Secreted protein acidic and rich in cysteine like 1(SPARCL1) is a secreted glycoprotein with important physiological functions and is involved in the proliferation and differentiation of various cells. However, the role of the SPARCL1 gene in sheep preadipocytes and its regulatory mechanism is still unclear. In this study, we explored the effect of SPARCL1 on the proliferation and differentiation of sheep preadipocytes. The results showed that the expression level of the SPARCL1 gene is higher in fat tissue than in other tissues, and the gene was significantly increased on the 6th day of preadipocyte differentiation. In the preadipocyte proliferation stage, interference of SPARCL1 gene reduced cell viability and increased cell apoptosis. In preadipocyte differentiation stage, SPARCL1 overexpression significantly inhibited lipid droplets accumulation and triglyceride content by increasing Wnt10b, Fzd8, IL6, and β-catenin and inhibiting PPARγ, C/EBPα, LPL, and IGF1 genes expression, whereas SPARCL1 deficiency significantly promoted cell differentiation by inhibiting β-catenin and increasing GSK3β, PPARγ, C/EBPα, and LPL. The results of this study suggest that SPARCL1 plays a negative role during preadipocyte differentiation and may become a novel target for regulating preadipocyte differentiation and improving IMF.Important candidate genes that regulate lipid metabolism have the potential to increase the content of intramuscular fat (IMF) and improve meat quality. Abbreviations:IMF: Intramuscular fat SPARCL1: Secreted protein acidic and rich in cysteine like 1 PPARγ: Peroxisome proliferator-activated receptor γ C/EBPα: CCAAT/enhancer-binding protein-α LPL: Lipoprotein lipase IGF1: Insulin-like growth factor 1 Wnt10b: Wnt family member 10B Fzd8: Frizzled class receptor 8 IL6: Interleukin 6 β-catenin: Catenin beta interacting protein 1 GSK3β: Glycogen synthase kinase 3 beta LRP5/6: Low-density lipoprotein receptor-related protein 5/6 Mutton is becoming more and more popular with consumers in China. Small Tail Han sheep is a unique breed in China, with high fecundity and strong viability, and it was widely bred in Northeast China to meet the local demand for mutton in the past . HoweverC/EBPα) also plays a vital role in adipocyte differentiation [PPARγ positively regulates C/EBPα and co-initiates preadipocyte differentiation [LPL) is a key enzyme of fat deposition, hydrolysing triglycerides and promoting lipoprotein uptake [IGF1) also promotes adipocyte proliferation and differentiation [β-catenin gene has a key regulatory function as a second messenger [β-catenin can affect downstream genes (PPARγ and C/EBPα) to regulate adipocyte differentiation [Adipocyte formation is a complex physiological process involving a large number of genes, non-coding RNAs, growth factors, and signal pathways ,5. The tntiation . PPARγ pntiation . Lipopron uptake . In addintiation . These gessenger . β-catenntiation .SPARCL1 has been extensively studied in cancer and could be a potential target for cancer therapy [SPARCL1 regulates adipogenesis in mice [SPARCL1 gene on the proliferation and differentiation of sheep preadipocytes and its mechanism, providing a new target for increasing IMF content and a new research direction for other fields.Secreted protein acidic and rich in cysteine like 1 (SPARCL1), a member of the SPARC family, is a glycoprotein that mediates cell-matrix interactions and is involved in many physiological processes, including cell adhesion, proliferation, differentiation, migration, and maturation , as well therapy , and it  in mice , but theThe experimental animals were three two-month-old and three six-month-old healthy male Small Tail Han sheep from the Institute of Animal Biotechnology, Jilin Academy of Agricultural Sciences. The groin adipose tissue of two-month-old sheep was used to extract preadipocytes. Heart, muscle, small intestine, stomach, liver, duodenum, and fat tissues of six-month-age male sheep were isolated for qPCR validation. Animal experiments were performed following animal use protocols approved by the Committee for the Ethics of Animal Experiments .3 pieces with surgical scissors, and the blocks were digested collagenase II(Sigma-Aldrich) in a water bath at 37°C for 1 h, mixing every 15 minutes, and the undigested tissue and miscellaneous cells were filtered out using 200 mesh(75 μm) and 400 mesh filters(38 μm), and then the supernatant was removed by centrifuging at 1500 rpm for 15 min to obtain preadipocyte. Next, the preadipocytes were cultured using a complete medium containing DMEM-F12 (Sigma-Aldrich), 10% foetal bovine serum , and 1% penicillin/streptomycin solution in 60-mm Petri dishes  in a 37 oC and 5% CO2 incubator, and change the culture medium every 48 h. When the cells overgrew the petri dish, some preadipocytes were digested with trypsin (Sigma-Aldrich) to subculture, and the remaining preadipocytes were induced by exogenous inducer I (10 mg/mL insulin (Sigma-Aldrich), 1 mM dexamethasone (Sigma-Aldrich), 0.5 mM isobutylmethylxanthine (Sigma-Aldrich) and complete medium) and inducer II solution (10 mg/mL insulin (Sigma-Aldrich) and complete medium) into mature adipocytes. The inducer I and II have respectively cultured the cells for 48 h and then exchanged a fresh complete medium to culture the cells until becoming mature adipocytes.The adipose tissue was washed with PBS containing 1% penicillin/streptomycin solution , and the connective tissue and blood clots in the adipose tissue were removed using sterile tweezers. The pure tissue was cut into mmWhen preadipocytes are differentiated into mature adipocytes, the intracellular lipid droplets can be stained red by oil red O dye to verify the maturation of the adipocytes. Adipocytes were washed three times with PBS and fixed with 4% paraformaldehyde  in a closed environment for 30 min, then washed three times with PBS and stained with 0.5% oil red O (Sangon Biotech) in an incubator for 30 min. Finally, the cells were washed a time with PBS and observed and photographed under the microscope. Isopropyl alcohol can dissolve intracellular lipid droplets, and the absorbance value at 490 nm detects lipid droplet content.Intracellular triglyceride content was determined by the triglyceride detection kit , following the supplier´s instructions. Differentiated cells were washed three times with PBS and digested with trypsin (Sigma-Aldrich) and centrifuged at 1500 rpm for 15 min. Lysis solution was then added to the adipocytes pellet at room temperature for 10 minutes, followed by heating at 70°C for 10 minutes and centrifuging at 2000 rpm for 5 minutes. The supernatants were detected triglycerides by measuring ODs at 550 nm wavelength by an enzyme-labelled instrument and calculating triglyceride content.SPARCL1 mRNA sequence available on the NCBI website (accession number: XM_004009982.3), we designed the following small interference sequences and a negative control sequence: si-1217-SPARCL1, si-1937-SPARCL1 , si-2244-SPARCL1 , Negative control . These sequences were synthesized by Shanghai GenePharma Gene Co. Ltd. . We verified the optimal siRNA sequence by cell transfection and qPCR for further experiments.Based on the sheep SPARCL1 mRNA sequence available on the NCBI website, we designed primer sequences to clone the CDS region of the SPARCL1 gene. The primers were synthesized by Suzhou Jin Weizhi Co. Ltd. . PEX4 vector and the restriction endonucleases XhoIII and EcoRI were used to construct recombinant plasmid completed by Shanghai GenePharma Gene Co. Ltd. pEX4 vector was used as the negative control.Based on the sheep th h. FuGENE  was a transfection reagent of the overexpression vector. 5 uL FuGENE solution was added to 200 uL Opti-MEM in an Eppendorf tube and placed at room temperature for 5 min, 3 mg plasmid was added to 200 uL Opti-MEM in another Eppendorf tube and placed at room temperature for 5 min. The two solutions were mixed well and placed at room temperature for 20 min. Each 6-well plate was spiked with 200 ul of the above mixture and 1.6 ml of fresh culture medium for 6 h. After this, we replaced the mixture with a complete culture medium. The recombinant plasmid contains green fluorescent protein, so we used fluorescence microscopy to detect the efficiency of cell transfection and qPCR to detect the results of plasmid transfection at 48th h.When the cells grew to 70% of the culture dish, we started transfection of plasmids or interference sequences to the cells with Lipofectamine 2000  transfection reagent. Lip2000 needs to be placed at room temperature for 5 min in advance, and 100 pmol siRNA and 5 uL lip2000 were added to 200 uL Opti-MEM  forming a mixed solution and placed at room temperature for 10 min. Each 6-well plate was spiked with 200 ul of the above mixture and 1.8 ml of fresh culture medium for 6 h. After this, we replaced the mixture with the complete culture medium, then detected the transfection efficiency by qPCR at 48The plasmid or siRNAs were transfected to the cells in 96-well plates for 48 h, then removed the culture medium and added the 10 mL/well of Cell Counting Kit-8 solution to each well in a 37°C incubator for 4 h. The OD value at 450 nm was read for each well and then the cell survival rate was calculated. Untransfected cells were used as a negative control and CCK8 alone was used as a low OD value.After 48 hours of cell transfection, cells were digested with 0.25% trypsin  without EDTA for 5 min, then collected in Eppendorf tubes and centrifuged at 300 x g and 4°C for 5 min, next were washed twice with PBS and centrifuged at 300 x g and 4°C for 5 min, after which we resuspended the cells with 100 uL of apoptosis detection kit buffer  with 4 uL of annexin V and 5 uL of PI working solution and incubated it in darkness at room temperature for 15 min. Finally, 400 uL PBS was added to each tube and immediately evaluated under flow cytometry. Annexin V emits spectra at 530 nm (FITC channel) and 617 nm (PI channel). Untransfected cells were used as a negative control.2O up to 10 μL, and reaction conditions were 37 oC for 15 min, 85 oC for 5 sec, and storage at 4 oC, and then the reaction products were diluted 10 times by double-distilled water. The quantitative fluorescent PCR detects the relative expression of mRNA levels in a Roche LightCycler® 480 (Roche Applied Science), and the Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH) gene was used as an internal control. The reaction system consisted of 10 μL of 2× Light Cycler 480 SYBR Green I Master, 8 μL of ddH2O, 1 μL of cDNA, and 0.5 μL of sense and antisense primers. The reaction conditions were 40 cycles of 95 oC for 5 min, 95 oC for 10 s, 60 oC for 15 s, and 72°C for 20 s, followed by 95 oC for 5 s, 65 oC for 1 min, and 40 oC for 10 s. The number of experimental repetitions was three times. The primer sequences are in We used TRIzol reagent (Thermo Fisher Scientific) to extract cellular total RNA extracts on days 2, 4, 6, 8, and 10 during cellular proliferation and differentiation, and the experimental steps are described in the instructions. The cells growing to 75% of the culture dish were on the 2nd day; The cells growing to 100% of the culture dish and added with induction solution I were on the 4th day; The cells were replaced with induction solution II on the 6th day; Replaced with fresh culture medium were on the 8th day; many cells differentiated into the mature adipocytes on the 10th day. Monitoring RNA degradation and contamination of cells and tissues using 1% agarose gels, followed by measurement of RNA concentration using a Quawell Q5000 spectrophotometer . Next, we reversed the RNA to the cDNA using a reverse transcription kit . The reverse transcription system consisted of 2 μL of 5× PrimeScript RT Master Mix, 500 ng of total RNA, and RNase Free ddHWe used a protein extraction kit  to extract total cellular