Umbilical cord-derived mesenchymal stem cells (UCMSCs) have been illustrated for their roles in immunological modulation and tissue regeneration through the secretome. Additionally, culture conditions can trigger the secretion of extracellular vesicles (EVs) into extracellular environments with significant bioactivities. This study aims to investigate the roles of three EV sub-populations released by UCMSCs primed with transforming growth factor β (TGFβ) and their capacity to alter dermal fibroblast functions for skin aging. Results show that three EV sub-populations, including apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXs), were separated from conditioned media. These three EVs carried growth factors, such as FGF-2, HGF, and VEGF-A, and did not express noticeable effects on fibroblast proliferation and migration. Only EX from TGFβ-stimulated UCMSCs exhibited a better capacity to promote fibroblasts migrating to close scratched wounds than EX from UCMSCs cultured in the normal condition from 24 h to 52 h. Additionally, mRNA levels of ECM genes  were detected with lower levels in fibroblasts treated with EVs from normal UCMSCs or TGFβ-stimulated UCMSCs compared to EV-depleted condition. On the contrary, the protein levels of total collagen and elastin released by fibroblasts were greater in the cell groups treated with EVs compared to EV-depleted conditions; particularly elastin associated with TGFβ-stimulated UCMSCs. These data indicate the potential roles of EVs from UCMSCs in protecting skin from aging by promoting ECM protein production. Skin aging is gradual progress caused by internal and external factors. Degradative activities happen in both cells and proteins that are components of the extracellular matrix (ECM), which are important for skin morphology and function ,2. DurinRecent studies have demonstrated that exosomes from mesenchymal stem cells (MSCs) promoted skin regeneration and rejuvenation, had anti-aging/wrinkle effects, and inhibited pigmentation in the skin ,8. ExosoTransforming growth factor β (TGFβ), which is a multifunctional cytokine, is a key regulator of ECM assembly and remodeling . Specifiv/v) in T75 cell culture flasks  surface-coated with CTSTM CELLstartTM substrate  under 5% CO2 and at 37 °C. Cells were split when they reached 80% confluency using CTSTM TrypLETM Select Enzyme .Primary umbilical cord-derived mesenchymal stem cells (UCMSCs) were supplied by the EV group (Vinmec Center for Applied Sciences and Regenerative Medicine). Cells were expanded to passage five in DMEM/F12  supplemented with 10% FBS  (2) with EV-depleted culture media (DMEM/F12 supplemented with 10% EV-depleted FBS). EV-depleted FBS was prepared by centrifuging FBS at 120,000× g/18 h/4 °C to remove FBS EVs. The UCMSCs were designated as the control when UCMSCs were cultured in normal conditions, and as the cytokine treatment when UCMSCs were treated with 10 ng TGFβ/1 mL culture media for two days. After two days of incubation, UCMSCs from both groups reached 80% confluency, and the conditioned medium was collected for EV isolation.At P5, UCMSCs were seeded in T75 flask , microvesicles (MV), and exosomes (EX), were collected sequentially at 2000× g/20 min/4 °C for AB, 16,500× g/30 min/4 °C for MV, and at 100,000× g/90 min/4 °C for EX . All EVs were resuspended in PBS and stored at −80 °C for further use.Conditioned media collected from cell culture were centrifuged 300× g for 20 min at 4 °C. The supernatant was aspirated and transferred to a new 1.5 mL centrifuge tube and stored at −20 °C for further experiments. Protein concentration was determined using the PierceTM BCA Protein Assay kit  and an optical densitometry method  at 560 nm.A volume of each EV population was mixed with an equivalent amount of RIPA buffer , incubated for 30 min at 4 °C, and then centrifuged at 16,000× TM, GE Healthcare Life Science, Piscataway, NJ, USA) at 200 mA for 2 h at 4 °C. After that, membranes were incubated with primary antibodies diluted in TBST solution for anti-CD9 (1:100), anti-CD63 (1:200), and anti-GAPDH (1:100)  overnight at 4°C. Then, primary antibodies were washed before being incubated with secondary antibody Mouse IgG . Antibody binding was detected by ECL chemiluminescent substrate  and imaged on an ImageQuant LAS 500 .Total exosome proteins (15 μg/well) were separated using a 4–12% NuPAGE gel  at 200 V for 60 min at 4 °C prior to being transferred to a PVDF membrane . Samples were then washed before being stained. The EVs were dried at room temperature and photographed by a Transmission Electron Microscopy JEOL 1100  at 80 kV at the National Institute of Hygiene and Epidemiology (NIHE).TM Multiplex Immunoassays . Frozen EV suspension was thawed and kept on ice for sample preparation, following the manufacturer’s instruction. The luminescent signal was detected using a LuminexTM 100/200TM system with xPONENT 3.1 software .Growth factors such as epidermal growth factor 2 (EGF-2), hepatocyte growth factor (HGF), and vascular endothelial growth factor A (VEGF-A), were measured by the Luminex assay using ProcartaPlex2 for 48 h before proliferation analysis using a 3--2,5-diphenyl tetrazolium bromide (MTT) assay kit . Step by step analysis was performed following the protocol described by the manufacturer. The cell proliferation rate was measured equivalent to the absorbance at 560 nm  at 0 h and 48 h.Human dermal fibroblasts were seeded into a 96-well plate (2500 cells/well) with EV-depleted media (5% EV-depleted FBS in DMEM/F12) containing 10 μg/mL EVs  derived from (1) normal UCMSC culture, or (2) TGFβ-stimulated UCMSCs. The EV-depleted medium was used as the negative control (No-EV). Cells were incubated at 37 °C and 5% COHuman dermal fibroblast cells were seeded at 150,000 cells/well in a 24-well plate to achieve 100% confluency. After achieving cell adhesion, cell proliferation was inhibited by using Mitomycin C (10 μg/mL) for 2 h before making a scratch with a sterile 100 μL tip. Cells were treated with EV-depleted media (5% EV-depleted FBS in DMEM/F12) containing 10 μg/mL EVs  from (1) normal UCMSC cultures, or (2) TGFβ-stimulated UCMSCs. The EV-depleted medium was used as the negative control (No-EV). Cell migration images were observed and captured by inverse microscopy  with 10× magnification for different time points. The rates of cell migration to close the wounded areas were analyzed using ImageJ software (Version 1.52P) .® ND-1000 spectrophotometer. cDNA synthesis was performed using the SuperScript™ IV First-Strand Synthesis System Kit . The cDNA samples were amplified by real-time PCR using the Power SYBRTM Green PCR Master Mix  on an Applied Biosystems 7500 Real-Time PCR system with parameters of 3 min for amplification, and 40 cycles . Specific oligonucleotide primers were used to detect collagen type I (COL I), COL III, hyaluronic acid synthase 2 (HAS II), HAS III, elastin, and GDPDH mRNA . Purity was then quantified using a NanodropPDH mRNA .2 for 48 h before processing according to the manufacturer’s instructions. Briefly, cells were released using CTSTM TrypLETM Select Enzyme  and maintained in 300 μL of supernatant after centrifugation. Subsequently, an equal volume of Elastin Precipitating Reagent was added to each sample tube. The supernatant was discarded after centrifugation, and the pellets were resuspended in 1 mL Dye Reagent and incubated for 90 min. Pellets were then collected and resuspended in 250 μL of Dye Dissociation Reagent. A volume of 100 μL of suspension was transferred to a 96-well plate for reading OD at 513 nm using spectrometer SpectraMax M3 .The secretion of elastin by fibroblasts was determined using a Fastin™ Elastin Kit . Cells were seeded in a 12-well plate with 100,000 cells/well density and maintained in different experimental settings similar to the proliferation and migration assay. Cells were incubated at 37 °C in 5% COTotal collagen secreted by fibroblasts was quantified using a Sirius Red Total Collagen Detection Kit . Briefly, cells were seeded in a 6-well plate at a density of 32,000 cells/well and maintained in different experimental conditions similar to the proliferation and migration assay. After reaching 90% confluency, cells were collected and processed as recommended by the manufacturer. A volume of 100 μL of Sirius Red Solution was added to the sample tubes, and pellets were collected after centrifugation. The pellets were rinsed once with washing solution and then resuspended in 250 μL of extraction buffer. The solution was transferred to a 96-well plate for reading OD at 510 nm using a spectrometer .In order to stimulate UCMSCs to secrete EVs into the extracellular environment, we incubated UCMSCs with TGFβ (10 ng/mL cell culture media). After two days of cytokine incubation, conditioned media was collected for EV isolation. At the time of conditioned media collection, UCMSCs primed with TGFβ maintained the typical morphology of MSCs with fibroblast-like shapes A.Additionally, three EV sub-populations were separated, including ABs, MVs, and EXs. These vesicles were investigated for their markers, such as CD9, CD63, and internal control of GAPDH. The expression of GADPH and CD9 occurred in all three EV sub-populations, while CD63 was detected in only the EX fraction B. RegardTo investigate growth factors that affect healthy skin structure, we quantified growth factors of FGF-2, HGF, and VEGF-A using the Luminex assay. Results showed that in the AB population, there was no difference among the three factors analyzed in the same conditions, or the two conditions of normal UCMSCs and TGFβ-stimulated UCMSCs A. In thep < 0.001) . The EV-depleted fibroblast culture media was used as the negative control (NoEV). Proliferation results showed that EVs, including ABs, MVs, EXs, from TGFβ-stimulated UCMSCs seemed to have a higher capacity to induce fibroblast proliferation, but this was not significant compared to the normal EV (CT group) and negative control (NoEV). Only EXs from normal UCMSC culture (CT group) expressed higher capacity to promote dermal skin fibroblast proliferation than in the NoEV group (< 0.001) A.p < 0.01 and p < 0.0001), and EXs from TGFβ-stimulated UCMSCs showed a greater induction of cells migrating to close the wounds compared to the EXs from normal UCMSCs (p < 0.05) (p < 0.05 and p < 0.001)  expressed a greater healing rate compared to ABs from TGFβ-stimulated UCMSCs at 24 h A,B. In t < 0.05) D. At 44 < 0.001) D. HoweveAs collagens (type I and III), elastin and hyaluronic acid are the main factors responsible for tensile strength, elasticity, and smooth skin, we investigated mRNA expression levels of collagen type I (COL I), COL II, elastin, and hyaluronan synthase type II (HAS II), and HAS III under the induction of EVs from TGFβ-stimulated UCMSCs, normal MSCs (the control), and negative control (NoEV) samples. Results show that the expression of all genes, including COL I, COL III, Elastin, HAS II, and HAS III, was lower in fibroblasts treated with EVs from both normal UCMSCs and TGFβ-stimulated UCMSCs compared to the negative control (NoEV–fibroblasts treated with EV-depleted culture media) . AdditioTogether with mRNA level analysis of skin structure proteins, we examined the protein production of these genes by fibroblasts. Regarding protein production induced by EVs, we investigated the secretion of elastin and total collagen by dermal skin fibroblasts under three conditions: (1) EVs from TGFβ-treated UCMSCs, (2) EVs from normal UCMSCs, and (3) NoEV (EV-depleted media). Results indicate that there was no difference in the number of proteins detected from three EV sub-populations from TGFβ-stimulated UCMSCs and normal UCMSCs A,B. ThisIt has been reported that priming culturing cells with chemical agents can generate EVs with particular functions . This stHow these EVs can affect dermal fibroblasts is an important question concerning their use as potential agents for cutaneous wound healing and prevention of skin aging. We examined dermal fibroblast activities within many aspects of proliferation, migration, mRNA and protein expression. EVs from both normal UCMSCs and TGFβ-stimulated UCMSCs did not show a precise proliferation rate of fibroblasts A. This wAs mentioned, collagen, elastin, and hyaluronic acid are important components required for a young skin appearance. We quantified mRNA levels of these proteins in order to understand which EV populations, such as ABs, MVs, or EXs, are able to promote gene expression. Surprisingly, no EVs promoted the expression of COL I, COL III, Elastin, HAS II, and HAS III in fibroblasts , even thIn summary, we found that TGFβ-stimulated UCMSCs released three EV sub-populations, including ABs, MVs, and EXs, carrying FGF-2, HGF, and VEGF-A. In general, these cytokine-stimulated EVs did not affect fibroblast proliferation and migration, but cytokine-stimulated EXs showed a greater capacity to induce fibroblast migration. Even though gene expression of cutaneous ECM components was not altered by EVs, total collagen and elastin were increased by the stimulation of EVs from TGFβ-stimulated UCMSCs. Data from this study are important in the further development of EVs released by UCMSCs and TGFβ-stimulated UCMSCs for skin rejuvenation.


