivo nude mouse models, we demonstrated the important role of ERCC6L in breast tumorigenesis. Taken together, our results indicated that ERCC6L was a novel, potential target that urgently needed to be explored.−/− mice, as indicated by increased infiltration of T cells, especially macrophages, in splenic tissues. It is possible that ERCC6L deletion mediated the generation of cells with a complex karyotype (aneuploid), which was reported to induce self-elimination by the immune system [A few studies have indicated that the knockdown of ERCC6L could induce cell cycle arrest, chromosome instability, and cell death in breast cancer cell lines. However, to the best of our knowledge, there is currently no in vivo study that directly examined the role of ERCC6L in breast tumorigenesis using ERCC6L conditional knockout mice. In our study, we explored the effects of ERCC6L on mammary gland development using an ERCC6L conditional knockout mouse model for the first time. Our results first showed that ERCC6L deletion significantly reduced the number of TEBs and ductal branches and significantly inhibited the extension of mammary branch ducts into the fat pad in both 6-week-old and 8-week-old mice, which suggested mammary dysplasia. However, mammary gland development in 12-week-old mice was not affected by ERCC6L deletion, and it is speculated that the effect on mouse self-growth and hormone levels exceeded the regulatory effect of ERCC6L on development. Interestingly, splenomegaly was found in 8-week-old ERCC6Le system . However−/− and ERCC6L+/− mice did not develop mammary gland tumors, which suggested that ERCC6L knockout does not induce tumorigenesis alone and might not be a breast tumor susceptibility gene such as BRCA1/2. However, when crossed with MMTV-PyMT mice, ERCC6L knockout significantly suppressed mammary tumorigenesis. It is possible that, because PyMT itself could induce breast tumorigenesis, the loss of ERCC6L caused PyMT-mediated mammary gland tumor cells to undergo G2/M phase checkpoint disorder. Therefore, they could not repair the damaged DNA and directly enter mitosis, resulting in “mitotic catastrophe”, which caused aneuploidy, micronuclei, etc., and eventually led to tumor cell death. This was consistent with the results verified by Huang et al. [Notably, ERCC6L, a key mitotic checkpoint-associated protein, is theorized to induce increased chromosome mis-segregation and chromosomal instability (CIN) when absent, which can contribute to tumorigenesis . Howeverg et al.  in breasLung metastasis has been observed in a high proportion of MMTV-PyMT mice . TherefoAnother interesting finding in our study was that there was a direct interaction between ERCC6L and KIF4A. However, it remains unclear what consequences could be mediated by the interaction between ERCC6L and KIF4A. Moreover, ERCC6L and KIF4A may synergistically promote breast cancer cell proliferation, migration and invasion. However, how KIF4A regulates ERCC6L levels remains unknown. It is worth mentioning that Mazumdar et al.  found thMechanistically, ERCC6L has been reported to act as a binding partner and substrate of polo-like kinases (PLKs), including PLK1, which is involved in centromeric chromatin remodelling , 34. PLKIn summary, we first demonstrated the role of ERCC6L in breast tumorigenesis and development in vivo using the ERCC6L conditional knockout mouse model, that is, ERCC6L is required for mammary gland tumorigenesis induced by MMTV-PyMT. In addition, ERCC6L, as a tumor promoter, could promote the proliferation, migration and invasion of breast cancer cells and accelerate cell cycle progression by activating the PLK/CDC25C/CDK1/Cyclin B and p53/p21/CDK1/Cyclin B signalling pathways. It is worth mentioning that ERCC6L was also demonstrated to have a close relationship with KIF4A, including interactions and regulatory relationships, which might be correlated with the mitotic process. Overall, targeting ERCC6L could be a promising strategy for the treatment of BC.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8


Clostridium perfringens epsilon toxin (ETX) is the third most lethal bacterial toxin and has been suggested to be an environmental trigger of multiple sclerosis, an immune-mediated disease of the human central nervous system. However, ETX cytotoxicity on primary human cells has not been investigated. In this article, we demonstrate that ETX preferentially binds to and kills human lymphocytes expressing increased levels of the myelin and lymphocyte protein MAL. Using flow cytometry, ETX binding was determined to be time and dose dependent and was highest for CD4+ cells, followed by CD8+ and then CD19+ cells. Similar results were seen with ETX-induced cytotoxicity. To determine if ETX preference for CD4+ cells was related to MAL expression, MAL gene expression was determined by RT-qPCR. CD4+ cells had the highest amount of Mal gene expression followed by CD8+ and CD19+ cells. These data indicate that primary human cells are susceptible to ETX and support the hypothesis that MAL is a main receptor for ETX. Interestingly, ETX bindings to human lymphocytes suggest that ETX may influence immune response in multiple sclerosis. Clostridium perfringens (C. perfringens) toxinotypes B and D and is the third most lethal bacterial toxin known to date. Recently, ETX has been identified as a possible environmental cause of multiple sclerosis (MS) in humans, an immune-mediated, demyelinating disease of the central nervous system (CNS) [C. perfringens toxinotypes versus healthy controls (HC), 61% versus 13%, respectively [Epsilon toxin (ETX) is produced by em (CNS) ,2,3. Ourectively ,5, demyeectively , on CNS ectively ,9 (murinectively ). Importectively ,12. HoweETX is secreted as a relatively inactive 33 kDa protoxin (pETX), and proteolytic cleavage at the N and C terminal by trypsin and chymotrypsin  results in 1000 fold increase in ETX’s toxicity . CytotoxAlthough a handful of proteins have been identified as possible receptors of co-receptors for ETX ,31,32, MMAL is a proteolipid, tetraspan protein with two extracellular loops. MAL cDNA was first cloned from human T cell lines and is indicated to play an important role in T cell differentiation, membrane protein sorting, and formation of immunological synapses ,36,37,38In human lymphocytes, MAL expression has been reported to be confined to the T cell lineage; expression of MAL in healthy B cell lineages is less clear ,57,58,59Mal gene expression in the T cell lineage, real-time quantitative PCR (RT-qPCR) for human Mal was performed on isolated CD4+, CD8+, and B cells  for 2 h, and binding to CD4+, CD8+, and CD19+ cells evaluated by multicolor flow cytometry  inclusion via flow cytometry A,B. CellMal were more susceptible to ETX-induced cytotoxicity, cell death was evaluated in CD4+, CD8+ and CD19+ cells by flow cytometry  signaling and