protein extracts on days 2, 4, 6, 8, and 10 during cellular proliferation and differentiation. The cells were washed with PBS and treated with lysis solution for 30 min on ice and centrifuged at 12,000 rpm at 4°C for 30 min. The protein concentration was determined BCA solution, and lysate regulates the protein concentration to make the same concentration of total protein in each group. The supernatant with proteins was then boiled at 100°C for 10 minutes to denature proteins and mixed with SDS-PAGE buffer . Preparing separation and concentrated gels using the PAGE Gel Fast Preparation kit . After the gel solidification, 20 ug of proteins and 5 uL of protein markers (Thermo Fisher Scientific) were injected into the gel hole at 80 voltages for 20 min. Voltage was changed to 120 V for 40 min after proteins reached the separation gel. We transferred the proteins from the gel to a 0.45 mm Immobilon poly-vinylidene difluoride membrane  at 200 mA for 1 h, after which the PVDF membrane was washed with TBST buffer for 5 min and sealed with 5% skimmed milk powder blocking solution for 2 h. The membrane was then washed three times with TBST buffer for 5 minutes each. The membrane was incubated with antibodies and shaken overnight at 4°C. On the second day, the membrane was washed three times with TBST buffer for 5 minutes each, incubated with secondary antibodies  at room temperature for 2 h, and washed three times with TBST buffer for 5 minutes each. The chromogenic reaction of proteins used ECL hypersensitive photoluminescence solution . The protein bands data were detected using the ChemiScope 6000 Touch imaging system , and were quantified using the ImageJ software. β-actin was used as an internal reference protein. The antibody dilution ratio was in P < 0.05.All the results are expressed as means ± SEM. Comparisons between two groups using an unpaired, two-tailed Student’s t-test. Multiple group comparisons used one-way ANOVA. Data analysis was in Graph Pad Prism 6.0 software. The original image and data are in the supplementary file. The statistical significance levels were set at SPARCL1 gene expression was highest in adipose tissue . As shown in P < 0.01).We synthesized overexpression plasmids and interference sequences to detect the effect of the th day to determine the effect of SPARCL1 on the sheep preadipocytes proliferation. The results showed that interference with SPARCL1 inhibited cell proliferation and reduced cell viability (P < 0.01), but overexpression had no significant effect on cell proliferation and viability , while SPARCL1 overexpression had no significant effect on apoptosis  in the Wnt/β-catenin pathway during adipocyte differentiation to explore its possible mechanisms. As shown in th day, after which the expression level decreased (P < 0.05). Based on an understanding of their expression patterns, we chose day 6 of cell differentiation to detect the effect of SPARCL1 on these genes. The results showed that SPARCL1 overexpression significantly increased Wnt10b, Fzd8, β-catenin, and IL6, whereas SPARCL1 interference significantly decreased β-catenin and increased GSK3β (P < 0.05; SPARCL1 may affect Wnt/β catenin pathway genes regulating PPARγ, C/EBPα, and LPL to affect preadipocyte differentiation.Studies showed that the ntiation ,18. We eSPARCL1 inhibits tumour cell growth and serves as a marker for cancer detection and a therapeutic target. As the direction of research has expanded, some reports have shown that the SPARCL1 gene has additional physiological functions, e.g, SPARCL1 inhibits adipogenesis in 3T3-L1 cells [SPARCL1, which also strongly suggests that SPARCL1 may affect cellular adipogenesis. However, the function and regulatory mechanisms of lipid metabolism in SPARCL1 are not known, particularly for sheep lipid metabolism, and we are therefore keen to conduct in-depth studies. In the present study, SPARCL1 expression was the highest in the subcutaneous adipose tissue, suggesting that SPARCL1 may be involved in the process of sheep adipogenesis. SPARCL1 expression was unchanged by the addition of exogenous inducer and early preadipocyte differentiation. However, SPARCL1 expression increased more than 20-fold on the 8th day. This implies that SPARCL1 plays a role in late cell differentiation and in lipid metabolism. PPARγ, C/EBPα is known to be a key transcription factor for cell differentiation. PPARγ has a decisive role in the early stages of cell differentiation. When PPARγ is activated, it induces C/EBPα and targets to promote cell differentiation [PPARγ expression was significant when exogenous inducers were added. Subsequently, C/EBPα expression was initiated. In the present study, the expression trends of PPARγ and C/EBPα were consistent with other reports. The results indicate that the experimental data are reliable and accurate.Studies have shown that L1 cells ; SPARCL1L1 cells , 20. Alsntiation . Thus, PSPARCL1 genes on sheep preadipocytes at two different stages of proliferation and differentiation. First, we observed that SPARCL1 expression was low in cell proliferation and did not alter cell viability and apoptosis rates. when SPARCL1 expression was increased. However, some studies have shown that SPARCL1 inhibits cancer cell proliferation [SPARCL1 overexpression inhibits renal cancer cell migration and invasion, which is different from our study, where overexpression of the SPARCL1 gene did not affect sheep adipocyte proliferation [SPARCL1 interference reduced cell viability and increased the rate of apoptosis, yet in other studies, SPARCL1 inhibited cancer cellular proliferation [SPARCL1 may reduce cell proliferation, whereas SPARCL1 overexpression does not affect cell proliferation.Preadipocytes become mature adipocytes through proliferation and differentiation , so we eferation ; SPARCL1feration . It is pferation . It is pSPARCL1 plays a negative role in preadipocyte differentiation and the results are consistent with other studies [PPARγ and C/EBPα have a decisive effect on adipocyte differentiation, SPARCL1 inhibition or overexpression can affect the expression of PPARγ, C/EBPα, LPL, and IGF1, thereby regulating intracellular triglyceride and lipid droplet content. What is the mechanism by which the SPARCL1 gene regulates preadipocyte differentiation? Some studies have shown that SPARCL1 can promote C2C12 cell differentiation [Y. β-catenin signalling pathway-related genes have similar functions, so we linked SPARCL1 and WNT/β-catenin signalling pathways, and also some studies have shown that SPARCL1 is associated with WNT/β-catenin signalling pathways [SPARCL1 has a similar function with Wnt/β-catenin signalling. Also, both SPARCL1 and Wnt are highly expressed in the cytoplasm [Wnt10b is lowly expressed in adipocyte differentiation, and overexpression of SPARCL1 significantly increases Wnt10b, which is then enriched in membranes and binds Fzd8 in the present study [GSK3β and increases β-catenin suppresses PPARγ and C/EBPα [D. GSK3β translocates to the nucleus, phosphorylates PPARγ and C/EBPα, and promotes adipocyte differentiation [SPARCL1 overexpression decreased GSK3β, PPARγ, C/EBPα. The results are consistent with Wnt/β-catenin signalling regulating adipocyte differentiation [SPARCL1 gene expression started to rise at day 6, it did not affect the conclusion of this experiment, and the results just indicate that SPARCL1 is one of the factors that can affect Wnt/β-catenin-related genes. Wnt/β-catenin is affected by a variety of factors during preadipocyte differentiation. SPARCL1 can regulate Wnt/β-catenin-related genes to influence preadipocyte differentiation.At the stage of adipocyte differentiation,  studies . Becauseation Y. , which iation Y. , and thepathways . Wnt sigpathways . Wnt sigpathways . Competipathways . SPARCL1ytoplasm , so we ent study . Wnt10b-/EBPα D. . GSK3β tntiation . In the ntiation ,37. AlthSPARCL1 in sheep adipocyte differentiation, and the study has practical implications. On the one hand, this study provides a new research direction to explore the regulatory mechanism of sheep preadipocyte differentiation. The newly identified regulatory genes can be used as molecular markers for screening sheep with good meat quality, and the application of genetic engineering technology can improve the meat quality of Small Tail Han sheep. On the other hand, SPARCL1 may provide new therapeutic targets for metabolic diseases caused by obesity, which can help us better understand adipose differentiation and metabolism.The present study is the first to show the role of SPARCL1 is involved in the process of sheep preadipocyte differentiation and plays a role in late differentiation. In addition, interference with SPARCL1 leads to apoptosis. SPARCL1 may be a negative regulator of adipocyte differentiation and lipid droplet accumulation. Thus, SPARCL1 may be a new potential target for increasing IMF content in sheep.Click here for additional data file.


Our previous study with human BMMSCs showed that megalin is required for the 25(OH)D3-DBP complex to enter cells and for 25(OH)D3 to stimulate osteoblast differentiation in BMMSCs. Furthermore, we reported that leptin up-regulates megalin in those cells. Leptin is a known inhibitor of PI3K/AKT-dependent chaperone-mediated autophagy (CMA). In this study, we tested the hypothesis that leptin acts synergistically with 25(OH)D3 to promote osteoblastogenesis in rat BMMSCs by a mechanism that entails inhibition of PI3K/AKT-dependent CMA.Vitamin D is important for the mineralization of bones by stimulating osteoblast differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are a target of vitamin D action, and the metabolism of 25(OH)D2D3 was measured by a Rat 1α,25(OH)2D3 ELISA Kit.BMMSCs were isolated from rat bone marrow ; qRT-PCR and western immunoblots or immunofluorescence were used to evaluate the expression of megalin, ALP, COL1A1, RUNX2, OSX, OSP, and CMA in rBMMSCs. The osteoblast differentiation was evaluated by ALP activity, ALP staining, and calcium deposition. The viability of rBMMSCs was assessed with the CCK-8 kit. Biosynthesis of 1α,25(OH)3 treatment significantly enhanced osteoblast differentiation as shown by ALP activity, ALP staining, and calcium deposition, the expression of osteogenic genes ALP, COL1A1, RUNX2, OSX, and OSP by qRT-PCR and western immunoblots in rBMMSCs. Leptin enhanced the expression of megalin and synthesis of 1α,25(OH)2D3 in rBMMSCs. Our data showed that leptin inhibited CMA activity of rBMMSCs by activating PI3K/AKT signal pathway; the ability of leptin to enhance 25(OH)D3 promoted osteoblast differentiation of rBMMSCs was weakened by the PI3K/AKT signal pathway inhibitor.The combination of leptin and 25(OH)D3 promote osteoblast differentiation in rBMMSCs. Leptin promoted the expression of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. PI3K/AKT is at least partially involved in the regulation of CMA. These data indicate the importance of megalin in BMMSCs for vitamin D’s role in skeletal health.Our data reveal the mechanism by which leptin and 25(OH)DThe online version contains supplementary material available at 10.1186/s13287-021-02623-z. The maintenance of bone mass in the human body results from the dynamic balance of bone resorption and bone formation, osteoclasts absorb calcified bone matrix, and osteoblasts synthesize new bone matrix. When the rate of bone resorption is faster than bone formation, the bone mass decreases gradually, which eventually leads to osteoporosis and increases the risk of fracture, which usually occurs in older people and postmenopausal women as well as in many secondary osteoporosis cases, e.g., cortisone, malnutrition, renal failure, immobilization, side effect of drugs –3. Bone 2D3, the active metabolite of vitamin D, in which 1α,25(OH)2D3 induces BMMSCs to differentiate into osteoblasts [3) needs to be hydroxylated to 1α,25(OH)2D3 by 25(OH)D3-1α-hydroxylase before it has a physiological role [3 is more stable than 1α, 25(OH)2D3 in blood; the half-life of 25(OH)D3 is about 3 weeks, and 1α, 25(OH)2D3 is only about 4 h. Vitamin D mainly in the form of 25(OH)D3 exists in the blood, and 25(OH)D3 has a high affinity with vitamin D binding protein (DBP) [3-1α-hydroxylase, which hydroxylates 25(OH)D3 into the more active form, 1α, 25(OH)2D3 [3 is related to osteogenesis [Vitamin D is essential for the overall mineralization of bones and the speed of bone conversion . Vitamineoblasts . 