Alphavirus and is transmitted to humans by infected Aedes aegypti and Aedes albopictus bites. In humans, CHIKV usually causes painful symptoms during acute and chronic stages of infection. Conversely, virus–vector interaction does not disturb the mosquito’s fitness, allowing a persistent infection. Herein, we studied CHIKV infection of Ae. aegypti Aag-2 cells (multiplicity of infection (MOI) of 0.1) for 48 h through label-free quantitative proteomic analysis and transmission electron microscopy (TEM). TEM images showed a high load of intracellular viral cargo at 48 h postinfection (hpi), as well as an unusual elongated mitochondria morphology that might indicate a mitochondrial imbalance. Proteome analysis revealed 196 regulated protein groups upon infection, which are related to protein synthesis, energy metabolism, signaling pathways, and apoptosis. These Aag-2 proteins regulated during CHIKV infection might have roles in antiviral and/or proviral mechanisms and the balance between viral propagation and the survival of host cells, possibly leading to the persistent infection.Chikungunya virus (CHIKV) is a single-stranded positive RNA virus that belongs to the genus  Alphavirus, family Togaviridae  is an Old World mosquito-borne virus that belongs to the genus aviridae . This viorldwide . The worlanguage  since CHlanguage . Howeverlanguage . The laclanguage . Furtherlanguage . In Brazlanguage .CHIKV single-stranded positive RNA genome has approximately 11.8 kb, with polyA-tail and 5′ cap, and it exists as a single copy in each virion. It encodes two polyproteins that are cleaved in the course of infection by host and viral proteases . This geDengue virus 2 (DENV2) entrance in Ae. aegypti and Ae. albopictus cells  organized in a T = 4 geometry . Spikes Aedes spp. mosquitoes. Previous studies have addressed the proteomics of whole Ae. aegypti mosquitoes and their specific organs, such as midgut and salivary glands, using two-dimensional electrophoresis and mass spectrometry (MS) . Proteomics and microscopy data revealed several regulated proteins that are possibly involved in host cell metabolic shifts that could assist viral replication.Historically, it has been extremely difficult to perform control of arbovirus vectors as a mitigation strategy . Thus, itry (MS) . The knoAe. aegypti Aag-2 cells (RRID : CVCL_Z617) were kindly provided by Gorben Pijlman, PhD  and cultured in Schneider’s medium  supplemented with 10% fetal bovine serum (FBS) and 100 U/ml of penicillin/streptomycin at 28°C. Ae. albopictus C6/36 cells were maintained in TC-100 medium  supplemented with 10% FBS at 28°C. African green monkey kidney-derived Vero cells were grown in high-glucose Dulbecco’s modified Eagle‘s medium  supplemented with 10% FBS and 100 U/ml of penicillin/streptomycin in an incubator at 37°C under 5% CO2. Cell passages were performed once a week. The CHIKV isolate  accession number MH823667) was supplied by the Central Public Health Laboratory of the Federal District (LACEN-DF).Ae. albopictus C6/36 cells were infected with virus-containing supernatant from Vero culture to increase the virus titer, since these mosquito cells have a defective antiviral RNAi machinery, being advantageous for arbovirus propagation  during CHIKV infection. Three days postinfection (dpi), the onset of CPE was noted, and the supernatant was collected. Viral RNA was extracted from Vero cells using TRIzol , and replication was confirmed by RT-PCR. SuperScript IV reverse transcriptase (Thermo Fisher Scientific) was used for cDNA synthesis, while PCR was performed with Platinum Taq DNA polymerase (Thermo Fisher Scientific). The primers forward 5′-CGAAGAGTGGAGTCTKGCATYCCAG-3′ and reverse 5′-GCCTCYTGGTATGTGGCCGCTTTAGC-3′ amplified the set of E3–E2 genes, generating an amplicon about 1.5 kb. Then, pagation . C6/36 c−1 to 1 × 10−9) of virus stocks in a 1:1 volume ratio (90 µl cell suspension:90 µl virus dilution). In 60-well Terasaki plates , 10 µl per well was added in 6 replicates per dilution. The CHIKV titration was achieved based on CPE visualized by light microscopy at 3 dpi and TCID50 calculation. Each condition was titrated twice, and the mean number of viral particles was considered.Viral titer was obtained by End-Point Dilution Assay in Terasaki plates. Confluent Vero cells grown in a T25 flask were detached with 1 ml of trypsin-EDTA and diluted in 4 ml of DMEM supplemented with 10% FBS and 100 U/ml of penicillin/streptomycin. The cells in suspension were incubated with serial dilutions  of 0.1 and 1 were used to infect Aag-2 cells in 6-well plates in biological triplicates. After 90 min, the supernatant was replaced with fresh Schneider’s medium supplemented with FBS and 100 U/ml of penicillin/streptomycin. The CHIKV-infected supernatants were titrated at 24, 48, and 72 hpi by End-Point Dilution Assay. The virus growth curve was set, and the harvesting time points 0, 12, and 48 hpi were determined for the following experiments, with a chosen MOI of 0.1.g for 3 min. After two rounds of phosphate-buffered saline (PBS) washing, pellets were transferred to microtubes, and fixation occurred overnight at 4°C in a solution containing 2% (v/v) paraformaldehyde, 2% (v/v) glutaraldehyde, and 0.1 M of sodium cacodylate buffer, pH 7.2. Next, whole preparations were post-fixed, for 30 min in 2% (w/v) osmium tetroxide, 1.6% potassium ferricyanide, and 10 mM of CaCl2 in 0.2 M of sodium cacodylate buffer, pH 7.2. Samples were washed in distilled water and stained in a block with 0.5% (w/v) uranyl acetate for 24 h at 4°C. The material went through dehydration in a graded acetone series (50%–100%) for 10 min each, and it was embedded in Spurr resin. Ultrathin sections were obtained using an ultramicrotome UC6  that were contrasted with uranyl acetate and lead citrate. The final step was to examine and photograph the sections under a Jeol 1011 transmission electron microscope  at 80 kV.Aag-2 cells were grown in three independent T25 flasks and cultured in Schneider’s medium (Sigma-Aldrich) supplemented with 10% FBS and 100 U/ml of penicillin/streptomycin at 28°C. Each flask was harvested at a different time point: 0 (uninfected control), 12, and 48 hpi. The samples were transferred to 15-ml tubes and centrifuged at 1,200 ×6 Aag-2 cells were seeded. After 16 h, cells were infected with CHIKV stock at MOI of 0.1 in biological triplicates. Mock cells (with Schneider’s medium only) were harvested at t = 0 h (uninfected), and CHIKV-infected cells were harvested at 12 and 48 hpi. Aag-2 pellets were lysed with 8 M of urea in 20 mM of ammonium bicarbonate, pH 7.9, containing a complete mixture of protease and phosphatase inhibitors . Then, 100 μg of protein was reduced with 5 mM of dithiothreitol (DTT) for 1 h at 32°C and alkylated with 14 mM of iodoacetamide for 40 min at room temperature in the dark. Samples were then applied to a filter  and centrifuged for 10 min at 14,000 ×g. Next, a washing step was performed with 8 M of urea followed by centrifugation for 10 min at 14,000 ×g. A total of 2 μg of modified trypsin  was used for tryptic digestion following the proportion ratio of 1:50 (enzyme:substrate) for 18 h at 37°C under 600-rpm agitation. Tryptic peptides were acidified by adding trifluoroacetic acid (TFA) to a final concentration of 0.1% (v/v), desalted with POROS R2 resin , and packaged in micropipette tips. Desalted peptides were vacuum dried and suspended in 0.1% (v/v) formic acid (FA), and aliquots corresponding to 0.5 µg/µl were used for MS analysis.In T25 flasks, 2 × 10m/z 200 and automatic gain control (AGC) set to 3 × 106). The 20 most abundant precursor ions from each scan  were sequentially subjected to fragmentation by higher-energy collisional dissociation (HCD). Fragment ions were analyzed at a resolution of 15,000 using an AGC set to 1 × 105. Data were acquired using Xcalibur software (version 4.2.47).Tryptic digests were analyzed by reversed-phase nanoscale liquid chromatography coupled to high-resolution nanoelectrospray ionization MS. Chromatography was performed using a Dionex Ultimate 3000 RSLCnano system coupled to the HF-X Orbitrap MS (Thermo Fisher Scientific). All samples (1 μg per run) were initially applied to a 2-cm guard column, followed by fractionation on a 25.5-cm PicoFrit™ Self-Pack column (New Objective) packed with 1.9-μm silica, ReproSil-684 Pur 120 Å C18-AQ . Each sample was loaded in 0.1% (v/v) FA and 2% (v/v) acetonitrile (ACN) onto the trap column at 2 μl/min, while chromatographic separation occurred at 200 nl/min. Mobile phase A consisted of 0.1% (v/v) FA in water, while mobile phase B consisted of 0.1% (v/v) FA in ACN. Peptides were eluted with a linear gradient from 2% to 40% eluent B over 32 min, followed by up to 80% B in 4 min. Lens voltage was set to 60 V. Full-scan MS mode was acquired with a resolution of 60,000 (FWHM at Ae. aegypti (UP000008820), with one protein sequence per gene, were loaded into MetaMorpheus. Both FASTA files were downloaded in May 2021. Then, calibration was performed with default values, with file-specific tolerances set to each MS run. Next, global post-translational modification discovery (G-PTM-D) was used to search for common biological, common artifacts, and metal modifications in the files. Identification and quantification were done allowing a maximum of two missed cleavages, two modifications per peptide, and a peptide minimum length of 7. Carbamidomethylation of cysteine and selenocysteine was set as fixed modifications. Oxidation of methionine was set as a variable modification. Protein parsimony was applied, two peptides were required to identify a protein group, and modified peptides were treated as different peptides to remove ambiguities in the quantification of proteins. Quantification was performed without match between runs (MBR), and results were normalized by FlashLFQ. Peptide-spectrum matches (PSMs) were considered valid if the q-value <0.01 and the MetaMorpheus minimum score was set to 5.Protein quantification and identification were performed by using MetaMorpheus , which tpost-hoc Bonferroni multiple test correction with a p-value <0.05 cutoff were used to identify proteins and peptides regulated between groups. CHIKV peptides were related to the mature proteins through a comparison of their position in the CHIKV protein sequence and annotation of the structural polyprotein (Q8JUX5) of the CHIKV strain S27-African prototype. Variance-sensitive clustering through VSClust  network was constructed by using STRING , also using MOI of 1. As a negative control, we used mock-infected Vero cells. From these assays, we could notice that our CHIKV isolate was more virulent than the other arboviruses, producing higher levels of CPE (syncytia formation) and cell death  rather than at 24 hpi (2.32 × 104/ml) and at 72 hpi (1.7 × 104/ml) , 12, and 48 hpi.We determined the times 24, 48, and 72 hpi to harvest supernatant and perform viral titration. As expected, the growth kinetics showed a higher CHIKV particle production at all harvesting times using MOI of 0.1, which was chosen for the next steps. Supernatant titration showed a slightly higher viral particle production at 48 hpi (2.86 × 10 104/ml) Figure 2 104/ml) . AccordiTEM analysis was performed using the established MOI of 0.1 and the harvesting time points 0, 12, and 48 hpi, to visually support the biological effects of CHIKV infection. Uninfected Aag-2 cells (Control) presented a well-defined morphology, with apparently intact mitochondria, nucleus, autophagosome vesicle, and a structure that appears to be a Golgi complex Figure 2A total of 2,583 protein groups with at least two unique peptides were identified  and structural (Q8JUX5) polyproteins. None of them were found to be regulated. Both