Primary endothelial cells (ECs), especially human umbilical vein endothelial cells (HUVECs), are broadly used in vascular biology. Gene editing of primary endothelial cells is known to be challenging, due to the low DNA transfection efficiency and the limited proliferation capacity of ECs. We report the establishment of a highly efficient and selection-free CRISPR gene editing approach for primary endothelial cells (HUVECs) with ribonucleoprotein (RNP) complex. We first optimized an efficient and cost-effective protocol for messenger RNA (mRNA) delivery into primary HUVECs by nucleofection. Nearly 100% transfection efficiency of HUVECs was achieved with EGFP mRNA. Using this optimized DNA-free approach, we tested RNP-mediated CRISPR gene editing of primary HUVECs with three different gRNAs targeting the HIF1A gene. We achieved highly efficient (98%) and biallelic HIF1A knockout in HUVECs without selection. The effects of HIF1A knockout on ECs’ angiogenic characteristics and response to hypoxia were validated by functional assays. Our work provides a simple method for highly efficient gene editing of primary endothelial cells (HUVECs) in studies and manipulations of ECs functions. Endothelial cells (ECs) form a single layer of cells lining the vascular systems . Blood ECultured ECs are widely used in vascular cell biology and for studies of ECs functions/dysfunctions. Genetic modification of cultured cells is a widely used method for studying biological processes, but the approach has been difficult to apply in primary ECs . Small iHIF1A) gene, which plays an important role in endothelial adaptation, to establish the method.Currently, successful CRISPR-Cas9 gene editing has been reported in primary ECs, with efficiencies ranging from 40% to 80% depending on the used ECs model and CRISPR-Cas9 methods ,23,24,252 atmosphere at 37 °C. The M199 medium with supplements was replaced three times per week. HUVECs were split every 14 days, the trypsination  was stopped by M199 medium with supplements. The cells were centrifuged at 250× g for 3 min and seeded at 5–6 × 104 cells per cm2 in 150 µL per cm2 combined medium of 50% M199 medium with supplements and 50% endothelial cell growth medium . All materials were thoroughly sterilized, and all reagents were filtered through 0.20 µm filters  to avoid contamination. The HUVECs used in these experiments are mycoplasma free , CD45- and CD31+  in M199 medium  supplemented with 2 mM L-glutamine , 0.4% Endothelial Cell Growth Supplement (ECGS)/Heparin , 20% fetal bovine serum (FBS)  in a 5% COnd CD31+ , and havnd CD31+  which arIn vitro transcription (IVT) EGFP plasmid was made by associate professor Rasmus O. Bak, Department of Biomedicine, Aarhus University. EGFP mRNA was generated by IVT. First, the EGFP plasmid was linearized by mixing 15 µL nuclease-free water , 2 µL 10× Fast Digest Green Buffer , 2 µL (1 µg/µL) EGFP plasmid, and 2 µL Fast Digest restriction enzyme BbsI  and stored on ice. The reaction mixture was incubated at 37 °C for 3 h. Next, the linearization of the EGFP plasmid was visualized on a 1% agarose gel with a 1 kb marker . NucleoSpin Gel and PCR Clean-up  was used to elute the EGFP DNA from the agarose gel by following the manufacturer’s protocol. Finally, IVT was performed by using MEGAscript kit  following manufacturer’s protocol with one improvement as 3 µL CleanCap AG (6 mM)  was added directly to the reaction mix to increase the stability of the mRNA and its translation. The RNA concentration was measured by a Nanodrop 1000 Spectrophotometer. The EGFP mRNA was stored at −20 °C.2 atmosphere at 37 °C. After 48 h incubation, the M199 medium with supplements was changed. The cells were incubated until optimal confluency of 90% before nucleofection.First, the HUVECs were split as described above and incubated for 48 h in a 5% CO2 atmosphere at 37 °C until use.Preparations for the nucleofection: All reagents were filtered through 0.20 µm filters. The nucleocuvette  was thoroughly washed. 24-well culture plates  were coated with 0.1% gelatin. The coated plates were filled with 50% M199 medium with supplements, 50% endothelial cell growth medium, and additional 1% penicillin/streptomycin , and incubated in a 5% CO5 cells in each group. The cells were gently washed twice in PBS, centrifuged at 500× g for 5 min, and resuspended in 60 µL OptiMem . EGFP mRNA was thawed on ice. The given concentration of EGFP mRNA was added to the appropriate groups and mixed thoroughly by pipetting. Each test group was divided into three wells in the nucelocuvette. Immediately upon transfer to the nucleocuvette, the HUVECs were nucleofected by the 4D-Nucleofector X Unit  with the nucleofection program: CM138. After nucleofection, 150 µL prewarmed M199 medium with supplements was added to each well in the nucelocuvette. Finally, the nucleofected HUVECs were seeded in the prepared 24-well plate with a total volume of 500 µL culturing medium and incubated for 24 h in a 5% CO2 atmosphere at 37 °C.Nucleofection: The 90% confluent HUVECs were divided into six groups (0.0 µg EGFP mRNA (control), 0.4 µg EGFP mRNA, 0.8 µg EGFP mRNA, 1.6 µg EGFP mRNA, 3.2 µg EGFP mRNA, and 4.8 µg EGFP mRNA) with 2.4 × 10g for 5 min. The cell pellet was washed twice in PBS + 5% FBS and resuspended in 200 µL PBS + 5% FBS. All samples were incubated in the dark on ice until FC analysis: 100 µL of each sample was acquired and EGFP detected off the 488 nm laser (100 mW) in the B530/30 detector. The FC data were analyzed in NovoExpress v. 1.5.6 using the following gating strategy: ① A forward-scatter-area to side-scatter-area density plot was made to exclude debris, ② A forward-scatter-height to forward-scatter-area density plot was followed by a side-scatter-height to side-scatter-area density plot to exclude doublets, ③ A single parameter histogram was made to identify the cells expressing EGFP. This experiment was performed with one HUVECs doner at p. 5 (n = 3).Visualization: After 24 h of incubation the expression of EGFP was visualized by fluorescent microscopy and quantified by FC analysis. The cells were visualized by a Leica DMi1 fluorescent microscope, DE, obtaining three brightfield and FITC microscopy images per well at 10× magnification, with the same laser intensities and camera exposures. Image visualization and analysis was done in Fiji (ImageJ) v. 2.9.0. The background signal was adjusted based on averaging background values from four areas negative for signal in each channel and subtracting the mean values from the final image. NovoCyte Quanteon 4025 flow cytometer was used to quantify the percentage of EGFP positive cells. The HUVECs were washed in PBS twice, trypsinized, and centrifuged at 400× The nucleofection of HUVECs protocol, described above was used to nucleofect HUVECs with 3.2 µg EGFP mRNA or 1.312 µg EGFP plasmid. The concentration EGFP plasmid had to be lower than EGFP mRNA since high plasmid concentrations are toxic to the cells. This experiment was repeated to achieve both biological- and technical triplicates, by using three different HUVECs donors at p. 2–5.HIF1A gene. The online CRISPR web tools “CRISPor” (http://crispor.tefor.net accessed on 10 October 2020) [https://rth.dk/resources/crispr/crispron/ accessed on 10 October 2020) [Streptococcus pyogenese CRISPR associated protein 9). The sequence of the three target regions’ exons was set as input in the web tools and submitted with default settings. The final three gRNAs targeting three different exons of HIF1A were all chosen by analyzing the CRISPRon/CRISPor output tables, by choosing the gRNA with the highest predicted efficiency   and “CRIer 2020) ,34 were HIF1A gene were analyzed by nucleofecting HUVECs with three different HIF1A gRNAs, as described in the nucleofection protocol above with few alterations. Before nucleofection of the HUVECs, the ribonucleoprotein (RNP) complex was prepared. The synthesized gRNAs  were dissolved to be 3.2 µg/µL in nuclease-free water mixed by vortexing and stored at −20 °C. The RNP complex was prepared in three groups: ① HIF1A gRNA 1 + spCas9 protein, ② HIF1A gRNA 2 + spCas9 protein, and ③ HIF1A gRNA 3 + spCas9 protein. All three groups were prepared by mixing 1.8 µL gRNA (3.2 µg/µL), 1.8 µL spCas9 Nuclease V3 , and 3.6 µL nuclease-free water in PCR-tubes and kept at room temperature for 10–60 min. The sample groups in this experiment were: wild type (untreated), 3.2 µg EGFP mRNA treatment (positive control), HIF1A gRNA 1 treatment, HIF1A gRNA 2 treatment, and