25-Hydrcal role . 25(OH)Din (DBP) –13. It i5(OH)2D3 . The levogenesis , 15.3 into BMMSCs [3 entered cells as the 25(OH)D3-DBP complex via megalin receptors on the cell membrane and hydroxylated to 1α, 25(OH)2D3 under the action of 25(OH)D3-1α-hydroxylase. We also found that there were significant differences in constitutive expression of megalin in samples from different human subjects [3, which arises the hypothesis in this study that up-regulation of megalin improves to enhance 25(OH)D3 to stimulate the osteoblast differentiation of BMMSCs. Leptin directly affects osteoblasts and indirectly affects bone mineral density through the change of body weight, in which leptin levels are higher in obese people, and obesity is positively correlated with bone mineral density [3 on osteoblast differentiation remains unclear; in this study, we investigated whether leptin enhances the sensitiveness of BMMSCs to 25(OH)D3 by promoting the expression of megalin.Our previous studies showed that megalin  is one of the key receptors for 25(OH)Do BMMSCs . In vitrsubjects . The sam density . It was  density . The relChaperone-mediated autophagy (CMA) is selective autophagy, which can degrade soluble cellular proteins through the lysosome; the proteins were degraded in the lysosomal cavity through lysosomal-associated membrane protein 2A (LAMP2A), which is the key rate-limiting receptor of CMA and can affect the activity of CMA; various organelle fragments, misfolded proteins, and pathogens are degraded by CMA pathway, and CMA maintains the stable expression of some large proteins such as cell membrane receptors . It is r3 promote osteoblast differentiation in rBMMSCs via the up-regulation of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. We demonstrated that PI3K/AKT is at least partially involved in the regulation of CMA. Our data indicate that megalin in BMMSCs is vital for vitamin D’s role in skeletal health.This study revealed the mechanism by which leptin and 25(OH)DAll procedures were approved by the Shandong University Committee on the Use and Care of Animals and conducted per the Guidelines for the Care and Use of Laboratory Animals. Sprague Dawley (SD) rats were purchased from SPF (Beijing) Biotechnology Co. Ltd. .3 was purchased from Aladdin . PI3K/AKT signaling pathway inhibitor LY294002 was purchased from Sigma-Aldrich . Antibodies against phosphorylated-p38 , total-p38 , β-actin (#3700), phosphorylated STAT3  and glyceraldehyde-3-phosphate dehydrogenase , anti-mouse IgG (H + L) (DyLight™ 680 Conjugate or DyLight™ 800 4X PEG Conjugate), and anti-rabbit IgG (H + L) (DyLight™ 680 Conjugate or DyLight™ 800 4X PEG Conjugate) secondary antibody were purchased from Cell Signaling Technology . Antibodies against phosphorylated-AKT , total-AKT , total STAT3 , and megalin (sc-515772) were purchased from Santa Cruz Biotechnology . Antibodies against runt-related transcription factor 2 , collagen type I , and LAMP2A (ab125068) were obtained from Abcam . Antibodies against HSC70 (AF5187) and leptin receptor (DF7139) were obtained from Affinity Biosciences . Fluorescent second antibody rhodamine (TRITC)-conjugated goat anti-mouse IgG (H + 2) was purchased from ZSGB-BIO . Alkaline phosphatase (ALP) activity kit, BCIP/NBT alkaline phosphatase (ALP) color development kit, enhanced cell counting Kit-8 (CCK-8), and Alizarin Red Staining (ARS) were purchased from Beyotime . Dexamethasone, β-glycerophosphate, and ascorbate-2-phosphate were purchased from Solarbio Science and Technology Co., Ltd. . Rat 1α,25(OH)2D3 ELISA Kit was purchased from Bioswamp Life Science Lab . All primers were synthesized by BioSune .Recombinant rat leptin (CYT-227) was purchased from Prospec-Tany TechnoGene Ltd (Israel). 25(OH)D2 at 37 °C. Nonadherent cells were removed by medium change after 48 h to 72 h; then, the medium was replaced every 3 days. When the cell density reached 80–90% confluency, the cells were trypsinized and subcultured at the ratio of 1: 3. Passage (P) 3 to 5 rBMMSCs were used in this study.Four-week-old (100–150 g) male SD rats were selected as the source of primary rBMMSCs for culture in vitro. The rBMMSCs were isolated from bone marrow with the method as follows. SD rats were euthanized with pentobarbital sodium  followed by cervical dislocation and bathed in 75% ethyl alcohol for 15 min. The muscles were removed from all femurs and tibias, and the bones were soaked and rinsed with aseptic phosphate-buffered saline . Both epiphyses were removed to expose the marrow cavity. The contents of the marrow were flushed into a sterile 100-mm tissue culture dish  with Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). The flushing was repeated 3 times; the process was repeated in the opposite direction. The pooled mixture was centrifuged at 1000 RPM for 5 min, the supernatant was discarded, and the cells were suspended with a complete culture medium and plated into a 100-mm tissue culture dish (NEST Biotechnology Co.LTD). The rBMMSCs were cultured in a saturated humidity incubator with a volume fraction of 5% CO−△△Ct method was used to calculate gene relative expression levels. The primers for qRT-PCR are shown in Table The total RNA was extracted from rBMMSCs with TRIzol reagent  according to the manufacturer’s instructions. Complementary DNA was synthesized with ReverTra Ace Qpcr RT Kit  according to the manufacturer’s instructions. The qRT-PCR was performed on ABI 7900HT Fast Real-Time PCR system  using SYBR Green Realtime PCR Master Mix (Toyobo Life Science) to evaluate the expression levels of megalin, RUNX2, ALP, COL1A1, OSX, OSP, LAMP2A, HSC70, and β-actin . The 2Total protein was extracted from rBMMSCs with radioimmunoprecipitation assay lysis buffer (Beyotime) and protease inhibitor cocktail . Protein concentration was measured with the BCA kit . After denaturation, 40 ug aliquots of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane . Membranes were blocked with 5% nonfat milk at room temperature for 1–2 h. Membranes were sequentially incubated overnight at 4 °C with the following primary antibodies: GAPDH , β-actin , RUNX2 , COL1A1 , t-AKT , p-AKT , t-p38 , p-p38 , p-STAT3 , t-STAT3 , LAMP2A , HSC70 , or leptin receptor . The membranes were washed with TBST three times and incubated with the anti-mouse IgG (H + L) (DyLight™ 680 Conjugate or DyLight™ 800 4X PEG Conjugate) or anti-rabbit IgG (H + L) (DyLight™ 680 Conjugate or DyLight™ 800 4X PEG Conjugate) secondary antibody for 30 min at room temperature. The fluorescent signal on membranes was detected by Odyssey two-color infrared fluorescence imaging system ; band gray values were quantified with ImageJ software.3 on the viability of rBMMSCs were assessed with the CCK-8 kit (Beyotime). Different concentrations of leptin , 25(OH)D3 , or the combination of leptin (300 ng/ml) and 25(OH)D3 (100 nM) was added to each well, with 5 replicate wells, at 24 h after seeding in 96-well plates (5000 cells/well). After 24, 48, and 72 h, 10 ul CCK-8 solution was added to each well according to the manufacturer’s instructions. Absorbance at 450 nm was measured with Varioskan flash .Effects of leptin and/or25(OH)D5/well); after reaching confluence in basal medium, cultures were changed to osteoblast differentiation medium ; leptin (300 ng/ml) and/or 25(OH)D3 (100 nM) were added to the ODM, 24 h after medium changes. The concentration of 25(OH)D3 used in the present study was chosen according to the optimal physiological concentrations [The rBMMSCs were plated into 6-well plates (2 × 10trations . The ODM3 (100 nM), 7 days in ODM. For ALP staining, the cells were washed with PBS 3 times and fixed with 4% paraformaldehyde (Solarbio Life Sciences) for 20 min. They were washed 3 times with PBS and stained with BCIP/NBT ALP color development kit (Beyotime). For the measurement of ALP activity, the rBMMSCs were collected and incubated at 37 °C for 30 min according to the manufacturer’s instructions of ALP Activity Assay Kit (Beyotime). The ALP activity was detected at 405 nm with Varioskan flash (Thermo Scientific).ALP staining and activity assays were performed in rBMMSCs in 12-well plates with/without leptin (300 ng/ml) and/or 25(OH)D3 (100 nM), 21 days in ODM, calcium deposition was quantified with the ARS Kit (Beyotime). In brief, cells were washed 3 times with distilled water, fixed with 4% paraformaldehyde (Solarbio) for 20 min, and washed 3 times with distilled water. Cells were then incubated with 1% ARS solution for 30 min at room temperature and rinsed with distilled water. For measure of the relative value of ARS, the stain was dissolved with 10% cetylpyridinium chloride  for 1 h. Aliquots (200 μl) were transferred to 96-well plates and detected at 405 nm with Varioskan flash (Thermo Scientific).After the rBMMSCs were cultures with/without leptin (300 ng/ml) and/or 25(OH)D4/well in 24-well plates) were seeded onto alcohol-washed coverslips  with 500 ul basal medium; after reaching 60–70% confluence, mediums were changed to ODM with different concentrations of leptin , 25(OH)D3 (100 nM), and leptin (300 ng/ml) + 25(OH)D3 (100 nM) for 24 h. The coverslips were washed with PBS 3 times, each time for 3 min; then, rBMMSCs were fixed with 4% paraformaldehyde for 20 min at room temperature and were washed with PBS 3 times, each time for 3 min. Triton Xmuri 100  was used to permeate the cells. They were washed 3 times with PBS and blocked with 5% BSA for 30 min at room temperature. First megalin antibody  was applied to each coverslip and incubated overnight at 4 °C in a humidified chamber. Thereafter, they were washed 3 times and incubated with fluorescent second antibody rhodamine (TRITC)-conjugated goat anti-mouse IgG (H + 2) , in the humidified chamber at room temperature for 1 h. Subsequent steps were carried out in darkness. The coverslips were washed with PBS 3 times for 3 min each time. For staining nuclei, the coverslips were incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride  for 5 min; images were collected with the Lionheart FX . The optical density of more than 6 cells was randomly measured with ImageJ software, and the average optical density was calculated to represent the fluorescence intensity.The rBMMSCs (5 × 103 (100 nM), with or without leptin (300 ng/ml), supernatants were collected and stored at − 80 °C for subsequent determination of 1α,25(OH)2D3. The cells were collected for western immunoblotting. 