Pleurotus ostreatus remains unclear. To investigate the function of PoMYB12, PoMYB15, and PoMYB20 TFs in P. ostreatus, mutant strains of PoMYB12, PoMYB15, and PoMYB20 were generated using RNA interference (RNAi) and overexpression (OE) techniques. The results indicated that the mycelia of OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains exhibited positive effects under heat stress at 32 °C, 36 °C, and 40 °C. Compared to wild-type strains, the OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains promoted the growth and development of P. ostreatus. These mutant strains also facilitated the recovery of growth and development of P. ostreatus after 24 h of 36 °C heat stress. In conclusion, the expression of PoMYB12 and PoMYB20 supports the mycelium’s response to heat stress and enhances the growth and development of P. ostreatus, whereas PoMYB15 produces the opposite effect.MYB transcription factors (TFs) have been extensively studied in plant abiotic stress responses and growth and development. However, the role of MYB TFs in the heat stress response and growth and development of  OsNAC1 and OsNAC6 [Arabidopsis thaliana’s AtMYB70, shows increased tolerance to drought and salt stress upon overexpression (OE) of the coding gene [FtMYB22 in Fagopyrum tataricum decreased transgenic A. thaliana’s tolerance to both drought and salt stress [Santalum album significantly participate in the cold stress response [OsMYB55 in rice enhances the amino acid metabolism of transgenic rice, thereby bolstering plant heat tolerance and mitigating the impact of high temperature on grain yield [Lilium LlMYB305 activates the LlHSC70 promoter activity under heat stress, contributing to plant heat tolerance [MYB transcription factors (TFs) are widely distributed in eukaryotes and feature highly conserved MYB binding domains that regulate diverse physiological processes in organisms . In pland OsNAC6 . The tobresponse . Within response . Moreovein yield . Meanwhiolerance .A. thaliana interacts with HY5/HYH to regulate seed germination and seedling development [Leymus chinensis promotes seed germination and root growth during drought stress [Nicotiana benthamiana [Fusarium graminearum and Acremonium chrysogenum [MYB TFs also assume significant roles in growth and development, as evidenced by their participation in seed germination, seedling development, root growth, leaf development, and anther development. For example, the MYB protein RSM1 in elopment , while Aelopment . Notablyt stress . Similarthamiana . These iysogenum ,17. AmonFlammulina velutiper [F. velutiper [hsp70, hsp90, and fes1 contribute to the regulation of heat stress during the initial stages of fruiting body development in F. velutiper [pal genes were discovered to be implicated in fruiting body development and the mycelial heat stress response in P. ostreatus studies [aox gene participates in regulating reactive oxygen species and enhancing resistance to heat stress in P. ostreatus [Lentinus edodes, research has revealed that the OE of the hsp20 can enhance mycelial heat resistance [Edible fungi possess significant edible and medicinal values. Currently, research into their abiotic stress response mechanism and growth and development has emerged as a research hotspot. For example, the transcription factor FvHmg1 has been identified as a negative regulator of fruiting body development in elutiper . Similarelutiper . Furtherelutiper . Notably studies . Additiostreatus . In the sistance . In summP. ostreatus is among the most widely cultivated edible mushrooms globally, valued for its nutritional and medicinal attributes. In China, the cultivation of P. ostreatus predominantly occurs within horticultural facilities, enabling year-round production. Among these practices, the price of P. ostreatus produced in summer is the highest [P. ostreatus’s fruiting body. The formation of the fruiting body depends on the interlacing of the mycelium, with the mycelium serving as a vital nutrient repository for P. ostreatus, facilitating its growth and development. As such, robust mycelial growth forms the bedrock for ensuring optimal yields. Moreover, P. ostreatus boasts a short production cycle and notable conversion efficiency [P. ostreatus. Consequently, in-depth investigations into P. ostreatus’s responses to heat stress and the mechanisms underlying its growth and development remain of utmost significance. highest . This prficiency . EnhanciP. ostreatus’s response to heat stress and its growth and development have revealed that PoMYB12, PoMYB15, and PoMYB20 may be involved in heat stress response and growth and development [P. ostreatus. This work aims to provide a new theoretical basis for studying the heat stress response and growth and development mechanisms of P. ostreatus.Prior transcriptome studies regarding elopment . NeverthPoMYB12, PoMYB15, and PoMYB20 in response to heat stress in P. ostreatus hyphae, mycelia from the WT were collected following 24 h of heat stress at 32 °C, 36 °C, and 40 °C. Specific primers were devised based on the nucleotide sequences of these three MYB genes, and they were cloned for PoMYB12, PoMYB15, and PoMYB20, respectively. Their open reading frames had total lengths of 1272 bp, 1440 bp, and 1947 bp, respectively  strain, respectively  medium, cultured at 25 °C in the dark for 4 d, transferred to 32 °C, 36 °C, and 40 °C in the dark for 24 h, and then transferred to 25 °C in the dark to resume growth for 24 h. The WT strain was used as the control. The mycelia of OE strains of PoMYB12 and PoMYB20 had stronger recovery ability than the WT strain after 32 °C, 36 °C, and 40 °C heat stress, while RNAi strains were weaker than WT strains . The Agrobacterium tumefaciens GV3101 strain was preserved in our laboratory.The ® One-Step gDNA Removal and cDNA Synthesis SuperMix Kits . The coding sequences (CDS) of the PoMYB12, PoMYB15, and PoMYB20 genes were obtained from the NCBI database . Primer Premier 5.0 was used to design PoMYB12-, PoMYB15-, and PoMYB20-specific primers . cDNA was synthesized by reverse transcription of total RNA using EasyScript primers  to cloneequences  were insectively ,41. The equences  were insequences  were insplasmids ,41.A. tumefaciens GV3101-competent cells were prepared according to the method in reference [PoMYB12, OE-PoMYB15, OE-PoMYB20, RNAi-PoMYB12, RNAi-PoMYB15, and RNAi-PoMYB20 were added to the competent cells, mixed, and incubated on ice for 5 min, in liquid nitrogen for 1 min, and in a water bath at 37 °C for 5 min, respectively. Subsequently, 600 μL of nonresistant lysogeny broth (LB) medium was added and expanded at 28 °C for 3 h. Finally, the cells were coated into culture dishes containing kanamycin (kan) 50 μg/mL and rifampicin (rif) 20 μg/mL resistance and incubated upside down at 28 °C for 2 d. After picking spots, colonies were PCR and expanded. Subsequently, 600 μL of nonresistant LB medium was added, shaken at 28 °C for 3 h, applied to kan (50 μg/mL) and rif (20 μg/mL) in a resistant petri dish, and cultured upside down at 28 °C for 2 d. A single colony was selected for colony PCR, and the culture was expanded for standby.eference . RecombiPoMYB12, OE-PoMYB15, OE-PoMYB20, RNAi-PoMYB12, RNAi-PoMYB15, and RNAi-PoMYB20 plasmids were expanded, centrifuged and collected, added to induction medium (IM) liquid medium, mixed, and induced for 5 h at 28 °C, 90 r/min and protected from light. The blocks in CYM were transferred into A. tumefaciens induced with OE-PoMYB12, OE-PoMYB15, OE-PoMYB20, RNAi-PoMYB12, RNAi-PoMYB15, and RNAi-PoMYB20 plasmids and incubated at 28 °C for 5 h in the dark. Then, they were transferred to IM solid medium, covered with cellophane, and cultured in the dark at 28 °C for 3 d. They were transferred to CYM containing Hyg and cefotaxime (Cef) resistance for 30 d. The surviving mycelia were transferred into PDA medium and collected. DNA was extracted, and Hyg gene fragments were amplified. RNA was extracted from the strain with the amplified Hyg fragment and reverse transcribed into cDNA, and the expression levels of PoMYB12, PoMYB15, and PoMYB20 were detected to obtain the transformed strain.The mycelium of P. ostreatus was inoculated into PDA medium, cultured in the dark at 25 °C for 5 d, and then inoculated into complete yeast medium (CYM) by punching 0.5 cm blocks with a hole puncher, which were left at 28 °C and cultured in the dark for 2 d. A. tumefaciens containing the OE-PoMYB12, PoMYB15, and PoMYB20  kits. The Bio-Rad CFX Connect TM Real-Time PCR System was used. Reaction system: 0.4 μL 10 μM upstream and downstream primers, 10 μL ChamQ Universal SYBR qPCR Master Mix (2×), 4 μL cDNA template, and 5.2 μL ddH2O. The reaction procedure was as follows: predenaturation at 95 °C for 30 s, then 40 cycles of 10 s at 95 °C and 30 s at 60 °C, and finally, extension at 72 °C for 30 s. β-Actin and β-tubulin were used as internal reference genes [−ΔΔCt method with three biological replicates [The mycelia of WT strain cultured at 25 °C in the dark for 4 d and subjected to 32 °C, 36 °C, and 40 °C heat stress for 24 h were collected. Then, RNA from mycelium was extracted and reverse transcribed into cDNA. The qPCR primers were designed using Primer Premier 5.0 for  PoMYB20 . The gence genes , and theplicates .P. ostreatus, the obtained mutant strains were inoculated into PDA medium, cultured in the dark at 25 °C for 4 d, transferred to 32 °C, 36 °C, and 40 °C for 24 h, and then placed back in the dark at 25 °C for 24 h. The recovery of mycelia was observed. Mycelial recovery growth length was measured by the cross-cross method [To simulate mycelium scald encountered in the summer production of s method . MeasurePoMYB12, PoMYB15, and PoMYB20 during the growth and development of P. ostreatus, samples of mycelium, primordium, young fruiting body, and mature fruiting body were collected. RNA from samples was extracted and reverse transcribed into cDNA. The procedure for gene expression measurement was the same as that described in the material method above.To detect the gene expression levels of The obtained transformants were cultured in PDA medium for 7 d at 25 °C in the dark, respectively. Then, the seed blocks were inoculated into the culture bottle (each bottle was inoculated with 5 evenly sized seeds 1 cm in diameter). The cultivar formulation was 78% cotton seed hulls, 2% lime, 18% bran, 1% glucose, 1% gypsum, and 60% water content and incubated at 25 °C for 16 d in the dark. With the WT strain as the control, the mycelial growth rates of transformed strains were observed. Additionally, mushroom emergence experiments were conducted. During the catalytic primordium stage, the temperature was alternated at 15 °C and 26 °C for alternating temperature difference stimulation, with a humidity of 85%, and scattered light alternated with dark conditions. During the growth stage of the fruiting body, the temperature was controlled alternately between 23 °C and 26 °C, with a humidity of 85%, and scattered light alternated with dark conditions. Mycelial growth length and cap length were measured by the cross-cross method . Among tThe transformed strains were inoculated into cultivation flasks, respectively. After the mycelia were cultured for 10 d, they were transferred to dark conditions at 36 °C for 24 h of heat stress. Following the heat stress, the mycelium was transferred to dark conditions at 25 °C for further cultivation. The subsequent conditions for catalytic primordium and growth of fruiting bodies remained consistent with the parameters described in the materials and methods section above. The mycelial growth length and cap length measurement was the same as that described in the material method above.All analyzed data were biologically replicated at least three times. Data represent the mean ± standard deviation. IBM SPSS Statistics 26 software was used to determine significant differences. Analysis of variance (ANOVA) and Duncan’s multiple range test were performed to assess differences between samples.