HIF1A gRNA 3 treatment. The HUVECs sample groups were resuspended in 60 µL OptiMem  and 2.4 µL of each RNP complex was added to the appropriate groups. This was followed by nucleofection and 48 h of incubation, in penicillin/streptomycin free medium, before genotyping. The EGFP fluorescence was visualized by a Leica Dmi1 fluorescent microscope to ensure efficient nucleofection after 24 h of incubation. This experiment was performed with one HUVECs donor at p. 4 (n = 3). Experiments with HIF1A gRNA 1 were repeated to achieve three biological replicates at p. 2–4, each with technical triplicates.The CRISPR-Cas9 editing efficiencies for the 2 1.5 mM, Tris/HCL pH8.5 10 mM) + 25 µL Tween20, 20%  + 50 µL NP-40 Surfact-Amps Detergent  + 20 µL proteinase K , and collected in PCR strips. The lysed HUVECs were incubated at 65 °C for 30 min and 95 °C for 15 min. The cell lysate was stored at −20 °C. PCR was used to amplify the DNA region of interest before Sanger sequencing.Genotyping: The HUVECs were washed twice with PBS and the cell pellet was lysed with 200 µL of the lysis master mix  + 1.2 µL forward primer, 5 µM,  + 1.2 µL reverse primer, 5 µM,  + 0.16 µL AccuPrime PFX DNA polymerase  + 4 µL betaine (5M)  + 9.44 µL ddHhttps://ice.synthego.com/#/ accessed on 8 December 2020) was used to calculate the overall CRISPR gene editing efficiency and determine the profiles of all different types of edits present in the Sanger sequencing data. Prism 9, US, was used to plot the gene editing efficiency for each HIF1A gRNAs Sanger sequencing: The PCR products were sent to Sanger sequencing at Eurofins Genomics, DK, by following the manufacturer’s protocol to Mix2Seq Kit . The Sanger sequencing results were analyzed by Snap gene viewer and the web tool “ICE”  and mouse anti-CD45 BV421  for 30 min on ice in the dark, After removing debris and doublets, density plots were used to gate the CD45-CD31+ stained HUVECs, .5 cells per replicate (n = 3) in 50% M199 medium with supplements and 50% endothelial cell growth medium and incubated for 24 h in a 5% CO2 atmosphere at 37 °C. The culturing medium was replaced to M199 medium with supplements and treated with 10 µg/mL Alexa Fluor 594 conjugated acetylated low-density lipoprotein (LDL)  and incubated for 4 h in a 5% CO2 atmosphere at 37 °C. The cells were washed trice with PBS and fixed with 4% paraformaldehyde (PFA)  for 15 min and washed trice with PBS for 5 min. At the final wash, the cells were stained with 1:1000 Hoechst for 10 min. Cover slides  were mounted with 3 µL mounting buffer . Microscopy images (5 images/well) were obtained with an Olympus BX63 fluorescent microscope equipped with CoolLED pE-300ultra fluorescence microscopy Illumination System and Sensitive Andor Zyla 5.5 camera using 40× (Plan Fluorite) objective. The same intensity of illumination and the same exposure time settings were used for comparative image acquisition as follows; UV excitation (maximum excitation irradiance at 345 nm wavelength) 20% with 10 ms exposure time (emission maximum at 455 nm), GR excitation (maximum excitation irradiance at: 595 nm wavelength) with 200 ms exposure time (emission maximum at 615 nm). Image visualization and analysis was done in Fiji (ImageJ) v. 2.9.0. The background signal was adjusted based on averaging background values from four areas negative for signal in each channel and subtracting the mean values from the final image  were coated with 0.1% gelatin. HIF1A KO and WT HUVECs were seeded  were cultured for 1 h in a normoxic- (21% oxygen) or hypoxic (1% oxygen) environment. The cells were fixed with 4% PFA for 15 min and washed trice with PBS for 5 min. The samples for staining were covered with blocking solution  and 1:100 Human/Mouse/Rat HIF1A antibody , and incubated at 4 °C overnight on plate shaker. The secondary antibody control samples were left untreated in the blocking solution. All the samples were washed twice with PBS for 5 min and resuspended in blocking solution with the secondary antibody 1:500 Alexa flour 594 Donkey anti goat IGG . The samples were incubated in the dark on a plate shaker for 2 h at room temperature. All the samples were washed trice with PBS for 5 min. At the final wash the cells were stained with 1:40 Alexa Fluor 488 Phalloidin (actin)  in PBS and incubated for 20 min at room temperature. Followed by 1:1000 Hoechst staining for 10 min in the dark. Cover slides were mounted with 3 µL mounting buffer. Image acquisition was performed (5 images/well) with a Zeiss LSM800 laser scanning confocal microscope, 63X oil objective equipped with diode lasers and three GaAsP detectors. The same laser intensity photo-detector sensitivity and exposure time were applied for comparative image acquisition as follows; lasers: 405 nm: 4%, 488 nm: 5%, 561 nm: 16% with the exposure time of 930.91 ms. The background signal was adjusted based on averaging background values from four areas negative for the signal in each channel and subtracting the mean values from the final image. To assess HIF1A expression in the HIF1KO and WT HUVECs, maximum intensity z-projections of the Hoechst and actin channels were used. ROIs of the nuclei were acquired, and a mask was subsequently created and subtracted from the maximum projection of the actin channel to eliminate the areas comprising the nuclei. In the resulting image, the wand tool was used to trace all areas negative for actin signal and the derived mask was inverted to create a ROI comprising the cell cytoplasm. The mean fluorescence intensity in a grey scale sum projection of the HIF1A channel was calculated using the mean grey values from the nuclei and actin ROIs, respectively. Image analysis was done in Fiji (ImageJ) v. 2.9.0.2, and evenly distributed in the wells. The GMS was allowed to solidify for 30 min at 37 °C. Every 5 min the slides were gently tapped against the flow bench table to minimize the formation of a strong concave meniscus. When the GMS had solidified, 0.3·105 cells (HIF1A KO and WT HUVECs) were seeded per well in 50% M199 medium with supplements and 50% endothelial cell growth medium and incubated for 24 h in a 5% CO2 atmosphere at 37 °C. Finally, the TFA HIF1A KO and WT HUVECs were incubated in a normoxic or hypoxic environment for 2 h. The cells were fixed with 4%PFA for 15 min and washed trice in PBS. Bright field images (6 images/well) were taken with a Leica DMi1 microscope, DE. Image visualization and analysis was done in Fiji (ImageJ) v. 2.9.0. The background signal was adjusted based on averaging background values from four areas negative for signal in each channel and subtracting the mean values from the final image. The tube formation analysis program WimTube from Wimasis.com, ES, was used to make a quantitative analysis of the six TFA images. Gaphpad Prism 9, US, was used to plot the WimTube analysis data  was performed by thawing the Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix  in an ice bath at 4 °C overnight. Four 8-well chamber slides were pre-cooled on ice, to avoid the Geltrex Matrix Solution (GMS) from immediately solidifying. The GMS was mixed by gentle pipetting and kept on ice until added, 100 µL/cmn = 3 per group). The bright field images showed that the morphology and confluency of the HUVECs after nucleofection with mRNA, appeared normal with few dead cells in all groups. The HUVECs receiving 4.8 µg mRNA were less confluent compared to those receiving lower doses  mRNA encoding an Enhanced Green Fluorescent Protein (EGFP). The expression of EGFP in the HUVECs was quantified by fluorescence microscopy and flow cytometry (FC) analysis. High concentrations of the transfection reagent could cause cellular toxicity . Hence, er doses a, which er doses a. FC anaer doses b,c, sugger doses c. We nexer doses d. Althouer doses c,d. We aer doses . We obseer doses a, thus 3HIF1A gene  complex comprising the SpCas9 protein and chemically modified synthetic guide RNA (gRNA). Three gRNAs were designed to target the early consecutive exons of the F1A gene a. SangerF1A gene b and ICEF1A gene c. The hiF1A gene d, which F1A gene . The domF1A gene . This unTo validate if HIF1A gRNA 1 had successfully disrupted gene expression at the protein level, we cultured the HIF1A gRNA 1 KO (hereafter referred to as HIF1A KO) and wild type (hereafter referred to as WT) HUVECs in normoxic (21% oxygen) or hypoxic (1% oxygen) conditions and assessed HIF1A expression by antibody-based protein staining . We had tubes). Both HIF1A KO and WT HUVECs cultured