possibly antigen trafficking . MAL may+ and Cl− [+ and Ca2+ [It is generally accepted that ETX causes cell death via the formation/oligomerization of a heptameric pore. ETX pore formation occurs in three sequential steps: (1) ETX binding to its receptor, (2) oligomerization of the pre-pore complex on the cell surface, and (3) the pore insertion into the cell membrane . Pore fo and Cl− ,86. Thisand Ca2+ ,87. ETX In our current experiments, the ETX treatment of PBMNCs resulted in ETX oligomerization/pore formation, as detected by Western blot. However, the majority of the ETX detected in the PBMNC lysates was observed as bound monomers, not in the pore complex. It is interesting to note that 90% of CD4+ cells were positive when PBMNCs were probed with 50 nM of pETX-647 for 2 h. However, when cells were treated with 50 nM of active ETX for 4 h, only 35% of the CD4+ cells died, indicating a large discrepancy in ETX binding versus cell death in CD4+ cells for this dose and time point. Alternatively, in rMAL-CHO cells, a highly ETX-susceptible cell line, we saw a closer correlation of ETX binding and ETX cytotoxicity . When rMBased on the limited amount of ETX-induced cell-death observed in human lymphocytes, despite a significantly higher degree of ETX binding, we postulate that ETX binding to lymphocytes may influence other cellular behaviors in addition to cell death, including various immune functions. This raises the interesting hypothesis that ETX bindings to MAL on human lymphocytes at sublethal doses may modify immune function, possibly influencing lymphocyte activity in immune-mediated diseases such as MS. CD4+, CD8+, and B cells have all been implicated in MS pathogenesis, although the exact mechanisms by which they influence MS pathogenesis is still unclear ,91,92,93In conclusion, we have demonstrated that ETX binding and cytotoxicity to human lymphocytes positively associates with MAL gene expression, supporting the hypothesis that MAL is the main receptor for ETX.Peripheral blood samples were collected from healthy controls via the cubital vein using BD Vacutainer K2 EDTA 7.2 mg Blood Collection tubes in accordance with our Institutional Review Board, protocol number 1003010940. At the time of donation, healthy controls were free of any chronic or acute disease, were both male and female, ranged in age from 18 to 59 years old, and lived in the New York City metropolitan area.Peripheral blood samples were collected from healthy controls via the cubital vein using BD Vacutainer K2 EDTA 7.2 mg Blood Collection tubes. Subsets were isolated using RossetteSep immunodensity negative selection cocktails . CD4+, CD8+, and B cells were isolated using RosetteSep™ Human CD4+ T Cell Enrichment Cocktail, RosetteSep™ Human CD8+ T Cell Enrichment Cocktail, and RosetteSep™ Human B Cell Enrichment Cocktail, respectively, per manufacturer’s instructions.TM SYBRTM Green Master Mix . Briefly, PCR was carried out in a 10 μL volume in a final concentration of 1X SYBRTM Green Master Mix containing 300 nM forward and reverse primers and 10 ng cDNA. The primer sequences were as follows: human MAL, F 5′-GGGTGATGTTCGTGTCTGTG-3′, R 5′-ACTGAGGCGCTGAGGTAAAA-3′; human b-actin, F 5′-CACCAACTGGGACGACAT-3′, R 5′-ACAGCCTGGATAGCAACG-3′. The PCR reaction steps were as follows: 50 °C for 2 min, 95 °C for 2 min, and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. A subsequent dissociation curve measurement from 60 °C to 95 °C was carried out. All samples were run in triplicate. PCR data were analyzed using the 7900 SDS v2.4.1 software . Relative gene expression was quantified by performing double delta Ct analysis (2−ΔΔCt). B-actin Ct values were used as internal controls, and the gene of interest (GOI) expression was normalized to CD4+ cell expression.Comparative RT-qPCR was performed in an ABI Taqman 7900HT Fast Real-Time PCR machine , using the PowerUppETX was provided by BEI . pETX was labeled with Alexa Fluor 647 Protein Labeling Kit  per manufacturer’s instructions. Labeled toxin was stored in a 50% glycerol stock (10 uM) at −20 °C until use.pETX provided by BEI was activated in house using immobilized trypsin, TPCK Treated, agarose resin . Briefly, 125 μL resin was washed three times in sodium phosphate buffer pH 7.98). Resin was suspended in 200 μL sodium phosphate buffer and combined with 500 uL of BEI pETX (0.5 mg/mL) for two hours at 37 °C with gentle agitation. The solution was centrifuged at 18,000 rcf for 10 min, and the supernatant containing the activated ETX was collected. ETX activation was confirmed by the treatment of rMAL-CHO cells [.98. Resi6 cells/mL. Cells were kept on ice until use.Blood samples were collected from healthy controls via the cubital vein using BD Vacutainer K2 EDTA 7.2 mg Blood Collection tubes. Samples reached room temperature and were then diluted with an equal volume of Phosphate Buffered Saline (PBS) +2% Fetal Bovine Serum (FBS). Diluted blood was layered on top of Ficoll-Paque PLUS . Tubes were centrifuged at 1200 rcf for 20 min at room temperature (without brakes). Buffy coat containing peripheral blood mononuclear cells (PBMNCs) was collected using a sterile transfer pipet. Buffy coat was washed with 20 mL of PBS + 2% FBS and centrifuged at 250 rcf for 10 min at 4 °C. The supernatant containing platelets was removed by aspiration. Cell pellet was washed with 20 mL of PBS + 2% FBS and centrifuged at 500 rcf for 10 min at 4 °C. The supernatant was aspirated, and cells were re-suspended in CTS™ OpTmizer™ T Cell Expansion Media  supplemented with Glutamax and 5% FBS. Cells were enumerated using a hemocytometer and adjusted to 1.5 × 106 cells/mL) were incubated with pETX-647 at 0 nM, 1 nM, 5 nM, 10 nM, 25 nM, and 50 nM for 15, 30, 60, and 120 min at 37 °C. In select experiments, media containing 50 nM pETX-647 was pretreated with or without an anti-ETX antibody (JL004) [To determine ETX binding, PBMNC (1.5 × 10 (JL004)  for 30 m6 cells/mL) were treated with activated ETX at 0 nM, 1 nM, 5 nM, 10 nM, 25 nM, and 50 nM for 30, 60, 120 and 240 min at 37 °C. In select experiments, media containing 50 nM ETX was pretreated with or without an anti-ETX antibody (JL008) [To determine ETX-induced cytotoxicity, PBMNC (1.5 × 10 (JL008)  for 30 m (JL008) . Cells w6 cells/mL) were incubated with activated ETX at 0 nM, 10 nM, 25 nM, and 50 nM for 120 min at 37 °C. Alternatively, cells were treated with 50 nM of ETX for 30, 60, or 120 min. As a positive control for pore formation, rMAL-CHO cells treated with or without 50 nM active ETX for 30 min were used as controls [PBMNC (1.5 × 10controls . After tOne-way ANOVA with post hoc Tukey HSD test was used to determine significance when comparing three or more data points. Unpaired Student’s t-tests were used to determine significance when comparing only two data points. These instances are indicated in figure legends.


Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-β (TGF-β)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-β. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-β-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies. Curcumin (Cur) is a natural polyphenolic pigment that is extracted from turmeric with a broad safety profile . This ph4-induced liver fibrosis mouse model by the direct inhibition of fibrosis-mediating proteins  have a great impact on future medical treatments for many diseases. Compared to conventional therapies, drug-loaded NPs possess several advantages, including a prolonged circulation half-life and the elimination of side effects ,6. Over nd TGF-β . AlthougTGF-β is a cytokine that modulates several cellular processes, including proliferation, migration, invasion, angiogenesis, and immune responses . During The main purpose of this work was to prepare and characterize a water stable, including the freely dispersible nanoformulation of curcumin, and to study the proteome modulation that it induces in a well-established model of in vitro TGF-β-stressed cholangiocytes .v/v (CurNPa), 1:158 v/v (CurNPb), 1:316 v/v (CurNPc), 1:625 v/v (CurNPd), and 1:1280 v/v (CurNPe). In all cases, these distributions were monodispersed with a relatively narrow distribution. The size and dispersion  obtained for the three preparations were 158 nm ± 3 nm (CurNPa), 120 nm ± 1 nm (CurNPb), 97 nm ± 2 nm (CurNPc), 95 nm ± 1 nm (CurNPd), and 97 nm ± 9 nm (CurNPe), indicating a slow, yet noticeably decreasing trend in CurNPs diameter with an increasing water/DMSO ratio. CurNPs were stabilized at approximately 97 nm for any water/DMSO ratio greater than 316:1, while larger sizes were obtained at lower ratios. The size distributions for the synthesized nanoparticles are reported in First, we sought to set up an automated, robust method for the reproducible preparation of CurNPs. To this end, we adopted a nanoprecipitation approach using DMSO-dissolved curcumin and water as a non-solvent. A microfluidic-based protocol was implemented to allow for the preparation of CurNPs with a reproducible, uniform size and with the possibility to scale up the process. Different volume ratios between DMSO and water were tested, leading to CurNPs preparations 1:79 b, CurNPc, and CurNPd. In fact, CurNPe was too diluted to be of practical use, while CurNPa was too polydisperse.With the aim of using smaller, homogeneous, and highly concentrated CurNPs in subsequent experiments, attention was focused on CurNPThe solutions of curcumin in DMSO maintained the same absorbance spectra over at least 48 h, while fluorescence decreased over time, reaching about 70% of the initial value in 48 h . Curcumib, CurNPc, and CurNPd in PBS was evaluated, and an “equivalent concentration” [µM] of curcumin was calculated from the standard curve of Curcumin in DMSO. CurNPd (1:615) preparation was 13.1 µM, CurNPc (1:316) was 55.6 µM and CurNPb (1:158) 187.4 µM. CurNPb was used as a stock solution to obtain the desired amount of CurNPs for the experiment with cells.CurNPs were produced instead of a more stable dispersion without any sign of precipitation over the hours. In order to exactly quantify the number of CurNPs per volume unit of the stock solution for each preparation, the absorbance of serial dilutions of CurNPWith the aim of assessing the behavior of CurNPs in the culture medium, the optical properties of their dispersions in the culture medium, with different amounts of the fetal bovine serum (FBS), were evaluated over time. In all the conditions tested, a reduction in the fluorescence emission was to be less than 40%; this is slightly higher than the reduction in fluorescence of curcumin in DMSO (30%) in the same interval . The admPrimary human cholangiocytes were treated for 48h with TGF-β at a concentration of 10 ng/mL, in accordance with our previous study . AnotherThe anti-inflammatory effect exerted by curcumin in multiple pathologic conditions, including liver diseases, is well known ,20. HencHuman cholangiocytes were subjected to protein extraction, reduction, alkylation, and digestion with trypsin, and the resulting peptide mixture was prepared for LC-MS/MS analysis, thus quantifying 2479 proteins. To obtain an overview of biological significance, these proteins were categorized according to their biological function using a David bioinformatic database. As reported in To identify the dysregulated proteins during TGF-β and TGF-β/CurNPs treatments, a comparative analysis with the control cells and TGF-β-treated cholangiocytes was performed, respectively.In TGF-β-treated cholangiocytes, a total of 28 proteins were significantly dysregulated with respect to the control cells greater than ±2.5-fold. In particular, data analysis showed 24 upregulated and 4 downregulated proteins following 48 h of treatment. Among them, fibronectin and dynamin-3 were the most deregulated proteins, with a fold-change (FC) of 295.85 and 0.01, respectively .The data analysis of TGF-β/CurNPs-treated cholangiocytes highlighted 10 significantly deregulated proteins compared to TGF-β-treated (48 h) cells. In particular, we showed eight upregulated and two downregulated proteins . Among tIn order to understand if some deregulated proteins in TGF-β-treated cells were also modulated by curNPs treatment, a comparative analysis was performed. Surprisingly, only three proteins  were shown to be common between the treatments . As showNutraceuticals play an important role in the management of multiple pathological conditions . Among tIn the present study, we synthesized, characterized, and identified a phototherapy formulation that was CurNPs-based . FollowiIn addition, TGF-β induced a more than 290-fold increase in the fibronectin level compared to the control cells, which could be related to the well-documented pro-fibrotic properties of this cytokine . GloballIn our proteomic analysis, we also identified cornifin-A as a marked down-regulated protein FC = 0.25) compared to the control cells. Cornifin-A is part of a small proline-rich protein (SPRR) and was recently identified in the biliary epithelium. Demetris and colleagues reported that SPRR2A overexpression within in vitro biliary epithelial cells increased resistance to oxidative injury and promoted wound restitution by enhancing the migration and acquisition of mesenchymal characteristics . Similar comparedThe global proteome analysis of TGF-β/CurNPs-treated cholangiocytes highlighted interesting information regarding curNPs activity. As reported in CurNPs induced the up-regulation of histone H1.2 (FC = 2.94), which plays several important functions in multiple cellular processes, including apoptosis, autophagy, senescence, cell cycle control, and gene transcription, regulating chromatin dynamics ,27. Acco2+ homeostasis in human cholangiocytes under TGF-β treatment through the downregulation of multiple associated proteins such as calumin, calcium/calmodulin-dependent protein kinase type II subunit delta, caveolin-1, and NipSnap1 [In our previous work, we reported the impairment of CaNipSnap1 . Human cNipSnap1 ,29,30,31NipSnap1 . The kinNipSnap1 ; howeverNipSnap1 . In thatNipSnap1 . AlthougNipSnap1 . In TGF-NipSnap1 . RecentlNipSnap1 . MoreoveConsidering the well-documented anti-inflammatory properties of curcumin in liver diseases , in the In conclusion, we highlighted several aspects of curNPs activity on human cholangiocytes whose functions were perturbed during TGF-β stress, as reported in In particular, we confirmed the anti-inflammatory properties of this nutraceutic and the induction of programmed cell death to counteract the cellular damages induced by TGF-β. In human cholangiocytes, this cytokine is also responsible for the fibrotic processes, and CurNPs could attenuate this condition by reverting the expression of key mediators. In our study, TGF-β seemed to perturbate the cholangiocyte cilia functions and endocytic processes that could be restored by CurNPs. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies , our res®, Carlsbad, CA, USA), Dulbecco’s Modified Eagle Medium , Penicillin-Streptomycin , Trypsin-EDTA , Sirius Red/Fast Green Staining Collagen , fertilized chicken eggs , distilled water , ethyl acetate , ascorbic acid, PBS pH 7.4, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich . TGF-β (T7039) was purchased from Merck. Formic acid (5.33002), trifluoroacetic acid (80457), and ammonium bicarbonate , all eluent additives for LC-MS, were purchased, respectively, from Merck , Sigma-Aldrich  and Fluka Analytical . Acetonitrile (1.00029) hypergrade solvent LiChrosolv for LC-MS was bought from Merck , while sodium deoxycholate (D6750) and albumin from bovine serum (A7906) from Sigma-Aldrich . Reagent A (23228) and reagent B (1859078) for Pierce BCA (bicinchoninic acid) Protein Assay were purchased from Thermo Scientific . Iodoacetamide (RPN6302V), dithiothreitol (D1 309.0010), and trypsin modified sequencing grade (11418033001) were obtained, respectively, from GE Healthcare , Duchefa Biochemie  and Roche . Mobicol spin columns (M1003) and their filters (M2110) 10 µm pore size were purchased from Mo Bi Tec , while VersaFlash spherical C18 bonded flash silica from Supelco Analytical . Milli-Q deionized water was filtered on a Millipak filter  and purified on an LC-Pak cartridge ; all Millipore, Bedford, MA, USA). Beta-galactosidase digested (4333606) was obtained from AB Sciex .Primary human cholangiocytes  and their specific growth medium without serum were purchased from Celprogen . Curcumin , dimethyl sulfoxide , Ethanol absolute , fetal bovine serum . Briefly, a Cur solution in DMSO (10 mM) was mixed with deionized water at 1:158, 1:316, and 1:625 volume ratios using a 250 μL microfluidic mixer chip. The total flow rate was maintained at 50 μL/min, and the temperature was controlled at 25 °C. An eluted suspension was collected and stored at 4 °C until analysis. DLS analyses were performed on a Malvern Zetasizer ZS90 dynamic light scattering, using default settings. Nanoparticles were stable for more than 80 days without appreciable signs of degradation, as indicated by the DLS analyses performed on aged samples.The concentration and the stability of CurNPs, once added to the PBS or culture medium (with different percentages of FBS), were evaluated by UV-VIS spectrophotometric and spectrofluorimetric analysis performed on a Tecan Infinite M200 PRO plate reader, using Corning® 96 Well Clear Flat Bottom UV-Transparent Microplates and 200 μL of each solution.2 for 24 h. The culture medium was then replaced with CurNPs stock dispersion diluted in a medium supplemented with 1% FBS to achieve a final concentration of 50 µM, 35 µM, 20 µM, 10 µM, 5 µM and 1 µM. The cells were treated for 24 h and 48 h. Finally, 10 μL of the CCK-8 reagent  was added into each well, and OD at 450 nm was measured using a microplate reader  after 2 h incubation at 37 °C.In total, 2000 human cholangiocytes per well were seeded in 96-well cell culture plates and incubated in a humidified atmosphere with 5% COg for 10 min at 4 °C. The bicinchoninic acid assay was used to determine the protein concentration by using serum albumin as a standard. For each sample, 100 µg of protein was reduced with dithiothreitol  and alkylated using iodoacetamide . Protein digestion was performed using trypsin (w/w ratio 1:50) at 37 °C for 18 h. The samples were incubated with 10% trifluoroacetic acid for 10 min at 37 °C to quench the trypsin reaction and remove sodium deoxycholate by acid precipitation. The samples were centrifuged at 16,000× g for 10 min and subsequently desalted with Mobicol spin columns equipped with 10 µm pore size filters and filled with a C18 stationary phase. The peptide mixtures were evaporated to dryness under a vacuum at 36 °C  before being resuspended in 50 µL of 5/95 CH3CN/0.1% HCOOH to achieve a final peptide concentration of 2 µg/µL and were finally transferred in plastic vials for LC-MS/MS analysis.The cell pellets were treated with 1% sodium deoxycholate in Ambic 50 mM, were sonicated for 5 min , and then clarified by centrifugation at 16,000× 2O and CHCN3, both with 0.1% HCOOH. The elution program was: 0 min, 5% B; 1 min, 5% B; 51 min, 22% B; 51.5 min, 90% B; 53.5 min, 90% B; 54 min, 5% B; 60 min, 5%B. Chromatographic performances and TOF accuracy were evaluated using an intra-run injection (5 µL) of beta-galactosidase 100 fmol/µL. The mass spectrometer was set in the positive ion mode, and the operation conditions of the ion source were the following: ion spray voltage floating 5.5 kV, probe temperature 150 °C, curtain gas 25 psi, ion source gas 1 and gas 2, respectively 30 and 20 psi, declustering potential 100 V. N2, as an inert gas, was used for CID (Collision Induced Dissociation) experiments.Sample analyses were performed using a micro-HPLC (Eksigent Ekspert microLC 200) combined with a Triple TOF 5600 mass spectrometer equipped with a Turbo Ion Spray probe as the ion source . Five µL of each sample/pool, set in an autosampler at 8 °C, was injected onto a C18 Jupiter column , thermostatted at 30 °C and equipped with a micro trap C18 (10 mm × 0.3 mm) . The flow rate was set at 5 µL/min, and the mobile phases A and B were, respectively, Hm/z and 100–1500 m/z, with an accumulation time of 250 and 100 milliseconds, respectively. CID experiments were carried out using rolling collision energy that was automatically calculated according to the m/z and the charge in of the candidate ion, with a collision energy spread (CES) of 5 V. Precursor ions with a charge state from 2+ to 5+ were chosen to trigger the MS/MS experiments. The isolation width for precursor ion selection was set at 0.7 m/z on the Q1.A sample pool was analyzed (double injection) with an information-dependent acquisition (IDA) tandem mass spectrometry method based on an MS1 survey scan from which the 20 most abundant precursor ions were selected for subsequent CID fragmentation. The MS1 survey and MS2 scans were acquired over a mass range of 250–1250 m/z. Precursor ion activation was performed by CID as described before; however, an acquisition mass range of 230–1500 m/z and a charge state of 2+ were chosen. An accumulation time of 100 milliseconds for MS1 and 50 milliseconds for MS2 scans resulted in an overall duty cycle of 2.6 s (~15 points per elution peak).IDA results were used to generate the SWATH data-independent acquisition (DIA) method for protein quantitation. The SWATH acquisitions for each sample were performed over 50 overlapping isolation mass windows of variable length  depending on the peptide density distribution along the entire mass range of 250–1250 According to the manufacturer’s protocol, IL-6 levels were determined using the Roche Elecsys Kit (09015604190). In total, 200 µL of the culture media underwent the first incubation with IL-6 specific antibodies, followed by a second incubation with IL-6 specific antibodies labeled with ruthenium complexes. After that, complexes were magnetically captured, and an electric voltage application determined the chemiluminescent emission that was directly proportional to the IL-6 concentration. https://github.com/vdemichev/DiaNN) version 1.8, the free universal software for DIA proteomics data processing by V. Demichev, M. Ralser, and K. S. Lilley .SWATH data analysis was carried out with DIA-NN ; use isotopologues, MBR (match between run) and remove likely interferences all enabled; neural network classifier = single-pass mode; protein inference = genes; quantification strategy = any LC (high accuracy); cross-run normalization = RT-dependent; library generation = smart profiling; where