polyproteins are proteolytically cleaved along the viral infectious cycle into non-structural (nsP1–4) and structural proteins  . RegardiVolcano plot Figure 6Regulated proteins at 48 hpi compared to Control Figure 6de novo synthesis of virions could explain the progressive detection of viral structural components over 48 h , and the enrichment analysis of the KEGG pathway ribosome gene set is enriched with negative level change at 48 hpi . On the other hand, measles virus, rabies virus, and foot-and-mouth disease virus target eukaryotic initiation factor 3 (eIF3), also resulting in host protein translation inhibition  . RPs mayt 48 hpi Figure 6t 48 hpi . Despitet 48 hpi . Moreovehibition . In the Ae. aegypti ATP synthesis-related proteins with an elevated abundance at 48 hpi (c (cyt c) as part of electron transport chain step of oxidative phosphorylation. This protein complex is essential for the respiratory chain functioning  have strongly diverged in structure and function . MitoribFlaviviridae members such as West Nile virus (WNV) and hepatitis C virus (HCV)  . In healus (HCV) . The virus (HCV) . It has us (HCV) . This imInfluenza-infected mammalian cells, a mitochondrial membrane disruption with a consequent decrease in ATP levels has been observed, requiring glycolysis activation  form a highly evolutionary conserved family of proteins that can act in different cell compartments, such as the plasmatic membrane, nucleus, and inner mitochondrial membrane, having specific site-associated functions . Moreovell types . A previll types . OverallIn addition to bioenergetic metabolism, mitochondria also play a central role in innate immune signaling and cell survival . The fisc, another compound well known to trigger apoptosis  to form a complex that activates caspase 9, which is cleaved into caspases 3 and 7 , A0A1S4EZS3 , and Q178X5 (FC 12 hpi vs. Control +0.49 and FC 48 hpi vs. Control +0.69). The rest of them were downregulated over time points: A0A1S4F0V4 (FC 12 hpi vs. Control −0.43 and FC 48 hpi vs. Control −0.71), Q16XX4 (FC 48 hpi vs. Control −0.62), Q16RY3 , A0A6I8TAB4 , and Q17CT5 (FC 48 vs. 12 hpi −0.91) . Recent Ae. aegypti DDX6 has antiviral properties against ZIKV and WNV. In both cases, DDX6 binds to the 3′ UTR subgenomic flavivirus RNA (sfRNA), inhibiting viral replication in Aag-2 cells . Moreover, the mitochondrial elongated morphology observed at 48 hpi may reflect a mitochondrial net imbalance that could affect energy metabolism and regulate apoptosis timing. It is widely accepted that different virus species modulate mitochondrial bioenergetics to enhance viral replication. As part of a central role in the viral replication and oxidative stress, the (dys)function of host mitochondria seems to be an essential element for many viruses , Coordenação de Aperfeiçoamento de Pessoal de Nível Superior , Conselho Nacional de Desenvolvimento Científico e Tecnoloígico , and Fiocruz (INOVA Program). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Cystophora cristata). Here, we studied antioxidant gene expression and enzymatic activity in the visual cortex, cerebellum and hippocampus of harp seals (Pagophilus groenlandicus) and hooded seals. Moreover, we tested several genes for positive selection. We found that antioxidants in the first line of defence, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione (GSH) were constitutively enhanced in the seal brain compared to mice (Mus musculus), whereas the glutaredoxin and thioredoxin systems were not. Possibly, the activity of the latter systems is stress-induced rather than constitutively elevated. Further, some, but not all members, of the glutathione-s-transferase (GST) family appear more highly expressed. We found no signatures of positive selection, indicating that sequence and function of the studied antioxidants are conserved in pinnipeds.While foraging, marine mammals undertake repetitive diving bouts. When the animal surfaces, reperfusion makes oxygen readily available for the electron transport chain, which leads to increased production of reactive oxygen species and risk of oxidative damage. In blood and several tissues, such as heart, lung, muscle and kidney, marine mammals generally exhibit an elevated antioxidant defence. However, the brain, whose functional integrity is critical to survival, has received little attention. We previously observed an enhanced expression of several antioxidant genes in cortical neurons of hooded seals ( Leptonychotes weddellii) and northern elephant (Mirounga angustirostris) seals endure arterial oxygen tensions well below 20mmHg   also represents a major challenge. ROS are radical or non-radical oxygen species produced by the partial reduction of oxygen. Mitochondria have been recognized as an important intracellular source of ROS, which can arise during oxidative phosphorylation that produces energy in the form of ATP. ATP production is accomplished by a tetravalent reduction of oxygen. In normal physiological conditions, 1–4% of the oxygen is incompletely reduced and leaks from the electron transport chain (ETC) in the form of superoxide radical (O2•–) . However2•–) . ROS pla2•–) . Acknowl2•–) .2•– is increased , and glutathione peroxidase (GPX) transforming H2O2 into H2O and limiting the hydroxyl radical (OH•) formation. Glutathione-S-transferase (GST) binds toxic products to glutathione and the resulting glutathione conjugates can then be removed from the organism , uric acid, melatonin, vitamins C and E and others . Promineorganism . Moreoveorganism .Trichechus manatus) and several cetacean species, have generally revealed higher antioxidant levels in diving compared to non-diving mammals (Tursiops truncatus) and the dwarf sperm whale (Kogia sima) (Cystophora cristata) and mice (Mus musculus), we previously observed a significantly higher expression of antioxidant genes in hooded seals (Bos taurus)  , the anted seals . Similar taurus) .Pagophilus groenlandicus), to investigate whether an increased antioxidant defence of the brain might be relevant in pinniped species other than the hooded seal. We studied antioxidant gene expression in the visual cortex, cerebellum and hippocampus of hooded seals, harp seals and mice. Further, we determined the enzymatic activity of SOD, GST, GPX and glutathione reductase (GSR) and the concentration of reduced glutathione (GSH) in these brain regions. We further tested a set of antioxidant genes in seven pinniped species for positive selection, to test whether selection pressure has favoured changes in gene sequences that might also lead to functional changes.In this study, we extend the results of via laser-capture microdissection of the visual cortex were available from via RNA-seq and the differential expression analysis were performed as described in p-values of differentially expressed genes were corrected for multiple testing using the false discovery rate (FDR)  ≥ 2 or ≤−2 were considered as significantly differentially expressed genes (DEGs). From these DEGs, genes of the GO terms “antioxidant activity” (GO:0016209) were taken from http://www.informatics.jax.org) and a list of human antioxidant genes (HMOX2) and Paraoxonase 2 (PON2), which are both known to be involved in the antioxidant defence  . Only gent genes . Additio defence .Cystophora cristata; n = 4 adult females, March 2019) and harp seals  were captured in the pack ice of the Greenland Sea under permits from relevant Norwegian and Greenland authorities. The hooded seals were euthanized immediately following live-capture, by sedation with an intramuscular injection of zolazepam/tiletamine (1.5–2.0 mg per kg of body mass), followed by catheterization of the extradural intravertebral vein and i. v. injection of an overdose of pentobarbital . The harp seals were all shot to the head and bled, after which brain tissue was immediately sampled from intact brain regions. For the repetition of the GST and GSH/GSSG assays that were performed at a later point in time, hooded seal tissues  were collected in March 2021, using the same procedure as described above for this species. All animal handling was in accordance with the Norwegian Animal Welfare Act and with approvals from the Norwegian Food Safety Authority (permits no. 12268 and 22451). Adult female mice  were a gift by Prof. Dr. Christian Lohr  and were anaesthetized with 1 ml isoflurane  in a chamber  and decapitated. All animals were handled according to the EU Directive 63 (Directive 2010/63/EU). This mouse strain has served as model organism in studies investigating oxidative stress, e.g., during aging  or genes that represent a component of important antioxidant systems . Only genes with a pFDR ≤ 0.05, TPM-value ≥1 in either species and a fold change (FC) ≥ 3 or ≤−3 in the transcriptomic data were considered for qPCR. Since we aim to determine the antioxidant defence of neurons, we used RBFOX3 as a neuronal marker to account for different numbers of neurons in every tissue sample. Primer sequences (https://www.ncbi.nlm.nih.gov/genbank/) and hooded seal sequences were extracted from via gel electrophoresis and/or sequencing.For expression analyses using qPCR, we selected essential antioxidant genes . The qPCR was performed with a 7,500 Fast Real-Time PCR System and the Power SYBR Green master mix  using a standard PCR protocol . Primer efficiencies  according to the manufacturer’s instructions, including an on-column DNA digestion with RNase-free DNase . The quantity and integrity of the isolated total RNA were assessed using the Agilent 4,200 TapeStation System and RNA ScreenTape Assay . First-strand cDNA was synthesized from 1 µg of total RNA with Oligo (dT)ciencies  were ass protein . Fold ch protein  and the  protein . Fold ch protein .2HPO4, 1.5 mM KH2PO4, pH7.4) to remove blood before assay-specific buffers were used for homogenization.For all assays, ∼20 mg of tissue from every brain region was washed twice in phosphate buffered saline (PBS) . Tissue samples (20 mg) were homogenized in 75 µL cold buffer  according to the manufacturer’s instructions. Total protein contents were measured with the Bradford assay  and adjusted with homogenization buffer to the sample with the lowest concentration (17.94 mg/ml). Of all adjusted samples a 1:5 dilution was prepared and used in the assay. Absorbance was read every minute at 340 nm using a DTX 880 Multimode Detector . The decrease in absorbance was measured for 25 min. The decrease was linear up until minute 10 and thus, the first 10 data points were used for statistical analysis and to calculate the GPX-activities (nmol/min/ml) according to the manufacturer’s instructions.The tissues samples (20 mg) were homogenized in 100 µL of cold 20 mM HEPES buffer . Total protein concentration was measured with the Bradford assay  and adjusted with HEPES buffer to the sample with the lowest concentration (1.97 mg/ml).All samples were further diluted 1:300 in HEPES buffer. The total SOD activity was measured with the Superoxide Dismutase Assay Kit  according to the manufacturer’s instructions. The absorbance was determined at 450 nm and SOD activities are expressed as U/ml.All samples were diluted 1:5 in HEPES buffer. The total GST activity was determined using the Glutathione S-Transferase Assay Kit  according to the manufacturer’s instructions. The absorbance was recorded every minute at 340 nm for 60 min and the linear range from minute 1 to 9 was used for calculation of the GST activities (nmol/min/ml). Measurement of the GST activity was repeated using a different GST assay kit  and newly sampled hooded seal tissues (March 2021). The tissue was homogenized in Assay buffer and the concentration of all samples was adjusted to 3.9 mg/ml. The assay was performed