in normoxic or hypoxic conditions can form a tube-like network. WT HUVECs formed tube-like networks with long branches, more tight junctions, and a clear mesh pattern under both conditions  is a commonly used method to study angiogenesis . We perfnditions . In contnditions . This dinditions . WimTubenditions a. The tunditions . Quantitnditions b. The HInditions c, and sinditions d. In addnditions e. The HInditions , which wnditions f. In conHIF1A gene, which to our knowledge has not been published to date.Primary ECs models, like HUVECs, most closely represent the tissue of origin compared to secondary or immortalized cell lines, but are challenging as they are hampered by a limited life span, low transfection efficiencies, and high contamination risks ,19,20. TFirst, an efficient protocol for nucleofection of HUVECs with EGFP mRNA was established, which resulted in transfection efficiencies of nearly 100%. Previous studies like Moradian et al. transfected primary cells with EGFP mRNA, which resulted in 70% EGFP positive macrophages, quantified by FC, with no significant change in the cell viability . Hunt etThe gene editing delivery method has a great impact on the gene editing efficiency of the primary ECs, thus the optimization of the nucleofection protocol has been of great importance for our experiments. Previous studies have used lentiviral vector mediated CRISPR as a delivery system and report gene editing efficiencies of 40–58% ,25. GongHIF1A gene in primary HUVECs. The HIF1A gene is well studied, found to control transcription of over 40 genes, playing an important role in endothelial adaption, vascular development, and angiogenesis [This study accomplished the creation of a highly efficient protocol for RNP-mediated CRISPR gene editing of primary ECs, with a gene editing efficiency of 98% of the ogenesis ,38. The ogenesis . This miogenesis ,47.HIF1A in ECs by cross-breeding Tie2-Cre transgenic mice and found that deletion of HIF1A in primary murine lung ECs disrupt vascular endothelial growth factor (VEGF)-dependent signaling pathway in vivo, which resulted in impaired angiogenesis [HIF1A) knockout in primary ECs allows us to investigate how gene (HIF1A) disruption affects the EC transcriptional machineries, functions, plasticity, and heterogeneity using, e.g., single cell RNA sequencing.Our results show that the HIF1A KO results in impaired angiogenesis, as we see many branching points, short tube lengths, and less tube covered area. The impaired angiogenesis might be caused by an imbalance in the proportion of tip and stalk cells, thus resulting in “split ends” in the sprouting of the ECs. Similarly, Tang et al. deleted ogenesis . Impaireogenesis ,50,51. Uogenesis ,53. New ogenesis . AlthougHIF1A in primary ECs, resulted in gene editing efficiencies up to 98%. HIF1A gRNA 1-based editing created a one base pair insertion leading to an early stop codon. The functional validation assays show that the HIF1A KO HUVECs are functional ECs as they have an uptake of LDL and express CD31 but the KO results in insufficient angiogenesis.In summary, we established an RNP-mediated CRISPR gene editing protocol for primary ECs allowing extremely efficient HIF1A KO in primary HUVECs. The RNP-mediated CRISPR gene editing of


In this work, we employ natural halloysite nanotubes (HNTs) and poly(vinylidene fluoride) (PVDF) to fabricate composite polymer electrolytes (CPEs). CPE-5 (HNTs 5 wt%) shows an ionic conductivity of ∼3.5 × 10−4 S cm−1, which is ∼10 times higher than the CPE-0 (without the addition of HNTs) at 30 °C. The greatly increased ionic conductivity is attributed to the negatively-charged outer surface and a high specific surface area of HNTs, which facilitates the migration of Li+ in PVDF. To make a further illustration, a solid-state lithium-ion battery with CPE-5 electrolyte, LiMn2O4 cathode and Li metal anode was fabricated. An initial discharge capacity of ∼71.9 mA h g−1 at 30 °C in 1C is obtained, and after 250 cycles, the capacity of 73.5 mA h g−1 is still maintained. This study suggests that a composite polymer electrolyte with high conductivity can be realized by introducing natural HNTs, and can be potentially applied in solid-state lithium-ion batteries.Solid composite electrolytes have gained increased attention, thanks to the improved safety, the prolonged service life, and the effective suppression on the lithium dendrites. However, a low ionic conductivity (<10 + transfer.The special structure of HNTs and the further formation of amorphous PVDF contribute to the enhancement of the Li Among various solid electrolytes, composite polymer electrolytes (CPEs) have shown great potential as alternatives to liquid electrolytes, thanks to the flexibility, as well as the tunability to enhance the ionic conductivity and electrochemical performance.10,11 Various efficient modifications have been employed, such as branching or cross-linking polymers,18 adding active fillers19 and blending plasticizers.20 Meanwhile, lots of polymers have been developed as electrolytes such as polyethylene oxide (PEO),21,22 polyacrylonitrile (PAN),23 polymethyl methacrylate (PMMA)–PEO,24 and PVDF.25 PEO is one of the most extensively investigated substrates, but PEO has several critical issues for practical applications such as the poor film formation ability due to its high viscosity.26 Fluorine-containing polymers such as PVDF have exhibited relatively high electrochemical stability and affinity as electrolytes.27 However, there are still several problems to be solved before the application of PVDF, such as the low ionic conductivity (<10−5 S cm−1) at room temperature. Recently, adding inorganic fillers to PVDF has been widely applied, such as ZrO2,23 SiO2,28 Al2O3,29etc. An effective filler can reduce the crystallinity of PVDF and increase the segmental motion, thereby boosting the ionic conductivity of PVDF. Very recently, it was reported that the addition of garnet-type active filler (e.g. Li6.75La3Zr1.75Ta0.25O12) could induce the partial dehydrofluorination of PVDF and lead to the enhancement of the ionic conductivity to 5.0 × 10−4 S cm−1.19 However, it has been recognized that the cubic garnet-type Li7La3Zr2O12 with high ionic conductivity is difficult to prepare, and Li7La3Zr2O12 is sensitive to H2O and CO2.30All-solid-state LIBs are considered to be one of the most promising solutions for future advanced LIBs,2Si2O5(OH)4·nH2O) is an environmentally friendly and low-cost clay material in nature.31,32 HNTs are readily available and abundant in raw materials, so it is suitable for large-scale applications. Thanks to the special 3D nanotube structure, HNTs have been widely applied in various important applications, such as antibacterial coatings,33 transport agents in drug delivery,34 catalyst supports,35 capacitors36 and so on. In this work, CPEs of PVDF–HNTs were prepared via a simple solution casting method. The obtained PVDF–HNTs films exhibit greatly enhanced ionic conductivity, as well as promising thermal stability and mechanical properties. Furthermore, a solid-state lithium battery with the PVDF–HNTs electrolyte, LiMn2O4 cathode and Li metal anode was assembled, exhibiting encouraging electrochemical performance.Halloysite nanotubes  were pretreated for the further usage. 