not specified, the standard settings were used.First, SWATH raw file.wiff were converted into .wiff.dia and then used to generate the spectral library utilizing the following parameters: FASTA digest for library-free search/library generation enabled; Human Swiss-Prot FASTA database of 20404 reviewed, non-redundant protein species (downloaded on 4 January 2023); deep learning-based spectra, RTs and IMs prediction enabled; protease = trypsin; missed cleavages = 2; maximum number of variable modifications = 3; N-term M excision, C carbamidomethylation as fixed modification and M oxidation as variable modification all enabled; peptide length range = 7–30; precursor charge range = 1–4; precursor For the protein quantitation step on files.wiff.dia, we kept the same parameters used above for spectral library generation, with only these exceptions: in the spectral library, the report-lib.tsv file generated was selected in the previous step; generate the spectral library, FASTA digest for library-free search/library generation, and deep learning-based spectra, RTs, and IMs prediction were all disabled; quantities matrices were enabled.2 transformed cross-run normalized protein areas , was used for further data analysis.The report-quantification.pg_matrix output file of DIA-NN, reporting the logp-value ≤ 0.05 was considered statistically significant. Biological significance and pathway analysis were conducted using DAVID Bioinformatics Resources 6.8 and the Reactome database, respectively. For each protein, the FC value was calculated as the ratio between the mean expression in TGF-β-treated cells and the mean expression in the control cells; and the mean expression in TGF-β/CurNPs-treated cells and the mean expression in TGF-β-treated cells. Proteins were considered differentially expressed when FCs were higher than 2.5 (FC ≤ 1/2.5 or FC ≥ 2.5). The values between different conditions were analyzed by two-way ANOVA followed by post hoc multiple comparison tests using GraphPad Prism version 8.0.0 for Windows , and a


P < 0.05). Furthermore, the levels of SEP1, IL-6, TGF-β1, Glu, and LD differed considerably between groups of the same ethnicity (P < 0.05). It was found that 33 kinds of lipid metabolites were significantly different in patients with hyperuricemia, which mainly involved 5 metabolic pathways. According to the results of further studies, it is speculated that CPT1, TGF-β1, SEP1, IL-6, Glu and LD may increase fatty acid oxidation and mitochondrial oxidative phosphorylation in patients through glycerophospholipid pathway, reduce the rate of glycolysis, and other pathways to change metabolic patterns, promote different cellular functions, and thus affect the disease progression in patients with hyperuricemia.A multiomics study was conducted to investigate how lipid metabolism disorders affect the immune system in Xinjiang patients with hyperuricemia. The serum of 60 healthy individuals and 60 patients with hyperuricemia was collected. This study used LC–MS and HPLC to analyze differential lipid metabolites and enrichment pathways. It measured levels of immune factors tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), carnitine palmitoyltransferase-1 (CPT1), transforming growth factor-β1 (TGF-β1), glucose (Glu), lactic acid (LD), interleukin 10 (IL-10), and selenoprotein 1 (SEP1) using ELISA, as well as to confirm dysregulation of lipid metabolism in hyperuricemia. 33 differential lipid metabolites were significantly upregulated in patients with hyperuricemia. These lipid metabolites were involved in arachidonic acid metabolism, glycerophospholipid metabolism, linoleic acid metabolism, glycosylphosphatidylinositol (GPI)—anchor biosynthesis, and alpha-Linolenic acid metabolism pathways. Moreover, IL-10, CPT1, IL-6, SEP1, TGF-β1, Glu, TNF-α, and LD were associated with glycerophospholipid metabolism. In patients with hyperuricemia of Han and Uyghur nationalities, along with healthy individuals, significant differences in CPT1, TGF-β1, Glu, and LD were demonstrated by ELISA ( A study found that HUA prevalence is 13.5%  when age and sex are standardized3. Uric acid (UA) is the only diagnostic indicator of hyperuricemia and is only produced by the xanthine oxidoreductase (XOR) oxidation of xanthine and hypoxanthine4. Furthermore, adipocytes produce uric acid through XOR5, indicating that adipose tissue may be another vital source of uric acid. Researchers have revealed that metabolic disorders, oxidative stress, and renin-angiotensin system dysfunction contribute to HUA progression9. Therefore, hyperuricemia may induce disorders of lipid metabolism.Hyperuricemia (HUA) is resulted from reduced uric acid excretion or abnormal purine metabolism and is inextricably linked to chronic kidney disease10. Metabolism and lipidomics can be efficiently combined to study changes in lipids during various biological processes, clarify the processes and mechanisms that underlie related biological activities, and identify markers in different organisms11. A recent study found that potential differential metabolites of numerous lipids are associated with HUA and participate in lipid metabolism pathways, such as glycerophospholipid metabolism, sphingolipid metabolism, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis12. Moreover, the CXC ligand 13 (CXCL-13) pathway can induce lipid metabolism disorder in hyperuricemia12. Chicory, plantaginis semen, and dendrobium officinalis may reduce uric acid levels by regulating lipid metabolism16. Studies on lipid metabolism have focused primarily on screening biomarkers associated with HUA and their metabolic pathways. The underlying molecular mechanisms by which hyperuricemia leads to lipid metabolism disorders remain unclear.By utilizing mass spectrometry-based metabolic approaches, it is possible to profile cell metabolism directly and uncover the mechanisms of metabolic pathway transcriptional activationIt is hypothesized that immune factors TNF-α, CPT1, IL-6, SEP1, TGF-β1, IL-10, Glu and LD are involved in the regulatory pathway of lipid metabolism. The regulation of metabolic homeostasis by these signaling molecules may be participated in the HUA's pathogenesis. Now, UPLC and LC–MS were used to analyze lipid metabolism in patients with hyperuricemia, identify different lipid metabolites, and clarify lipid-related metabolic pathways. At the same time, ELISA was utilized to measure the changes of the important regulatory components IL-10, CPT1, TNF-α, SEP1, TGF-β1, IL-6, Glu and LD levels. The study aims to identify potential targets for further intervention in HUA.18.120 persons participated in this cross-sectional study, 60 patients with hyperuricemia were treated between January 2021 and December 2022 to the Xinjiang Uyghur Autonomous Region Hospital of Traditional Chinese Medicine. The hospital’s physical examination center recruited 60 healthy controls of matched age and gender. The Ethics Committee of the Xinjiang Uyghur Autonomous Region Hospital of Traditional Chinese Medicine (No.20XE0115-1) approved the study, which was conducted with the informed consent of all participants. All programs related to human participants were developed according to the Declaration of Helsinki (updated in 2013)Participant inclusion criteria included a 30–70-year-old age range and living more than three generations in Xinjiang. Patients with cardiovascular and cerebrovascular diseases, severe liver dysfunction, severe nephropathy, tumors, and psychosis were excluded.19: Over 420 mol/L of uric acid is considered hyperuricemia.Based on the Guidelines for Diagnosis and Treatment of Hyperuricaemia and gout in China (2019)A fast of 12 h was followed by collection of 5 mL of venous blood, followed by separation of serum. UA concentrations were measured using an enzyme uricase method. This study used a