according to the manufacturer’s instruction using a 1:5 dilution of the samples.The tissues samples (20 mg) were homogenized in 100 µL of cold buffer . Total protein concentration was measured with the Bradford assay  and adjusted with Sample buffer  to the sample with the lowest concentration (2.3 mg/ml). The GSR activity was measured according to the manufacturer’s instructions using the Glutathione Reductase Assay kit . The absorbance was read at 340 nm once every minute for 6 min and GSR activity was calculated in nmol/min/ml.The ratio of reduced glutathione (GSH) and oxidized glutathione (GSSG) was determined using the Amplite Fluorometric Glutathione GSH/GSSG Ratio Assay Kit Green Fluorescence . Tissues were homogenized in HEPES buffer, protein concentrations adjusted to 5.2 mg/ml and diluted 1:50 in Assay buffer. The assay was run according to manufacturer’s instructions. The results were verified at a later point in time using a different assay, the GSH/GSSG Ratio Detection Assay Kit , and newly sampled hooded seal tissues from March 2021. Tissues were homogenized in 1xPBS (pH = 6) with 0.5% NP40 . Protein concentrations were adjusted to 3 mg/ml and samples were diluted 1:50 in Assay buffer. Before both assays were run, samples were deproteinized with the ReadiUse™ TCA Deproteinization Sample Preparation Kit .p-values were corrected with the False Discovery Rate , the Weddell seal (Leptonychotes weddelli), the southern elephant seal (Mirounga leonina), the Hawaiian monk seal (Neomonachus schauinslandi), the gray seal , the harbour seal (Phoca vitulina) and the walrus (Odobenus rosmarus divergens) were compared with the dog (Canis lupus familiaris), ferret (Mustela putorius furo), giant panda (Ailuropoda melanoleuca), grizzly bear (Ursus arctos horribilis) and the polar bear (Ursus maritimus). The nucleotide sequences were retrieved from GenBank  providing a peptide alignment generated in MAFFT   . In bothFDR ≤ 0.05, a TPM-value ≥1 in either species and a fold change (FC) ≥ 2 or ≤−2. We extracted the antioxidant gene expression values from the cell-type specific transcriptome of visual cortex neurons in hooded seals and mice . We founGPX1, FC = 2.2) and 3  and the glutathione-S-transferase kappa 1 , omega 1 , omega 2  and the microsomal glutathione-S-transferase 1 .Of the 49 genes, 28 were more highly expressed in hooded seal compared to mouse neurons. The annotated GO term “glutathione peroxidase activity” was represented by eight of these 28 genes and thus, was the most frequently represented term. For example, genes assigned to this function were the glutathione peroxidase 1 , superoxide dismutase 1 , peroxiredoxin-like 2 A , peroxiredoxin 2  and selenoprotein W . Further, within antioxidant genes, SOD1 was among the top five genes with the highest TPM value (TPM = 481) in hooded seal neurons, followed by PRDX2 (TPM = 388), GPX1 (TPM = 140), heme oxygenase 2  and glyoxalase 1 . Even when all genes (not only antioxidants) in the transcriptome were considered, SOD1 had a high expression, being the gene with the 22nd highest TPM value. Antioxidant genes with the top five F C were S100A9 (FC = 99.7), GSTK1 (FC = 26.9), arachidonate 5-lipoxygenase activating protein , selenoprotein T  and GSTO1 (FC = 15.1).The GO term “antioxidant activity” was represented by six genes and all of them were more highly expressed in neurons of hooded seals compared to mice. Among these genes were S100 calcium binding protein A9 , although the difference in expression was not always significant (GPX3 and TXNRD3). As in the transcriptomic data, GLRX2, was less expressed in hooded seals than in mice (pGLRX2 = 0.002). Similarly, expression in harp seals was significantly  or insignificantly higher  and insignificantly lower for GLRX2.In the visual cortex, expression of all six antioxidants confirmed the transcriptomic data, i.e. the expression was higher in hooded seals than in mice (pp = 0.0008) and harp seals (p = 0.0087) than in mice. There were no significant differences in the expression of the other antioxidants between species.In the cerebellum, GLRX2 was less expressed in hooded seals  and harp seals  with exception of an insignificantly higher expression of TXNRD3 in hooded seals.In the hippocampus, we observed no difference in GLRX2 expression. However, the other antioxidants were all significantly more highly expressed in hooded seals (pp = 0.003) and 45.9 ± 1.6 U/ml (p = 0.006) in the harp and hooded seal, respectively, while mice showed a mean activity of 37.3 ± 1.1 U/ml. The cerebellum exhibited SOD activities similar to the visual cortex, with harp and hooded seals reaching 47 ± 2.1 U/ml (p = 0.006) and 47.7 ± 3 U/ml (p = 0.003) compared to mice, which showed a mean activity of 37.6 ± 1.2 U/ml. The hippocampus displayed the overall lowest SOD activity levels, with 46.4 ± 2.4 U/ml (p ≤ 0.001) and 40.4 ± 2.6 U/ml (p = 0.007) in harp and hooded seals, respectively, and 32.3 ± 0.6 U/ml in mice.In all three brain regions tested , SOD activity was significantly higher in hooded seals and harp seals than in mice ; Table 2p = < 0.001) and 355 ± 37.2 nmol/min/ml (p = < 0.001), respectively. Across species, the cerebellum displayed the highest GPX activity, with mice reaching 385 ± 22.1 nmol/min/ml, while hooded seals displayed a significantly higher activity of 504 ± 37.1 nmol/min/ml (p = 0.003) whereas the highest activity of 610 ± 17 nmol/min/ml was noted in the harp seal cerebellum (p < 0.001). In contrast, the hippocampus showed the overall lowest GPX activity, where mice had a lower rate (162 ± 11.2 nmol/min/ml) than either the hooded seal or the harp seal (309 ± 22.2 nmol/min/ml (p = < 0.001) and 376 ± 39.4 nmol/min/ml (p = 0.001), respectively). With exception of the visual cortex, harp seals displayed the overall highest GPX activity levels, even compared to hooded seals, in both the cerebellum (p = 0.007) and the hippocampus (p = 0.023).The total GPX activity was significantly higher in hooded seals and harp seals than in mice across brain regions ; Table 2p = 0.03), while hooded seals showed significantly lower activity , compared to mice (64 ± 3.1 nmol/min/ml). In the cerebellum, harp seals  and hooded seals , had significantly lower enzyme activities than mice (150 ± 1.6 nmol/min/ml). In the hippocampus, we found no difference between harp seals (65 ± 7 nmol/min/ml) and mice (65 ± 0.6 nmol/min/ml), while hooded seals showed significantly lower GSR activities .The results of the GSR enzymatic activity assay were mixed, but overall pinnipeds displayed a lower activity than mice ; Table 2The GST activity was measured using two assays. The results were similar and thus, only the results of the Glutathione S-Transferase Assay Kit  are presented. Total GST activity did not confirm the significantly higher expression of GST observed in the neuronal transcriptomic data . Across The results of two different assays were similar and thus, only the results of the Amplite Fluorometric Glutathione GSH/GSSG Ratio Assay are presented. In order to calculate the GSH/GSSG ratio, both the amounts of reduced glutathione (GSH) and total glutathione (GSH + GSSG) must be determined, whereby the concentration of total glutathione is expected to be equal to or higher than the GSH amount. However, total glutathione levels (GSH + GSSG) of both assays were lower than GSH concentrations, in spite of repeated trials with different sample preparation and different individuals used. Therefore, GSSG determination failed and the specific GSH/GSSG ratio could not be calculated.p < 0.001) in harp seals and 103 ± 8.3 μM (p < 0.001) in hooded seals, compared to only 34 ± 2.9 μM in mice. Further, while harp seals and hooded seals had cerebellar GSH concentrations of 90 ± 10.1 μM (p < 0.001) and 80 ± 3.2 μM (p < 0.001), respectively, mice had only 16 ± 0.4 μM. Similarly, the hippocampi of harp and hooded seals had GSH levels of 86 ± 17.7 μM (p < 0.001) and 100 ± 5.5 μM (p < 0.001), while mice had 30 ± 1.5 μM.As for GSH, both seal species exhibited significantly elevated levels compared to mice ; Table 2To explore whether positive selection was acting on genes coding for antioxidants in the pinniped lineages, we selected seven pinnipeds and five terrestrial carnivores and fitted two branch-site models (BUSTED and aBSREL). We found no genes for which any positive selection pressure was indicated, for either seal species  and paraoxonase 2  in hooded seal neurons from the visual cortex was higher than in corresponding cells from mice . Concord2•−), which is the primary free oxygen radical produced in mitochondria (2•− to the more stable hydrogen peroxide (H2O2)  . SOD1 wae (H2O2) . Even whMitochondrial energy metabolism is quantitatively the most relevant source of ROS in eukaryotic cells . EarlierOur results are in line with previous studies in diving mammals: Blood, heart, kidney and lung tissue of several cetacean, pinniped and manatee species have generally higher SOD activities as compared to domestic pigs and/or other non-diving mammals . Among d2•− by SODs to hydrogen peroxide (H2O2), H2O2 can be reduced to water by glutathione peroxidase (GPX)  has elevated GPX activity compared to domestic pig tissues (Sus scrofa domesticus) (After the conversion of Ose (GPX) . GPX alsse (GPX) . GPX1, aesticus) . Blood oesticus) . Similaresticus) , while oesticus) .2O2 to water by GPX goes along with the oxidation of the reduced glutathione (GSH) to glutathione disulphide (GSSG) (The conversion of He (GSSG) . The gluα-tocopherol (vitamin E) protecting lipids from ROS damage (Lontra longicaudis annectens)) compared to terrestrial mammals  and thus, could not calculate the GSH/GSSG ratio in spite of using two different assays, varying preparation of samples, and testing different individuals of seals and mice. In mice, the expected GSSG concentration should be ∼0.7% of the GSH concentration, as was found in the cerebral cortex, cerebellum and brain stem in mice . Filho eOften, the GSH/GSSG ratio is considered an indicator of the redox state and, thus, the wellbeing of a cell. Drastic changes in this ratio indicate that there is an imbalance in the redox metabolism. However, enzymatic reactions involving GSH depend on the GSH-concentration, not on GSSG, as predicted by the Nernst equation, and are typically not affected by GSSG . Even thGlutathione reductase (GSR) mediates the transition of GSSG to GSH that is necessary for the recovery of the GSH pool . Since GApart from GSR, other enzymes and factors also contribute to the GSH level. Since GST conjugates GSH to electrophilic compounds, a reduced activity of GSTs - as found in pinnipeds-might draw relatively less GSH from the GSH pool, although to the best of our knowledge we do not know the effect size of GST activity on GSH concentration. In hooded seal neurons, glutathione synthase , nuclear factor erythroid 2-related Factor 2 (NFE2L2), a transcription factor activating glutathione biosynthesis and the expression of other antioxidants , and sollower GST activity in both seal species. We therefore extracted gene expression values for all GSTs present in our neuronal transcriptome (The glutathione-S-transferases (GSTs) are enzymes that conjugate GSH to electrophilic reactive compounds that would otherwise bind to proteins or nucleic acid, leading to cellular damage. Some GSTs are also able to detoxify hydroperoxides  and certcriptome  and founcriptome  makes itMirounga leonina), marine manatee (Trichechus manatus) and 3 dolphin species, when compared to terrestrial mammals  having 16 GSTs, while mice have 30 copies and Weddell seals have an intermediate number of 22 GSTs  has a high number of the cytosolic mu subclass of GSTs responsible for cellular detoxification (10 