10 g of HNTs were added in 100 mL of ethanol solution (20 wt%). Then 1 mL of hydrochloric acid (36 wt%) was added for the purification for 24 h. After the centrifugal separation and washing with deionized water to neutral, it was dried at 80 °C for 12 h to remove the residual water. The dried HNTs were further treated in 100 mL of ethanol solution (20 wt%) with 0.1 g of sodium hexametaphosphate (Macklin). After mechanically stirring for 1 h and standing for 24 h, the mixture was centrifuged. Finally, the HNTs were dried in a vacuum oven at 80 °C for 12 h to remove the residuals.4 (Aladdin) were firstly dried at 80 °C for 24 h in a vacuum oven to remove the trapped water. After that, PVDF and LiClO4 were dissolved in an appropriate amount of N,N-dimethylformamide (DMF) solvent with a mass ratio of 3 : 1. The treated HNTs were also added into the PVDF–LiClO4 solution via controlling the weight percentage of HNTs to 0, 2, 5, 10, and 20 wt% in the total amount of HNTs and PVDF to form CPE-0, CPE-2, CPE-5, CPE-10 and CPE-20, respectively. Then the mixture was stirred at 50 °C for 6 h to obtain a milky mixture for casting. After homogeneously casting onto a glass dish, it was dried in a vacuum oven at 60 °C for 24 h to remove the residual DMF in order to finally form CPEs with the thickness of ∼100 μm.To prepare CPEs, PVDF (Shenzhen Tiancheng Technology) and LiClO2.2λ = 1.5406 Å) at 40 kV and 40 mA, collected from 10° to 90° with a step size of 0.02°. Attenuated total reflectance Fourier transforming infrared spectroscopy (FT-IR) was obtained on a Bruker Vertex 70 spectrometer. The morphology and element distribution was recorded using a field emission scanning electron microscope  and energy-dispersive X-ray spectroscopy (EDX). The N2 adsorption/desorption isotherms were measured using an Autosorb Quantachrome apparatus , and the specific surface area was derived from the Brunauer–Emmett–Teller (BET) method. Thermogravimetry (TG) analysis was performed from room temperature to 600 °C at a heating rate of 10 °C min−1 in the flowing N2 atmosphere on a TG209F3-ASC instrument. The stress and strain curves of CPEs with a size of ∼15 mm × 50 mm × 0.1 mm were recorded by a universal testing machine (UTM-1422).X-ray diffraction (XRD) patterns were obtained by an X-ray diffractometer  with Cu-Kα  were also assembled. EIS and direct-current (DC) polarization with a DC voltage of 10 mV were recorded.Electrochemical performance of CPEs was measured with three configurations. CPEs sandwiched between stainless steel (SS) disks (SS|CPEs|SS) were assembled for the measurement of the ionic conductivity, which could be derived from the obtained electrochemical impedance spectra (EIS). EIS were performed over the frequency of 1 MHz to 0.1 Hz using a Princeton Applied Research (VersaSTAT 4) instrument. To evaluate the electrochemical stability of CPEs, Li|CPEs|SS coin cells (2032) were assembled. The corresponding linear sweep voltammetry (LSV) from 3 V to 8 V 2O4|CPEs|Li coin cells (2032). To prepare the cathode, Super-P carbon black, PVDF and LiMn2O4 with the mass ratio of 1 : 2 : 7 as a conductive agent, a binder, and an active material were mixed with an appropriate amount of N-methyl-2-pyrrolidone (NMP). After mechanically stirring the mixture, a black viscous paste was obtained, which was coated on an aluminum foil and dried at 120 °C in a vacuum oven for 12 h. After drying, a disk-shape electrode with a diameter of 14.0 mm was cut from the aluminum foil. The active material loading in the electrode is ∼1.4 mg cm−2. Then, the LiMn2O4 cathode layer, CPEs and Li foil anode layer were assembled into a battery. To lower the interfacial resistance, dry CPEs of 17.0 mm in diameter were wetted with 5 μL of liquid electrolyte (1.0 M LiPF6 dissolved in ethylene carbonate (EC), ethyl methyl carbonate (EMC), and dimethyl carbonate (DMC) in a volume ratio of 1 : 1 : 1). The charge–discharge performance and cycling stability were then measured from 3.4 to 4.5 V using a battery test system . All preparations were carried out inside a glovebox  filled with ultrapure Ar (99.999%) when lithium was participated.The charge–discharge performance and cycling stability was evaluated on LiMn3.3.1via a simple solution-casting method. XRD patterns were recorded to analysis the structure evolution after the film-formation, as well as with the addition of lithium salt and HNTs. The corresponding XRD patterns of the HNTs, PVDF, and CPEs are displayed in 37 For the pure PVDF film and CPEs, a slight peak at ∼19.0° corresponds to the α-phase (nonpolar) of PVDF, while a clear and broad peak located at ∼20.3° suggests the formation of the polar β- or γ-phases of PVDF.38 The predominant presence of polar phases in CPEs is beneficial for the ion transportation.39 It is worth noting that the crystallinity of CPEs decreases with the increase of HNTs amount. In addition, the crystallinity could also be obtained from the differential scanning calorimetry (DSC) thermal analysis  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C due to the partial dehydrofluorination.36,47 The detailed assignment of FT-IR is summarized in −1 of CPEs indicates the formation of CC double bonds.19 As suggested by Nan et al., the partial dehydrofluorination of PVDF could be beneficial for the interaction between components.193.2L, S and R are the membrane thickness, effective membrane area, and the bulk resistance, respectively. Typical EIS plots of CPEs measured at 30 °C are shown in −4 S cm−1, which is ∼10 times higher than the CPE-0 at the same temperature. The decrease of the ionic conductivity when increasing the content of HNTs beyond 5 wt% may be due to the aggregation of HNTs , and the corresponding coulombic efficiency is ∼102.0%. After 250 cycles, the capacity is ∼73.5 mA h g−1. The initially increased capacity is due to the gradual activation between HNTs and PVDF.22 The increase of the impedance (see 19 As shown in −1 is obtained at 0.5, 1, 1.5, 2, and 3.5C. Moreover, The cell performance using CPE-5 as an electrolyte was further evaluated in a Li|CPE-5|LiMnance see  is attri4.via a simple solution casting method. The obtained CPEs show promising thermal and mechanical properties, as well as high conductivity thanks to the special structure. The negatively charged outer surface of HNTs provides a facile channel for Li+ transfer. In addition, the special nanotube structure and the high specific surface area could prevent PVDF segments recombination, leading to the reduction of the crystallinity and the formation of the amorphous zone, thus increasing the ionic conductivity. The interaction between HNTs, lithium salt and PVDF may also contribute to the enhancement of the ionic conductivity. Among CPEs, CPE-5 shows an ionic conductivity of as high as ∼3.5 × 10−4 S cm−1 with a transference number of 0.37 at 30 °C. The discharge capacity of a LiMn2O4|CPE-5|Li battery reaches ∼71.9 mA h g−1 at 1C in the initial cycle. After 250 charge–discharge cycles, a capacity of ∼73.5 mA h g−1 can be still maintained. This study suggests that a composite polymer electrolyte with high conductivity can be realized by introducing natural HNTs.In this work, CPEs containing PVDF as the polymer matrix and HNTs as the filler were prepared There are no conflicts to declare.RA-008-C8RA06856A-s001


N6-methyladenosine (m6A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m6A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood–testis barrier (BTB) through cell junctions between adjacent Sertoli cells.RNA 6A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m6A immunoprecipitation–quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation–qPCR, western blot, coimmunoprecipitation, and polysome fractionation–qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure.ALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m6A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice.ALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the mCdh2 mRNA translation.Our study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting The online version contains supplementary material available at 10.1186/s11658-022-00404-x. As reogenesis , mitosisogenesis . In the ogenesis . ALKBH5 t manner . HoweverAdjacent Sertoli cells near the basal lamina of the seminiferous tubules can form the blood–testis barrier (BTB), which is critical to maintain the unique microenvironment for normal spermatogenesis via cell junctions . Cell ju6A level of Cdh2 mRNA in basal endoplasmic specializations and regulates the translation of its corresponding protein, N-cadherin, via IGF2BP1/2/3 complexes and YTHDF1 and thus participates in the regulation of BTB integrity.In this study, we mainly explored the relationship between ALKBH5 in Sertoli cells and the integrity of the BTB. Ultimately, we found that ALKBH5 in Sertoli cells is involved in regulating the mAlkbh5-deficient mice were used and genotyped as described previously [The TM4 mouse testicular Sertoli cell line (4201MOU-CCTCC00624) was purchased from the National Infrastructure of Cell Line Resource  and cultured with DMEM/F12  containing 5% horse serum  and 2.5% fetal bovine serum . Two-month-old wild-type C57BL/6 mice were purchased from Vital River. Four-month-old Gapdh-forward: 5′-ACAACTTTGGCATTGTGGAA-3′, Gapdh-reverse: 5′-GATGCAGGGATGATGTTCTG-3′; Cdh2-forward: 5′-CACACCCTGGGGATATTGGG-3′, Cdh2-reverse: 5′-GCTGCCCTCGTAGTCAAAGA-3′; GLuc-forward: 5′-CGACATTCCTGAGATTCCTGG-3′, GLuc-reverse: 5′-TTGAGCAGGTCAGAACACTG-3′; CLuc-forward: 5′-GCTTCAACATCACCGTCATTG-3′, CLuc-reverse: 5′-CACAGAGGCCAGAGATCATTC-3′.In this study, all antibodies, including primary and secondary antibodies, are listed in Additional file The protein was extracted, and