Mindray Co, Shenzhen, China, automatic biochemical analyzer to measure high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), Very Low-Density Lipoprotein Cholesterol (VLDL-c), creatinine (CR), blood urea nitrogen (BUN), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), as well as total cholesterol (TC) levels.We stored the total serum samples at − 80 °C before performing the experiments.Sodium heparin blood collection was used to collect 5 mL of whole blood, which was immediately inverted to ensure homogenization. Using a low temperature and elevated speed centrifuge , we centrifuged whole blood for 10 min at − 1 °C using 3000 rpm at 3000 rpm after plasma was stored at − 80 °C. 240 L of precooled methanol and 200 L of water were vortexed with 100 L of plasma. For 20 min, 800 L of MTBE were vortexed and sonicated in a water bath at low temperatures. Following 30 min at room temperature and incubation at 10 °C, samples were centrifuged for 15 min at 14,000 g. Nitrogen was used as a drying agent for the organic phase. A mixture of 200 L of 90% Isopropanol/acetonitrile was mixed with it and centrifuged at 14,000 g for 15 min at 10 degrees Celsius. Lastly, mass spectrometric analysis was performed on the supernatants. We took it by a positive/negative ion fingerprints analysis, respectively. Extracts from all samples were mixed in equal volumes, and QC samples were prepared with the same sample volume. A − 20 °C refrigerator was used to store the extraction reagents.Lipids were separated using an HPLC system (Thermo Scientific). The chromatographic analysis was conducted with a 3 L injection volume, a 45 °C column temperature, and a flow rate of 300 L per minute.During phase A (ACN/H2O [6:4 v/v]), 10 mM ammonium formate and acetonitrile solutions were combined. As mobile phase B, ACN:IPA (2:9 v/v) was used with 10 mM isopropyl ammonium formate. The chromatography gradient started at 30% mobile from 0 to 2 min phase B, increasing to 100% from 2 to 25 min; mobile phase B was then left at 30% from 25 to 35 min. Throughout the entire analysis, temperatures of 10 °C were maintained in an autosampler and analyzed in random order using continuous analysis. An electrospray ionization mass spectrometer with Q active plus was used to perform untargeted lipidomic analyses. According to the source conditions, the heater temperature was 300 °C, the sheath gas flow rate was 45 ARB, the auxiliary gas flow rate was 15 ARB, the sweep gas flow rate was 1 ARB, the spray voltage was 3.0 kV, the capillary temperature was 350 °C, and the s-lens rf level was 50%. There was a scanning range of 200–1800 for MS1. Negative: The heater tempo was 300 °C, sheath gas flow rate was 45 ARB, aux gas flow rate was 15 ARB, sweep gas flow rate was 1 ARB, spray voltage was 2.5 kV, the sweep gas flow rate was 1 ARB, the spray voltage of 2.5 kV, the capillary tempo was 350 °C, and the s-lens rf level was 60%. After each full scan (full scan), 10 fragment profiles  were collected to determine the mass-to-charge ratios of lipid molecules and fragments. As of M/Z 200, 70,000 pixels were used in MS1, while MS2 had a resolution of 17,500. We purchased chemical components from Thermo including ethylene, isopropyl alcohol, and methanol. In addition to the ammonium formate, a spectrograph (Q-Exactive Plus from Thermo Scientific) and a chromatography column (ACQUITY UPLC CSH C18 from Waters) were purchased. All chemicals and solvents were analytically pure or chromatographic grade.Serum samples were read from plates using a Versa Max microplate reader . Then, using SoftMax pro 6.2.2 measured the levels of interleukin 6 (IL-6), carnitine palmitoyltransferase-1 (CPT1), selenoprotein 1 (SEP1), tumor necrosis factor-α (TNF-α), lactic acid (LD), interleukin 10 (IL-10), glucose (Glu) and transforming growth factor-β 1 (TGF-β1). The contents of these factors were calculated using the following formulas. Pattern recognition was done using SIMCA-P 14.1, an application software package.2Y and Q2 were determined. There was a t-test as well as a fold-shift analysis included in the univariate statistical analysis. R software created a volcano plot, hierarchical clustering analysis, bubble map, and correlation analysis. For path enrichment analysis, KEGG(www.kegg.jp/kegg/kegg1.html) was used.LipidSearch data were normalized by removing any molecules with missing values over 50% during data extraction. The research treated the data with Pareto-scale before performing pattern recognition with SIMCA-P 14.1 which was from Umetrics in Umea, Sweden. The data analysis was conducted using partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), and principal component analysis (PCA). Using seven cycles of linear regression interactions, R2 test was used for comparison of counts. Correlations were analyzed using Pearson’s correlation. Each test was statistically significant if P < 0.05 or P < 0.001. Logistic regression results are expressed as exp (B)  and 95%CI.Statistics were analyzed and processed using SPSS statistics (Version 25) . Normally distributed measurements were expressed as normal data. In both comparisons, t tests were used for two groups of measurement data, and comparisons between multiple groups were based on analysis of variance. A XEthics Committee of the Hospital of Traditional Chinese Medicine, Xinjiang Uygur Autonomous Region, has approved the project (No. 20XE0115-1).P < 0.05 or P < 0.001).Table Logistic regression was performed with Uric Acid Levels dependent variables. We added relevant variables to the regression model to build three regression models, as shown in the following Table In QC samples, the study detected various substance peaks, demonstrated in the total ion current diagram (TIC). A QC sample was characterized by positive and negative ions QC1  -anchor biosynthesis, arachidonic acid metabolism, and alpha-linolenic acid metabolism.As shown in Table 19 reported similar results. Confounding factor analysis in the Table 20 et al. showed a strong correlation between SUA and triglyceride values (the strongest correlation with triglycerides) for lipid indexes and adipose indexes. Elevated uric acid triggers an inflammatory response in the body, causing oxidative stress and promoting excessive fat metabolism in the liver, thus allowing the accumulation of free fatty acids 6, further indicating that elevated uric acid levels and lipid metabolism have a close relationship.The metabolic syndrome, hyperlipidemia, diabetes, and nephropathy can be linked to disorders of uric acid metabolism. In addition, these diseases can possibility increase blood uric acid levels, which can create a vicious cycle in the body. Due to body weight and BMI increases, elevated uric acid levels may be closely linked to obesity and insulin resistance in the research. As can be seen from Table 21. The LPCs can be activated by UA, and recombinant lyso-phosphatidylcholine acyltransferase-3 (LPCAT3) can be activated in vitro and in vivo, catalyzing PC generation19,22. This indicated that uric acid participates in metabolism and biosynthesis.In contrast to healthy persons, the study identified nine different lipids in HUA patients by analyzing non-targeted lipid metabolism, including fatty acid (FA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC), ceramide (Cer), triglyceride (TG), diacylglycerol (DG), phosphatidylserine (PS), and lysophosphatidylinositol (LPI). It was worth noting that 33 differential lipid molecules were significantly upgraded in HUA patients identified through heatmaps, among which were FA(20:4), PE, PS(42:8), TG[(12:1e_6:0_18:3)/(16:0_18:1_24:0)/(16:0_16:0_18:2)/(16:0_18:1_22:1)/(16:2e_20:0_22:6)/(4:0_10:2_22:5)/(18:3_10:2_18:2)/(18:0_18:0_18:1)/(18:0_18:0_18:0)/(16:0_18:1_22:0)/(20:0_16:0_20:0)/(16:2e_20:2_22:6)/(20:4e_18:4_20:2)], PI(18:0_22:4), PC[(23:1_11:2)/(18:0_22:6)], PG(28:1_18:2), LPI(12:0), Hex3Cer(d34:6) and