1α,25(OH)2D3 levels in the supernatants were measured by a Rat 1α,25(OH)2D3 ELISA Kit  according to the manufactures’ protocol.The rBMMSCs were cultured in 6-well plates in growth medium until confluent; 24 h after the medium was changed to ODM with 25(OH)Dt test, one-way ANOVA test, and Tukey’s multiple comparisons test, and the Kolmogorov–Smirnov test was used to analyze the normal distribution of data. A value of P < 0.05 was considered significant.All experiments were repeated at least three times. All data are presented as means ± standard deviation, and statistical analyses were performed with SPSS 20. Significant differences between the groups were determined by Student’s 3 treatment during osteoblast differentiation Dtin Fig. a. At thetin Fig. d. IF dat3 had cytotoxicity on proliferation/viability of rBMMSCs, different concentrations of leptin and/or 25(OH)D3 were used to stimulate rBMMSCs for 3 days, and then, CCK-8 analysis was used to measure cell proliferation/viability. The results showed that leptin at concentrations  has no effect on the proliferation of rBMMSCs at day 3 and, however, promoted the proliferation of rBMMSCs at doses of 100, 300, 500 ng/ml  promoted the proliferation of rBMMSCs at 48 h and 72 h D3, we measured mRNA expression level of osteoblast genes , 12 h, 3, and 5 days after rBMMSCs were transferred to ODM D3 was gradually increased with the extension of time. Moreover, leptin or 25(OH)D3 had no significant effect on the mRNA and protein expression of leptin receptors D3 compared with 25(OH)D3 group in ODM for 24 h, as assessed by ELISA D3 with or without leptin on the ALP activity of rBMMSCs. The results showed that there was no significant change in ALP Activity of rBMMSCs at 7 days after 25(OH)D3 or leptin was added to ODM alone. The combination of 25(OH)D3 and leptin resulted in significantly increased ALP at day 7 D3 did not affect CMA in rBMMSCs with or without leptin /p38, and PI3K/AKT signaling pathways. Our results showed that the expression level of p-AKT protein, but not p-STAT3, t-STAT3, p-P38, t-P38, and t-AKT, increased significantly, 1 h after rBMMSCs was stimulated with the increase in leptin concentration in ODM , to inhibit the expression of p-AKT and to measure the expression of CMA-related genes and megalin by qRT-PCR, IF, and western immunoblot. The PI3K/AKT signal pathway inhibitor LY294002 was added to the ODM 2 h earlier before leptin and 25(OH)D3 on the stimulation of osteoblast differentiation via PI3K/AKT signal pathway, we used PI3K/AKT signal pathway inhibitor . The increased mRNA expression levels of ALP, COL1A1, RUNX2, OSP, and OSX promoted by leptin synergistically with 25(OH)D3 were significantly inhibited by PI3K/AKT signal pathway inhibitor  at 12 h, day 3 and 5 D3 group  D2D3 plays an essential role in the osteoblast differentiation of BMMSCs, which may prevent age-related osteoporosis [3 by 25-hydroxylase (CYP2R1) in the liver and thereafter to active 1α,25(OH)2D3 by 1α-hydroxylase (CYP27B1) in the kidney [3 binds to vitamin D binding protein (DBP) in blood and enters into the proximal tubular cell via the megalin receptor [3-DBP complex [3 in human BMMSCs and osteoblasts [3. We found that the megalin expression levels in BMMSCs of some donors were relatively low, and 25(OH)D3 has little effects on the MSCs, which poor expression megalin [1α,25(OH)oporosis . There ae kidney . Many ste kidney –26. Evidylase CYPB1 in the complex . Many ex complex . Our preeoblasts , 30, and megalin . In this3 induces the osteoblast differentiation of BMMSCs at 250 nM, some even up to excess 500 nM, but not at 100 nM [3. At present, there is controversy about the normal physiological safe concentration of 25(OH)D3 in humans, which high concentration of 25(OH)D3 (> 125 nM) is thought to have side effects on the body [3. The concentration of 25(OH)D3 (100 nM) used in our study that did not significantly induce osteoblast differentiation of BMMSCs in vitro, but the co-stimulation of leptin and 25(OH)D3 significantly promoted the osteoblast differentiation of rBMMSCs showed with the results of qRT-PCR and western blots that the expression levels of osteoblastogenic genes ALP, COL1A1, RUNX2, OSX, and OSP are increased; ALP activity and ARS enhanced significantly after co-stimulation of leptin and 25(OH)D3.Studies have shown that 25(OH)Dt 100 nM –34; we hthe body –37; ther3 on the osteoblast differentiation of rBMMSCs. The results of qRT-PCR and IF showed that leptin enhanced the expression of megalin in rBMMSCs, but 25(OH)D3 had no effect on megalin; the ELISA results showed that leptin promoted 25(OH)D3 to enter into rBMMSCs. Our studies showed the importance of megalin expression in rBMMSCs for their osteoblast differentiation potential. Synergistic actions of 25(OH)D3 with leptin on osteoblast differentiation required up-regulation of megalin, the key receptor of the 25(OH)D3-DBP complex. When the megalin was up-regulated, more 25(OH)D3 was utilized to induce more osteoblast differentiation of rBMMSCs. These results are consistent to our previous report with human BMMSCs [We explored the mechanism of synergistic stimulation of leptin and 25(OH)Dn BMMSCs .3 had no significant effect on the expression of leptin receptor. PET imaging showed that 15% labeled leptin concentrated in red bone marrow in rhesus monkeys and binding at high level in bone marrow tissue of mice via leptin receptor [Leptin regulates energy metabolism and appetite through the hypothalamic leptin receptor . Some evreceptor .3 into rBMMSCs was decreased, and the synergistic effect of leptin and 25(OH)D3 on osteoblast differentiation of rBMMSCs was inhibited. There are three types of autophagy, including macroautophagy, microautophagy, and CMA; similar to the results of our study, leptin is an inhibitor of autophagy to promote apoptosis of chondrocytes through PI3K/AKT signal pathway during osteoarthritis pathogenesis [Most importantly, we discovered that leptin inhibited CMA through the PI3K/AKT signal pathway to promote the expression of megalin, and the expression level of genes (LAMP2A and HSC70) was suppressed. The results of qRT-PCR and western blots showed that PI3K/AKT inhibitor could reverse the inhibited effect of CMA caused by leptin, and the expression of megalin was down-regulated. ELISA results showed that the entry of 25(OH)Dogenesis . In a chogenesis . These rCMA maintains the physiological function of cells, which can degrade specific cellular proteins in time through the lysosome. These proteins contain specific amino acid sequences KFERQ; about 40% of the proteins in the mammalian contain typical KFERQ sequences. Heat-shock protein HSC70 recognizes and binds the specific amino acid sequence of these proteins, unfolds the substrate protein, then enters the lysosome through the LAMP2A receptor on the lysosomal membrane for subsequent degradation . LAMP2A 3 to promote osteoblast differentiation. Leptin promoted the expression of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. PI3K/AKT is at least partially involved in the regulation of CMA. These data indicate the importance of megalin in BMMSCs for vitamin D’s role in skeletal health.In sum, these data suggest the mechanism by which there is synergy between leptin and 25(OH)DAdditional file 1. Identification of rBMMSCs and morphological and microstructure changes during osteoblast differentiation. a Morphology of BMMSCs at P3 under the inverted microscopy . b Flow cytometry analysis of surface markers  of BMMSCs. c The morphological changes of rBMMSCs during osteoblast differentiation at day 10 under the inverted microscopy . d The microstructure changes of rBMMSCs during osteoblast differentiation at day 10 under the transmission electron microscope Additional file 2. The cytotoxicity effects of leptin and 25(OH)D3 on rBMMSCs proliferation. a Leptin with different concentrations on the cytotoxicity effects of rBMMSCs proliferation at day 3. b 25(OH)D3 with different concentrations on the cytotoxicity effects of rBMMSCs proliferation at day 3. Additional file 3. Leptin synergistically with 25(OH)D3 to induce osteoblast differentiation of rBMMSCs and was inhibited by inhibitor LY294002. a, b Relative mRNA expression of osteoblastogenic genes  at days 3 and 5. c, d The expression of protein of leptin receptor at day 3. e, f The effect of inhibitor on the mRNA expression of osteoblastogenic genes at day 3 and 5. D3 group.)


Here, we report the biochemical characterization of two hNEIL2 variants found as polymorphisms in the general population, R103W and P304T. Arg103 is located in a long disordered segment within the N-terminal domain of hNEIL2, while Pro304 occupies a position in the β-turn of the DNA-binding zinc finger motif. Similar to the wild-type protein, both of the variants could catalyze base excision and nick DNA by β-elimination but demonstrated a lower affinity for DNA. Steady-state kinetics indicates that the P304T variant has its catalytic efficiency (in terms of kcat/KM) reduced ~5-fold compared with the wild-type hNEIL2, whereas the R103W enzyme is much less affected. The P304T variant was also less proficient than the wild-type, or R103W hNEIL2, in the removal of damaged bases from single-stranded and bubble-containing DNA. Overall, hNEIL2 P304T could be worthy of a detailed epidemiological analysis as a possible cancer risk modifier.Human NEIL2 DNA glycosylase (hNEIL2) is a base excision repair protein that removes oxidative lesions from DNA. A distinctive feature of hNEIL2 is its preference for the lesions in bubbles and other non-canonical DNA structures. Although a number of associations of polymorphisms in the  N-glycosidic bond ATP  and phage T4 polynucleotide kinase, as specified in the text. The 32P-labeled oligonucleotides were purified by reverse-phase chromatography on an Isolute C18 sorbent . The AP site-containing substrate was obtained by treating 1 μM duplex 23U//23C , and escribed . Oligonu23U//23C  with UnghNEIL2 insert was kindly provided by Dr. Murat Saparbaev (Paris-Saclay University). The insert was PCR-amplified and re-cloned into pLATE31 using the aLICator ligation-independent cloning kit  according to the manufacturer’s protocol. The resulting construct codes for a full-length hNEIL2 equipped with a C-terminal -GHHHHHH peptide. Site-directed mutagenesis was carried out using the QuikChange Site-Directed Mutagenesis Kit . The sequences of the inserts were confirmed by Sanger sequencing. E. coli BL21(DE3) cells carrying the plasmids were grown overnight at 37 °C with vigorous shaking in 3 mL of LB broth containing 50 μg/mL carbenicillin, inoculated into 50 mL of the same medium, and the growth continued until A595 = 0.8. Synthesis of the recombinant protein was induced by adding isopropyl β-D-1-thiogalactopyranoside to 1.5 mM and ZnSO4 to 10 μM for 5 h. The bacteria from several cultures grown in parallel were pelleted by centrifugation , and the cell paste (~12 g) was frozen at −70 °C until further use. For purification, the thawed pellet was resuspended in 20 mM sodium phosphate buffer (pH 7.4) supplemented with 1 mM phenylmethylsulfonyl fluoride and lysed by ultrasound . The lysate was clarified by centrifugation  and loaded on a 5 mL SP-Sepharose column  equilibrated in 20 mM sodium phosphate (pH 7.4). The proteins were eluted with a 0–1000 mM NaCl gradient in the same buffer and analyzed by 12% polyacrylamide gel electrophoresis (Laemmli system). The fractions containing the protein band with the expected mobility were loaded on a 5 mL Ni–NTA agarose column  equilibrated in 20 mM sodium phosphate (pH 7.4) supplemented with 500 mM NaCl and 5 mM imidazole. The proteins were eluted with a 5–500 mM NaCl gradient in the same buffer. The fractions containing the target protein were collected, dialyzed against the storage buffer  and kept at −20 °C.pET-21b plasmid carrying a 2, 1 mM DTT, and 0.5 mg/mL bovine serum albumin. For the enzyme activity experiments, the substrate concentration was 10 nM or 100 nM, and the enzyme concentration and incubation time was varied as needed. The reactions were allowed to proceed at 37 °C, 10 μL aliquots were withdrawn and terminated by mixing with an equal volume of gel loading solution  and heating for 2 min at 95 °C. For the kinetic experiments, the substrate concentration was 10–1800 nM, of which radioactively labeled substrate constituted 10 nM, and the enzyme concentrations were 1.5 nM for WT hNEIL2, 0.75 nM for hNEIL2 R103W, and 5.5 nM for hNEIL2 P304T. The reactions were allowed to proceed for 60 min at 37 °C and terminated as above. The reaction products were separated by electrophoresis in 20% polyacrylamide/7.2 M urea, and the gels were imaged and quantified using the Typhoon FLA 9500 imager .The reaction