an appropriate amount of denatured protein was used for electrophoresis and electrically transferred to a blotting membrane. After incubating the membrane with 5% blocking solution for 1 h, the primary antibody against each specific protein of interest was incubated with the membrane overnight at 4 °C. Then, the appropriate secondary antibody was incubated with the membrane at room temperature for 1 h or overnight at 4 °C. Finally, the membrane was visualized with an enhanced chemiluminescence reagent .After fixation with 4% paraformaldehyde (PFA) Sigma, #158127), testis tissue was embedded in paraffin for IHC or optimal cutting temperature compound (OCT) for IF and sliced. For IHC, sections were dewaxed with xylene and rinsed sequentially with 100%, 95%, and 75% ethanol. Then, the sections were heated in citric acid at 95 °C for 10 min for antigen retrieval. Subsequently, endogenous catalase was blocked by treatment with 3% hydrogen peroxide at room temperature for 10 min. Sections were then incubated with primary antibodies . Briefly, after cells were seeded in a dish and cultured for 24 h, an appropriate volume of RFect was mixed with serum-free medium and incubated at room temperature for 5 min. An appropriate amount of siRNA was mixed with serum-free medium and incubated with the diluted RFect at room temperature for 20 min and then added to the culture dish. The transfection efficiency was evaluated after 24–72 h. The siRNA sequences used in this study were as follows: scramble: 5′-GGCUCUAGAAAAGCCUAUGC-3′, siThe tissue was fixed with an appropriate amount of 2.5% glutaraldehyde and incubated overnight at 4 °C. Ultrathin sections were stained with 1% uranyl acetate and 0.1% lead citrate and then observed under a transmission electron microscope.6A antibody at 4 °C for 4 h, Dynabeads Protein A was added to mix for immunoprecipitation  at 4 °C for 2 h. Beads were washed five times with IPP buffer , and immunoprecipitated RNA was recovered through elution with m6A nucleotide followed by ethanol precipitation. When m6A-seq was performed, precipitated RNA was used with SMARTer Stranded Total RNA-Seq Kit v2—Pico Input Mammalian  for library construction, followed by second-generation sequencing on an Illumina X Ten platform. Two sets of biological duplicate samples were subjected to m6A-seq. When qPCR was performed, the precipitated RNA was reverse-transcribed into cDNA using ReverTra Ace qPCR RT master mix with gDNA remover , and qPCR was performed using Thunderbird SYBR qPCR mix .Total RNA was extracted according to the instructions of the TRIzol reagent manufacturer . Total RNA (20 µg) was fragmented into ~ 100–200 nt fragments using RNA fragmentation agents . Fragmented RNA was incubated with anti-m2FC|> 0.58, P < 0.05) between the scramble and siAlkbh5 samples were identified using the DESeq2 R package [6A peaks in each sample, and HOMER software was used to determine the conserved motifs within these regions. Furthermore, we divided the 3′ untranslated region (UTR), coding sequence region (CDS), and 5′UTR regions of the longest transcript of each gene into 100 equally sized bins separately to characterize the distribution patterns of m6A peaks. The percentage of m6A peaks in each bin was calculated to represent the occupancy of m6A along with the transcripts. Differentially methylated regions between the scramble and siAlkbh5 samples were further identified using exomePeak software by taking the cutoff of |log2FC|> 0.58 and false discovery rate (FDR) < 0.05. Gene Ontology (GO) analysis of the differentially expressed or modified RNAs was conducted on the basis of the DAVID online annotation database. [Alkbh5 samples were utilized to detect alternative splicing (AS) events using the rMATS tool [Differential methylation analysis and alternative splicing analysis were performed in a similar way as described in our previous study . The cle package . The exo package  was usedatabase. , 18 VisuATS tool . The incIP − CtIgG), and then Student’s t-test was used to analyze statistical differences between the groups.RIP was performed as previously reported . BrieflyCoimmunoprecipitation was performed using an immunoprecipitation kit  according to the manufacturer’s instructions. Briefly, the cells were lysed and then incubated with the primary antibody at 4 °C overnight; then, Protein A + G beads were added to the cell lysate and primary antibody mixture for 1 h at room temperature, and the beads were washed with 1× TBS. After that, SDS–PAGE was applied to elute protein from the beads.7 cells were taken from each sample and lysed. The cell lysate was added to a 20–50% sucrose gradient and centrifuged at 4 °C and 38,000 rpm for 3 h. The supernatant was fractionated into 38 tubes through a gradient fractionator. The efficiency of fractionation was determined through the detection of RPS6 and RPL11 expression in different pipe fractions by WB. RT–qPCR was performed to detect the abundance of the target gene in each fraction.After treatment with cycloheximide D  at 37 °C for 10 min, approximately 2 × 10Alkbh5 knockdown model by infection with AAV8-shAlkbh5-GFP (shAlkbh5: ACAAGTACTTCTTCGGCGA) or AAV8-scramble-GFP generated by WZ Biosciences Inc. After anesthesia, the abdominal cavity was opened to expose the testis, and the output tubule was found under a postural microscope. A certain volume of the aforementioned viruses was taken up with a glass pulling needle and injected into the seminiferous tubule. The testis was placed into the scrotum, and the abdominal incision was sutured. The mice were housed in a specific-pathogen-free environment for more than 1 month before used for subsequent analysis.Two-month-old C57BL/6 male mice were used to establish an in vivo t-test. P < 0.05 was considered statistically significant.Data are expressed as the mean ± standard deviation (SD). GraphPad Prism 7  and SPSS 22.0  were used to conduct statistical analyses. Differences between the groups were analyzed using Student’s To understand the expression levels and distribution of ALKBH5 in adult testes, we first detected the expression of ALKBH5 in adult mouse testes. Similar to previous studies , ALKBH5 + and some CD68+ cells appeared. In Alkbh5-knockout mice (Alkbh5-KO), around the seminiferous tubules, the extent of their infiltration into the stroma with normal morphology was similar to that in the wild-type mice (Fig. + and CD68+ cells were increased significantly in the stroma near the morphologically damaged seminiferous tubules (Fig. Alkbh5-KO mice, biotin was distributed not only in the stroma but also in the seminiferous tubules, regardless of the morphological integrity of the seminiferous tubule.The lumen of the seminiferous tubule is an immune-privileged environment dependent on a tight BTB . In the ice Fig. a, b. Howles Fig. a, b. NexAlkbh5 being knocked down specifically in Sertoli cells by microinjecting AAV8-shAlkbh5-GFP particles into seminiferous tubules (Fig. Alkbh5-GFP particles but not those infected with AAV8-scramble-GFP (Fig. To further clarify that ALKBH5 in Sertoli cells is related to BTB integrity, we constructed a mouse model with les Fig. a. One moles Fig. b, indicaGFP Fig. c. These Alkbh5 in mouse Sertoli cells TM4 and performed m6A-seq (Fig. 6A peaks showed that all samples had conserved consensus motifs as reported in previous studies (Additional file Alkbh5, we observed an increase in the overall m6A level and identified 1416 hypermethylated RNAs and 615 hypomethylated RNAs (Fig. 6A levels showed some gene sets associated with the cytoskeleton for forming the BTB (Fig. Alkbh5 caused a significant increase in the m6A level of Cdh2 mRNA, which encodes an important structural protein, N-cadherin, in the BTB (Fig. 6A IP-qPCR results confirmed that the m6A level on Cdh2 mRNA increased after silencing Alkbh5 (Fig. Cdh2 mRNA was bound to ALKBH5 protein (Fig. Cdh2 mRNA is a demethylation substrate of ALKBH5 in Sertoli cells.To investigate the intrinsic mechanism, we knocked down NAs Fig. . BiologiCdh2 mRNA. We knocked down Alkbh5 but observed no change in the expression of Cdh2 mRNA (Additional file Alkbh5-knockdown cells (Additional file Alkbh5 knockdown. Although multiple RNAs exhibited significant changes in alternative splicing events (Additional file Cdh2 mRNA after silencing Alkbh5 (Additional file Cdh2 mRNA translation efficacy after silencing Alkbh5 had an upward trend, although there was no statistical difference (Fig. 6A-binding proteins and Cdh2 mRNA. As shown