copies). Further, some GSTs have signatures of positive selection (GSTP2) in five mammalian lineages, while others (GSTP1) are conserved across mammals  is an antioxidant enzyme belonging to the glutaredoxin family, which consists of small redox proteins of the thioredoxin superfamily. It catalyses the transfer of electrons from GSH to disulfides , which mSince other essential components of the antioxidant system, such as SOD and GPX, appear to be constitutively more highly expressed in seals, it might not be necessary for the glutaredoxin system to be constitutively more active. Instead, upregulation of GLRX2 might be triggered by oxidative stress, as observed in mouse cardiomyocytes upon hypoxia/reoxygenation treatment . FurtherThioredoxin reductase 3 (TXNRD3) is a representative of the thioredoxin system. Among the three thioredoxin reductase isoenzymes known in mammals, TXNRD3 is the only one that contains an additional N-terminal glutaredoxin domain, which enables this isoenzyme to be involved in both the thioredoxin and the glutaredoxin systems . TXNRD3 Tursiops truncatus) and both GSR and GPX2 show cetacean-specific amino-acid substitution  have been subject to positive selection in pinnipeds when compared to non-diving carnivores . Since wtitution . Interestitution  and BUSTIn this study, pinniped antioxidant capacity was compared to that of mice. While mice are a well-accepted organism for scientific purposes with clear advantages in availability and handling, it would be ideal to compare pinnipeds to non-diving mammals of similar body size, since metabolic rate correlates with body mass e.g.,  which miWe conclude that the brains of harp and hooded seals have an overall constitutively enhanced antioxidant defence system, as has been generally found in other tissues of diving mammals. We found that not the antioxidant system as a whole, but some of its essential components, such as SOD and GPX, were constitutively elevated, whereas others, like the glutaredoxin and thioredoxin systems, were not enhanced . Since w


Polymer electrolytes (PEs) have been thoroughly investigated due to their advantages that can prevent severe problems of Li-ion batteries, such as electrolyte leakage, flammability, and lithium dendrite growth to enhance thermal and electrochemical stabilities. Gel polymer electrolytes (GPEs) using in situ polymerization are typically prepared by thermal or UV curing methods by initially impregnating liquid precursors inside the electrode. The in situ method can resolve insufficient interfacial problems between electrode and electrolyte compared with the ex situ method, which could led to a poor cycle performance due to high interfacial resistance. In addition to the abovementioned advantage, it can enhance the form factor of bare cells since the precursor can be injected before polymerization prior to the solidification of the desired shapes. These suggest that gel polymer electrolytes prepared by in situ polymerization are a promising material for lithium-ion batteries. Lithium-ion batteries (LIBs) are now extensively used everywhere in our lives, from small personal mobile electronics to electric vehicles (EVs) and large energy storage systems (ESSs) ,2. The d−3–10−2 S cm−1) and can form a good interfacial contact with the electrode active materials to function as a good pathway for lithium-ion during the charge/discharge cycling process  have high ionic conductivity 3 (LAMP), and this type typically shows ionic conductivity of 10−3–10−4 S cm−1 and low stability to Li metal. The electrochemical stability of NaSICON-type changes according to property of metal ions. LiSICON-type electrolyte possesses γ–Li3PO4 structure with low ionic conductivity of 10−5 S cm−1. However, LiSICON-type demonstrates outstanding stability to bare lithium, which is appropriate to use with Li metal electrode. Li7La3Zr2O12 (LLZO) electrolytes with garnet structure have good ionic conductivities (10−3–10−4 S cm−1) despite the possible point contact of LLZO with Li metal electrode. Li3xLa2/3−x□1/3−2xTiO3 (LLTO) with a composition of ABO3 is a perovskite structure that possesses mild ionic conductivity of ~10−4 S cm−1, although the side effect to Li metal exists. Overall, oxide-based inorganic solid electrolytes possess advantages, such as high ionic conductivity of ~10−3 S cm−1, good thermal stability, mechanical strength, and air stability. However, they have the disadvantages of high interfacial resistance with electrodes, high resistance between electrolyte particles, undesired reactivity when used with Li metal electrodes, and especially, they require high processing temperatures over 1000 °C, making it difficult to fabricate large area devices ,7977,79]ctrolyte ,81.The precursor used in in situ polymerization is composed mainly of monomer with functional group, lithium salt, solvent, and initiator. Monomers typically used in in situ polymerization are trimethylolpropane ethoxylate triacrylate (ETPTA), poly(ethylene glycol) diacrylate (PEGDA), trimethylolpropane propoxylate triacrylate (TPPTA), and di(trimethylolpropane) tetraacrylate (DTPTA)  82,83,8,85. WhenIn situ polymerization is commonly initiated by thermal or UV light photo-illumination. The thermal initiation can have an advantage that the thermal energy can be sufficiently transferred to the inside of the cell after the assembly-sealing procedure of the battery cell. However, thermal curing length of at least ~20 to 30 min is required for sufficient polymerization inside the cell which might damage the cell component inside. Furthermore, the relatively longer time will affect the manufacturing lead time, which restricts mass production of batteries. On the other hand, the UV illumination has the advantage of a short preparation time of tens of seconds for full polymerization, though initiation can be possible only before the cell assembly that restricts the facile manufacturing and fabrication conditions. An initiator that can create radicals by thermal treatment, such as benzoyl peroxide (BPO), lauroyl peroxide (LPO), and azobisisobutyronitrile (AIBN), are the typically used initiators. The precursor solution with the thermal initiator was injected into the electrode and thermally cured at 60 to 100 °C depending on the types of initiators ,86,89,906 in EC/DMC/EMC = 1/1/1 (v/v/v) solvent through thermal curing for 3 h. PEGPEA–GPEs demonstrated high ionic conductivity (3.35 × 10−3 S cm−1) at 25 °C and wide electrochemical stability of 4.9 V (vs. Li+/Li). A rapid mass loss was observed from 323 °C with the TGA measurement that confirmed good thermal stability. Even though PEGPEA–GPEs were rotated, stretched, and tied like rope, PEGPEA-GPEs were still physically not damaged and showed good flexibility. PEGPEA–GPEs were prepared using EGPEA, AIBN, and 1.0 M LiPF−3 S cm−1) were fabricated by thermal polymerization using polyvinyl formal (PVFM) and 1.0 M LiPF6 in EC/DMC = 3/7 (v/v) solvent without the use of an initiator. In −1 indicate that the C-O-C-O-C bond to the ether ring in PVFM completely disappeared in this GPE. In this system, GPEs had interfacial resistance of 64 Ω cm2. Compared with interfacial resistance (40 Ω cm2) of LEs, GPEs had relatively higher resistance; still, it is noticeable to improve safety despite this slight increase of resistance [GPEs having high ionic conductivity , lithium bis (trifluoromethane) sulfonimide (LiTFSI), dimethyl carbonate (DMC) with 2,2′-Azobis- (ABVN), and ionic liquids (ILs)-tributylmethylammonium bis(trifluoromethanesulfonyl) imide  [GPEs, which have high ionic conductivity of 6.15 × 104,4TFSI) . The GPE6 in EC/EMC = 3/7 (v/v), AIBN, and PAN membrane with mesoporous SiO2 are reported [−3 S cm−1). Methacrylate-functionalized SiO2 (MA-SiO2) provided a channel through which Li+ could move because of the inner pore due to the mesoporous structure. Since MA-SiO2 has a reactive methacrylate group as a cross-linking site, it directly reacted with the precursor to fabricate CPEs. Due to the reactive methacrylate group, the adhesion between the PAN membrane and MA-SiO2 became strong, thereby preventing the SiO2 particles from detaching from the PAN membrane. CPEs with the abovementioned properties delivered better tensile strength compared with CPEs with non-porous SiO2. MA-SiO2 also acted as an HF scavenger, in which the prepared CPEs showed reduced HF contents at high temperatures (CPEs composed of tetra(ethylene glycol) diacrylate (TEGDA), 1.15 M LiPFreported . The faberatures a. The ceeratures b.−3 S cm−1 at 20 °C), and the high Li+ transference number of 0.75. Superior mechanical properties of PCEE were confirmed from tensile and adhesion tests. PCEE showed high extensibility of approximately 300% and high adhesion energy of 21.5 J m−2. Generally, PEs have a trade-off relationship between ionic conductivity and mechanical properties, but PCEE did not compromise the relationship. PCEE had remarkable Li reversibility since the stability of PCEE was outstanding, even after 100 cycles at 10 mA cm−2 and Li plating/stripping cycle for 1500 h. PCEE maintained a dense and homogeneous surface, resulting from no dendritic and dead lithium.Plastic–crystal-embedded elastomer electrolyte (PCEE) composed of succinonitrile (SN) and LiTFSI as plastic crystal, PEGDA and butyl acrylate (BA) as elastomer, and AIBN as thermal initiator are reported . PCEE ac−5 S cm−1 to ~10−4 S cm−1 at room temperature. Poly-DOL GPE showed high anti–oxidation potential above 5 V, while liquid-state DOL electrolyte showed electrochemical decomposition at 4.2 V. The cycling performance was measured at 1 C for 1000 cycles. After the cycling test, capacity of the full cell using pretreated Li metal as anode maintained 105.7 mAh g−1, with the superior retention capacity (83.55%), and the coulombic efficiency of cell was ~99%, even after 1000 cycles. On the other hand, capacity of the full cell with unpretreated Li anode showed 79.4 mAh g−1, with low-capacity retention of 66.89% and coulombic efficiency of 94.15%.Poly-DOL GPE composed of lithium difluoroborate (LiDFOB), 1,3–dioxolane (DOL), LiTFSI, and SN were prepared by in situ polymerization . Poly-DO6 in EC/DEC/EMC = 1/1/1 (v/v/v) poly(ethylene glycol) dimethacrylate (PEGDMA), and pentaerythritol tetraacrylate (PETEA) through thermal in situ polymerization [−3 S cm−1. As shown in −1 after 100 cycles at 0.5 C, and after 500 cycles, GPE 1:1 showed 132 mAh g−1, with the capacity retention ratio of 90%. When the mass loading was 3.0 mg cm−2, LEs showed higher discharge capacity than GPEs at 60 cycles, presumably due to the good wettability. However, GPEs enabled by in situ polymerization have superior coulombic efficiency and capacity retention of 90% after 60 cycles.  light is illuminated. The precursor is injected onto and into the electrode, followed by exposure of UV light to incur initiation.