Cer[(m19:0_23:1)/(t18:0_25:3)/(m22:0_16:1)/(m18:1_22:0)]. These findings indicate that HUA patients have altered blood levels of glycerol phospholipids and their metabolites, and the disturbance of phospholipid metabolism mainly involves phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylinositol (LPI), and phosphatidylinositol (PI). In addition to forming biofilms and recognizing cell membranes, these lipid molecules play a key role in signal transduction as well23, such as free fatty acids (FFA), low-density oxidized low-density lipoprotein (OxLDL), phospholipid oxide (OxPL), ceramides (Cer), and UA. A chronic low-grade inflammation in HUA is caused by these endogenous danger signaling molecules, which activate pro-inflammatory signaling pathways and release pro-inflammatory mediators. This study's results were also related to the finding that metabolic overload induces aseptic inflammation24. The study speculated that different lipid molecules and lipid metabolites in the inflammatory process of HUA could function as proinflammatory mediators by stimulating various immune receptors to initiate the downstream inflammatory cascade. Furthermore, it is involved in signal transduction in lipid metabolism.It is speculated that uric acid may decompose and synthesize various lipids and lipid molecules through the main lipid metabolism pathway in the human body, promoting the patient's body's lipid metabolites. Increasing the risk of HUA is increased by disordered lipid metabolism, which accelerates the accumulation of body uric acid. To further explore the influence of clinical indicators and interaction between lipids on the progression of HUA, the study used correlation analysis to analyze the interaction between clinical indicators of HUA and 33 different lipids and found that TG [(16:0_18:1_22:0)/(16:0_18:1_22:1)/(16:0_18:1_24:0)/(16:0_16:0_18:2)/20:4e_18:4_20:2)/(20:0_16:0_0)/(18:0_18:0_18:1)] and DG [(38:0e)/(36:1e)/(38:3e)] were positively related to VLDL-C and TG. CR was negatively related to TG (4:0_10:22:5), PE (16:0_18:3), PC [(18:0_22:6)/ (23:1_11:2)], and DG (36:1). BUN was negatively related to DG (18:2_18:2), LPI (12:0), PE (16:0_18:3), and TG (12:1e_6:0_18:3) levels. HUA was negatively related to DG (18:2_18:2) and TG (4:0_10:22:5) levels. HDL-C was negatively related toTG (16:0_16:0_18:2), PS (42:8), Hex 3Cer (d34:6), DG [(36:1)/(36:1e)], and Cer (m19:0_23:1). In the above study, the abnormal expression of lipid molecules were closely related to blood glucose, glomerular function, and dyslipidemia, which may explain the occurrence of hyperuricemia. These findings could be attributed to the different diets, lifestyles, and metabolism of HUA patients promoting excess nutrients and their metabolites25 to study pathways related to lipid changes. The influence values of glycosylphosphatidylinositol (GPI) -anchor biosynthesis, glycerophospholipid metabolism, arachidonic acid metabolism, alpha-linolenic acid metabolism, and linoleic acid metabolism pathways in HUA patients were 0.216, 0, 0, 0.004, and 0, respectively, indicating that elevated uric acid mainly leads to changes in glycophosphatidylinositol-anchor biosynthesis and glycerophospholipid metabolism in the human body. A similar finding was observed in rats with an elevated uric acid level by Liu Mingyu et al.26 suggesting that excess uric acid enhances the glycosylphosphatidylinositol-anchor biosynthesis, immunity, inflammation, and autophagy-related pathways. The study speculate that the lipid metabolism of HUA could be regulated through a metabolic microenvironment composed of various vital enzymes, transcription factors, and lipids. Furthermore, abnormal lipid metabolism also shifts the metabolic signaling pathway in cells of patients with hyperuricemia and affects the normal cell population of the human body through lipid substances. This complexity indicates that it is important to examine not only the HUA lipid metabolic network, but also the pathways by which HUA interacts with lipids and the influence of cellular lipid metabolism on its progress and outcomes.The research conducted pathway analysis using LIPEA based on KEGG database sources28. Therefore, liver fat can be prevented from accumulating by oxidizing fatty acids, resulting in a lipid metabolism disorder28. However, inhibition of mitochondrial FAO affects the expression of key regulatory factors in the energy metabolism pathway and modifies the levels of intracellular metabolites, thus reshaping intracellular energy metabolism homeostasis29. CPT1 is a crucial enzyme and regulatory factor in the fatty acid β-oxidation that can regulate lipid metabolism and improve body fat content. As can be seen from Tables 30 confirmed these findings, and they believed that this process mainly promoted fatty acid oxidation of endogenous lipids through the release of TGhydrolase, denutrient/fatty acid triglyceride lipase (ATGL), fatty acids, and glycerol. A study found that increased fatty acid oxidation31 can affect glycolysis-related cell metabolism. Glu and LD are critical intermediates in gluconeogenesis, and glucose conversion produces lactic acid to pyruvate via glycolysis and LDH. Increased oxidation of fatty acids decreases pyruvate dehydrogenase (PDH) activity, inhibits Glu glycolysis, inhibits glucose transporter (Glut-1) expression and Glu uptake, and increases glucose utilization by activated T cells. Meanwhile, It reduces intracellular glucose and 6 monophosphate glucose, while increasing glucoselytic intermediaries 1 and 2, as well as LD and pyruvate. The increase in LD levels may also symbolize the metabolic migration to glycolysis related to inflammation33, suggesting that LD, the final product of aerobic glycolysis, maybe the medium that causes the disorder of lipid metabolism in HUA to produce an inflammatory state. TGF-β1, a transforming glycoprotein growth factor, can participate in cell proliferation, differentiation, behavioral induction, and survival. According to research, hyperuricemia injury can increase the expression and phosphorylation of TGF-β1 in the kidney34. Due to uric acid stimulation, the TGF- β signaling pathway is activated35 and the mTOR signaling pathway is downregulated35, leading to increased oxidative stress, IL-6, IL-10 levels, and other inflammatory factors36. According to Table The above studies demonstrated that glycerophospholipids accounted for a considerable proportion of all the differential metabolites in patients with hyperuricemia, and the metabolic pathway of glycerophospholipid metabolism has interfered. Glycerol, fatty acids, and phospholipids are vital components of glycerophospholipids, among which dietary fatty acids can enhance fatty acid oxidation (FAO) in the mitochondria, accelerating production of TG in fat droplets, and provide more energy to liver cellsThe advantage of this study lies in the differential metabolites identification and metabolic pathways in patients with hyperuricemia by lipid metabolomics technology, and objective analysis of possible pathogenesis of abnormal expression of immune factors in differential metabolic pathways combined with ELISA. The disadvantage is that there is a limited number of participants in this study, and further large sample detection is needed to improve the reliability and universality of the research results. Second, the validation tests in this study were too simplistic, and a comprehensive analysis of lipid metabolism in hyperuricemia by transcriptome sequencing and proteomics is necessary to determine whether these indicators contribute to predicting HUA risk, and to illuminate downstream and HUA disease progression using regulatory metabolites.This study screened for differential metabolites of HUA lipids and their