There were 193 differentially expressed proteins in the EEP group compared with the control group (p < 0.05), of which 103 proteins (53.37%) were upregulated, and 90 proteins (46.63%) were downregulated. The main three activated and suppressed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were extracellular matrix (ECM)-receptor interaction, amoebiasis, cell adhesion molecules (CAMs), nonalcoholic fatty liver disease (NAFLD), retrograde endocannabinoid signaling, and Alzheimer’s disease. The tumor volume of the 100 mg/kg EEP group was significantly different from that of the control group (p < 0.05). These results provide a theoretical basis for the potential treatment of human CSCC A431 cell tumors using propolis.Propolis is a gelatinous substance processed by western worker bees from the resin of plant buds and mixed with the secretions of the maxillary glands and beeswax. Propolis has extensive biological activities and antitumor effects. There have been few reports about the antitumor effect of propolis against human cutaneous squamous cell carcinoma (CSCC) A431 cells and its potential mechanism. CCK-8 assays, label-free proteomics, RT–PCR, and a xenograft tumor model were employed to explore this possibility. The results showed that the inhibition rate of A431 cell proliferation by the ethanol extract of propolis (EEP) was dose-dependent, with an IC Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from keratinocytes of the epidermis or appendages and the primary cause of death among nonmelanoma skin tumors . In recePinus spp., Prunus spp., Acacia spp., Betula pendula, Aesculus hippocastanum, Salix alba, Baccharis dracunculifolia, and Dalbergia ecastaphyllum (L.) Taub., and the collections are mixed with the secretion of worker maxillary glands and beeswax [Baccharis dracunculifolia, and red propolis is from the resin of Dalbergia ecastophyllum, while poplar propolis (brown color) is from the resin of Populus nigra L. [Propolis (bee glue) is a kind of viscous substance processed by Western honeybee workers that collect mucilage, gums, resins, and lattice from plants, such as  beeswax ,6. The c beeswax . There anigra L. . Propolinigra L. ,8.The antitumor activity of propolis has attracted much attention. In recent years, the antitumor effects of propolis on cancer and the relevant mechanisms have been reported in in vitro studies on colorectal, lung, breast, melanoma, gastric, lymphoma, tongue, and skin ,13,14,15In this study, Cell Counting Kit-8 (CCK-8) assays, label-free proteomics, RT–PCR, and a xenograft tumor nude mouse model were employed to determine the effect of propolis on the proliferation, differentially expressed proteins, related pathways in A431 cells, and tumor volume in an animal xenograft tumor model.The total flavonoid content of ethanol extract of propolis (EEP) was 32.04 ± 0.89/100 g. The chromatogram for UPHLC-MS/MS of propolis is shown in 50 of 5-FU and EEP against A431 cells for 48 h was 6.57 and 39.17 µg/mL, respectively.There were no significant differences between the viability of A431 cells in the solvent control group and the blank control group. EEP inhibited the proliferation of A431 cells. DMSO (0.05%) had no effect on A431 cells. The inhibition rates are shown in The morphology of A431 cells after EEP treatment for 48 h is shown in p < 0.05). Partial DEPs (p < 0.01) are shown in There were 103 upregulated and 90 downregulated differentially expressed proteins (DEPs) between the EEP group and the control group (screened by FC > 2.0 or FC < 0.5 and p < 0.05) and Kyoto Encyclopedia of Genes and Genomes (p < 0.05) analyses are shown in The volcano plot of proteins in the two groups is shown in p < 0.05) of upregulated and downregulated proteins were separately analyzed, as shown in These DEPs played roles in different pathways. The significantly enriched pathways  data of selected genes is shown in p < 0.05), which indicated that the 100 mg/kg propolis group had in vivo inhibitory effects on A431 cell tumors.The tumor volumes of nude mice in the control group, solvent group, 50 mg/kg propolis group, and 100 mg/kg propolis group after 12 days of gavage are shown in The HE staining results of the tumor tissue of the EEP, solvent control, and control groups are shown in 50 values against Hep-2 cells and HeLa cells were 63.48 ± 3.30 μg/mL and 81.40 ± 6.40 μg/mL, respectively [50 values of EEP in the KB cell line and A431 cell line were 40 ± 8.9 μg/mL and 98 μg/mL, respectively, after 48 h of incubation [50 values of EEPs  against tongue cancer cells treated for 24 h were approximately 88 µg/mL, 110 µg/mL, and 150 µg/mL, respectively [50 values of EEPs range from 26.33 to 143.09 μg/mL against the human colon cancer cell line HCT-16 [50 values of EEP  against A549 cells were 106 ± 0.004 µg/mL, 199 ± 0.009 µg/mL, and 87 ± 0.012 µg/mL, respectively, and for HeLa cells were 81 ± 0.006 µg/mL, 116 ± 0.023 µg, and 54 ± 0.005 µg/mL, respectively [50 of EEP  against the 5 × l05/mL DLBCL SU-DHL-2 cell line for 24 h was 5.729 μg/mL [50 of EEP  can inhibit the growth of cancer cells, and after 24 h of treatment, the ICectively . EEP  components, such as laminin, fibronectin, and collagen, plays a crucial role in tumor invasion and metastasis. The genes d COL6A3  or COL6Ad COL1A1 . There wd COL1A1  and non-d COL1A1 ,27. Mostd COL1A1 ,29. Simid COL1A1 . The difd COL1A1  and gastAnother important pathway of the upregulated DEPs was the amoebiasis pathway. The amoebiasis pathway was significantly enriched and identified as one of the important processes or signaling pathways of melanoma metastasis ,32, the The third pathway of the upregulated DEPs was the cell adhesion molecule (CAM) pathway. CAMs, having four main groups including cadherins, integrins, selectins, and immunoglobulins, are primarily glycoproteins on cell surface membranes and can promote homeostasis between cells and between cells and the extracellular matrix. With higher levels of neural cell adhesion molecule expression, neuroblastoma cells have more intense homophilic tumor binding . KnockdoThe most significantly downregulated pathway was the nonalcoholic fatty liver disease (NAFLD) pathway. NAFLD affected the cell cycle and p53 pathways. SNORA71A knockdown in HT-29 cells led to significant inhibition of cell migration and invasion ability, which targeted LBP to participate in NAFLD in colorectal cancer cells . CorosolFaecalibacterium prausnitzii [Another suppressed pathway was retrograde endocannabinoid signaling. This pathway was also suppressed in retinoblastoma , glioblausnitzii . This pausnitzii . The DEPusnitzii . Other sThe expression levels of genes subjected to RT–PCR were not completely consistent with the protein expression levels. Some studies have pointed out that transcription levels alone are not sufficient to predict protein levels in many cases ,57 becauThis experiment showed that EEP inhibited tumor growth in nude mice. It was also found that the growth of gastric pyloric tumors and colorectal cancer was inhibited by a diet containing propolis ,60. PropThe limitation of this study is that metabonomics, cancer stem cell, molecular docking, or other methods can be employed to explore more accurately the regulation of the EPP antitumor approach against the A431 cells for new drug development. More research about the cell cycle of A431 cells inhibited by EEP and the active components, such as caffeic acid, dihydro cinnamic acid, and p-coumaric acid, or others, who play the main antitumor effect can be determined in the future.The crude poplar propolis sample and extraction procedure of ethanol extract of propolis (EEP) were the same as in our previous report .The total flavonoid content determination of EEP was performed using the spectrophotometer method according to the national standards for propolis in China (GB/T 24283-2018). Rutin wg, 4 °C for 15 min. The supernatant was diluted to 53% methanol final concentration by LC-MS grade water. The solution was transferred to an Eppendorf tube and subsequently centrifuged at 15,000× g, 4 °C for 15 min. The elution conditions and processes were the same as in reference [The chemical components of EEP were determined using a UHPLC-MS/MS system  coupled with an Orbitrap Q ExactiveTMHF-X mass spectrometer ) by Untargeted Metabolomics mothed and performed by Novogene Co., Ltd. . Simply, The EEP samples (1 mL) were resuspended with prechilled 80% methanol by well vortex and then incubated on ice for 5 min and centrifuged at 15,000× eference . Q Exact2 humidified incubator at 37 °C .The human skin squamous cell carcinoma A431 cell line  was cultured in a special complete culture medium  in a 5% COv/v; equal to DMSO in the 100 µg/mL propolis group) was added to the complete medium as a solvent control.EEP (0.1 g) was dissolved in 0.5 mL DMSO and diluted with a complete culture medium at a concentration of 1 mg/mL. Propolis solution was diluted with a complete medium to 100, 75, 50, and 25 µg/mL. DMSO . At a beginning concentration of 5 × 104 cells/mL, 100 µL cell suspension was added in 96-well plates with 6 repetitions per treatment. After 48 h of cell culture with EEP 100, 75, 50, and 25 µg/mL, and 0.05% DMSO, the viability of cells was determined by a CCK8 kit  at 450 nm using a microplate reader . The IC50 of EEP on A431 cells for 48 h was calculated using GraphPad Prism 8.4.3 for Windows . 5-Fluorouracil  at 0, 4, 8, 16, 32, and 64 µg/mL were employed to determine the IC50 value against A431 cells.The A431 cells were irrigated with PBS buffer  twice and then digested with 1 mL 0.25% trypsin solution . The digested solution was centrifuged at 137× 5 cells/well. After 24 h of cell culture, the cells were treated with control, and IC50 EEP solution for 48 h, and then the morphological changes of the cells were observed using an inverted microscope .A431 cells in the logarithmic growth phase were digested with trypsin and seeded in 6-well plates at 1.5 × 1050 EEP solution. After these cells were treated with propolis or non-propolis for 48 h, the culture medium was removed. Then, the cells were irrigated twice with precooled PBS buffer, digested, and irrigated twice again. Cells were collected in a centrifuge tube (1.5 mL) after centrifugation. Therefore, these cells were frozen in liquid nitrogen and further stored in a refrigerator at −80 °C .The A431 cells were treated as described for the morphology observation experiment, which was also treated with control and ICThe extraction and concentration determination of total proteins and spectra of proteins were performed as described in our previous report .50 EEP groups using RT–PCR assay. The primers were designed through NCBI’s free online primer design platform, which is given in According to the proteomics results, the genes coded NDUFS1, NDUFV1, and SDHA proteins involved in oxidative phosphorylation and LAMC1, SDC1, and THBS1 ECM-receptor interaction were selected to determine the gene expression levels between the control and IC7 cell/mL) (0.1 mL) was injected subcutaneously into the axilla of the right forelimb of nude mice. The small nodules at the inoculation site mean the heterologous tumor model in nude mice was successful. After 1 week, the tumor sizes were 4–7 mm3. Then, 20 nude mice were randomly divided into 4 groups: the control, solvent, 50 mg/kg EEP, and 100 mg/kg EEP groups  were experimental animals. The A431 cell suspension (1 × 10rding to ). They wThese nude mice were sacrificed after the final treatment. The tumor tissue was immediately peeled off with scissors and tweezers, washed with normal saline, and fixed in 4% paraformaldehyde  for paraffin section preparation and hematoxylin-eosin staining .p < 0.01: extremely statistically significant differences between treatment and control groups, p < 0.05: statistically significant differences). The relative gene expression was represented by the ratio of gene expression in propolis-treated cells to that of control cells. Differences in tumor volumes among groups were analyzed using repeated-measures ANOVA using Stat View 5.0.1 .All experiments were performed in triplicate. All these data are expressed as the mean ± standard error. One-way ANOVA was used to analyze the significance of differences  to perform peak alignment, peak picking, and quantitation for each metabolite, whose main parameters were set as follows: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance,30%; signal/noise ratio, 3; and minimum intensity.A431 cancer cells can be inhibited by poplar propolis via the main pathways enriched DEPs were ECM-receptor interaction, amoebiasis, cell adhesion molecules (CAMs), nonalcoholic fatty liver disease (NAFLD) related pathway, retrograde endocannabinoid signaling, and other pathways. The inhibition effect was also found in a xenograft tumor for nude mice. Poplar propolis has the potential to be a new treatment strategy for CSCC patients.


Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the role of liver-specific CPT1a on hepatic lipid metabolism.Plin2, Cidec, G0S2) and in polyunsaturated fatty acid metabolism , while only female LKO mice increased genes involved in inflammation . Kinase profiling showed decreased protein kinase A activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice.Male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet  for 15 weeks. Mice were necropsied after a 16 h fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging, kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis  in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.  •Sexually dimorphic NAFLD is driven, in part, by CPT1a-mediated fatty acid oxidation.•CPT1a-deficiency drives microvesicular steatosis in periportal hepatocytes.•DHA-containing phospholipids are selectively reduced in lipid droplets from CPT1a LKO mice.•PKA activity and triglyceride turnover is impaired in CPT1a LKO mice. Over The carnitine palmitoyltransferase 1 (CPT1) gene family consists of three isoforms  that differ in subcellular localization, tissue specificity, and function . The CPTWe characterized the contribution of hepatic CPT1a to hepatic lipid metabolic phenotypes in male and female mice. Using a CPT1a liver-specific deficient mouse model, we show female mice depend on CPT1a for protection from the high-fat diet (HFD)-induced liver dysfunction. Male mice are more susceptible to HFD-induced liver dysfunction than female control mice, a condition that is not worsened by the absence of CPT1a. Utilizing a combination of lipidomic approaches, we demonstrate that hepatic CPT1a-deficiency alters the total phospholipid pool and promotes the incorporation of monounsaturated (MUFA)-containing phospholipids at the expense of docosahexaenoic acid (DHA)-containing phospholipids in lipid droplets (LD). The shift in MUFA from DHA-containing phospholipids within LDs associates with increases in PUFA-biosynthetic and peroxisome proliferator activated receptor (PPAR)-target gene expression. Mechanistically, kinase profiling of liver samples shows reductions in protein kinase A (PKA) activity, which coincided with impaired triglyceride hydrolysis in CPT1a-deficient livers. These studies reveal new mechanisms by which impairments in long-chain fatty acid oxidation drive sexually dimorphic SLD and alter LD dynamics in mice.22.1Animal protocols were in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee at the University of Kentucky. Mice were housed in individually ventilated cages at 20–22 °C on a 14 h light/10 h dark cycle with ad libitum access to food and water. CPT1a-floxed mice were obtained from the laboratory of Dr. Peter Carmeliet , backcroFor intraperitoneal glucose tolerance tests, mice were fasted overnight and were injected into the peritoneal cavity with a 20% glucose solution at a volume of 10 μL per gram of body weight. For insulin tolerance tests, baseline glucose levels were measured in the fed state (at 8 AM), then mice were then injected into the peritoneal cavity with 1 U/kg of insulin. Serum glucose levels were measured using the INFINITY glucometer and glucose strips (ADW). Serum insulin  and β-hydroxybutyrate (Cayman Chem) levels were quantified using commercial assays according to manufacturers' instructions.2.22 prior to adding 6 mL of 2:1 chloroform to methanol. To separate the phases, 1.2 mL of 0.05% H2SO4 was added, samples were vortexed then centrifuged at 2000 rpm for 15 min. The bottom (organic) phase was recorded, and 0.5 mL of the organic phase was added to 1 mL of 1% TritonX-100 in chloroform. The samples were dried under N2 and 0.5 mL H2O was added prior to quantification of triglycerides (Wako), total cholesterol (Pointe Scientific), and choline-containing phospholipids (Sigma), per manufacturer's instructions. All standards and blanks were prepared in a similar fashion, and data were presented normalized to initial liver weights.Extraction of liver lipids and subsequent quantification of hepatic triglycerides, total cholesterol, and phosphatidylcholine were conducted using enzymatic assays as previously described ,17. Live2.3Hematoxylin and eosin (H&E) staining of formalin-fixed paraffin-embedded liver sections was performed as previously described ,17. LD n2.4To determine the degree of liver injury in control and LKO mice, alanine aminotransferase (ALT) levels were measured in serum using enzymatic assays , as previously described ,17.2.52.5.1Liver samples on dry ice were spiked with 10 microliters of an internal standard and calibration mixture consisting of 500 μM each of di-myristoyl phospholipids , 500 μM SM (30:1) and 50 μM TG (14:1/14:1/14:1). To each sample, 300 μL of −20 °C chilled 75% methanol containing 0.01% BHT (butylated hydroxytoluene) were added along with 0.5 mm zirconium oxide beads. Samples were homogenized briefly in a Bullet Blender tissue homogenizer and placed on ice. Sixty microliters of methanol and 1 mL of MTBE were added to each sample, and samples were then vortexed for 60 min at room temperature. Water (170 μL) was added, and the samples were vortexed for an additional 15 min and then centrifuged for 15 min. The supernatants were collected to new test tubes and precipitated proteins were re-extracted as above. Pooled extracts were dried overnight in a speedvac, and resuspended in 400 μL of isopropanol containing 0.01% BHT.2.5.2m/z 400) were collected on a Thermo Scientific LTQ-Orbitrap Velos mass spectrometer in both positive and negative ionization modes. Scans were collected from m/z 200 to m/z 1200. For each analysis, 10 μL of sample was directly introduced by flow injection [Immediately prior to analysis, aliquots of each lipid extract were diluted 50-fold in isopropanol: methanol  containing 20 mM ammonium formate. Full scan MS spectra at 100,000 resolution  v.1.0 software linear fit algorithm, in conjunction with an in-house database of hypothetical lipid compounds, for automated peak finding and correction of escribed . Peak ar2.6High-performance liquid chromatography (HPLC)-methanol, HPLC-grade water and N-(1-Naphthyl)ethylenediamine dihydrochloride (NEDC) matrix were obtained from Sigma-Aldrich. Slides were prepared similar as previously described . After d2.7Male and female control and LKO mice were fed a HFD for 15-weeks. After 15-weeks of feeding, mice were necropsied in the fasted state and LDs were isolated by sucrose gradient centrifugation as we have previously described . LD PC a2.8A targeted lipidomic assay for PC and PE lipids was developed using HPLC on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), as previously described .2.8.1The standards (PC_36:4 and PE_34:2) and the isotope labeled internal standards (PC_33:1-d7 and PE_31:1-d7) were purchased from Avanti Polar Lipids . The Standard solution at concentrations of 0, 10, 50, 200, 1000, 5000 and 20000 ng/ml were prepared in 80% methanol containing the internal standard at the concentration of 500 ng/ml. Tissue homogenate with a volume of 20 μl was mixed with 80 μl methanol containing 2 internal standards at the concentration of 625 ng/ml, vortexed for 30 sec and then centrifuged at 18000 rcf, 4 C for 12 min. After centrifugation, 50 ul supernatant was transferred into a HPLC vial for injection. The volume of 5 μl standard solution and extracted sample was injected into the Vanquish UHPLC system  for lipid separation.2.8.2m/z) of molecular cation to the daughter ion m/z 184, the specific phosphocholine group. The PE species were monitored using Selected Reaction Monitoring (SRM) and the SRM transitions were the mass to charge ratio (m/z) of molecular cation to the daughter ion at m/z minus 141, the specific ethanolamine group.A C18 column  was used for the separation of the lipid species. Mobile phases were A (water containing 0.1% acetic acid and 0.3% ammonium hydroxide) and B ) containing 0.1% acetic acid and 0.3% ammonium hydroxide). Mobile phase B at 80% was used from 0 to 2 min at the flow rate of 0.3 mL/min and then a linear gradient from 80% B to 100% B from 2 to 8 min, kept at 100% B from 8 to 20 min, 100% B to 80% B from 20 to 20.1 min, kept 80% B from 20.1 to 29 min. The HPLC eluent was directly injected into a triple quadrupole mass spectrometer  and both the PC and PE lipids were ionized at the positive mode. The PC species were monitored using Selected Reaction Monitoring (SRM) and the SRM transitions were the mass to charge ratio  PamChip4 porous 3D microarrays and measured using the PamStation12 . Substrates contained in each array are listed in 2.10https://github.com/The-Hinds-Lab/Liver-Specific-Cpt1a-KO-Kinome-Analysis).The images taken during the run were analyzed using BioNavigator (PamGene). Signal ratios are used to calculate fold change (FC) for each phosphopeptide sequence averaged across three replicates. Minimum threshold values were selected using cutoffs cited in previous literature . These t2.11RNA extraction, cDNA synthesis, and quantitative real-time PCR was performed as previously described ,17. A sm2.12RNA was isolated using the RNeasy Mini kit (Qiagen) from livers of male and female, WT and LKO mice fed HFD for 15-weeks. The quantity and quality of the samples were determined using the Cytation 5 (BioTek) plate reader and Agilent 4150 Tape Station System, respectively, prior to submission to Novogene. The mRNA-seq libraries were prepared and sequenced on an Illumina HiSeq2500 platform, at a depth of 20 M read pairs (or 10 M/sample) by Novogene. After sequencing, the paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene, and the fragments per kilobase of transcript (FPKM) of each gene was calculated based on the length of the gene and read counts mapped to the gene.https://www.genome.jp/keg/).Differential expression analysis was performed using the DESeq2 R package (1.20.0). The resulting p-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted p-value ≤0.05 found by DESeq2 were assigned as differentially expressed. Gene ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package. GO terms with corrected p-values <0.05 were considered significantly enriched by differential expressed genes. The clusterProfiler R package was also used to test the statistical enrichment of differentially expressed genes in KEGG pathways (2.13bimake.com). After two sequential centrifugation steps , the supernatant was collected and protein was quantified using the Pierce™ Bicinchoninic Acid Protein (BCA) assay (ThermoFisher). For SDS-PAGE, 10 μg of protein was loaded and separated on 4–15% criterion TGX gels. Proteins were blocked with 5% milk