−5 S cm−1 for PA3-PEO and 7.76 × 10−6 S cm−1 for PA4-PEO at 25 °C. The PEs were analyzed to have lower crystallinity due to possessing endothermic peak at a lower temperature with A3 and A4 with PEO compared with pure PEO film. PA3/4-PEO resulted in being more stable at high potential. Especially, PA3-PEO showed a stable oxidation potential up to 4.7 V (vs. Li+/Li). Li+ can be easily transported due to the lower interfacial resistance of CSPEs which was prepared in situ compared to the ex situ method. PA3-PEO CSPEs also showed a good coulombic efficiency of 96% after 100 cycles. (Two composite SPEs (CSPEs) using PA3-PEO and PA4-PEO are reported as photo-initiated PE matrix structure . CSPEs w cycles. a,b−5 S cm−1 and electrochemical stability window of 4.96 V (vs. Li+/Li) were studied. UV-SPEs composed of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA), poly(ethylene glycol) diacrylate (PEGDA), and LiTFSI with HMPP were used as an initiator for PE precursor. The fabricated UV-SPEs had initial thermal decomposition (5% weight loss) temperature of polymer at 321.94 °C, which is an outstanding thermal stability. For UV-SPEs paired with a lithium metal electrode, the potential value was stable at approximately 0.06 V at current density of 0.1 mA cm−2 and no short circuit appeared up to 800 h. Although the current density became 0.15 mA cm−2 at 60 °C, hard short of both electrodes in Li|LEs|Li cell occurred at 220 h, and the Li plating/stripping process lasted more than 250 h with UV-SPEs. This result suggests that the UV-SPEs can well suppress side reaction that might occur with LEs and demonstrates that UV-SPEs are significantly more stable than LEs. The cell using LiMnxFE1-xPO4 (LMFP) as the cathode obtained a maximum discharge capacity of 158.8 mAh g−1 at 0.2 C and a discharge capacity of 134.4 mAh g−1 after 240 cycles at 60 °C [UV-SPEs with ionic conductivity of 2.95 × 10at 60 °C .6 in EC/DEC = 1/1 (v/v) with HMPP initiator along with the incorporation of montmorillonite (MMT) showed high ionic conductivity (1.60 × 10−3 S cm−1) and good thermal stability [+ transference number of 0.78, presumably due to the incorporation of MMT with a high dielectric constant so that Li+ could be more easily dissolved into the MMT layer. The polymer matrix interferes with the coordination of Li+ and PF6− to have freer Li+  at 25 °C and a lithium transference number of 0.55. An ionic conductivity of 3.31 × 10−5 S cm−1 was achieved with CPEs using only PCLA, LiFSI, and DMAP; however, the value increased to 6.62 × 10−5 S cm−1 at 30 °C when HNTs were added. Since the charged surface of HNTs supports the dissolution of lithium salts, more free ions could be added, and consequently, the increased quantity of mobile charge carriers improved ionic conductivity. With the Li|CPEs|Li symmetric cell, the interfacial stability of CPEs to Li metal could be confirmed. The cell showed a cycling life of 1600 h at a current density of 0.1 mA cm−2. In LFP|CPEs|Li, an initial discharge capacity of 161 mAh g−1 was obtained at 0.2 C and 60 °C and good electrochemical performance with excellent capacity retention value (92%) after 600 cycles at 1 C [CPEs were also prepared via solvent-free in situ polymerization using acrylate functionalized poly-ε-caprolactone (PCLA), lithium bis(flourosulfonyl)imide (LiFSI), halloysite nanotubes (HNTs), and 4-(Dimethylamino)pyridine (DMAP). CPEs showed stable thermal stability up to 190 °C by TGA measurement with a two-step thermal decomposition process. The first decomposition occurred with LiFSI at ~190 °C and the next with the polymer matrix at ~350 °C. CPEs exhibited electrochemical stability of 5.4 V  PEL-0.1 was measured at various angles; resistance values at 0°, 45°, 90°, 135°, and 180° twisted angles were 491, 518, 465, 467, and 703 Ω, respectively, which suggests that the developed PEL electrolyte exhibited outstanding flexibility, and the ion conduction was not be greatly impacted during the deformation process. PEL-0.1 with good cycling performance presented capacity retention of 94.3% and ~100% coulombic efficiency after 250 cycles. PEGDA, LiTFSI, butyl acrylate (BA), 1-ethyl-3-methylimidazoline bis(trifluoromethylsulfonyl)imide (EMIM TFSI), and diphenyl  phosphine oxide (TPO) were used for fabricating elastomeric PEL electrolyte (PEL-0.1) enabled by UV induced in situ polymerization . PEL-0.1−4 S cm−1 at room temperature and was electrochemically stable up to 5.2 V. SGPE also showed uniform lithium deposition property since the overvoltage of SGPE was still approximately 0.04 V after the cycle for 2000 h, indicating small voltage polarization in the symmetric cell. In the LFP half-cell, SGPE showed remarkable cycling performance; the specific capacity of the SGPE cell hardly deteriorated and showed average coulombic efficiency of 99.7% after 750 cycles. Additionally, SGPE showed a capacity retention value of 94.7 and average coulombic efficiency of 99.8% after 450 cycles. As shown in The solvated ionic liquid-based gel polymer electrolyte (SGPE) is reported, which was prepared by ETPTA, TPO, LiTFSI, and tetraethylene glycol dimethyl ether (TEGDME) . The hig−4 S cm−1 were fabricated and demonstrated [4), and (ethylene glycol) methyl ether acrylate (mPEGA) exhibited a good elastic tensile performance, which can be stretched above 200%, and good electrochemical stability, up to 4.6 V. The all-solid-state full cells consisting of SN-SPE as an electrolyte, LFP as a cathode, and LTO as an anode were fabricated and measured at 0.2 C. The all-solid-state LIBs (ASSLIBs) enabled by in situ polymerization showed a discharge capacity of 145.93 mAh g−1 and good capacity retention of 93.62 % after 100 cycles. However, ASSLIBs fabricated by the ex situ method exhibited a discharge capacity of 98.67 mAh g−1 and low capacity retention value of 65.84% after 100 cycles. To investigate interfacial properties between electrode and electrolyte, interface morphology and impedance were measured. The cross-sectional SEM image demonstrated the strong coupling between the cathode and SN-SPE without any discernible delamination . Solid polymer electrolytes (SPEs) are composed of a solid polymer matrix that contains an ionic species that can conduct ions. SPEs are typically used in thin film batteries and other applications where the electrolyte must be flexible and conform to the shape of the device. Gel polymer electrolytes (GPEs) are like solid polymer electrolytes, but they are made by suspending the ionic species in a polymer matrix rather than dissolving it in a solvent. This creates a gel-like material that is flexible and can easily conform to the shape of the device. Gel polymer can provide good ionic conductivity as well as mechanical durability and is easy to fabricate. Composite polymer electrolytes (CPEs) are formed by combining a gel polymer electrolyte with inorganic fillers, such as ionic liquid or inorganic nanocomposites, to improve the conductivity of the electrolyte or decrease the crystallinity of the polymer matrix. Composite polymer electrolytes are typically used in high-performance batteries and other electrochemical devices where a high level of conductivity is required.The promising method of preparing a gel polymer electrolyte is in situ polymerization. A liquid precursor solution is injected into electrodes and then polymerized by thermal- or photo-initiation using a radical initiator. The precursor of liquid phase easily immerses into the internal pores of electrode, and finally, GPEs can be created inside the pores and on the surface of electrode active materials. The GPEs using in situ polymerization can significantly decrease the contact resistance due to the good interfacial contact formed between the electrode and polymeric electrolyte compared with the ex situ preparation method.In summary, GPEs prepared by in situ polymerization are promising solid phase polymeric electrolytes for Li-ion batteries with increased safety as well as high performance. The in situ polymerized GPEs possess various advantages, including reduced interfacial resistance due to excellent interfacial contact, increased stability of lithium metal-based batteries due to good suppression property of lithium dendrite growth, and the possibility of expansion, such as flexible batteries, owing to good feasibility of PEs. Nevertheless, further studies are needed to improve both electrical and mechanical characteristics of PEs simultaneously to achieve safe and reliable cell performance.


Phosphorus commonly reduces lipid deposition in the vertebrates. However, the underlying mechanisms involved in the process remain unclear.− 1, respectively, for 8 weeks. The contents of triglyceride, non-esterified free fatty acids, adenosine triphosphate, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, enzymatic activities, mRNA and protein expression were determined in the intestinal tissues. Hematoxylin and eosin, Oil Red O staining, and transmission electron microscope were performed for intestinal tissues. Primary intestinal epithelial cells were isolated from yellow catfish intestine. Western blot analysis, Immunoprecipitation assays, Immunofluorescence staining, and RNA extraction and quantitative real-time PCR were decided. Luciferase reporter assays and electrophoretic mobility shift assay were used to evaluate the function of Sirt3, PPARα and Lcad promoters.Yellow catfish were given three experimental diets with dietary phosphate levels of 3.22, 6.47 and 7.99 g Pi kgkrüppel like factor 4 (klf4), silent mating-type information regulation 2 homolog 3 (sirt3), peroxisome proliferator activated receptor alpha (pparα) and long chain acyl-CoA dehydrogenase (lcad) in the intestinal epithelial cells (IECs), and klf4 knockdown attenuated the phosphate-induced increase of protein levels of Sirt3, Pparα and Lcad. Further investigation found that Klf4 overexpression increased the activity of sirt3 and pparα promoters, which in turn reduced the acetylation and protein level of Lcad.High dietary phosphate intake activated intestinal phosphate absorption and excretion, and reduced lipid deposition through increasing lipolysis in the intestine. Moreover, phosphate incubation increased the mRNA and protein expression of Dietary Pi excess induced lipid degradation by the activation of the Klf4-Sirt3/Pparα-Lcad pathway in the intestine and primary IECs.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-022-01008-w. Phosphorus is a predominant mineral element within the body of animals and plays many vital roles in diverse biological processes, including skeletal formation, the maintenance of cell membrane integrity, ATP synthesis and lipid metabolism . The intOn the other hand, inorganic phosphorus (Pi) is a key component such as adenosine tri-phosphorus (ATP) and plays important roles in maintenance of mitochondrial homeostasis , 7. MitoMultiple signaling molecules and pathways participate in the regulation of mitochondrial lipid metabolism. Among these signaling molecules, krüppel-like factor 4 (KLF4), an important zinc-finger protein, mediated the regulation of mitochondrial homeostasis and lipid metabolism by PPARα pathway , 14. SIR20. As some duplicated genes will evolve the new functions, new regulatory mechanisms have emerged [Pelteobagrus fulvidraco, a widely distributed freshwater teleost fish species in China and several other Asian countries, is economically important for aquaculture because of its good fillet quality and easy culture. However, under the intensive aquaculture, yellow catfish is easy to deposit excessive lipid in the intestine, which influences its growth performance and health. Gong et al. [Fish have more than 30,000 species and accordingly are the most abundant vertebrate in the world. Studies pointed out that fish underwent the fish-specific genome duplication (FSGD) events during the evolution, compared with terrestrial animalsg et al.  publishe2PO4·2H2O at the addition levels of 0 , 1.5%  and 3.0%  , and cultured in indoors circular fiberglass tanks for a 14-day acclimatization. After the acclimatization, 270 juvenile yellow catfish  were stocked in nine tanks and there were 30 fish each tank (300-L water volume). Each experimental diet was fed to triplicate tanks randomly. During the feeding, yellow catfish were fed to satiation twice daily . All tanks were placed under indoors controlled conditions with a 12-h day/night cycle. The feeding experiment continued for 8 weeks. Water quality were monitored twice one week, and the values were followed: water temperature, 28.0 ± 1.0 °C; pH, 7.89 ± 0.15; dissolved oxygen, 6.01 ± 0.12 mg L+ and NADH, and the