related lipid molecules and identified key lipid molecules that participate in glycerophospholipid metabolism, arachidonic acid metabolism, linoleic acid metabolism, glycosylphosphatidylinositol (GPI)—anchor biosynthesis, and alpha-linolenic acid metabolism pathways. Furthermore, the study identified that the glycerophospholipid metabolic pathway is crucial for the progression of HUA. By detecting changes in the expression levels of key regulatory factors in the metabolic pathway, we speculated that the regulatory factors CPT1, TGF-β1, Glu, and LD cytokines could promote fatty acid oxidation, inhibit glycolysis, and promote oxidative phosphorylation metabolism through the glycerophospholipid metabolic pathway of glycerophospholipids, and then regulate and affect metabolism microenvironment in patients with hyperuricemia after reprogramming. This may be a target for future intervention or a key point for further studies of HUA. Different metabolite markers can provide evidence linking lipid metabolism disorders with HUA at the molecular level and provide hypotheses for future research. This study suggests that regular monitoring of lipid indexes in patients with hyperuricemia should be conducted in clinical practice, and lipid-lowering drug intervention is necessary for patients with hyperuricemia with obvious dyslipidemia, which may delay the development of HUA to a certain extent.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


Mitochondria play a key role in regulating host metabolism, immunity and cellular homeostasis. Remarkably, these organelles are proposed to have evolved from an endosymbiotic association between an alphaproteobacterium and a primitive eukaryotic host cell or an archaeon. This crucial event determined that human cell mitochondria share some features with bacteria, namely cardiolipin, N-formyl peptides, mtDNA and transcription factor A, that can act as mitochondrial-derived damage-associated molecular patterns (DAMPs). The impact of extracellular bacteria on the host act largely through the modulation of mitochondrial activities, and often mitochondria are themselves immunogenic organelles that can trigger protective mechanisms through DAMPs mobilization. In this work, we demonstrate that mesencephalic neurons exposed to an environmental alphaproteobacterium activate innate immunity through toll-like receptor 4 and Nod-like receptor 3. Moreover, we show that mesencephalic neurons increase the expression and aggregation of alpha-synuclein that interacts with mitochondria, leading to their dysfunction. Mitochondrial dynamic alterations also affect mitophagy which favors a positive feedback loop on innate immunity signaling. Our results help to elucidate how bacteria and neuronal mitochondria interact and trigger neuronal damage and neuroinflammation and allow us to discuss the role of bacterial-derived pathogen-associated molecular patterns (PAMPs) in Parkinson’s disease etiology. Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by the progressive loss of movement control, gaiting and bradykinesia, in consequence of substantia nigra pars compacta (SNpc) neuronal degeneration . The patMitochondria are distinctive organelles that possess their own DNA (mtDNA) and replication processes independent of their host cellular division. Furthermore, mitochondria harbor unique ribosomes and contain cardiolipin  in the inner membrane, a diphosphatidylglycerol lipid also found in the membrane of most bacteria ,20,21. TIn PD neurons, the mitochondrial network is fragmented, which leads to prolonged exposure to mitochondrial DAMPs, triggering a chronic inflammatory response denominated as “sterile inflammation” . Indeed,Innate immunity activation generates a multitude of cellular responses. TLR and NLR activation not only trigger the inflammatory cascade, but also induce the expression of antimicrobial peptides, a crucial response against bacterial infections. For instance, it was observed that TLR2 is essential to drive the expression of antimicrobial peptide human β-defensin 2 . LikewisHerein we show that an environmental proteobacterium strain can activate innate immunity in mesencephalic neurons, partly through the activation of TLR4 and NLRP3 signaling pathways. α-Syn is expressed upon exposure of neurons to the bacteria and aggregates inside mitochondria, whose dysfunction leads to the fragmentation of their network to promote their degradation by mitophagy. Although we observed activation of the autophagic pathway, we also detected a decrease in the autophagic flow, which potentiated the accumulation of defective mitochondria, thus contributing to the exposure of additional DAMPs and further activation of innate immunity in a self-amplified cycle with obvious deleterious effects.n = 7). TLR4 can activate NF-κB signaling pathway and regulate pro-inflammatory cytokine expression. We observed that bacteria led to the activation of NF-kB, although at the time point selected for the study, we do not see statistical significance (n = 3). However, we observed caspase-1 activation (n = 3) associated with NLRP3 inflammasome, which promotes pro-IL-1β cleavage into its mature form (n = 5–6) to be released (n = 4–5). Indeed, NF-κB is required for the induction of a large number of inflammatory genes  under aseptic conditions. Brains were dissected and the mesencephala were carefully harvested and submerged in HBSS. Collected mesencephala were trypsinized (0.5 g/L) for 10 min at 37 °C. Trypsin action was halted with the addition of trypsin inhibitor  in HBSS containing DNase I (0.04 g/L), followed by mechanical dissociation. Cells were then centrifugated at 1000 rpm for 5 min at 4 °C. The pellet was resuspended and washed in HBSS and centrifugated again at 1000 rpm for 5 min at 4 °C. The pellet was then suspended in fresh Neurobasal medium supplemented with 2 mM L-glutamine, 2% B-27 supplement, penicillin  and streptomycin (100 mg/L) and 1% heat-inactivated FBS and seeded on poly-L-lysine (0.1 g/L)-coated dishes at a density of 1.3 × 106 cells/mL. For mitochondrial membrane potential and mitochondrial ROS production experiments, neurons were seeded on poly-L lysine (0.1 mg/mL)-coated 24-well plates at a density of 1.3 × 106 cells/mL. Cultures were grown at 37 °C in a fully humidified air atmosphere containing 5% CO2. Half of the medium was changed every other day to serum-free and antibiotic-free medium. At DIV3, cultures were treated with 1:2000 5-Fluoro-2′-deoxyuridine (FDU) to inhibit glial cell proliferation. For autophagy experiments, 20 mM NH4Cl and/or 20 μM Leupeptin  were added to the culture medium 4h prior to protein extracts preparation. NH4Cl in combination with Leupeptin allows for the blockage of several types of autophagy by increasing lysosomal lumen pH. This increase halts the activity of lysosomal proteases activity, maintaining the activity of the intracellular proteolysis systems -propanesulfonic acid (CHAPS), 10% (w/v) sucrose, 2 mM DTT) with 100 μM of the colorimetric substrate for caspase-1 from Sigma Chemical Co.  for 2 h at 37 °C. Reaction extent was measured at 405 nm using a Spectramax Plus 384 spectrophotometer .Caspase.1 activation was assessed as described in . BrieflyTo determine the cytokine levels in neuronal extracts, 25 μg of protein were used for each ELISA kit. NFκB p65, IL-1β, TNF-α, IL-6 and α-Syn oligomers ELISA kits were used per the manufacturer’s instructions as described in . Absorbat-test was used, and significant values are shown as: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.All the results were obtained from at least 3 independent experiments done in duplicates. All data are represented as the mean ± SEM. Normality distribution analysis (Shapiro-Wilk’s test) was applied to choose the subsequent parametric or non-parametric tests. Unpaired Student’s