and probed with antibodies listed in Whole liver (∼20 mg) homogenates were solubilized in 1 mL of 1X ice-cold RIPA buffer  supplemented with 1X protease and phosphatase inhibitor cocktail  was prepared in 0.3 mg/mL of assay buffer, and 20 uL was then added to all samples. Kinetic readings were taken every 2 min at 530 nm excitation and 590 emission. Moles of resorufin ester hydrolyzed are calculated from a standard curve generated using free resorufin.Triacylglycerol hydrolysis experiments were conducted as previously described . A small2.15A 1 mm × 3 mm piece of liver was fixed with 2.5% glutaraldehyde/4% paraformaldehyde in 0.2 M cacodylate buffer overnight at 4 °C. The sample was then washed 3 times with sodium cacodylate buffer  at 5 min/wash. The buffer was removed, the samples were post-fixed with 1% Osmium Tetroxide (in H2O) for 60 min at 4 °C. The samples were washed 2 times with sodium cacodylate buffer for 5 min/wash, then rinsed with 2% Maleate buffer (pH 5.1) for 5 min. The buffer was changed to 1% uranyl acetate in Maleate buffer and stained for 60 min at room temperature (RT). After staining, the uranyl acetate was removed and washed with maleate buffer 3 times at 5 min each. Dehydration is followed by different concentrations of cold ethanol 30%, 50%, 75%, 95% 1X, 5 min/wash followed by 100% ethanol at RT 3 times for 10 min/wash. For the infiltration step, the 100% ethanol was removed, and 1:1 ethanol/eponate12 medium was added at RT overnight. The media was then removed and changed to pure eponate 12 medium for 4–6 h at RT. For embedding, the sample was placed into pure eponate 12 in a rubber mold and allowed to polymerize for 24 h in an oven at 62 °C. Ultra-thin sections (85 nm) were cut with diamond knife, stained with 10% uranyl acetate and lead citrate, and then observed with a Tecnai G2 SpiritBT electron microscope operated at 80 kV.2.16post hoc analysis. When comparing two groups, a Student's t-test was utilized. Energy expenditure was calculated by analysis of covariance (ANCOVA) using body weight as a covariate [All graphs and statistical analyses were completed using GraphPad Prism 9.3.0. Data are expressed as ±SEM, unless otherwise noted in the figure legends. Differences were computed using two-way ANOVAs followed by a Tukey's multiple comparison ovariate . P-value33.1Cpt1a-liver specific knockout (LKO) and littermate Cpt1aF/F (Control) mice a semipurified low-fat  or high-fat diet  for 15 weeks. An experimental scheme has been provided in Cpt1a in the liver, we measured Cpt1a RNA and protein levels in livers collected from LFD- and HFD-fed mice. Real-Time PCR (qPCR) analysis revealed a 70–80% reduction in Cpt1a RNA in male and female LKO mice fed LFD . In response to LFD, male and female LKO mice  and products (long chain acylcarnitines) of CPT1a have been associated with insulin resistance ,37. ThisLKO mice  had imprLKO mice . During LKO mice C and D. LKO mice ; howeverLKO mice . These rLKO mice , a surroLKO mice D and E.Cpt1a on metabolic parameters in male and female mice in response to HFD-feeding. Notably, control and LKO mice were challenged with an overnight fast during night 4 of the five-day experiment. Over the course of the 5-day experiment, male control and LKO mice exhibited no differences in cumulative food intake during the light and dark cycles  are exposed to oxygen-rich blood flow and undergo greater rates of FAO ,43. Hepa3.4The LD core is surrounded by a phospholipid monolayer consisting of primarily (>90%) phosphatidylethanolamine (PE) and phosphatidylcholine (PC) ,45. GiveP = 2.1E-08; P = 2.9E-04; Aside from the total phospholipid pool, we next asked if CPT1a deficiency would alter the acyl-CoA composition of PE and PC. Male and female LKO mice exhibited increased MUFA -, ω6-PUFA--, and EPA (38:5)-containing PE F and PC 3.5Given the shift towards MUFA-containing PE and PC in whole cell lysates , we askePhospholipids exhibit varying spatial intensities across the periportal–pericentral axis in response to genetic- and dietary manipulation ,48. Whil3.6Cpt1a deficiency, we completed bulk RNA-sequencing on whole livers from HFD-fed male and female mice. We identified 577  and 748  genes that met pre-determined statistical  and effect size cut-offs . Partial least squares-discriminant analysis showed that the transcriptome of LKO mice was profoundly different from that of WT mice , accounting for up to 12.16% on PC2  and carbohydrate metabolism  were significantly downregulated compared to control mice (Lamb3), which is involved in Akt-mediated tumorigenesis [2 FC = 2.39; Padj = 2.05E-73) gene in response to LKO in male mice. In female LKO mice, the most significantly upregulated gene  was cell death-inducing DFFA-like effector C (Cidec), a gene encoding a LD-tethering protein involved in the fusion and growth of LDs [Plin2, Hilpda, G0S2) and fatty acid metabolism  were collectively upregulated in LKO mice across both sexes  were selectively upregulated only in female LKO mice  enrichment analysis for both male and female mice. The top 5 dysregulated pathways comparing male control and LKO mice were related to peroxisome proliferator-activated receptor (PPAR) signaling, fatty acid degradation, arachidonic acid metabolism, fatty acid elongation, and retinol metabolism  and elongase gene families . We examined normalized read counts of PUFA biosynthetic genes in livers collected from female control and LKO mice fed a HFD. Despite significant reductions in DHA-containing phospholipids in both whole liver lysates  gene facilitates the synthesis of long-chain PUFA-CoAs for esterification into phospholipids, albeit with a preference for arachidonic acid and ω3-PUFAs [Acsl4 gene expression was significantly elevated as compared to control mice (sn-glycerol-3-phosphate acyltransferase (Agpat3) or lysophosphatidylcholine acyltransferase (Lpcat3), two genes shown to facilitate DHA [sn-1 position of PE contains saturated fatty acids (SFAs) or MUFAs, and recent work identified transmembrane proteins containing TRM-Lag1p-CLN8 domains 1 and 2  as selective inducers of MUFA incorporation into sn-1 position of PEs [Tlcd1 and Tlcd2 increase MUFA-PEs and drive worsened liver injury in mice [Tlcd1 RNA levels as compared to female control mice  and its downstream targets, some of which are involved in LD hydrolysis  and in MUFA incorporation into PE (Tlcd1) were also elevated in female LKO mice , which show the confidence of our bioinformatics analysis relative to the number of random sampling over 2000 iterations and overall kinase activity across all PKA-peptides [Cpt1a in the liver  in liver samples from HFD-fed male and female mice using PamGene technology in real-time ,25. We dpeptides ,25  LD accumulation in periportal hepatocytes while total hepatic lipids  across the lobule were not different compared to male control mice. In fact, male LKO mice were protected from liver dysfunction (via ALT levels) compared to control mice. However, female control mice were largely protected from diet-induced liver dysfunction, which was completely lost in female mice lacking CPT1a in the liver. The exacerbation of SLD in female LKO mice was characterized by the accumulation of LDs within periportal and pericentral hepatocytes, consistent with significant elevations in triglycerides, cholesterol, and circulating ALT levels. Estrogen has been shown to stimulate FAO  and Cpt1ceptor α . It is pCpt1a deficiency in female mice is associated with alterations in the total phospholipid pool from PC to PE, as well as the acyl-CoA composition of these two major phospholipids. The PEMT enzyme facilitates the methylation of PE to generate PC, accounting for ∼30% of total liver PC biosynthesis in mammalian hepatocytes, while the remaining ∼70% of PC is synthesized by the conversion of choline to PC [Pemt gene expression in the liver [Next, we show that ne to PC ,62. PEMTne to PC ,63. A rene to PC , promotene to PC , and assne to PC ,69. Notahe liver . These pCpt1a shapes phospholipid metabolism, we measured the expression of genes involved in the PUFA-biosynthetic pathway. Despite reductions in DHA-containing phospholipids, we observed significant increases in the fatty acid elongase gene family . The ELOVL2 enzyme facilitates the elongation of 20- and 22-carbon PUFAs [de novo lipogenesis [Srepf1 and its downstream target genes, but Srebf1 was consistently downregulated across male and female LKO mice, as were its canonical target genes Acly and Fasn. Given that this pathway is also positively regulated by insulin levels [To further understand how on PUFAs , while Eon PUFAs . Depletiogenesis ,73. On togenesis . Given tn levels , the lowAside from reduced SREBP1-mediated lipogenesis, our studies show that loss of CPT1a in the liver of male and female mice stimulates PPAR-signaling. We found that the PPARα isoform, which transcriptionally regulates genes involved in peroxisomal biogenesis and fatty acid metabolism, was elevated across male and female LKO mice. This is consistent with other reports in skeletal muscle showing compensatory increases in peroxisomal FAO in response to impaired mitochondrial FAO . FurtherPparγ1 RNA, which coincided with significant upregulation of Cidec, an established PPARγ target gene [Cidec [Cidec in mediating microvesicular steatosis, a phenotype observed in individuals with impaired FAO [Another key observation in female LKO mice was the significant elevation in get gene . While Pget gene ,81, whice [Cidec . CIDEC ie [Cidec . Countere [Cidec . Future ired FAO .To assess which signaling pathways might be altered with CPT1a ablation, we performed an unbiased characterization of STK activity in male and female mice. This analysis revealed significant suppression of cyclic adenosine monophosphate (cAMP)-dependent protein kinases, including protein kinase X (PRKX) and PKA in LKO mice. PKA functions as a heterotetramer which consists of 2 regulatory and 2 catalytic subunits, one of which is PRKX. An increase in intracellular cAMP, as observed during fasting, results in a conformational change of the protein leading to activation and subsequent phosphorylation of lipolytic proteins, including PLIN5  and HSL It is important to note that some of the results highlighted here are not in complete agreement with a previous study showing the impact of liver-specific CPT1a-deficiency on SLD in mice. The manuscript by Sun et al. showed male CPT1a LKO mice had significantly lower body weight, and improved glucose and insulin tolerance in response to a 45% kcal from fat HFD . While wIn this study, we show that female mice are