mixture included 25 mM potassium phosphate (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.5 mg/mL bovine serum albumin, 10% glycerol, 35 nM bubble DNA ligand or 100 nM duplex oligonucleotide ligand, and varying enzyme concentrations. The mixtures were incubated on ice for 5 min and loaded on an 8% non-denaturing polyacrylamide gel. The gels were run at 10 V/cm, thermostated at 4 °C. The gels were imaged and quantified as above. The apparent binding constants were obtained by fitting to a sigmoidal binding model using SigmaPlot v11.0 .The reaction mixture included 25 mM potassium phosphate (pH 7.5), 5 mM MgClhttp://fathmm.biocompute.org.uk/, “Inherited disease” prediction mode; all URLs accessed on 15 December 2021), Mutation Assessor [http://mutationassessor.org/r3/, accessed on 15 December 2021), MutationTaster [https://www.mutationtaster.org/, accessed on 15 December 2021), PolyPhen-2 [http://genetics.bwh.harvard.edu/pph2/, accessed on 15 December 2021), and Provean [http://provean.jcvi.org/, “PROVEAN Protein” tool, accessed on 15 December 2021). The output from Mutation Assessor was converted to the binary output by classifying the “neutral/low” prediction as “no effect”  and the “medium/high” prediction as “affected” . The binary outputs “tolerated”, “polymorphism” or “neutral” from other algorithms were classified as “no effect”, and “damaging”, “disease causing” or “deleterious” as “affected”. The mean of all predictions was taken as an overall measure of the effect.To predict the possible effects of amino acid changes in hNEIL2, we have used online versions of the following five classifiers: FATHMM  (http://Assessor  (http://onTaster  (https:/lyPhen-2  (http:// Provean  (http://The homology models of hNEIL2 were built using SwissModel suite . The str


The phase structure, micro structure morphology, and electrical properties of BSI–PT–PMN–xCo were systematically studied. The introduction of Co ions exerted a significant influence on the structure and electrical properties. The experiment results demonstrated that Co ions entered the B-sites of the lattice, resulting in slight lattice distortion and a smaller lattice constant. The average grain size increased from ~1.94 μm to ~2.68 μm with the increasing Co content. The optimized comprehensive electrical properties were obtained with proper Co-modified content 0.2 wt.%. The Curie temperature (Tc) was 412 °C, the piezoelectric constant (d33) was 370 pC/N, the remnant polarization (Pr) was 29.2 μC/cm2, the relatively dielectric constant (εr) was 1450, the planar electromechanical coupling coefficient (kp) was 46.5, and the dielectric loss (tanδ) was 0.051. Together with the enhanced DC resistivity of 109 Ω cm under 300 °C and good thermal stability, BSI–PT–PMN–0.2Co ceramic is a promising candidate material for high-temperature piezoelectric applications.Cobalt-modified 0.40Bi(Sc Tc), which means their piezoelectric properties decrease quickly with rising environmental temperatures, restrict their high-temperature application in petrochemical, aerospace, and other industries [Lead zirconate titanate (PZT) systems represent most of the market share of piezoelectric materials because of their excellent piezoelectric performance, simple preparation process, and low cost ,2,3. Howdustries ,5,6.x)Bi(Me)O3–xPbTiO3, where Me3+ refers to Sc3+, Y3+, In3+, Yb3+, etc. Preferably, (1−x)BiScO3–xPbTiO3 (BS–PT) ceramics exhibit a morphotropic phase boundary (MPB) at x = 0.64, with a high Curie temperature of 450 °C and excellent piezoelectric performance (d33 = 460 pC/N), comparable to commercial soft PZT ceramics [3+ by other +3 valence cations, such as In3+, Co3+, et al. Unfortunately, although the Tc is still over 400 °C, the piezoelectric properties decrease significantly. For example, for the In3+-modified, BS–PT based solid solutions Bi(Sc3/4In1/4)O3–PbTiO3 (BSI–PT), the Tc reaches 457 °C, whereas the d33 reduces to only 201 pC/N [3+ by composition ions such as (Mg1/2Ti1/2)3+, (Ni1/2Ti1/2)3+, (Zn1/2Zr1/2)3+, (Ni1/2Zr1/2)3+, etc. With proper composition ion modification, obtained improved piezoelectric properties can be obtained, together with considerable Tc. For example, BS–PT–0.025Bi(Ni1/2Zr1/2)O3 solid solutions exhibit d33 of 480 pC/N and Tc 439 °C for MPB composition [3 ternary solid solutions. The electrical properties can be regulated due to the different chemical composition of ABO3. For example, the piezoelectric properties  and Curie temperature, of 408 °C, were obtained for the MPB composition in the BS–PT–Pb(Sn1/3Nb2/3)O3 system [3 system possesses an enhanced d33 of 551 pC/N and reduced Tc of 337 °C in the vicinity of the MPB [3 system exhibits an enhanced Tc of 511 °C and a reduced d33 of 102 pC/N [In 2001, Eitel et al.  first receramics . Later, ceramics ,13,14,15ceramics ,20,21,22ceramics ,28,29,30201 pC/N  for the 3 system . Further3/4In1/4)O3–PbTiO3–Pb(Mg1/3Nb2/3)O3 to modify the electrical properties of the BS–PT solid solution [x)BSI–xPT–0.02PMN ceramics at the MPB x = 0.58 of piezoelectric constant (d33) 403 pC/N, planar electromechanical coupling factor (kp) 47.2%, and remnant polarization (Pr) 36.4 μC/cm2. The Curie temperature (Tc) remained at 421°C, making the system much more suitable for high-temperature piezoelectric applications.In our previous study, we combined the first method and the third method to form a new ternary system BiO3–0.58PbTiO3–0.02Pb(Mg1/3Nb2/3)O3 ternary ceramics, aiming to generate good electrical performance while still providing a relatively high Curie temperature.Furthermore, high electrical resistance is so necessary for high-temperature application that a large electric field can be applied during poling without breakdown or excessive charge leakage. As a common doping element in various piezoelectric material systems, cobalt is often used to improve the electrical properties of piezoelectric materials ,35,36,373/4In1/4)O3–0.58PbTiO3–0.02Pb(Mg1/3Nb2/3)O3–xCo  ceramics were produced through conventional two-step solid state processing. The reagent-grade materials of Bi2O3 (99.8%), Sc2O3 (99.99%), In2O3 (99.99%), PbO (99%), TiO2 (99.5%), MgO (99%), Nb2O5 (99.9%), and Co2O3 (99.9%) were utilized as the raw materials and weighted by the stoichiometric amount. First, the powder MgO and Nb2O5 were mixed and calcined at 1100 °C for 4 h to synthesize the columbite precursor MgNb2O6. Next, the precursor MgNb2O6 and other raw materials were mixed together for 12 h by ball milling with alcohol. The powders were then dried, ground, granulated by milling with polyvinyl alcohol (PVA) as the binder, and then pressed into tablets with a diameter of 12.5 mm and a thickness of 1.5 mm under 120 MPa of pressure. Before sintering, a cold isostatic pressing process of 200 MPa was carried out on the tablets for 20 min. After burning out PVA at 650 °C, the tablets were sintered at 1120 °C for 3 h in a muffle furnace.The 0.40Bi. The P-E hysteresis loops of the samples were characterized using a ferroelectric analyzer . The temperature dependence of the DC resistivity was measured by a Digit Multimeter . When it was necessary to measure the thermal stability, the samples were heated to the set temperature and held for an hour. The piezoelectric coefficient d33 was measured when the samples cooled down to room temperature.The X-ray diffraction (XRD) patterns of the BSI–PT–PMN samples were obtained by using an X-ray diffractometer . The fresh fracture surface microstructure of the samples was observed by using scanning electron microscopy (SEM) . After polishing and ultrasonic cleaning, the ceramic samples were evenly coated with silver slurry and then fired at 600 °C. After cooling to room temperature, the ceramic samples were poled at 120 °C in a silicone oil bath under a DC electric field of 4.5 kV/mm for 20 min. The dielectric behavior as a function of temperature was analyzed using an impedance analyzer . The piezoelectric coefficient xCo . From the figure, one can clearly observe that all the Co-doped ceramic samples presented the perovskite phase with a secondary phase of Co2O3 (Index PDF#02-0770), indicating that the Co modification did not change the crystal structure of BSI–PT–PMN ceramics significantly. Furthermore the characteristic peak moved slightly, to a high angle, as the Co content increased, which can be seen in dsinθ = nλ), the lattice constant was calculated and listed in a changed slightly and the lattice parameter c decreased with the increasing Co content x. It has been reported that Co ions exist in the valence state as Co2+ if the sintered temperature exceeds 1080 °C [3+ and Co2+ were coexistent for Co2O3 used as dopants in this work. Furthermore, the radii of Co3+ and Co2+ were 0.63 Å and 0.72 Å, respectively, smaller than the radius of Ti4+ of 0.86 Å; Co3+ or Co2+ may have entered the lattice to replace Ti4+ in the B-sites for the BSI–PT–PMN ceramics and resulted in the decrease in the lattice constant. In addition, the c/a ratio decreased from 1.022 to 1.017 with the increasing Co content x, which exerted a significant effect on the electric properties. 1080 °C . On accoxCo  sintered at 1120 °C. All the samples exhibited a dense structure with no obvious porosity, in accordance with the high relative density listed in xCo samples were statistically analyzed and listed in in Nano Measurer software , free from the Internet), with the number of the grains over 50. It was found that the average grain size increased from ~1.94 μm to ~2.68 μm as the Co content increased, suggesting that the doping of Co ions can reduce the sintering temperature and facilitate grain growth, which has been reported in other Co-doped piezoelectric ceramics [ceramics .ε) for the BSI–PT–PMN–xCo ceramics. It can be seen that the dielectric peak of the BSI–PT–PMN–xCo ceramics became suppressed and broader when the measured frequency increased, and there was no frequency dispersion in any of the samples. The spectral line of the dielectric permittivity featured only one peak, which corresponded to the Curie temperature.t’ caused by the B-site substitution. For the perovskite ferroelectric structure, the tolerance factor ‘t’ can be described asrA, rB, and rO refer to the ionic radii of the A-site, B-site, and oxygen atom, respectively. As is already known, for the perovskite ferroelectric structure, there is a consistent relationship between the Curie temperature and the tolerance factor ‘t’: the lower tolerance factor ‘t’, the higher the Curie temperature. As discussed above, the introduced Co3+ and Co2+ replaced Ti4+ and entered into B-sites for BSI–PT–PMN ceramics. As a result, the tolerance factor ‘t’ enlarged and the Curie temperature declined due to the reduced B-sites effective ionic radius. Furthermore, typical dielectric diffuse phenomena can be found in this system, which can be described by the modified Curie–Weiss law asεm is the maximum value of the dielectric permittivity ε, Tm is the temperature of εm, C is the Curie–Weiss constant, and γ is the diffuseness coefficient value between 1 to 2, respectively. The values of γ of all the BSI–PT–PMN–xCo ceramics were calculated and presented in 1/3Nb2/3)O3 ), due to the slight change in the values of γ in line with the increasing Co content.The Curie temperature ranged from 421 °C to 398 °C with increasing Co ion-doped content, which can be attributed to the enlarged tolerance factor ‘P–E hysteresis loop behavior for the BSI–PT–PMN–xCo  ceramics at room temperature under 1 Hz. The samples featured better saturated P–E loops when the Co ion-doped content increased. For the undoped BSI–PT–PMN ceramic, Pr was 36.4 μC/cm2 and Ec was 24.7 kV/cm. By doping the Co ions, Pr decreased and reached a minimum value of 25.1 μC/cm2, while Ec increased and reached a maximum value of 32.3 kV/cm at x = 0.8. This phenomenon may have been due to the Co ions entering the lattice to replace the Ti ions in the B-sites, resulting in oxygen vacancy, which plays a role in domain wall pinning.S) versus the electric field (E) loop by applying an electric field of 40 kV/cm and 1 Hz of BSI–PT–PMN–xCo ceramics. The strain under a unipolar electric field of 40 kV/cm was 0.222, 0.205, 0.198, 0.188, and 0.182, with 0, 0.2%, 0.4%, 0.6% and 0.8% Co ion doping, respectively.d33* and strain hysteresis h were calculated and listed in h can be described asS is the strain difference at half the maximal driven electric field in a cycle and the Smax is the strain maximum. It was found the large-signal d33* were over 450 pm/V for all these BSI–PT–PMN–xCo ceramics and the strain curve exhibited a good linearity with small hysteresis h, which is advantageous for piezoelectric actuator applications.Furthermore, the relevant large-signal Tc), relatively dielectric constant (εr), piezoelectric constant (d33), remnant polarization (Pr), planar electromechanical coupling coefficient (kp), and dielectric loss (tanδ) monotonically decreased, while the coercive electric field (Ec) monotonically increased, when the Co ion content increased from 0 to 0.8 wt.