NAD+/NADH ratio. The intestinal tissues from another four fish each tank were sampled for the analysis of gene and protein expression. Before the analysis, all samples were fast frozen in liquid nitrogen and stored at − 80 °C freezer.At the end of the feeding experiment, all yellow catfish were fasted overnight. They were then euthanized (MS222 at 100 mg/l), counted, measured and weighed to determine the survival, weight gain (WG), specific growth rate (SGR) and condition factor (CF). The intestinal samples were randomly selected from two fish from each tank and used for H&E staining, Oil Red O staining and TEM analysis. Another three fish were collected randomly from each tank and their whole intestine tracts were used for the determination of phosphorus concentration. Another three fish from each tank were sampled for the intestine and used for analyzing TG contents, enzyme activities, the contents of NEFA, ATP, NAD2 at 28 °C. The analytical protocols were showed in Additional file 2 at 37 °C. To investigate the influences of Pi incubation on lipid metabolism in the IECs of yellow catfish, we used 0.9 mM Pi (the control) and 2.0 mM Pi (Pi-treated group) to incubate the primary IECs, respectively. The incubation continued for 48 h. We analyzed the parameters related to lipid metabolism , and the mRNA and protein expression of mitochondrial relevant genes .Primary intestinal epithelial cells (IECs) were isolated from yellow catfish intestine by two-step collagenase-protease enzymatic digestion, as described elsewhere . The IEC2 20 TWIN TEM .We performed the H&E and ORO staining, and TEM observation for the intestinal tissues, as described previously . In brieThe BODIPY 493/503  staining was performed for primary IECs, as described by Wei et al. . The lasThe carnitine palmitoyl-transferase 1 (CPT1) activity was determined based on the measurement of the initial CoA-SH formation by the 5, 5’-dithio–bis– (2–nitrobenzoic acid) (DTNB) reaction from palmitoyl–CoA by mitochondrial samples with L–carnitine at 412 nm and the protocols followed Wei et al. . One uni+ and NADH contents were measured via the NAD+/NADH quantification kit . Protein concentrations were analyzed the BCA protein assay kit .The ATP quantification kit  was used to analyze the ATP content, based on protocols described in Zhang et al. . Brieflyβ-actin, 18s rRNA, hprt, ubce, gapdh, tuba, b2m, rpl7, tbp and elfa) were measured as endogenous controls for their mRNA expression, and the best combination of two genes were analyzed by geNorm (https://genorm.cmgg.be/) [−ΔΔCt method was used to analyze the relative fold changes. The primers are listed in Additional file The qPCR assays were performed with SYBR Green PCR Master Mix (Applied Biosystems) and experimental protocols were described in Zhang et al. . Ten houmgg.be/) . The 2−ΔTo explore the underlying mechanisms for Pi-induced lipolysis, we constructed the Klf4, Sirt3, Lcad, Errα and Pgc1α expression vectors based on the published protocols . We subcklf4-siRNA sequences were transfected with the EntransterTM-R4000 Reagent kit  to primary IECs, respectively. The siRNA knockdown sequence was given in Additional file klf4-siRNA sequences were determined by the qPCR and Western blot analysis, respectively. The siRNA-1164 was chosen to perform the klf4-knockdown experiment because of its high efficiency for inhibiting the klf4 expression. After primary IECs were transfected with NC’s (negative control) siRNA and klf4 siRNA, cells were cultured for 48 h with two treatments: the control (without extra Pi addition) and Pi incubation (2mM), respectively.For Klf4-knockdown experiment, three sirt3 promoter-luciferase (− 1576/+44 bp), pparα promoter-luciferase (− 837/+63 bp) and lcad promoter-luciferase (− 1618/+20 bp) construct and their mutants were built, based on the protocols described in Xu et al [http://jaspar.genereg.net/). Finally, the plasmids containing the promoter sequences were subcloned into Sac I/Hind III sites of pGL3-basic vector. The specific primers were given in Additional file 2 and 37 °C in the DMEM containing 10% FBS. The 400 ng reporter plasmids were co-transfected into the 293T cells and the 20 ng pRL-TK plasmids was used as the control. After 4 h, the medium was replaced by the DMEM or DMEM with 2mM Pi incubation. After 24 h incubation, cells were collected to analyze the relative luciferase activities.Based on the hi-TAIL PCR  method , the sirXu et al . BrieflyThe EMSA was performed to analyze the direct binding sites at the promoters, as described previously . All oliWestern blot analysis was conducted to explore the protein expression of Slc34a2, Ucp2, Klf4, Sirt3, Pparα and Lcad, based on the protocols in Liu et al. . In brieThe immunoprecipitation assay was conducted according to our publication . The HA-The immunofluorescence staining was conducted, as previously described . BrieflyP-values < 0.05 were considered significant.All data were presented as the mean ± SEM (n ≥ 3). The statistical analyses were performed by the software SPSS version 19 . The data were analyzed for the normality with the Kolmogorov–Smirnov test. The Bartlett’s test was used to analyze the homogeneity of the variances among the treatments. The Student’s T test was utilized to analyze these data between two treatments. The one-way ANOVA and Duncan’s multiple range test were utilized to analyze the experimental data among more than three treatments. The GraphPad Prism 8.0 software  was used to draw figures. The survival was not influenced significantly by dietary phosphorus levels , complexes II  and ATP synthase genes  were analyzed [ndufa9 mRNA expression increased with increasing dietary phosphorus levels  Fig. A, B and  balance . The conAD+ Fig. F, the raADH Fig. G were hiADH Fig. H. The mRanalyzed . The nduels Fig. I. The mRoup Fig. I. The sdacads, acad8, acadsb, acadm, hadha1, hadha2, cpt1a, cpt2 and eci1) and peroxisomal β-oxidation genes  was significantly higher in the HP group than those in the LP group , and − 1444/−1457 bp Errα binding site (ERE) significantly attenuated the Pi-induced increase of sirt3 promoter activity  did not significantly change the sirt3 promoter activity, but the deletion of − 1444/−1457 bp Errα binding site (ERE) significantly attenuated the overexpression of Klf4 with ERRα and Pgc1α vectors-induced increase of the activity of sirt3 promoter  located at lcad promoter ; in contrast, klf4-knockdown abrogated the increase of acadm, lcad and eci1 mRNA abundances induced by Pi , short-chain specific acyl-CoA dehydrogenase (acads), short and branched chain specific acyl-CoA dehydrogenase (acadsb), and medium-chain specific acyl-CoA dehydrogenase (acadm) are the dehydrogenases that catalyze the first step of fatty acid β-oxidation in the mitochondria, and trifunctional enzyme subunit alpha 1 (hadha1), trifunctional enzyme subunit alpha 2 (hadha2), and enoyl-CoA delta isomerase 1 (eci1) catalyze the last step of fatty acid β-oxidation in the mitochondria [acox1 and acox3), and acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha 1 (acca1) involved in the rate-limiting step of peroxisomal β-oxidation of very long-chain fatty acids [acad8, acads, acadsb, acadm, hadha1, hadha2, cpt1a, cpt2 and eci1; peroxisomes: acox1, acox3 and acca1), suggesting that high dietary phosphorus activated the fatty acid β-oxidation. Taken together, for the first time, our data indicated that high dietary phosphate activated the mitochondrial fatty acid β-oxidation in the intestine.Studies reported that high dietary phosphorus increased lipid degradation in the liver , 12, butxidation . In the chondria . Peroxisty acids . In the klf4 knockdown attenuated the increase of the Sirt3 protein and mRNA levels induced by Pi and that Klf4 bound to the Errα/Pgc1-α transcriptional module and increased the activity of sirt3 promoter, which confirmed our speculation. PPARα is an important transcription factor that regulates fatty acid β-oxidation [klf4 knockdown attenuated the Pi-induced increase of the Pparα protein and mRNA levels, in agreement with the report in mice [pparα promoter and found that KLF4 directly bound to the KRE of pparα promoter and mediated Pi-induced changes of the promoter activity, indicating that Pparα is a target protein of Klf4 under dietary phosphorus addition.KLF4 is one of the crucial modulators for fatty acid β-oxidation in the mitochondria , 33. Thexidation . In our  in mice . Moreovelcad promoter. Moreover, Pi incubation activated the transcriptional activity of Lcad by the Pparα binding to the PPRE on the lcad promoter. Thus, our data demonstrated that Pparα directly binds to the lcad promoter, and mediates Pi-induced increase of lcad transcription levels, thereby controlling mitochondrial fatty acid β-oxidation.LCAD is a rate-limiting enzyme that catalyzes the first step of the mitochondrial fatty acid β-oxidation. Studies pointed out that the elevated deacetylation and transcription level of LCAD contributed to mitochondrial fatty acid oxidation , 41. Chesirt3, which in turn regulated the deacetylation of Lcad under Pi treatment. Klf4 also regulated the transcriptional level of Lcad which was mediated by Pparα. Thus, our study provided potential preventive targets against excess intestinal lipid deposition in the vertebrates, possibly including human beings.In this study, dietary Pi excess significantly increased lipid degradation in the intestine and IECs. Mechanistically, high phosphate induced lipid degradation via the up-regulation of mitochondrial fatty acid β-oxidation, and Klf4-Sirt3/Pparα-Lcad pathway was required for high phosphorus-induced mitochondrial fatty acid β-oxidation and lipolysis. Mechanistically, Klf4 bound to and cooperated with the Errα/Pgc-1α transcriptional module, and enhanced the promoter activity, transcription and protein levels of Additional file 1: Supplemental Text S1. Yellow catfish primary intestinal epithelial cells (IECs) isolation and culture. Supplemental Table S1. Feed formulation and proximate analysis of experimental diets. Supplemental Table S2. Primers used for quantitative real-time PCR analysis. Supplemental Table S3. Primers used for plasmid construction of expression vector. Supplemental Table S4. Primers used for plasmid construction of si-klf4. Supplemental Table S5. Primers used for plasmid construction of promoters. Supplemental Table S6. Primers used for site-mutation analysis. Supplemental Table S7. Primers used for electrophoretic mobility-shift assay. Supplemental Table S8. Effects of dietary phosphorus supplementation on growth performance and feed utilization of yellow catfish. Supplemental Figure S1. MTT assay of primary IECs under Pi incubation. Supplemental Figure S2. Immunofluorescent staining of KLF4 protein under Pi incubation. Supplemental Figure S3. Nucleotide sequence of yellow catfish sirt3 promoter. Numbers are relative to the transcription start site (+1). Supplemental Figure S4. ERRα response elements (ERE) located at −1443 bp to −1457 bp and KLF4 response elements (KRE) located at −406 bp to −419 bp of pparα promoter. Supplemental Figure S5. Relative luciferase activity of sirt3 promoter after the incubation with different overexpression vectors. Supplemental Figure S6. The immunoprecipitation experiment for the analysis of KLF4 and ERRα binding with SIRT3. Supplemental Figure S7. Nucleotide sequence of yellow catfish pparα promoter. Numbers are relative to the transcription start site (+1). Supplemental Figure S8. KLF4 response elements (ERE) located at −1443 bp to −1457 bp (KRE1) and −406 bp to −419 bp (KRE2) of pparα promoter. Supplemental Figure S9. Nucleotide sequence of yellow catfish lcad promoter. Numbers are relative to the transcription start site (+1).