dependent on CPT1a-mediated FAO for protection from HFD-induced SLD. The exacerbation of SLD observed with CPT1a deficiency in female mice is associated with significant reductions in hepatic PC synthesis via the PEMT-dependent pathway. Modulation of PPARα and PKA signaling pathways may serve as compensatory mechanisms to protect hepatocytes from lipotoxicity in the face of impaired FAO. Future studies should consider sexually dimorphic responses to FAO when designing more personalized therapeutics for the management of SLD.Mikala M. Zelows: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization. Corissa Cady: Investigation, Writing – Review & Editing. Nikitha Dharanipragada: Methodology, Formal analysis, Investigation, Data Curation, Writing – Review & Editing. Anna Mead: Methodology, Formal analysis, Investigation, Data Curation, Writing – Review & Editing. Zachary Kipp: Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing – Review & Editing. Evelyn Bates: Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing – Review & Editing. Venkateshwari Varadharajan: Formal analysis, Data Curation, Writing – Review & Editing. Rakhee Banerjee: Formal analysis, Data Curation, Writing – Review & Editing. Se-Hyung Park: Investigation, Writing – Review & Editing. Nathan R. Shelman: Formal analysis, Data Curation, Writing – Review & Editing. Harrison A. Clarke: Formal analysis, Data Curation, Writing – Review & Editing. Tara R. Hawkinson: Formal analysis, Data Curation, Writing – Review & Editing. Terrymar Medina: Formal analysis, Data Curation, Writing – Review & Editing. Ramon C. Sun: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization, Supervision. Todd A. Lydic: Formal analysis, Investigation, Data Curation, Writing – Original Draft, Writing – Review & Editing. Terry Hinds Jr.: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data Curation, Writing – Original Draft, Writing – Review & Editing, Supervision. J. Mark Brown: Data Curation, Methodology, Validation, Formal analysis, Resources, Data Curation, & Funding acquisition. Samir Softic: Resources, Writing – Original Draft, Writing – Review & Editing, Supervision, Project administration, Funding acquisition. Gregory A. Graf: Conceptualization, Resources, Data Curation, Writing – Original Draft, Writing – Review & Editing, Supervision, Project administration, Funding acquisition. Robert N. Helsley: Conceptualization, Data Curation, Methodology, Validation, Formal analysis, Investigation, Resources, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization, Supervision, Project administration, Funding acquisition.10.13039/100000002National Institutes of Health grants K01DK128022, UL1TR001998, an 10.13039/100000968American Heart Association Career Development Award (23CDA1051959), and an 10.13039/100000048American Cancer Society Award (IRG2215234) to R.N.H. This work was also supported in part by grants from the National Institute of Health 2R01DK113625 (G.A.G), 5R01DK130227 (J.M.B.), 5R01DK120679 (J.M.B.), R01DK121797 (T.D.H.J.), R01DA058933 (T.D.H.J.), and F31HL170972 (Z.A.K.). Research reported in this manuscript was also supported by an Institutional Development Award (IDeA) from the 10.13039/100000057National Institute of General Medical Sciences of the 10.13039/100000002National Institutes of Health under grant number P30 GM127211. Development of some of the lipid mass spectrometry methods reported here were supported by generous pilot grants from the 10.13039/100012729Clinical and Translational Science Collaborative of Cleveland (4UL1TR000439) from the 10.13039/100006108National Center for Advancing Translational Sciences component of 10.13039/100000002NIH and the 10.13039/100000002NIH Roadmap for Medical Research, the 10.13039/100008533Case Comprehensive Cancer Center (P30 CA043703).This work was supported in part by the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Given the potential impact of TiO2 on the gastrointestinal epithelial and parenchymal cells, including goblet cells, the public consumers may suffer the risk of diseases caused by its widespread dissemination in food products. We therefore set out to investigate the impact of TiO2 NPs on the course and prognosis of ulcerative colitis by oral gavaging TiO2 NPs at the doses levels of 0, 30, 100, and 300 mg/kg during the induction  and recovery  phases of colitis in mice.Titanium dioxide  and colonic mucosa damage index (CMDI) scores, shortened the colonic length, increased the inflammatory infiltration in the colon. The most significant changes occurred in the low dose (30 mg/kg) group of TiO2 NPs exposure during the development phase of UC and the high dose (300 mg/kg) group of TiO2 NPs during UC self-healing phase. Increased reactive oxygen species (ROS) level and upregulation of anti-oxidant enzymes including total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) and catalase (CAT), demonstrate that the TiO2 NP exposure has triggered oxidative stress in mice. Moreover, the upregulation of caspase-1 mRNA and increased expression of thioredoxin interacting protein (TXNIP) further demonstrate the involvement of the ROS-TXNIP-NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway in aggravating the development of UC.The ulcerative colitis (UC) disease model was established by administrating of 2.5% dextran sulfate sodium (DSS) solution. Our results show that TiO2 NPs could affect the course of acute colitis in exacerbating the development of UC, prolonging the UC course and inhibiting UC recovery.Oral intake of TiOThe online version contains supplementary material available at 10.1186/s12989-023-00535-9. 2 NPs can induce mild colitis and exacerbate colitis caused by DSS.Short-term intake of TiO2 NPs exacerbated development of UC, and a high dose of TiO2 NPs delayed the recovery of UC.Exposure to a low dose of TiO2 NP exposure could trigger the activation of the antioxidant system in colitis mice.TiO2 NPs might aggravate the development of UC through the ROS-TXNIP-NLRP3 inflammasome pathway.TiOThe online version contains supplementary material available at 10.1186/s12989-023-00535-9. To regulate the application of TiO2 in food, the US food and drug administration (FDA) has limited the weight of TiO2 to 1% of the total weight in food [2 can be used as food additives in food with a limit of TiO2 at 10 g/kg food [2 in populations reaches approximately 1 mg/kg/d, especially in children, where it can reach up to 3 mg/kg/d [2 nanoparticles (NPs) have been gradually used in food for better taste. Researchers have proven that the sizes of food additive TiO2 particles range from nanoscale to micronscale, of which approximately 19–39% is TiO2 NPs [2 NPs every day through food, which arouses extensive attention on the health effect of TiO2 NPs as food additives.Because of its superior whiteness, high covering power and strong whitening power, TiO mg/kg/d . In receTiO2 NPs . Therefo2 NPs enter the gastrointestinal tract (GIT) and interact with the gut directly. Meanwhile, the gut is an important biological barrier to prevent the transport of TiO2 NPs. This renders the gut a major target organ of ingested TiO2 NPs. It has been reported that ingested TiO2 NPs can be absorbed by microfold cells (M cells) covering the top of Peyer’s patches. Moreover, TiO2 NPs can also enter and accumulate in the mesenteric lymph nodes to further affect the intestines [2 [2 NPs [2 NPs could be passing through the gut barrier and causing more severe damages.In contrast to fine particles, nanosized particles have a smaller size range, larger specific surface area and higher surface activity, which make it easier to cross biological barriers and elicit severe biological effects. After ingestion, TiOtestines –9. It istines [2  or TiO2 2 [2 NPs . could i2, are known to be a common risk factor for IBD [2 and aluminosilicate could be seen aggregating in the Peyer’s patches of children with IBD, especially in children with UC [2 ingested through the diet plays important roles in the process of IBD, especially in UC.Inflammatory bowel disease (IBD) is a chronic, recurrent, nonspecific and common intestinal disease that consists of two conditions, i.e. ulcerative colitis (UC) and Crohn’s disease (CD) , 13. It  for IBD . Several with UC . The Ti  with UC . All the2 NPs on UC are limited. Chen et al. reported that no colitis-like symptoms or apparent disturbance of gut microbiota was found in mice ingested TiO2 NPs [2 NPs could worsen intestinal inflammation and exacerbate dextran sulfate sodium (DSS)-induced acute UC through the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome [2 NPs affect UC through the activation of NLRP3 inflammasome or by different mechanisms on different stages of UC is still not clear. Existing studies mostly explained the induction or aggravation of inflammation by TiO2 NPs from the perspective of oxidative stress. Exposure to TiO2 NPs can induce reactive oxygen species (ROS) production and influence the expression of enzymes associated with oxidative stress, including glutathione peroxidase (GSH-PX) and total superoxide dismutase (T-SOD), etc., in colorectal cancer cell and liver cell lines, or in the kidneys and intestines in vivo [2 NPs induced ulcerative colitis (UC).To date, studies regarding the impact of TiOTiO2 NPs . Ruiz etammasome . However in vivo –24. Oxid in vivo . There i in vivo , 27. The2 NPs at varying doses by gavage during the development or self-healing phase of acute UC .This study established an acute mouse ulcerative colitis (UC) model by using dextran sulfate sodium (DSS). The animals were exposed to TiO UC Fig. . We aimeA commonly used method to establish ulcerative colitis is by administering of dextran sulfate sodium (DSS) to animals –30. PracIn the UC development phase, the mice that were given DSS solution began to exhibit characteristics including dull hair, loose feces, apathetic, and decreased activity from the 3rd exposure day and showed hematochezia and weak adhesion caused by hematochezia on the 6th day. The DSS-induced acute UC model had a low mortality rate, and the existing studies reported that the mortality varied from 0 to 40% –33. In tIn the self-healing phase of UC, the condition of the mice was not improving at the beginning but progressively worsened after the DSS solution was replaced with standard tap water. It was shown that the mice in the DSS group continued to lose weight from the 8th to the 11th day, after which they regained weight Fig. b. The DA2 NPs. Nine-week-old mice were gavaged with 300 mg/kg/d TiO2 NPs for 7 continuous days in the UC development experiment, and 