%.The electrical properties of the Co ion-doped BSI–PT–PMN ceramics at room temperature are listed in Eac was calculated according to the Arrhenius law: σ= σ0exp(-Eac/kT), where σ, Eac, k, and T are conductivity, activation energy, Boltzmann constant, and temperature, respectively. The activation energy was 0.3 eV at 25 < T < 220 °C and 1.5 eV at 220 < T < 400 °C for the undoped BSI–PT–PMN, while the activation energy was 0.33 eV at 25 < T < 220 °C and 1.43 eV at 220 < T < 400 °C for 0.2% Co-doped BSI–PT–PMN. The value of 0.2% Co ion-doped BSI–PT–PMN ceramic was found to be slightly larger than the value of the undoped BSI–PT–PMN ceramic at high temperature.Eac for ionic conduction is to be ≥1 eV and for electronic conduction around 0.1 eV [T < 400 °C.It has been reported that for the BS–PT system, the activation energy d 0.1 eV ,41,42. T9 Ω magnitude to108 Ω magnitude) at 300 °C, which is advantageous for high-temperature actuator applications.In addition, as can be seen in the inset of d33 as a function of temperature for BSI–PT–PMN–xCo . All the piezoelectric properties were measured at room temperature after annealing the poled specimens at different temperatures for 1 h. All the samples displayed the same trend and the piezoelectric constant d33 remained almost unchanged from room temperature to 250 °C; it began to drop when the annealing temperature exceeded 250 °C, dropped quickly when the annealing temperature exceeded 300 °C, and finally dropped to zero near the Curie temperature point. For the ceramic with 0.2 wt.% Co ion doping, the value of the piezoelectric constant d33 at 325 °C could still reach 80% of room temperature. The results reveal that BSI–PT–PMN–0.2Co ceramics possesses better thermal stability than ceramics without Co ion doping. The harder domain switching [witching  may expl3/4In1/4)O3–0.58PbTiO3–0.02Pb(Mg1/3Nb2/3)O3 (abbreviated as BSI–PT–PMN) doped with different amounts of Co ions  were prepared. The influences of Co ion doping on the phase transition, micro structure morphology, and electrical properties of the prepared BSI–PT–PMN–xCo ceramics were studied. It was found that Co ions entered the B-sites of the lattice, resulting in slight lattice distortion and a smaller lattice constant. With the increase in the doping of the Co ions, the grain size became larger; Co ions can promote the grain growth. The high temperature resistivity and temperature stability of the BSI–PT–PMN ceramics can be improved through an appropriate amount of Co ion doping. When the Co ion-doped content was 0.2 wt.%, the BSI–PT–PMN exhibited optimized comprehensive electrical properties: the Curie temperature (Tc) was 412 °C, the piezoelectric constant (d33) was 370 pC/N, the remnant polarization (Pr) was 29.2 μC/cm2, the relatively dielectric constant (εr) was 1450, the planar electromechanical coupling coefficient (kp) was 46.5, and the dielectric loss (tanδ) was 0.051. BSI–PT–PMN–0.2Co ceramics feature the merits of relatively high Curie temperature, high resistivity at high temperature, large strain, good strain linearity, and good thermal stability. BSI–PT–PMN–0.2Co ceramics are suitable for high-temperature actuator applications.High-temperature piezoelectric ceramic 0.40Bi(Sc


Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli. The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNAfMet, implying that degradation of tRNAfMet might trigger growth arrest in E. coli. Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.Toxin–antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The  Toxin–antitoxin systems (TA systems) are genetic modules composed of a toxin, which is engaged in the regulation of vital processes, and an antitoxin that blocks the activities of the toxin . TA systToxins are always proteins, whereas an antitoxin can be either a protein or a regulatory RNA . TA systAmong the six types of TA systems, the type II TA system is ubiquitous in prokaryotic genomes . Based ovirulence associated proteins) TA systems, consisting of toxin VapC and antitoxin VapB, are the largest family of type II TA modules  domain protein family, which shares structural similarity with the bacteriophage T4 RNase H, and shows ribonuclease activity  toxin inhibited growth only at low temperatures by a bacteriostatic mechanism, and this growth inhibition was recovered by the coexpression of BoVapB1 (AXW83_01400). Furthermore, we demonstrated that BoVapC1 possesses cleavage activity against tRNAfMet using quantitative real time polymerase chain reaction (qRT-PCR) and northern blotting, which may cause growth inhibition and increase survivability of E. coli at 4°C.We hypothesized that the abundance of type II TA systems in sed tool . The groBosea sp. PAMC 26642 genome were identified using TAfinder  strain. All of the transformant colonies were scraped with M9 minimal media. They were diluted with the same media to an optical density at 600 nm (OD600) of 0.2 and serially diluted from 10−1 to 10−4. Each diluted bacterial suspension was spotted onto M9 agar plates with varying concentrations of arabinose, to determine the minimal concentration at which colony formation was inhibited by the expression of toxin. Eight of the 12 toxins produced a toxic effect on E. coli growth  at both 37°C and 18°C . Transformants expressing BoHigB3 or BoHigB4 could not form colonies, even in the absence of arabinose. The toxic activity of BoVapC1 was temperature-dependent, being toxic only at 18°C. In contrast, the remaining three putative toxins  did not inhibit colony formation at either temperature. Toxicity of these predicted toxins were further assessed in α-proteobacterium Rhodobacter sphaeroides 2.4.1. Expression of BovapC5 and BorelE inhibited the growth of R. sphaeroides at both 30°C and 15°C, and BoVapC1 and BoVapC4 produced a toxic effect only at 15°C . These results indicate that BoVapC1 possesses the temperature-sensitive activity in both strains.To investigate the functionality of these putative toxins, the growth-inhibitory effects of the toxins on d-1-thiogalactopyranoside (IPTG). Expression of these antitoxins did not influence the growth of E. coli, except for BoHigA3 and BoRelB, which have marginal negative effects on colony formation at the highest concentration of IPTG (0.1 mM) .To examine whether the cognate antitoxins could neutralize the activities of the toxins, nine cognate antitoxin genes were cloned into the pET21c vector. Prior to the neutralization assays, the inhibitory effects of the cognate antitoxins on cell growth were investigated. pET21c-antitoxin plasmids were transformed into BL21(DE3) cells, and the transformants were spotted on M9 agar plates containing various concentrations of isopropyl β-Next, to assess the ability of antitoxins to antagonize their cognate toxins, BL21(DE3) cells harboring pET21c or pET21c-antitoxin were transformed with pBAD33 or pBAD33-toxin and spotted on M9 agar plates with or without inducers. For the expression of toxins, different concentrations of arabinose were utilized as follows: BoVapC1; 0.1%, BoHicA, BoVapC4, BoHigB2, and BoVapC5; 0.05%, BoVapC3, BoHigB3, and BoRelE; 0.01%, and BoHigB4; 0.005%. The expression of the antitoxins was induced with 0.02 mM IPTG, except for BoHigA2, for which expression was induced with 0.1 mM IPTG.Supplemental Fig. S4). In the presence of both arabinose and IPTG, BL21(DE3) cells with pET21c and pBAD33-toxin showed defects in colony formation ability, which was reversed by coexpression of the toxin and the antitoxin in the cells harboring pET21c-antitoxin and pBAD33-toxin . The expression of the antitoxin BoVapB1 induced by 0.1 mM IPTG had no effect on the growth of E. coli . In M9 minimal media supplemented with 0.4% arabinose and 0.1 mM IPTG, the growth defect caused by the toxin BoVapC1 was recovered by coexpression with the antitoxin BoVapB1, indicating that BoVapB1 can antagonize the activity of BoVapC1 in vivo  of BoVapB1 were predicted to interact tightly with the hydrophobic core of the VapC toxin  cells harboring pBAD33 or pBAD33-BoVapC1 were grown in M9 minimal media supplemented with chloramphenicol. Arabinose (0.4%) was added for toxin expression during the early exponential phase. The growth was inhibited by BoVapC1 expression at 18°C but was not inhibited at 37°C . Of note, the control strain with pBAD33 exhibited reduced cell length after 24 h induction at 18°C , suggesting that the effect of BoVapC1 is bacteriostatic rather than bactericidal.This bacteriostatic phenotype was further verified using cell growth resumption experiments. BL21(DE3) cells cotransformed with pBAD33-BoVapC1 and pET21c-BoVapB1 were grown to the early exponential phase at 37°C, then transferred to 18°C. After the induction of BoVapC1 with arabinose for 24 h at 18°C, cells were harvested and resuspended with M9 minimal medium containing 0.1 mM IPTG, followed by further incubation at 18°C up to 60 h. As shown in S. flexneri, Salmonella enterica, nontypeable Haemophilus influenzae (NTHi), Leptospira interrogans, and a large number of VapCs from M. tuberculosis cleave tRNA site-specifically, between the anticodon stem and loop (M. tuberculosis target and cleave the sarcin-ricin loop of 23S rRNA (M. smegmatis and S. solfataricus cleave mRNAs in a sequence-specific manner (rrlA (23S rRNA) and rrsA (16S rRNA), five mRNAs of various sizes , and three tRNAs  . As shown in metV decreased 1.63-fold by BoVapC1 expression, while that of metU increased by 1.25-fold, and that of hisR was not affected in BoVapC1-expressing cells  cells harboring pBAD33 or pBAD33-BoVapC1 at 0 h and 3 h after induction. The full-length tRNAfMet (77 nt) was observed intact in BL21(DE3) cells carrying pBAD33 regardless of induction (fMet (<50 nt), which was not detected before induction, appeared with a concomitant reduction of full-length tRNAfMet . These results suggest that BoVapC1 specifically cleaves the initiator tRNAfMet only at low temperatures with a site-specific endoribonuclease activity. The decline in the level of tRNAfMet caused by BoVapC1 expression was neutralized by the expression of BoVapB1 antitoxin  cells harboring plasmid pairs  were prepared and spotted on M9 agar plates with or without 0.1% arabinose. As shown in metZWV partially restored the growth defect caused by BoVapC overexpression at 18°C.These results led us to examine whether the overexpression of fMet of E. coli can serve as a substrate for the toxin BoVapC1 at 18°C, which causes inhibition of translation, and subsequently growth arrest, and that BoVapB1 neutralized BoVapC1 by inhibiting the ribonuclease activity of BoVapC1, resulting in growth resumption. Furthermore, given the high sequence identity between E. coli and Bosea sp. PAMC 