We aimed to elucidate the roles of NMN in regulating HIF-1α and fibrosis in hypoxic adipose tissue.Fibrosis is increasingly considered as a major contributor in adipose tissue dysfunction. Hypoxic activation of hypoxia-inducible factor 1α (HIF-1α) induces a profibrotic transcription, leading to adipose fibrosis. Nicotinamide mononucleotide (NMN), a member of the vitamin BIn vitro, Stromal vascular fractions (SVF) cells were treated by hypoxia with or without NMN (200μM), sirtinol  and CoCl2 . The effects of NMN on hypoxia-associated adipose fibrosis, inflammation, NAD+/SIRT1 axis alteration, and HIF-1α activation were evaluated by real-time polymerase chain reaction (PCR), western blots, immunohistochemistry staining, immunoprecipitation, and assay kits.Mice were placed in a hypobaric chamber for four weeks to induce adipose fibrosis. NMN  was administered by intraperitoneal injection. +/SIRT1 axis and inhibited the activation of HIF-1α induced by hypoxia. In hypoxia-induced SVFs, the SIRT1 inhibitor sirtinol blocked the anti-fibrotic and anti-inflammatory effects of NMN, upregulated the HIF-1α and its acetylation level. The HIF1α stabilizer CoCl2 showed similar effects as sirtinol.Mice placed in a hypoxic chamber for four weeks showed obvious adipose fibrosis and inflammation, which were attenuated by NMN. NMN also restore the compromised NAD+/SIRT1 axis.NMN effectively attenuated HIF-1α activation-induced adipose fibrosis and inflammation by restoring the compromised NAD Obesity is commonly considered as a persistent low-level inflammatory state closely related to insulin resistance, metabolic syndromes, and cardiovascular diseases , 2. ThisHIF1α is considered as a significant initiating factor in adipocytes for fibrotic and inflammatory response, directly linked to metabolic dysfunction in AT under hypoxic conditions , 8. Unli3 family, is a defined biosynthetic precursor of nicotinamide adenine dinucleotide (NAD+). The effect of NMN on restoring NAD+ homeostasis and activating sirtuins has been extensively investigated in the past decades  and SIRT1 is severely compromised in white adipose tissue (WAT) by HFD, thus leading to diabetes, whereas NMN ameliorates these changes (Nicotinamide mononucleotide (NMN), a member of the vitamin B decades , 14. Threcretion –17. For ecretion . Moreoveecretion , 21. Howecretion . It has  obesity –25. A re changes . Althoug+/SIRT1 axis is compromised by hypoxia, thus leading to adipose fibrosis and inflammation; (b) whether NMN inhibits hypoxia-related adipose fibrosis and inflammation with an emphasis on regulating HIF1α by restoring the compromised NAD+/SIRT1 axis.In the present study, we aimed to find out: (a) whether NAMPT/NAD2 was obtained from Sigma-Aldrich Co . The reverse-transcription assay kit was obtained from Thermo Fisher Scientific (USA). Antibodies against the following proteins were obtained from Abcam: TGF-β1 (ab92486), SIRT1 (ab110304), adiponectin , and NAMPT(ab236874). Antibodies against HIF1α (NB100-105) were purchased from Novus Biologicals. Antibodies against acetyl-lysine (9441S) were purchased from Cell Signaling Technology. Antibodies against MMP9(27306-1-AP), TIMP1(16644-1-AP), IL-6 (66146-1-lg), GAPDH, and β-actin were obtained from the Proteintech Group.Nicotinamide mononucleotide (B7878) was purchased from APExBIO Technology  . Sirtinol was obtained from MedchemExpress . CoCl2) for four weeks. The chamber was opened daily for 30 min to clean and replenish food and water. NMN  was delivered to mice 5-days before hypoxic treatment and one week after hypoxic treatment finished  weighing 20.2 ± 2g were purchased from the Institute of Laboratory Animal Science, Hunan SJA Laboratory Animal Co., Ltd . All animal experiments were under the approval of the Animal Care and Use Committee of Second Xiangya Hospital of Central South University and were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed in a specific-pathogen-free (SPF) environment with a regular 12/12h day/night cycle, a humidity of 70%, and a temperature of 22°C for seven days before experiments. Mice were randomly divided into three groups (n = 8), namely, Control, hypoxia, and hypoxia + NMN. Hypoxia group mice were placed in a hypoxic chamber -stained sections. The extent of interstitial fibrosis in WAT was evaluated from Masson-stained sections. It was calculated as the mean ratio of the blue-stained fibrotic area to the total tissue area. For each section, five optical fields were analyzed using digital analysis software (Image J) in a blinded manner.We performed immunohistochemical staining to assess the level of SIRT1 expression and macrophage infiltration in the eWAT. After the sections were blocked using 8% goat serum in phosphate buffer saline (PBS), they were incubated with primary antibody against SIRT1 or the macrophage marker F4/80 at four °C overnight. Next, the sections were incubated with GT VisionTM+Detection System/Mo&Rb reagent for one hour at room temperature and developed using a peroxide-based substrate diaminobenzidine (DAB) kit . Eventually, we dehydrated and cleared these sections in ethanol and xylene, respectively, and took the fields of view at the magnification of 200 ×. For each adipose depot, five optical fields per section were analyzed.3 piece, then digested by type I collagenase (1 mg/ml) under agitation for 30-40 min at a 37 °C water bath. SVF cells were separated from the top layer of mature adipocytes by centrifugation , then suspended in DMEM/F12 (Gibco) with 10% FBS and 100 U/ml penicillin-streptomycin and filtered by the cell strainer. Next, we centrifuged and re-suspended the SVF pellets in fresh media. Two hours after culturing cells under normal conditions (at 37°C in 95% O2 and 5% CO2), we washed the cells with PBS twice to remove red blood cells, immune cells, and other contaminants, and fresh media were added. All cells between 3-5 passages were used in this experiment. After serum starvation for 24 h, cells were randomly placed in cell culture chamber with 1% O2 for 24h to conduct hypoxia. NMN was dissolved in sterile PBS and diluted to the desired final concentrations (200µM). NMN was added simultaneously with hypoxia treatment. Sirtinol (25µM) was used to block SIRT1 expression in SVFs. CoCl2 (100μM) was used to enhance HIF1α expression in SVFs. Sirtinol or CoCl2 was also added simultaneously with NMN.Stromal vascular fraction (SVF) cells in epididymal fat depots from male C57BL/6 mice were isolated and cultured as described previously , 28. BriImmunoblotting analysis and qPCR were performed according to previous articles . In brie-ΔΔCt value was calculated to compare the differences among groups.Total mRNA was extracted from eWAT or SVF cells with GeneJET RNA Purification Kit . Reverse transcribed into cDNA using RevertAid First strand cDNA Synthesis kit . The StepOne Real-Time PCR  was used for real-time qPCR analysis. The primers used are described in For immunoprecipitation experiments, total homogenates from adipose tissue and cultured cells were treated with RIPA lysis buffer , vortexed for the 30s, and centrifuged for 15 min at 12000 r/min. The tissue or cell extracts was subjected to immunoprecipitation with HIF1α primary antibody at 4°C overnight. The antibody-bound proteins were precipitated with 20 μL protein A/G PLUS-Agarose  and rotated for 1 h, then incubated overnight at 4°C. The beads were then gently centrifuged at 1000 r/min for 5 minutes at 4°C. After four RIPA buffer washes, the immunoprecipitates were diluted with 40 μL of 1 × SDS loading buffer  and boiled at 100°C for 2-3 min to separate complexes from the protein A/G PLUS-Agarose. The samples were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes . After blocking with QuickBlock™ Western , the membranes were incubated with an anti-acetylated-lysine antibody  overnight at 4°C, washed in PBST three times, and incubated with a secondary goat anti-rabbit polyclonal antibody  at room temperature for 1h. Finally, the signals were tested by WesternBright™ Sirius ECL kit .+, EnzyChrom NAD+/NADH Assay Kit  was used according to the manufacturer’s instructions. In brief, mice eWAT weighing 20 mg for each sample or cells pelleted about 105 for each sample were taken and homogenized in 100 μL NAD+ or NADH extraction buffer, respectively. Extracts were heated for 5 min at 60°C, and 20 μL of assay buffer was added into extracts, followed by 100 μL of the opposite extraction buffer . Mixtures were vortexed and centrifuged at 12,000 g for 5 min. Supernatants (40 μL) were then mixed with a working reagent (80 μL) in each well. The optical density of supernatants at 565nm were measured at 0 and 15min intervals using a 96-well plate reader spectrophotometer. NAD+/NADH concentration and their ratio were calculated using the manufacturers’ equation.In order to measure the NADpost hoc test. P < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism version 7.0 software . All values are expressed as mean ± standard deviation of the mean (SD). If the data fit normal distribution by taking Kolmogorov-Smirnov tests, the statistical comparisons between two groups were performed using Student’s t-test, and comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s vs. 1.445 ± 0.1626, P<0.05). In contrast, the observed abnormal collagen deposition was reduced in NMN-treated eWAT   and qPCR. NMN significantly reduced the amounts of macrophages in the eWAT . The protein levels of NAMPT were significantly decreased in hypoxia-induced SVF cells, and NMN restored the impairment independently of Sirtinol or CoCl2 treatment , Sirtinol  and CoClWe next examined the expression of fibrogenic and inflammatory genes associated with HIF1α signaling in SVF cells, including Col1a1, FN, TGF-β, IL-6, MIF, and TNF-α. The expression of the above genes was significantly increased in hypoxia-induced SVF cells, and this effect was reversed by NMN , indicating the involvement of SIRT1 in NMN treatment. As an energy sensor, SIRT1 regulates cellular homeostasis, and therefore, we wanted to know whether NMN inhibited HIF-1α accumulation by activating SIRT1. Gomes et al. have found that SIRT1 is constantly required to ensure the efficient degradation of HIF1α under normal oxygen condition (+ depletion reduces the activity of SIRT1, leading to the activation of HIF1α (in vivo or in vitro, our results showed that the hypoxia-induced HIF1α upregulation was accompanied by reduced NAD+ levels and SIRT1 activity. NMN treatment failed to reverse the above process with the disturbance of Sirtinol, indicating that NMN reduced HIF-1α accumulation by promoting proteasomal degradation in a SIRT1-dependent manner. Although the HIF-1α stabilizer CoCl2 treatment can enhance the HIF1α expression with the existence of NMN, it failed to block the activation of SIRT1, further demonstrating that HIF-1α is the downstream target of SIRT1. In addition, several studies have shown that SIRT1 inactivated HIF1α by deacetylating it and consequently inhibited HIF-1 target genes (NMN has been reported to be a multifunctional compound. In addition to replenishing NADammation , 26, 39.ondition . During of HIF1α , 41. Conet genes . In the + repletion and the regulation of SIRT1 (+ levels in healthy or obese volunteers (+ metabolism can be a potential target for pathophysiological processes, including mitochondrial metabolism, oxidative stress, inflammation, and fibrosis, NMN or NR may become a therapeutic option. The above evidence increases the possibility of NAD+ precursors’ clinical application, but its efficacy in patients with metabolic disorders remains unclear, and more studies are needed.In summary, our study showed that NMN inhibited HIF-1α activation-induced adipose tissue fibrosis and inflammation, while the underlying mechanism may be associated with the NADof SIRT1 Figure 6of SIRT1 , 39, 43.lunteers –48. BesiThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Ethical and Welfare Committee of Second Xiangya Hospital of Central South University.KW, BL, NL, and QML designed the research. KW and BL performed the experiments. KW was a major contributor in writing the manuscript. YM and BL revised the manuscript. QML and NL supplied financial support and revised the manuscript. All authors read and approved the final manuscript.