10-week-old mice were administered 300 mg/kg/d TiO2 NPs for 10 continuous days in the UC self-healing experiment. The results showed significantly decreased body weight of mice with the exposure time of TiO2 NPs, the obviously high DAI score and colon IL-1β level, the increasing trend of CMDI score, colonic mucosal thickness, colonic histopathological score, colon MPO, T-SOD, and CAT activity, serum IL-1β, IL 18, and MDA level, and the decreasing trend of colonic length in mice treated with TiO2 NPs, while these changes were less apparent than in the DSS group  on human colorectal adenocarcinoma Caco-2 cells could induce high release of proinflammatory cytokines IL-8, IL-1β, and IL-18 [2 NPs by gavage for 10 days, the levels of various inflammatory cytokines in the mouse jejunum and ileum were elevated, including IL-1β, IL-6, IL-8, IL-12, IL-4, IL-13, tumor necrosis factor γ (TNF-γ), and IFN-γ, suggesting the induction of small intestinal inflammation [2 NPs, the activities of SOD, CAT, GSH-Px, and GSH decreased, the content of MDA increased, the intestinal mucosa and recess structure was damaged, and the intestinal IL-1β level increased significantly, indicating that TiO2 NPs induced intestinal oxidative stress and inflammatory response in rats [2 by gavage for 100 days, the cytokines TNF-α, IL-1β, IL-18, IL-10, IL-8 and IL-6 increased in the colonic mucosa of rats, suggesting that food grade TiO2 induced slight inflammation of the rat colon [2 NPs will trigger an intestinal inflammatory response and then play a role in the occurrence and development of IBD.Some studies reported that short-term exposure 24 h) to different doses of TiOnd IL-18 , 36. Wheammation . After 3 h to dif2 NPs on the course of UC, we gave mice 30 (low dose), 100 (medium dose), and 300 (high dose) mg/kg/d TiO2 NPs through gavage during the development and self-healing of acute UC induced by DSS. The results showed that TiO2 NPs changed the severity of UC caused by DSS in mice, resulting in significant weight loss, an increase in DAI and CMDI scores, a shortened colon, inflammatory infiltration in the colon, and increased expression of IL-1β and IL-18 . TXNIP binds with TRX in general, but under conditions of oxidative stress, TXNIP will be separated from TRX and combine with the NLRP3 inflammasome. Therefore, the NLRP3 inflammasome is activated, and free TRX can reduce oxidized proteins and play an antioxidant role . So just2 NPs could aggravate the development of DSS-induced UC in mice, prolong the course of UC, and slow the self-healing process of UC. However, in different stages of UC, the effect of TiO2 NPs on colitis showed different dose–response relationships, manifesting as aggravation of UC development by low-dose TiO2 NPs and delay of UC self-healing by high-dose TiO2 NPs. Considering that the effect of TiO2 NPs on colitis is complex, it is recommended that IBD patients try to avoid using food containing TiO2 NPs.This study found that oral intake of TiO2 NPs were purchased from Shanghai Yunfu Nanotechnology Co. Ltd, China. The characterization of TiO2 NPs was performed in our previous studies [2 NPs were dispersed in absolute ethyl alcohol and ultrasound for 15 min . Two drops of the suspension were put onto carbon-coated transmission electron microscopy  grids prior to image capture. TEM was used to determine the primary size and shape of TiO2 NPs. X-ray powder diffractometry  was used to detect the crystal structure of TiO2 NPs. The Brunauer–Emmett–Teller (BET) method  was used to measure the specific surface areas of TiO2 NPs. Inductively coupled plasma–mass spectrometry  was used to determine the purity of TiO2 NPs. As described in our previous study [2PO4. TiO2 NPs were suspended in ultrapure water, AGJ or AIJ respectively and ultrasonicated for 15 min , then the hydrodynamic diameter and Zeta-potential were tested at 0–2 h post sonication using ZetaSizer Nano ZS90 . The results showed that TiO2 NPs were in anatase form and nearly spherical in shape with an average primary diameter of 33.6 ± 11.5 nm  and zeta potential of TiO2 suspension were changed with the interval time between tests after ultrasonic treatment  in 100 mL ultrapure water. The acute UC model was established by exposing the animals to 2.5% DSS in drinking water for 7 continuous days. After the successful establishment of acute UC, the DSS solution was replaced with standard tap water for another 10 days, and acute colitis healed itself.2 in the population reached approximately 1 mg/kg/d in adults and 3 mg/kg/d in children [2 NPs to 30, 100, 300 mg/kg/d according to uncertainty factor of 10–100 times.Based on the dietary exposure data of different countries in which the daily intake of TiO2 NPs  were added to 10 ml ultrapure water to prepare TiO2 NP suspensions in concentrations of 3, 10, and 30 mg/ml. These three TiO2 NP suspensions were sonicated for 15 min before they were used every time. Considering the changes in the hydrodynamic diameters, PDI and zeta potential of TiO2 NPs suspension at 0–2 h post sonication  group, 2.5% DSS (DSS) group, 2.5% DSS + 30 mg/kg/d TiO2 NPs (DSS + 30 NPs) group, 2.5% DSS + 100 mg/kg/d TiO2 NPs (DSS + 100 NPs) group, and 2.5% DSS + 300 mg/kg/d TiO2 NPs (DSS + 300 NPs) group. The exposure procedure is shown briefly in Fig. 2 NP suspension by gavage every day. Mice in the other 4 groups were UC model mice, and these mice drank 2.5% DSS solution freely during the 7 days and were gavaged with 0, 30, 100, 300 mg/kg/d TiO2 NP suspension. The UC self-healing experiment, which lasted for 17 days, was carried out during the induction and self-healing period of UC. In this part of the experiment, the exposure of TiO2 NPs by gavage was carried out in the last 10 days, which was the stage of UC self-healing. In both experiments, mice were fasted for 1 day after the end of exposure and then harvested for further detection.After five days of acclimation, 120 Balb/c mice were divided into 2 groups for 2 experiments: UC development and UC self-healing. In each part of the experiment, 60 mice were divided into 6 groups: the control (CT) group, 300 mg/kg/d TiODuring experiments, the general conditions of the mice were observed every day, including activity, mental states, appearance and mortality. Mouse weight and water and food intake were also recorded every day. The food utilization were calculated according to the formula food utilization (%) = weight increase (g) × 100/feed consumption (g). Compared with the CT group, significantly decreased food utilization rate , T-SOD, and GSH-PX activities and cytokine expression. Then, the mice were sacrificed through cervical dislocation, and the gut was removed.The duodenum, jejunum, ileum, and colon were carefully separated and cut in length. The intestinal content was washed out, and intestinal morphology was observed. For the colon, colonic length was detected from the cecum to the anus before cutting. After overall assessment, 1 cm pieces of each intestine were fixed in 4% formaldehyde solution for the preparation of pathological sections. The remaining intestine was stored at − 80 °C for further detection of MPO, T-SOD, and GSH-PX activities, cytokine expression and quantitative real-time polymerase chain reaction (RT-qPCR).Other organs, including the liver, kidney, and spleen, were also separated. The caudate lobe of the liver, right kidney, and lower third of the spleen were fixed in 4% formaldehyde solution for the preparation of pathological sections. Remained organs were stored at − 80 °C.All histopathological examinations followed standard laboratory procedures as follows. Intestines, liver, kidney and spleen fixed in formaldehyde solution were embedded in paraffin and cut into 5–8 μm thick slices. The slices were stained with hematoxylin–eosin (HE). The stained slices were observed under an optical microscope and evaluated blindly by the pathologist.The histopathological changes of the small intestine segments in the treated mice were mainly villi rupture, loss of lacteal, and inflammatory cells infiltration  score, colonic mucosa damage index (CMDI) score, and histological changes in the colon following the methods described in our published work .The DAI score was calculated according to the weight loss rate, fecal characteristics and fecal occult blood  NaCl solution. The colonic homogenate was centrifuged at 4000 r/min and 4 °C for 15 min, and the supernatant was retained. The protein content of colonic homogenate was detected by the Bradford method. To determine the status of oxidative stress in mice, T-SOD, GSH-PX, and catalase (CAT) activities and malonaldehyde (MDA) content in colonic homogenate and T-SOD and GSH-PX activities and MDA content in serum were detected according to the manufacturer’s instructions2O, moderate cDNA that made concentrate of final reaction system 0.2 μmol/L, and moderate cDNA that made volume of final reaction system 20 μl. All reactions were performed in triplicate. Then, the reactions were performed in a real-time fluorescence quantitative PCR instrument. Relative changes in gene expression were determined using the 2–ΔΔCt method (ΔΔCt = (CtA2 − CtB2) − (CtA1 − CtB1). CtA1 and CtA2 were the Ct values of the target genes of sample 1 and sample 2, respectively, and CtB1 and CtB2 were the Ct values of the internal reference genes of sample 1 and sample 2, respectively. The primer sequences were as follows:reverse: TCCCAGTCAGTCCTGGAAATGcaspase-1 forward: ACAAGGCACGGGACCTATG,reverse: GAAGGTCCACGGGAAAGACACIL-1β forward: TTCAGGCAGGCAGTATCACTC,reverse: AGCGCAAGTAGTCCAAAGTCTTXNIP forward: GGCCGGACGGGTAATAGTG,reverse: GGGGTCGTTGATGGCAACAGAPDH forward: AGGTCGGTGTGAACGGATTTG,TXNIP, cysteinyl aspartate specific proteinase 1 (caspase-1), and IL-1β mRNA expression in the colon was detected using RT–qPCR. Total RNA of colonic tissues was isolated using TRIzol reagent , and cDNA was synthesized using HiScript II Q RT SuperMix for qPCR  according to the instructions. The internal control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RT–PCR was performed using AceQ qPCR SYBR Green Master Mix. A mixed PCR system was prepared containing 10 μl qPCR SYBR Green Master mix , 0.4 μl PCR forward primer, 0.4 μl PCR reverse primer , 3 μl ddHSPSS 20.0 was used to carry out the statistical analysis. Data that exhibited a normal distribution according to the K-S test are expressed as the mean value ± standard deviation (Additional file 1. Oral intake of titanium dioxide nanoparticles affect the course and prognosis of ulcerative colitis in mice: Involvement of the ROS-TXNIP-NLRP3 inflammasome pathway.


Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ  domain and adjacent conserved regions involved in protein interactions. Here, we examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and dCTCF, interacted predominantly with the BTB domain of CP190. Due to the difficulty of mutating the BTB domain, we obtained a transgenic line expressing a chimeric CP190 with the BTB domain of the human protein Kaiso. Another group of architectural proteins, M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. We also found that the N-terminal of CP190 was sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes.CP190 is a co-factor in many  In higher eukaryotes, the regulation of gene expression is a complex process which ultimately leads to cell differentiation during embryonic development. Cell specialization is determined by differences in transcription factor (TF) repertoires, and the genes responsible for cell differentiation and development of organisms are typically regulated by multiple independent enhancers, each of which stimulates a promoter in a limited population of cells during a specific time interval ,6,7,8,9.A number of studies have shown that the promoter can act as an insulator in some cases ,19,20,21Drosophila melanogaster genome contains only four genes encoding C2H2 proteins that have BTB-forming stable homodimers [The modimers . In contmodimers .It has been shown that CP190 is recruited to regulatory elements via interactions with DNA-binding transcription factors ,39,40,41Ubx, abd-A, or Abd-B, in the appropriate parasegment/segment during Drosophila development [CP190 predominantly associates with gene promoters and is required to support the opening of the chromatin ,49,50,51elopment ,60,61,62elopment . It has elopment ,50,64,65elopment ,46, it shk chimeric protein to regulatory elements associated with dCTCF and Su(Hw) proteins. We also found that the N-terminal region of CP190 is sufficient to open chromatin and recruit the partner proteins Chromator (Chro/Chriz) and Z4 (Putzig/Pzg).The aim of this study is to finely map the CP190 domains that are responsible for interaction with various C2H2 proteins and to examine the functional role of these CP190 domains in vivo. We mapped two CP190 regions that interact with the ZIPIC, M1BP, and Opbp proteins. ZIPIC and Opbp interact with both the 210–245 aa and 309–390 aa regions, while M1BP interacts only with the 210–245 aa region. The deletion of these regions strongly affected CP190 binding to the chromatin. The BTBs of CP190 and human Kaiso showed structural similarity but low sequence homology. Kaiso BTB was able to partially substitute for the functions of the BTB domain in CP190, but it could not effectively recruit the CP190The CP190 protein A containIt was previously shown that CP190 interacts with a large group of architectural C2H2 proteins. Five C2H2 proteins, including the well-known insulator proteins Su(Hw), dCTCF, and Pita, primarily interact with the BTB domain ,47. ThreWe used a yeast two-hybrid assay to precisely map the domains in CP190 involved in the interaction with M1BP, Opbp, and ZIPIC B. A seriDrosophila BTB CP190 and the BTB of the human Kaiso protein appeared incomplete  promoter into the 38D region using the φC31-based integration system [Our previous results ,39,45,46complete . In the complete . To examn system  . In the 309–390 region, we deleted the conserved 308–373 aa in CP190Δ308–373–F. Western blots showed that CP190Δ210–245–F, CP190Δ309–390–F, and CP190hK–F were expressed at similar levels as the control CP190WT–F, suggesting that all CP190 variants displayed normal stability  survived to three-day-old adult flies that showed a normal phenotype and remained fertile. The homozygotes hKUbi:CP190/hKUbi:CP190; 2Cp190/3Cp190 survived longer: about 15% of the offspring reached the adult stage. The flies were viable and fertile, but their progeny died at the embryonic and first instar larval stages. These results suggest that CP190hK protein expression cannot compensate for the functions of the wild-type protein. Moreover, the hKUbi:CP190/hKUbi:CP190; 2Cp190/TM6,Tb, and hKUbi:CP190/hKUbi:CP190; 3Cp190/TM6,Tb lines could not be maintained for several generations due to low viability and fertility. Thus, a high concentration of CP190hK negatively affects the activity of the wild type CP190 protein, the amount of which is lower in a heterozygote for the Cp190 null mutation.To examine the functional activity of the CP190 variants, we crossed the transgenes into the d Cp1903 . The Cp1us state . The UbihKUbi:CP190, both Δ210–245Ubi:CP190 and Δ308–373Ubi:CP190 in the heterozygous state were insufficient to complement the 2Cp190/3Cp190 mutations; the adult flies died within days of hatching (−Cp190 flies homozygous for the Δ308–373Ubi:CP190 or Δ210–245Ubi:CP190 transgenes that developed to the adult stage were less viable than hKUbi:CP190 (less than 7%), and they died soon after hatching. Also, strong expression of Ubi:CP190Δ210–245 or Ubi:CP190Δ308–373 negatively affected the viability of flies heterozygous for the 2Cp190 or 3Cp190 mutations. Thus, each of the mutant proteins Ubi:CP190hK, Ubi:CP190Δ210–245, and Ubi:CP190Δ308–373 displayed similar effects on Drosophila development.Similar to hatching . The Cp1Recent studies have shown that the interbands of polytene chromosomes typically correspond to the promoter regions of broadly expressed housekeeping genes and display an “open” chromatin conformation . CP190 bUbi:CP190*/CyO) in 2/Cp1903Cp190 larvae. The degree of co-localization of CP190 variants with Su(Hw) or dCTCF (insulator and promoter sites) and M1BP (mainly housekeeping promoters) on the polytene chromosomes of third instar larvae was studied , dCTCF, and M1BP the same as the wild-type proteins, we analyzed the binding patterns of the CP190 variants expressed from one copy of the transgene , dCTCF, and M1BP on the polytene chromosome of WTUbi:CP190/CyO; 2Cp190/3Cp190 larvae  and dCTCF sites was considerably lower than with CP190WT   sites. At the same time, co-localization of CP190Δ308–373 and M1BP was significantly reduced (Comparison of polytene chromosomes in larvae expressing CP190 CP190WT . CP190Δ3d Su(Hw) . Thus, u reduced .hK to dCTCF/Su(Hw) sites was reduced, and that this was correlated with the main role of the BTB domain of CP190 in interaction with dCTCF and Su(Hw). At the same time, CP190Δ308–373 and CP190Δ210–245 bound more efficiently to the dCTCF and Su(Hw) sites and co-localized less efficiently with M1BP.Overall, our results suggest that the binding of CP190yellow gene and contains two Su(Hw) binding sites [gypsy insulator consists of the 12 binding sites for Su(Hw) [2/Cp1903Cp190 larvae heterozygous for either of the CP190 transgenes. For all tested sites, CP190WT bound more efficiently than the mutant versions of CP190 (Δ210–245 bound to the Su(Hw) sites with similar or better efficiency than CP190Δ308–373. Finally, CP190hK bound with the same or weaker efficiency than CP190Δ308–373. Thus, the results were consistent with the hypothesis that the BTB domain of CP190 is critical for binding to Su(Hw) sites [To further analyze the specificity of recruiting by CP190 variants, we compared the binding of CP190 variants by qChIP to the main well-described binding sites for the Su(Hw) protein A. The 50ng sites . The gypr Su(Hw) ,74. For of CP190 A. In accw) sites ,47.Δ210–245, and less efficient for CP190hK  B. The se CP190hK B.The CP190 protein binds to promoters of housekeeping genes and is involved in the organization of the opened chromatin ,50. InteWT) gave rise to a prominently decondensed zone and formation of the interband that splits the 10A1-2 band , DBD:CP190(1–470), and DBD:CP190(1–430) induced the formation of interbands, but Z4 and Chro recruitment was less efficient . It seemDrosophila insulators. The second group of architectural proteins, including M1BP, Opbp, and ZIPIC, interacted with one or two highly conserved regions located between 210–245 aa and 309–390 aa. The existence of several domains in CP190 involved in interactions with DNA-binding architectural proteins makes it possible to create an effective platform for in vivo recruitment of CP190 to regulatory elements. The regions involved in the interactions with architectural proteins are located close to the regions that are responsible for the association of CP190 with centrosomes. It is likely that such an organization of domains makes it possible to efficiently carry out the relocalization of CP190 from chromatin in the interphase to the centrosomes during mitosis.The architectural C2H2 proteins employed different strategies for interaction with CP190. The dCTCF, Su(Hw), and Pita proteins interact predominantly with the BTB domain of CP190. These proteins bind to promoters and are responsible for the organization of the most well-characterized Drosophila architectural proteins. Our results showed that in the chimeric CP190hK, the Kaiso BTB could partially functionally replace the CP190 BTB domain. This suggests that the BTB domains may not only have a common ability for homodimerization but may also interact with unknown protein complexes that are conserved among higher eukaryotes. Thus, the CP190hk chimeric protein can be used to identify conserved transcriptional complexes among higher eukaryotes that are recruited to the Kaiso BTB domain. Mammalian BTB-C2H2 proteins, including Kaiso, are important transcriptional regulators involved in organismal development and cell differentiation [The BTB domain of Kaiso has 67% sequence homology to the CP190 BTB domain ,79. Bothntiation ,33,80,81ntiation ,87,88,89ntiation ,95,96,97hK, CP190Δ210–245, or CP190Δ308–373 displayed similar phenotypes: strong reduction in viability and fertility of adults, and high levels of larval and pupal mortality. Thus, all three regions of CP190 may have similar essential functional roles as well as being highly conserved in different Dipteran species. In contrast, the 126-209 aa microtubule binding domain, the C2H2 domains, and the C-terminal D/E rich region are not essential for CP190 functions in vivo [Flies expressing chimeric CP190 in vivo ,28. ThushK binds less effectively to the regions that are associated with the dCTCF and