in Fig. Cdh2 mRNA and IGF2BP1, IGF2BP2, and IGF2BP3 proteins, respectively. Interestingly, coimmunoprecipitation showed that IGF2BP3 interacted with IGF2BP1 and IGF2BP2 (Fig. Igf2bp1, Igf2bp2 or Igf2bp3 led to a significant decrease in the expression of N-cadherin (Fig. 6A level of Cdh2 mRNA, which further affects its translation with the aid of IGF2BP1/2/3 complexes. Additionally, we also found that YTHDF1, another translation-related m6A binding protein, interacted with Cdh2 mRNA (Additional file Ythdf1 also led to decreased N-cadherin expression (Additional file Alkbh5 did not change YTHDF1 expression (Additional file 6A levels on Cdh2 mRNA and its translation via the IGF2BP1/2/3 complex and YTHDF1.Next, we aimed to elucidate the molecular mechanism underlying ALKBH5-mediated regulation of Cdh2, was indeed upregulated after silencing Alkbh5 (Fig. Alkbh5 knockout (Additional file Alkbh5 knockdown did not induce any change in other key BTB-related molecules (Fig. Alkbh5-WT mice, the actin bundles of basal endoplasmic specializations in the testis of Alkbh5-KO mice were severely disordered (Fig. Cdh2 mRNA translation to maintain blood–testis barrier integrity (Fig. Finally, we found that N-cadherin expression, which is encoded by Alkbh5 depletion in Sertoli cells led to the destruction of BTB integrity. Further studies found that ALKBH5 regulated the m6A level of Cdh2 mRNA and promoted its translation with the aid of the IG2BP1/2/3 complex and YTHDF1. Finally, ALKBH5 participates in the assembly of basal endoplasmic specialization through Cdh2-coding N-cadherin.To date, the role of ALKBH5 in spermatogenic cells and Leydig cells has been studied, but there is a lack of research on the role of ALKBH5 in testicular Sertoli cells. In this study, the BTB assay showed that Alkbh5-KO mice, hinting at BTB destruction. The BTB assay confirmed that Alkbh5 knockdown or knockout led to damage to the BTB. Additionally, it is known that the cytoskeleton, including actin and microtubules, is an important infrastructure for forming the BTB [6A upregulation after Alkbh5 knockdown were involved in the cytoskeletal function of actin or microtubules. Cdh2-encoding N-cadherin is the main structural molecule of basal endoplasmic specialization. In this study, we found that Cdh2 mRNA translation was upregulated by silencing Alkbh5 and that basal endoplasmic specialization in the testes of KO mice was seriously disturbed. Therefore, we deduced that ALKBH5 participates in BTB formation via basal endoplasmic specialization.The lumen of the seminiferous tubule is an immune-privileged environment . BTB can the BTB  and thatCdh2 translation. N-cadherin is known to be an important protein for BTB via basal endoplasmic specialization. Cumulative studies have shown that the downregulation of N-cadherin is one of the important reasons for the destruction of BTB integrity [Alkbh5 increased N-cadherin expression and that basal endoplasmic specialization was disrupted in Alkbh5-KO mice. It is known that gene expression in cells is under precise regulation, and the imbalance would cause functional disruptions. Therefore, we thought that ALKBH5 can enhance BTB integrity by inhibiting the overexpression of N-cadherin. Finally, we found that IGF2BP1, IGF2BP2, and IGF2BP3 bound to Cdh2 mRNA and regulated its translation. It has been shown that IGF2BP1, IGF2BP2, and IGF2BP3 promote the translation of target genes [6A binding proteins to regulate RNA metabolism [Cdh2 mRNA translation. However, in this study, we found that YTHDF1 also bound to Cdh2 mRNA, and knockdown of Ythdf1 also led to decreased N-cadherin expression. Cumulative studies have shown that YTHDF1 is responsible for the translation of target genes via m6A [Cdh2 mRNA translation. However, more experiments are needed to clarify the mechanism by which the above m6A regulators promote translation.ALKBH5 is expressed in mouse spermatogenic cells, Leydig cells, and Sertoli cells . In thisntegrity –28. In oet genes , 30. In tabolism . In this via m6A –35. TherLycium barbarum polysaccharide [ALKBH5 mRNA level was downregulated in patients with idiopathic azoospermia [Normal spermatogenesis depends on an intact BTB to prevent toxic substances and immune cells from invading the seminiferous tubules. Cumulative studies have shown that some adverse factors, such as new coronavirus , Marburgccharide  and tradccharide , 45, havospermia . Further6A level of Cdh2 mRNA in mouse testicular Sertoli cells and that Cdh2 mRNA translation is mediated by IGF2BP1/2/3 complexes and YTHDF1. Ultimately, we discovered that ALKBH5 participates in the regulation of basal endoplasmic specializations to regulate BTB integrity via N-cadherin.In conclusion, in this study, we found that ALKBH5 regulates the mAdditional file 1. Supplementary studies on the mechanisms of ALKBH5 on BTB integrity and list of antibodies used in this study.Additional file 2. Bioinformatic analysis results of m6A-seq data after sliencing Alkbh5 in TM4 cell line.Additional file 3. List of differentially expressed genes after sliencing Alkbh5 in TM4 cell line.


The prepared CuCo2O4@NiMn LDH core/shell nanoflowers (CCO@NM LDH NFs) possessed a high specific surface area (~181 m2·g−1) with an average pore size of ~256 nm. Herein, the CCO@NM LDH NFs exhibited the typical battery-type electrode material with a specific capacity of 2156.53 F·g−1 at a current density of 1 A·g−1. With the increase in current density, the rate capability retention was 68.3% at a current density of 10 A·g−1. In particular, the 94.6% capacity of CCO@NM LDH NFs remains after 2500 cycles at 5 A·g−1. An asymmetric supercapacitor (ASC) with CCO@NM LDH NFs//activated carbon (AC) demonstrates a remarkable capacitance of 303.11 F·g−1 at 1 A·g−1 with excellent cycling stability. The coupling and synergistic effects of multi-valence transition metals provide a convenient channel for the electrochemical process, which is beneficial to spread widely within the realm of electrochemical energy storage.Dandelion-like CuCo Supercapacitors have attracted extensive research attention due to the increasing demand for sustainable and reliable energy storage devices. Simultaneously, supercapacitors can bridge the gap between batteries and capacitors since they can provide power density and long cycle life ,2. Binar2O4@Ni(OH)2 core-shell flowers [2/NiCo-LDH core/shell [2@Co(OH)2 core/shell [2-containing Co(OH)2/Ni(OH)2 core/shell [2@α-Fe2O3/CNTF [2 core/shell [2@NHCSs electrode [2 core/shell nanoarrays [The electrode material with core/shell structures can provide more active sites to improve its conductivity, achieving unprecedented electrochemical performance. The main reason for this is that the core/shell structure can promote the rapid transfer of ions and electrons in the electrochemical process . Moreove flowers , re/shell , P-Ni2re/shell , MoS2@α-2O3/CNTF , MnS@Ni2lectrode , NiCoP@Cnoarrays , and so noarrays ,20. At tnoarrays ,22.−1 at a current density of 1 A·g−1. Activated carbon and CCO@NM LDH NFs are used as the anode and cathode, respectively, for the construction of the asymmetric supercapacitor. The hybrid supercapacitor demonstrates a remarkable capacitance of 303.11 F·g−1 at 1 A·g−1. It also provides a maximum energy density of 92.2 Wh·kg−1 at a power density of 730 W·kg−1; moreover, and it shows excellent stability. Therefore, this work can be used as a guide for preparing new functional materials with high-performance supercapacitors.Herein, a CCO core is shelled with NiMn LDH, which forms CCO@NM LDH NFs’ electrode material via two hydrothermal reactions. CCO@NM LDH NFs with core/shell structure can construct abundant oxygen vacancy defects to increase the large specific surface area of CCO@NM LDH NFs, which enables ions to freely insert and remove in the electrochemical process, thus improving the supercapacitor performance of composite electrode materials. As the anode material of supercapacitors, the discharge-specific capacitance of core/shell CCO@NM LDH NFs is as high as 2156.53 F·g3)2·6H2O and 2 mmol of Cu(NO3)2·6H2O were dissolved in 80 mL of distilled water and ethanol with a volume ratio of 1:1 as the starting materials. A piece of Ni foam  was submerged in the homogeneous solution and heated at 120 °C for 6 h using a hydrothermal reaction. Then, the CuCo precursor attached to NF surface was successfully obtained. the CuCo precursor powder was annealed at 350 °C for 6 h to obtain NF-loaded CuCo2O4. Secondly, Ni foam loaded with