26642 tRNAfMets . Of the eight Bosea species with whole genome sequence available, four do not have TA systems, and B. vestrisii and B. caraganae have two TA loci each. Interestingly, B. psychrotolerans, a psychrotrophic bacterium that can grow at 5°C  cells with pBAD33/pET21c-BoVapB1 or pBAD33-BoVapC1/pET21c-BoVapB1. After induction with 0.4% arabinose for 3 h at 18°C, the cultures were moved to refrigeration temperature (4°C), followed by static incubation for a further 48 h. The cells were harvested at 12-h intervals and the survival rates were analyzed as described in Materials and Methods. As the exposure time increased, the survival rates of cells harboring pBAD33 gradually reduced and reached 48.51% at 48 h  are induced from the metY-nusA-infB operon   E, suggesfMet target at a temperature that is this low; however, why tRNAfMet was cleaved by BoVapC1 only at 18°C  is still unclear. Several type II toxins have been reported to be functionally affected by temperature, in the same manner as BoVapC1. In Pseudomonas putida, deletion of the graA antitoxin gene causes a reduction in the colony formation rate, which becomes apparent at low temperatures  the activity of BoVapC1 may be controlled by conformational changes induced by temperature, or (iii) there may be another regulator protein inactivating BoVapC1 at 37°C or activating it at 18°C.Considering the temperature-sensitive phenotypes observed in our study and several other TA systems, we can propose three hypotheses to explain why BoVapC1 was active only at 18°C as follows: (i) BoVapC1 may aim to the target(s) such as tRNASupplemental Figure S8, it is likely that the stability and/or activity of BoVapC1 diminishes at 37°C with no tRNAfMet cleavage activity, and that the conformation at 18°C gains access and catalytic activity to the substrate. Nevertheless, the absence of BoVapC1 activity at 37°C does not appear to be a result of either reduced expression or instability of the BovapC1 mRNAs owing to the fact that a higher transcript level of BovapC1 was noted at 37°C compared with that at 18°C . Furthermore, we cannot rule out the possibility that a trans- or cross-regulator may modulate the function of BoVapC1 at either temperature  strain was grown at 37°C or 18°C in Luria–Bertani (LB) or M9 minimal media supplemented with 0.2% casamino acids and 0.4% glucose. When necessary, antibiotics were added at the following concentrations: ampicillin (Amp); 100 µg/mL and chloramphenicol (Cm); 50 µg/mL.The Bosea sp. PAMC 26642 were identified using TAfinder (https://bioinfo-mml.sjtu.edu.cn/TAfinder/TAfinder.php), and the open reading frames of those toxins were synthesized and cloned in pUCIDT (Cosmogenetech), yielding pUCIDT-toxin plasmids . The pUCIDT-toxin plasmids were digested by the appropriate restriction enzymes. The digested DNA fragments carrying toxin genes were cloned into the NdeI-HindIII or NdeI-SacI sites of the pBAD33 vector containing arabinose-inducible araBAD promoter, and we used LB medium supplemented with 0.2% glucose to strongly repress the promoter -inducible T7 promoter, yielding pET21c-antitoxin plasmids.Twelve putative type II toxin and antitoxin genes in promoter . Nine anSupplemental Table S1. The toxin DNA fragments carrying each mutation were ligated into pBAD33, yielding pBAD33-BoVapC1D7A, pBAD33-BoVapC1E42A, pBAD33-BoVapC1D98A, and pBAD33-BoVapC1E119A. The mutated BoVapB1 DNA fragments were ligated into pET21c, yielding pET21c-BoVapB1W53R, pET21c-BoVapB1F56SF57S, and pET21c-BoVapB1W53RF56SF57S. To clone the metZWV operon, the PCR-amplified DNA fragments were ligated with the SmaI site of pBR322, yielding pBR322-metZWV. All plasmids and primers used in this study are listed in Supplemental Tables S1, S2.Point mutations were introduced into BoVapB1 or BoVapC1 by site-directed mutagenesis PCR using pUCIDT-BoVapB1 or pET21c-BoVapC1 as templates, respectively, and the primer sets listed in 600) of 0.2, and serially diluted 10−1, 10−2, 10−3, and 10−4-fold. A total of 2 µL of each dilution was spotted on M9 agar plates containing Cm with various concentrations of arabinose, or without inducer. To assess the effect of antitoxin expression on E. coli growth, serial dilutions of BL21(DE3) cells harboring pET21c or pET21c-antitoxin were prepared as described above and spotted on M9 agar plates containing Amp with or without IPTG.To determine the minimal concentration of arabinose for the neutralization assay, BL21(DE3) cells were transformed with pBAD33 or pBAD33-toxin and plated on M9 agar plates containing Cm, followed by incubation at 37°C overnight. Then, each transformant was scraped from the agar plates, and resuspended into 5 mL of M9 minimal media. The resuspension was diluted in the same media to an optical density at 600 nm  harboring plasmid pairs  were prepared and spotted on M9 agar plates containing Amp and Cm with or without inducers. For the neutralization assay for the BoVapBC1 TA system in liquid medium, overnight cultures of BL21(DE3) transformants with plasmid pairs  were diluted 100-fold in M9 minimal media supplemented with Amp and Cm. After incubation at 37°C to the early exponential phase, the cultures were diluted fivefold with the same medium, and then inducers were added as follows: 0.4% arabinose, 0.1 mM IPTG, or both. The diluted cultures were incubated at 18°C for 24 h and continuously diluted fivefold every 6 h in fresh M9 minimal medium with or without inducers, and OD600 of 0.8 were harvested by centrifugation at each point in time  and washed twice with 1.2 mL of 0.85% NaCl. The pellets were resuspended in 1.2 mL of 0.85% NaCl. Then, the samples were stained using LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher Scientific), following the manufacturer's instructions. Briefly, 12 µL of the diluted dye mixture of SYTO9 and PI was added to each bacterial suspension. After incubation at room temperature in the dark for 15 min, the bacterial suspensions were concentrated 10-fold. A total of 5 µL of the stained bacterial suspension was placed between the slide and cover glasses, and cells were observed with an Axio Observer (Zeiss) at 1000× magnification.Overnight cultures of BL21(DE3) cells harboring pBAD33 or pBAD33-BoVapC1 were diluted 100-fold in M9 minimal media containing Cm at 37°C, followed by cultivation to the early exponential phase. The cell cultures were diluted fivefold in fresh M9 media supplemented with Cm and 0.4% arabinose and incubated at 18°C for 24 h. A total of 1.5 mL of cell cultures at the OD600 of 0.2 in 1 mL with 0.85% NaCl. Equal volumes of SYTO9 and PI dyes were mixed beforehand, following which 3 µL of dye mixture was added to each bacterial suspension. The stained samples were incubated at room temperature in the dark for 15 min and transferred to a cuvette. Fluorescence intensity was determined with a fluorescence spectrophotometer (Hitachi F-7000) at 470 nm for excitation and wavelength spectrums of 510–540 nm (for SYTO9) and 620–650 nm (for PI) for emission. The percentage of live bacteria was calculated by dividing the SYTO9 intensity into the sum of SYTO9 and PI intensities.To quantitate each staining, the bacterial suspensions were diluted to an OD600 was measured at every 10 h.Overnight cultures of BL21(DE3) cells cotransformed with pET21c-BoVapB1 and pBAD33-BoVapC1 were diluted 100-fold in M9 media supplemented with Amp and Cm and incubated at 37°C to the early exponential phase. Then, the cultures were diluted fivefold into fresh M9 media with or without 0.4% arabinose, and further incubated at 18°C for 60 h. At 24 h after arabinose addition, the cells were harvested by centrifugation and resuspended in the same volume of M9 minimal medium supplemented with 0.1 mM IPTG. OD600 of 1.0 were harvested by centrifugation.BL21(DE3) cells harboring pBAD33 or pBAD33-BoVapC1, grown to the early exponential phase, were diluted fivefold in M9 minimal media supplemented with Cm and 0.4% arabinose and incubated at 37°C or 18°C for 3 h. For the neutralized cells, BL21(DE3) cells cotransformed with pBAD33/pET21c, pBAD33-BoVapC1/pET21c, pBAD33/pET21c-BoVapB1, or pBAD33-BoVapC1/pET21c-BoVapB1 were grown to the early exponential phase and diluted fivefold in M9 minimal media supplemented with Amp, Cm, 0.4% of arabinose, and 0.1 mM IPTG, followed by incubation at 18°C for 3 h. At the indicated time point, 2 mL of cell cultures for OD600 of 1.0, and 1.5 mL of culture was harvested by centrifugation at 13,000 rpm for 1 min. The cell pellets were resuspended in 500 µL of Solution A , and mixed with equal volumes of phenol (pH 4.3). After incubation at 60°C for 10 min and on ice for 5 min, centrifugation at 13,000 rpm was performed to remove the phenol. The supernatants were transferred to new tubes and mixed with 50 µL of 3 M sodium acetate (pH 5.2) and 1 mL of 100% cold ethanol. Following incubation at −20°C for 1 h, ethanol was removed by centrifugation at 13,000 rpm for 10 min at 4°C. The pellets were dissolved in Solution A without SDS and precipitated with ethanol once more. Ethanol was removed by centrifugation, following which the pellets were washed with 75% ethanol. Finally, RNAs were dissolved in 100 µL of diethylpyrocarbonate (DEPC)-treated water. The concentration and purity of the RNA were determined using a NanoDrop OneC Spectrophotometer (Thermo Fisher Scientific), and the quality of RNA was validated by agarose gel electrophoresis. DNA contamination was removed by treatment with recombinant DNase I (RNase-free) (TaKaRa) according to the manufacturer's protocol. Then, cDNA synthesis was carried out using BioFACT RT Kit and 1 µg of total RNA. The qRT-PCR reaction was carried out as previously described . Next, the membrane was prehybridized with hybridization buffer  with 50 µg/mL denatured salmon sperm DNA for 4 h at 65°C. Then, the membrane was incubated at 52°C overnight with hybridization buffer supplemented with 500 ng/mL probes labeled with biotin at both ends . The membrane was washed sequentially with wash buffer I , with wash buffer II [1× SSC 0.1% SDS], and with 0.1× SSC at 42°C for 15 min each. The biotin signals were detected using a Biotin Chromogenic Detection Kit (Thermo Fisher Scientific) following the manufacturer's instructions.A total of 20 µg of total RNAs, denatured with formamide by heating at 70°C for 10 min, were separated on 15% denaturing polyacrylamide gel containing 8 M urea in 1× TBE . The gel was run at 100 V for ∼2.5 h until the bromophenol blue dye front reached to 0.5 cm above the bottom of the gel. Then, the RNAs were transferred to a nylon membrane  by electroblotting using Bio-Rad Semi-Dry electrophoretic transfer cell at constant 20 V for 1 h. After transfer, the membrane was cross-linked by exposure to ultraviolet light (254 nm) at 120 mJ/cm600: 0.1. The resuspension was further diluted 10−5-fold and plated on M9 agar plates with 0.1 mM IPTG, followed by incubation at 37°C. Colony forming units (CFU) were counted, and the survival rates were expressed as the percentage of the number of colonies in the treated samples , compared with that of the untreated samples (0 h).BL21(DE3) cells harboring pET21c-BoVapB1 were transformed with pBAD33 or pBAD33-BoVapC1 and grown in M9 minimal medium with Amp and Cm at 37°C to the early exponential phase. The cultures were diluted fivefold in M9 minimal media supplemented with Amp, Cm, and 0.4% arabinose and further incubated at 18°C for 3 h. Then, the cells were incubated at 4°C for 48 h. At the indicated time points, the cells were diluted in M9 minimal medium to an ODt-tests. The statistical analyses were performed with the following significance levels: NS, nonsignificant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.Results are presented as the mean ± SD of three independent experiments. The data were analyzed by two-tailed Supplemental material is available for this article.