Su(Hw) proteins. This result supported the hypothesis of a key role for the BTB domain in the interaction of CP190 with this group of architectural C2H2 proteins.On the polytene chromosomes, it was possible to study the efficiency of CP190 binding to the promoters of active genes and insulators located in the interbands. Only the most efficient CP190 binding sites are visible on polytene chromosomes, making it possible to assess the efficiency of CP190 variants binding to different architectural proteins. We obtained the clear result that CP190M1BP along with ZIPIC and Opbp form another group of architectural C2H2 proteins that predominantly bind to housekeeping promoters and interact with the CP190-Z4-Chromator complex. The 309–390 aa region is the most essential element for recruiting CP190 to polytene chromosomes. It is likely that the same or adjacent (located in 390–430 aa) region is required for interaction with Z4 and Chromator proteins. The Z4, Chromator, and CP190 proteins can form a platform on the housekeeping promoters for efficient recruitment of transcription and remodeling complexes such as NURF and dREAM ,53,55 thOn the whole, the obtained results are consistent with the hypothesis that the CP190 protein constitutes a platform for interaction with various proteins and transcription complexes. The BTB domain and several conserved N-terminal regions are responsible for specific interactions of CP190 with architectural proteins and Chro/Z4 partner proteins. Different combinations of the architectural proteins determine the ability of CP190 and its partners to bind to the regulatory regions—predominantly promoters. The binding of CP190/Z4/Chro to regulatory elements results in the attraction of transcriptional complexes and, as a result, in switching on of regulatory elements. There are also a number of experimental data indicating that CP190/Z4/Chro can also participate in the organization of TADs and distance interactions between regulatory elements ,99,100. Plasmids for the yeast two-hybrid assay (Y2H) were prepared using the full-sized M1BP, Opbp, Zipic, and Su(Hw) fused with pGAD424 vector .g aattcATGTCGAAATCGGCGC-3′ (introduced site for EcoRIenzyme) and 5′-aggtcgacGATGTGCAGGCTGTC-3′  primers were used to amplify cDNA, followed by digesting with EcoRI and SalI enzymes (recognition sites underlined) and cloning in pGAD424 vector digested by the same enzymes.To generate M1BP plasmid, 5′-tcggatcc aaATGTCATCGTCCAACCAGG-3′ (introduced site for BamHI enzyme) and 5′-ttgtcgacGTTATTACAGATTGGCTTAAG-3′  primers were used to amplify cDNA, followed by digesting with BamHI and SalI enzymes (recognition sites underlined) and cloning in pGAD424 vector digested by the same enzymes.To generate Opbp plasmid, 5′-ttg aattcATGAATTGCTGCATTTGTC-3′ (introduced site for EcoRI enzyme) and 5′-ttgtcgacAACTGGCGTGCAGTCGTC-3′  primers were used to amplify cDNA, followed by digesting with EcoRI and SalI enzymes (recognition sites underlined) and cloning in pGAD424 vector digested by the same enzymes.To generate ZIPIC plasmid, 5′-ttgtcgacTCAAGCTTTCTCTTGTTC-3′ ) and 5′-GTTAAACACATCAGCGGGCTC-3′ primers were used to amplify C part of Su(Hw) cDNA. Resulting PCR fragments were digested with EcoRI and SalI enzymes and cloned in pGAD424 vector digested by the same enzymes. Next, EcoRI-EcoRI fragment from Su(Hw) cDNA was cloned in obtained plasmid with orientation checking.To generate Su(Hw) plasmid, 5′-agTruncated versions of CP190 were fused with pGBT9 vectors .To generate CP190 derivatives 1–166 aa, 1–220 aa, 1–245 aa, and 1–293 aa, one common 5′ cATGGGTGAAGTCAAGTCCGTG 3′ and set of various primers were used for each construct with introducing for BamHI (underlined):ggatccttaTCGCGATTGCGGCGAAGG 3′166 rev 5′ tggatccttaATAACCCTTTCGCAGCTGCTC 3′220 rev 5′ tggatccttaCTTAACCTCTTCCAAACTGGG245 rev 5′ tggatccttaTGTAGATGGCTGCGGTGGC 3′293 rev 5′ tResulting PCR fragments were digested with BamHI and cloned in pGBT9 or pGAD424 vectors digested by SmaI and BamHI.To generate 1–293 CP190 derivatives with either 190–210 aa or 217–242 aa deletions, we used in-fusion cloning mutagenesis technology . PCR-fusrev190 5′ CTCGAATGGTGACGTTGTTCGGCTCATTGTG 3′,dir210 5′ ACAATGAGCCGAACAAGTCACCATTCGAGC 3′primers for 190–210 aa deletion; and:rev217 5′ TTCCTTAACCTCTTCTCGCAGCTGCTCGAAT 3′,dir242 5′ TTCGAGCAGCTGCGAGAAGAGGTTAAGGAAT 3′primers for 217–242 aa deletion. PCR fusion of the amplified fragments through a single overlap extension was carried out on gel-purified PCR fragments from the parallel reactions.To generate CP190 derivatives 309–470 aa, 309–390 aa, and 309–440 aa one common 5′ATCCACCACCATTATCTTGAAACAG 3′ and set of various primers were used for each construct with introducing site for BamHI (underlined):ggatccttaCCAGGGCCCAGTAGTATTC 3′470 rev 5′ tggatccttaAGTTGCGGATGACTGGTTCG 3′390 rev 5′ tggatccttaTGCTGGTGGATTCGCCC 3′440 rev 5′ tResulting PCR fragments were digested with BamHI and cloned in pGBT9 vector digested by SmaI and BamHI.To generate CP190 BTB, we digested pGBT9 CP190_1–166 or pGAD424 CP190_1–166 with BamHI and NcoI, then filled in with Klenow fragment and self-ligated plasmids.To generate hKaiso BTB, we used primers:hK-dir 5′ cATGGAGAGTAGAAAACTGATTTCTG 3′ggatccttaGCGCTGTACCTGAGATGC 3′hK-rev 5′ tResulting PCR fragments were digested with BamHI enzyme (underlined) and cloned in pGBT9 or pGAD424 vectors digested by SmaI and BamHI.Transgene line expressing full length CP190 protein was described previously .To generate hKaiso-BTB CP190 derivative, hKaiso-BTB was amplified with primeractagtATGGAGAGTAGAAAACTGATTTCTG 3′, including recognition site for SpeI enzyme (underlined) and 5′ GCTGTACCTGAGATGCTTTTAAC 3′ primer. Resulting PCR product was digestated with SpeI and cloned in pUbi-CP190-attB vector digested by NcoI followed by fill in with Klenow fragment and SpeI.5′ ttTo generate CP190 derivative with SV40 NLS positioned in 210–245 aa deletion, 5′ and 3′ sequences of SV40 NLS were introduced in 5′ ends of primers with recognition site for BsaI :ggtctcaCTTCTTGGGcagctgctcg aatggtgacgtt 3′CP190-NLS1-R 5′ ttggtctcag aagAAGAGGAAGGTGgaggtt aagg aattcgctgag 3′.CP190-NLS2-F 5′ ttAdditional two primers with recognition site for NcoI and AgeI enzymes were used (recognition sites underlined):ccatggag aa 3′CP190-NcoI-F 5′ gcaccgtcgcaaccggttgtc 3′CP190-AgeI-R 5′ ttccctggttpUbi-CP190-attB [PCR products from CP190-NcoI-F/CP190-NLS1-R and CP190-AgeI-R/CP190-NLS2-F reactions were purified and digested with BsaI followed by ligation. Ligated product was used as matrix for second round of PCR with CP190-NcoI-F/CP190-AgeI-R primers. Resulting product was digested with NcoI and AgeI and ligated in 190-attB  vector dpUbi-CP190-attB vector digested by BamHI followed by fill in with Klenow fragment and BstEII.To generate CP190 derivative with 308–373 aa deletion, we digested PCR product generated by 5′ AAAATCTGCTCCAGCGACAGT 3′ and 440 rev 5′ tggatccttaTGCTGGTGGATTCGCCC 3′ primers by BstEII, and cloned it in ggcgcgccaATGGGTGAAGTCAAGTCC 3′ primer with introduced recognition site for AscI enzyme and different specific primers with introduced recognition site for NotI enzyme (recognition sites underlined):To generate CP190 derivative 1–1096 aa, 1–755 aa, 1–590 aa, 1–470 aa, 1–430 aa, and 1–309 aa tested in interband organization, we performed several PCRs with common 5′ atgcggccgcttatagctcctccttcgccg 3′1096_rev 5′ tgcggccgcgTTCCAGGTTGTCAATGGAC 3′755_rev 5′ aagcggccgcgCTTGTGGTAGCTCTTCATGTG 3′590_rev 5′ aagcggccgcgCAGGGCCCAGTAGTATTC 3′470_rev 5′ aagcggccgcgACCGGTTGTCTGCGAGGTT 3′430_rev 5′ aagcggccgcgCATGTTCTAGTTGGGTCTGTG 3′309_rev 5′ aahsp70-GAL4DBD-MYC [Resulting PCR products were digested with AscI and NotI and cloned in 4DBD-MYC  digestedDrosophila strains were grown at 25 °C under standard culture conditions. The transgenic constructs were injected into preblastoderm embryos using the φC31-mediated site-specific integration system [1w1118y flies, and the progeny carrying the transgene were identified by y+ pigmented cuticle. Details of the crosses are available upon request.n system . The emeThe Y2H assay was performed with plasmids and protocols from Clontech . Yeast strain PJ69-4a  gal80(deleted) LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) was used. Yeast clones from fresh YPD  agar plate were inoculated into 0.5 mL YPD medium, incubated overnight with shaking at 30 °C and 250 r/min. Then, overnight culture was diluted ten times in new flask and grow in YPD medium until A600 0.4–0.6. Next, cells were precipitated 1000 g 5 min RT follow by supernatant discarding. Then, cells were resuspended in 25 mL of sterilized water, washed, and collected by centrifugation in the same conditions. Cells were resuspended in 1 mL fresh sterile 100 mM LiAC. These cells’ suspension was gently resuspended with 0.1 μg mix of BD/X and AD/Y plasmid previously prepared into each tube. Appropriate volume of 50% PEG 4000/100 mM LiAc was added following by gentle mix and incubation 30 °C, 200 r/min, with shaking for 0.5 h. Heat shock was performed for 15 min at 42C followed by 5 min on ice incubation. Then, cells were precipitated 2 min at 4000 g at room temperature. Resuspended in sterilized water and plated on first selective media , 2% glucose, 1.5 agar) with addition of all essential amino acids except tryptophan and leucine. After 3 days of growth at 30 °C, the cells were plated on the same selective media without tryptophan, leucine, histidine, and adenine, and their growth was compared after 2–3 days. Each assay was repeated three times.Chromatin was prepared from the third instar larvae stage, and the resulting chromatin preparation was used for ChIP experiments, as described previously . ImmunopAntibodies to Su(Hw), CP190, dCTCF, Z4, and Chriz were generated and described previously ,103,104.Protein extracts were prepared by homogenization of 15 third-instar larvae or 25 flies in 100 μL of hot 60 mM Tris-HCl (pH = 6.8), 4% SDS, 10% glycerol, and 0.7 M β-mercaptoethanol, followed by boiling for 10 min. Debris was precipitated by centrifugation and 25 μL of supernatant was subjected to 7% polyacrylamide gel electrophoresis . ProteinDrosophila 3rd instar larvae were cultured at 18 °C under standard conditions. Polytene chromosome staining was performed as described [escribed . The fol