CuCo2O4 was used as a substrate for secondary hydrothermal growth in the following solution. The solution contains 0.713 g of NiCl2·6H2O and 0.198 g of MnCl2·4H2O, and 0.7 g of hexamethylenetetramine (HMT) and the NF-loaded CuCo2O4 were rinsed in 70 mL of distilled water. The reaction was conducted at 90 °C for 6 h. After the reaction, the mixture loaded on NF was washed and dried at 60 °C to obtain CCO@NM LDH NF samples.The CCO@NM LDH is synthesized using a two-step hydrothermal reaction schematically shown in A field emission scanning electron microscope  and transmission electron microscope  were used to characterize the morphology of the samples. Energy dispersive X-ray spectroscopy (EDS) was used to display the distribution of elements. X-ray diffractometer  in Cu K target (α = 1.542 Å) was recorded at the 2θ scanning range of 10° to 80° Kα. The chemical states of elements were studied using X-ray photoelectron spectroscopy . The specific surface area was analyzed via BET JW-004 with nitrogen adsorption/desorption for 1 h.2O4 nanoflowers, as illustrated in 2O4 sample and CuCo2O4-NiMn LDH sample are shown in To better investigate the influence on the morphology of the dandelion-like CCO@NM LDH NFs, a series of FE-SEM images were carried out to determine the evolution of the CuCo precursors. 2O4-NiMn LDH material were further analyzed using high-resolution transmission electron microscopy (HRTEM) and XRD measurements. As shown in 2O4 (JCPDS No. 78-2177). Then, the diffraction peaks of LDH can be reasonably indexed to a series of crystal planes, such as (003), (006), (009), and (110). This demonstrates the successful preparation of spinel CuCo2O4 shelled with NiMn LDH. No peaks of impurities such as copper oxides and cobalt oxides were found, confirming the high purity of the product. The synergistic effect of dandelion-like CuCo2O4 and ultrathin NiMn LDH resulted in the successful preparation of CCO@NM LDH NFs with good crystallinity.The morphology and crystallinity of CuCo3/2 and Cu 2p1/2, respectively [3+, while peaks at 780.1 eV and 794.8 eV can be corresponded to Co2+. Moreover, Co2+ and Co3+ co-exist in the CuCo2O4 sample and CCO@NM LDH NFs. It should be noted that the peak at 528.45 eV in the O1s spectrum disappears in the obtained CCO@NM LDH compared with CuCo2O4. The peak in the O1s spectrum of CuCo2O4 is fitted into three subpeaks, corresponding to OL (528.4 eV), OOH (529.9 eV), and OV (531.1 eV). The OL peak is ascribed to the Cu−O and Co−O bonds in OH peak is indexed to the −OH group [L almost disappears with the synergistic effect of the core/shell structure. The appearance of OW, located at 532.4 eV, induced physically and chemically absorbed water molecules on the surface of CCO@NM LDH NFs [V increases due to the transformation of OL, and OV represents oxygen vacancy. Meanwhile, peaks at 855.6 eV and 873.2 eV correspond to Ni 2p3/2 and Ni 2p1/2 in the Ni 2p spectrum in 1/2. The peak of Mn 2p3/2 at 641.80 eV is divided into two peaks, which demonstrates that Mn is present in the compound, similar to Mn3+ in As shown in ectively . The Co OH group . OL almo LDH NFs . Obvious2·g−1) is 3.2 times that of the CCO NFs (~56.5 m2·g−1) in It can be speculated that an increase in oxygen vacancy provides more active sites for electrochemical processes. At the same time, the BET-specific surface area of the CCO@NM LDH NFs is more likely to be characterized as in −1. It is noted that the CCO@NM LDH NF electrodes show a better capacity than that of the CCO NFs and NiMn LDH electrodes. Similarly, the GCD plots of all samples at 1 A·g−1 are illustrated in −1, 2 A·g−1, 4 A·g−1,5 A·g−1, 6 A·g−1, 8A·g−1, 10 A·g−1, and 20 A·g−1 are 2156.53 F·g−1, 2003.85 F·g−1, 1826.88 F·g−1, 1756.58 F·g−1, 1692.79 F·g−1, 1591.68 F·g−1, 1472.79 F·g−1, and 882.97 F·g−1. Meanwhile, the obvious voltage platform at different current densities indicates that the CCO@NM LDH electrode is made of a battery-type capacitor. The GCD curves of each electrode show excellent reversible redox behavior and pseudo-capacitance characteristics, which is consistent with the corresponding redox reaction in CV. With an increase in current density, the rate capability retention is 68.3% at a current density of 10 A·g−1. For comparison, CV and GCD plots of CCO NFs and NiMn LDH electrodes are given in −1 at a current density of 1 A·g−1, which is far higher than that of the CCO (~164.51 F·g−1) and NiMn LDH samples (~1129.29 F·g−1). This is because CCO@NM LDH NFs with a large and specific surface area make better contact with the electrolyte as an active substance. The Nyquist plots of the CCO NF, NiMn LDH, and CCO@NM LDH NF electrodes are shown in S), charge transfer resistance (RCT), double layer capacitive (Cdl), and Warburg impedance (Zw) [−1 is still 94.6%, which demonstrates the outstanding cycle stability.Due to its abundant oxygen vacancy defects and highly specific surface area, the CCO@NM LDH NFs were speculated as a high-performance electrode material for supercapacitors. The capacitive properties of the as-obtained CCO NF, NiMn LDH, and CCO-NM LDH NF electrodes are measured using CV, GCD, and EIS tests. nce (Zw) ,31. It ii = avb. The CV curves of the CCO@NM LDH NF electrodes at a low scanning speed were tested to thoroughly understand the mechanism of charge storage inhibition in the system in ia represents the peak current density of the oxidation peak in the oxidation process, and v is the scanning rate. When b is close to 0.5, the electrochemical process mainly depends on Faradic intercalation limited by diffusion; when 0.5 < b < 1, the behavior is generally defined by the capacitance of the material in the electrode. Notably, the b value of the CCO@NM LDH NFs is the largest (0.748), reflecting that it has faster reaction kinetics in 2O4 sample as the core is 0.647. With an increase in scanning speed, the contribution of CCO@NM LDH NFs is almost entirely shown by capacitance in The storage methods of electrode charges are divided into surface capacitance control and diffusion control . The pro−1. When the potential window increases from 1.2 V to 1.5 V, the shape of the CV curve basically remains stable. We further confirm the rationality of the potential window through the GCD curve illustrated in −1 at a current density of 1 A·g−1. When the current density increases to 20 A·g−1, the specific capacitance of CCO@NM LDH//AC is still as high as 128 F·g−1, as shown in −1 at a power density of 730 W·kg−1. At the same time, the energy density is maintained at 39.55 Wh·kg−1 under the power density of 14,831 W·kg−1. This suggests that CCO@NM LDH NFs with a core-shell structure demonstrate a large specific surface area, thus providing more active sites for the electrochemical process. According to the XPS results, the NiMn LDH shell also increases abundant oxygen vacancies in CCO@NM LDH NFs, which means that more free ions and electrons can be oxidized and reduced conveniently to improve the supercapacitor’s performance. In addition, the device shows excellent stability in that its specific capacitance is 85.28% compared with that after 5000 cycles. Because of the advantages of traditional batteries and capacitors, supercapacitors not only share the high-energy density of batteries, but also express the high-power density of capacitors. In summary, the battery-type supercapacitor composed of CCO@NM LDH NFs is a promising energy storage device.In view of its excellent capacitance performance as three electrodes, CCO@NM LDH NF, as the positive electrode, is assembled into an asymmetric capacitor. Meanwhile, activated carbon (AC) is the negative electrode, as shown in 2·g−1) with more oxygen vacancy defects. Notably, the structures can provide a strong guarantee for the ions and electrons to enter and de-intercalate conveniently in the electrochemical process. As the anode material of supercapacitor, the discharge-specific capacitance is as high as 2156.53 F·g−1 at a current density of 1 A·g−1. Meanwhile, CCO@NM LDH NF electrodes also show excellent stability. When AC and CCO@NM LDH are used as the anode and cathode of the asymmetric hybrid capacitor, respectively, the device can provide a maximum energy density of 92.2 Wh·kg−1 at a power density of 730 W·kg−1. With impressive electrochemical performances, CCO@NM LDH NFs act as a battery-type electrode material on high-performance supercapacitors for energy storage.Dandelion-like CCO@NM LDH NFs with core/shell structure were synthesized via a two-step hydrothermal method. NiMn LDH was used as the shell to form the large and specific surface area of the CCO@NM LDH NFs (~181 m