The iron oxide nanoparticles encapsulated in the carrier maintained good saturation of magnetization (41.9 emu/g) and had superparamagnetism. The heating capacity test showed that the specific absorption rate (SAR) value was 18.9 ± 0.5 W/g, indicating that the ferromagnetic nanoparticles encapsulated in the gel still maintained good heating capacity. Moreover, the magnetocaloric temperature could reach 43 °C in a short period of five minutes. In addition, DOX-Fe3O4@agar reached a maximum release rate of 85% ± 3% in 56 min under a neutral pH 7.0 to simulate the intestinal environment. We found using fluorescent microscopy that DOX entered HT-29 human colon cancer cells and reduced cell viability by 66%. When hyperthermia was induced with an auxiliary external magnetic field, cancer cells could be further killed, with a viability of only 15.4%. These results show that agar is an efficient multiple-drug carrier, and allows controlled drug release. Thus, this synergic treatment has potential application value for biopharmaceutical carrier materials.The purpose of this study was to use agar as a multifunctional encapsulating material to allow drug and ferromagnetism to be jointly delivered in one nanoparticle. We successfully encapsulated both Fe Fe3O4 is a superparamagnetic material with high saturation magnetization capacity, good biocompatibility, and low toxicity [3O4 nanoparticles, which allows for in vivo deep penetration, has proven to be an alternative and promising approach for cancer treatment. When subjected to an alternating magnetic field, superparamagnetic Fe3O4 nanoparticles can convert electromagnetic energy into heat through the oscillation of their magnetic moments. In this study, agar was successfully used as a drug carrier to encapsulate Fe3O4 and DOX simultaneously by means of co-precipitation to form a DOX-Fe3O4@agar nanoparticle. Then, the DOX-Fe3O4@agar carrier particles were used to treat colon cancer HT29 cells, after which the cell viability was evaluated. We also explored the release effect of the chemotherapy drug as well as the cytotoxic effect on cancer cells in a controlled magnetic-induced hyperthermia test. To do so, we induced alternating magnetic field (AMF) to generate heat from the encapsulated iron oxide magnetic particles. This allowed us to understand whether the gel carrier particles can achieve a synergistic effect, improve carrier functionality, and reduce the drug dosage.The design and synthesis of effective drug delivery carrier materials holds critical importance in biomedicine. However, studies have shown that conventional chemotherapy may lead to drug resistance during treatment ,2. Theretoxicity , and is Agar was purchased from Fisher chemical, Pittsburgh, PA, USA. Iron (II) chloride and iron (III) chloride were from Merck, Germany. DOX was from Sigma-Aldrich, Saint Louis, MO, USA. MTT test was from EMD Millipore, USA. Isopropanol was from TEDIA, USA. Phosphate-buffered saline (PBS) solution (pH 7) was from UniRegion Bio-Tech, Taiwan and used as obtained without further purification. Milli-Q reagent-grade water (18.2 MΩ cm at 25 °C) was used for all synthesis processing steps that required water. 3O4@agar, refer to Hsieh et al. [3O4@agar NPs. Finally, Fe3O4@agar NPs were heated to 38 °C, mashed, and vacuum-dried after rapidly adding DOX cooling gel. The dried gel was ground into powder using a mortar to obtain DOX-Fe3O4@agar NPs before storing them at 4 °C protected from light before the experiment.For the co-precipitation synthesis method of Feh et al. . 0.05 g 3O4 and DOX-Fe3O4@agar were acquired using a JEOL JEM-2100  operated at 200 KV. The TEM sample were suspended in Deionized water and supported onto a carbon composite TEM grid with copper 200 mesh and allowed to dry. The average particle dimension of the Fe3O4 NPs was estimated by image J software (version Java 1.8.0). The crystalline structure was characterized using powder X-ray diffraction (XRD) with a Bruker D8 ADVANCE  diffractometer equipped with Ni-filtered Cu Kα (λ = 1.5406 Å) radiation source, over a 2θ range of 20° to 80° with scan step 0.1 degrees, scan rate 3 s/step. The average particle size (D) of the Fe3O4 NPs was estimated by the Scherrer’s formula using the highest intensity XRD peak.K is Scherrer constant related to crystallite shape (K = 0.9), λ is the wavelength of the X-rays, β2θ is full-width at half-maximum diffraction peak (FWHM), and θ is a diffraction angle.Transmission Electron Microscopy (TEM) images of Fe3O4 and DOX-Fe3O4@agar were measured using a Quantum Design MPMSXL7  superconducting quantum interference device (SQUID). The fluorescence microscopy images of HT-29 cells emitting appropriate fluorescence were recorded with the Olympus IX73  inverted fluorescence microscope equipped with the tetramethylrhodamine (TRITC) channel, at original magnification 100-fold.Room-temperature magnetization curves for Fe3O4@agar NPs (5 mg/mL) were prepared in Dulbecco’s Modified Eagle Medium  (pH 7) by sonication. Their heating capacity was measured by portable electric induction heat machine . The sample was placed at the center of the induction coil, and an alternating current of 450 A/m and a fixed frequency of 400 kHz were applied for up to 15 min. The temperature of the sample was recorded using an alcohol thermometer every 30 s. The experiments were repeated at least three times. We examined the hyperthermia effect of the DOX-Fe3O4@agar NPs on HT-29 cells. HT-29 cells were cultured in 5 mg/mL of DOX-Fe3O4@agar NPs for 24, 48, and 72 h, respectively. Then, treated HT-29 cells were exposed to the center of the induction coil for 15 min. Finally, the viability of the cells was studied by MTT assay.The DOX-Fe2 [6 cells) were seeded in 3-cm plates for 24 h and treated with 3 µg/mL, 6 µg/mL, and 12 µg/mL Fe3O4@agar or DOX-Fe3O4@agar for 24, 48, and 72 h. After the incubation periods, the cells were further incubated with MTT (0.1 mg/mL in medium) for 3 h at 37 °C to allow viable cells with active metabolism to produce a purple-colored formazan. The reaction was then stopped with 100 µL isopropanol, and the optical density (OD) was measured at 570 nm. The cell viability was expressed as a percentage of the viability of the control culture.HT-29 cell line (human colon adenocarcinoma cells) was cultured in Dulbecco’s modified eagle medium (DMEM) , at 37 °C in a humidified atmosphere containing 5% CO2 . Cell vi3O4@agar in phosphate-buffered saline (PBS) solution (pH 7). In a typical experiment, 5 mg of DOX-Fe3O4@agar was added to 1 mL of PBS solution. At the given reaction time interval (record every 2 min) for 60 min, the DOX-Fe3O4@agar were separated using an external magnetic field, then the supernatant was collected for analysis of DOX concentration. After each measurement, the DOX-Fe3O4@agar sample was placed back into the release solution to keep the solution constant. The release concentration of DOX was determined using a UV–vis spectrometer  at an absorption wavelength of 480 nm.The drug release test of DOX-FeCell viability data were analyzed using the statistical analysis software SPSS for Windows, version 12.0 . Data were subject to a one-way ANOVA test and where significant differences among means were found, these were separated by Duncan’s multiple range tests. OriginPro, version 8.1  was used for preparing figures.3O4@agar. The results show that DOX-Fe3O4@agar was uniformly distributed in granular form. The average particle size of encapsulated iron oxide calculated on TEM images was 9.2 ± 3.2 nm. The lattice structure of DOX-Fe3O4@agar was analyzed by X-ray diffraction (XRD), as shown in 3O4@agar at 2θ were 30.3°, 35.7°, 43.5°, 57.4°, and 62.9°, which were assigned to magnetite crystal planes (220), (311), (400), (511), and (440) (refer to JCPDS no. 85-1436). Therefore, biocompatible agar, as a drug carrier, has been confirmed to have the ability to encapsulate Fe3O4 magnetic particles. Simultaneously, suggesting that DOX exists in the amorphous form in DOX-agar and DOX-Fe3O4@agar [3O4 particle size calculated by Scherrer’s formula was 9.2 nm, which was consistent with the particle size observed in TEM image. The SQUID was used to measure the magnetic field of DOX-Fe3O4@agar nanoparticles (NPs)  , which o3O4@agar NPs to release the DOX drug was monitored by UV-vis spectrophotometer. In addition, based on the colonic pH value of 6.4–7.0 [n = 3). According to the literature, agar gel is a material of open porous network system and spontaneous sustained release can be achieved. That have mesopores with an average pore radius of 4.7–8.6 nm for 4–6% (v/v) gel [According to literature, DOX has a stable UV absorption wavelength (around 480 nm) and red fluorescence emission of about 590 nm . Therefo 6.4–7.0 , DOX relv/v) gel ,33. In a3O4@agar nanoparticles was monitored thanks to DOX’s red fluorescence property, on HT-29 cells after 1 h treatment by fluorescence microscopy -2,5-diphenyltetrazolium bromide (MTT) analysis to test the cell viability, as shown in 3O4@agar nanoparticles were used to treat cells, the more significant was the decrease in cell viability  was 23.6 ± 1.1 W/g, showing good heating capacity. However, compared with pure Fe3O4 (SAR 46.8 ± 2.0 W/g), the heating capacity was slightly lower. The reason for this may be that the outer gel layer of Fe3O4 NPs absorbed water, causing the particles to swell, and increasing the dipole-dipole interaction between the particles, which reduces the rotational ability of particles and inhibits the Brownian heating mechanism. We further measured the intracellular heating capacity of DOX-Fe3O4@agar NPs. It was observed that the SAR value of heating capacity decreased to 18.9 ± 2.0 W/g, which might be due to the restriction of drug carrier particles by more space obstacles of cells. Even so, DOX-Fe3O4@agar NPs could still make the heating temperature reach about 43 °C in 5 min. As shown by previous studies, this temperature is sufficient to overcome the heat resistance for the synthesis temperature of cancer cell proteins, and thus exerts a cytotoxic effect by hyperthermia [In order to increase the functionality of the carrier, the ability of DOX-Ferthermia ,35,36.3O4@agar NPs to HT-29 cells and cultured them for 24, 48, and 72 h, applied alternating magnetic field to induce hyperthermia for 15 min, and then carried out MTT test for cell viability, as shown in 3O4@agar was significantly reduced after hyperthermia. The viability of HT-29 cells decreased to 18.1% ± 10.7%, 16.9% ± 13.6%, and 15.4% ± 6.1% respectively after treatment for 24, 48, and 72 h.Finally, we added DOX-FeAgar drug carriers showed a high release rate of 85% ± 3% under simulated colonic environment (pH 7). When HT-29 cells were cultured with the carrier particles, significant red fluorescence present in the cells could also be observed. In order to determine the magnetocaloric functionality, we evaluated the heating capacity of magnetic particles carrying drugs, which reached 43 °C after 5 min. This temperature is sufficient to exert a hyperthermic effect on cancer cells. Therefore, biocompatible agar, as a drug carrier, has been confirmed to have the ability to simultaneously encapsulate drugs and magnetic particles. This allows for the simultaneous application of chemotherapy and physical hyperthermia, and has a significant additive effect in killing cancer cells. Therefore, the commercial cost can be reduced, and the complexity of synthesis can also be reduced, thus presenting excellent potential application value in biomedical applications.