The in vivo experiments demonstrated the significant potency of Ce6@THMSNs-based PDT in obliterating primary tumors and inducing persistent tumor-specific immune responses, thus preventing distant metastasis. Therefore, we offer a THMSNs-mediated and PDT-triggered nanotherapeutic system with immunogenic property, which can elicit robust antitumor immunity and is promising for future clinical development of immunotherapy.Nanomedicine has demonstrated great potential in enhancing cancer immunotherapy. However, nanoparticle (NP)-based immunotherapy still has limitations in inducing effective antitumor responses and inhibiting tumor metastasis. Herein, polyethylenimine (PEI) hybrid thin shell hollow mesoporous silica NPs (THMSNs) were applied as adjuvant-nanocarriers and encapsulated with very small dose of photosensitizer chlorine e6 (Ce6) to realize the synergy of photodynamic therapy (PDT)/immunotherapy. Through PEI etching, the obtained Ce6@THMSNs exhibited enhanced cellular internalization and endosome/lysosome escape, which further improved the PDT efficacy of Ce6@THMSNs in destroying tumor cells. After PDT treatment, the released tumor-associated antigens with the help of THMSNs as adjuvants promoted dendritic cells maturation, which further boosted CD8 The development of cancer immunotherapy is a promising strategy for the next-generation of cancer therapy, as it is based on teaching or exciting the immune system of the body to detect and kill tumor cells . Immuno-Photodynamic therapy (PDT) has obtained clinical approval and been successfully used against many solid tumors, , which cIn this study, we fabricated thin-shell polyethyleneimine (PEI)-hybrid hollow mesoporous silica nanoparticles (HMSNs) loaded with photosensitizer chlorine e6 (Ce6) (denoted as Ce6@THMSNs) to realize photodynamic-triggered immunotherapy (Scheme 1). HMSNs with large and uniform pore size, feasibility of surface functionalization and excellent biocompatibility are attractive nanocarriers . After P2@mSiO2 NPs) with core-shell structure were firstly formed, subsequently, etched for 30 min by sodium carbonate  with the concentration of 0.4 M, mesoporous core-shell silica (mCSiO2) NPs were obtained after removing CTAB micelles. Finally, the as-prepared mCSiO2 NPs were further etched by 2.0 mg/ml of branched PEI  for 2 h. After removing free PEI, THMSNs were obtained. The morphology, structure, and surface chemistry of THMSNs were investigated using transmission electron microscope , field emission transmission electron microscope , and dynamic light scattering .THMSNs were synthesized following our previous report. . Brieflyvia a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc.) under 400 nm excitation.Ce6@THMSNs were prepared by a solution-solvent evaporation method. 133 μl of Chlorin e6  dimethyl sulphoxide  solution with the concentration of 1.0 mg/ml was added into 5 mg THMSNs. After mixing by sonication for 10 min, the suspensions in liquid state were evaporated by negative pressure. Ce6@THMSNs were achieved after removing unabsorbed excess Ce6 by washing the deposits. Ce6 loaded HMSNs (Ce6@HMSNs) as control group was synthesis according to the above method. Ce6@THMSNs, Ce6@HMSNs, and Ce6 were evaluated by UV-Vis spectrophotometer . The concentration of Ce6 in the supernatant was analysed by UV-Vis spectrophotometer to measure the drug loading capacity (DL) and encapsulation efficiency (EE) of Ce6. Fluorescence spectrophotometer of the Ce6@THMSNs, Ce6@HMSNs, and Ce6 were analyzed 1O2) was proved by the diminished optical density at 440 nm.Singlet oxygen was detected as follows method . Solutio2). For confocal laser scanning microscopy (CLSM) analyzing, these cells were incubated with Ce6@THMSNs, Ce6@HMSNs, Ce6, or phosphate-buffered saline (PBS) for 6 h using the same 1 µg/ml Ce6 concentration across groups. Nuclei were stained on coverslips using DAPI per the manufacturer’s directions, mounted on microscopic slides, and imaged with a Leica SP5 confocal microscope. To study the intracellular localization of NPs, the cells were incubated with fresh medium containing LysoTracker . These results were imaged by confocal microscope (Olympus IX73) and the overlap coefficient between the red and green fluorescence signal were calculated by Image-Pro Plus 6.0 software. For flow cytometry evaluations, the cells were incubated with Ce6@THMSNs, Ce6@HMSNs, Ce6, or PBS  at different times and the harvested cells were assessed with flow cytometry .4T1 cells were cultured in a standard environment (37°C and 5% CO2). After irradiation, cells were incubated with Cell Counting Kit-8 (CCK-8) solution  for an additional 1 h, and their relative viabilities were determined using a microplate reader  to read absorbances at 450 nm.To evaluate photodynamic toxicity, 4T1 cells were stimulated with PBS or Ce6@THMSNs, Ce6@HMSNs, Ce6 with the concentration of Ce6 (1 μg/ml) across all groups at different times or several Ce6 concentrations for 6 h. The cells were subsequently subjected to irradiation from 655 nm laser  for 1 min  were obtained from 6-8-week-old BALB/c mice acquired from Beijing Huafukang Bioscience Co., Inc., as detailed in a previous report, , and co-5) were suspended in PBS and then subcutaneously injected into the right rear flank of mice. To set up the dual tumor model, 5 × 105 4T1 cells were subcutaneously injected into the left side of mice for the primary tumor and 5 × 105 4T1 cells were subcutaneously injected into the right side of the mice 7 days later to create the distant tumor.The BALB/c mice (6–8 weeks old) were purchased from Beijing Huafukang Bioscience Co., Inc., and mice experiments were performed per Chinese law and approved by the Animal Experimentation Ethics Committee of the Huazhong University of Science and Technology (IACUC Number: 2891). The mice were partitioned randomly into groups. To establish the orthotopic tumor model, 4T1 cells  and injected PBS (control), free Ce6, and the various nano-formulations intratumorally at the Ce6 dose of 6 μg, THMSNs dose of 60 μg, HMSNs dose of 60 μg per mouse on day 0, day 3rd, and day 6th. 6 h after the injection, the mice from four selected groups were irradiated by a 655 nm laser for 3 min (0.3 W/cm2 power density) and we monitored their tumor sizes [V= (length × width2)/2] and body weights every 2 days before sacrificing them at the end of day 12th. We harvested the tumors and major organs, photographed the tumors and weighed and fixed them with 4% PFA for staining with H&E, and Ki-67.Upon the volumes of tumors reaching about 80 mmTo examine immune cells after the antitumor study, TDLNs and tumors were harvested on day 9th for flow cytometry. The TDLNs were crushed and filtered through a 70 μm strainer and the obtained single cell suspension in each case was washed twice and re-suspended in PBS containing 2% FBS. The single cells in TDLNs were obtained and blocked with anti-CD16/32 (FcBlock) (Biolegend) for 10 min and further incubated with PerCy7-CD11c (Biolegend), PE-CD86 (Biolegend), Brilliant Violet 421™ MHC-II (I-A/I-E) (Biolegend), and FITC-CD80 (Biolegend) for 30 min at 4°C. The harvested tumors were digested with 1500 U/ml collagenase (Sigma) for 30 min and then 70 μm filters were used to get the single-cell suspensions. These cells were stained with PE-CD3 (Biolegend), APC-Cy7-CD8a (Biolegend), PerCP-Cy5.5-CD107a (Biolegend), and FITC-CD4 (Biolegend) antibodies for 30 min and evaluated utilizing flow cytometry. Data were analyzed with FlowJo V10.n = 4) and injected with the same parameters of the various NPs, as explained above, into the primary tumors of all the animals intratumorally. 6 h after the injections, three groups were chosen, which were irradiated by a 655 nm laser  and the distant tumors were monitored without treatment. At day 14th, the distant tumors and TDLNs were harvested for the assessment of immune cell populations using flow cytometry, as explained above.The day after the distant tumors were inoculated, and the mice were partitioned randomly into 6 groups  were employed for the statistical analyses and all statistical analyses were presented as the mean ± standard deviation (SD) of at least three separate independent tests. One-way ANOVA were performed for quantitative data comparisons between different groups. *via electrostatic interaction. The illustration of the synthesis of Ce6@THMSNs were shown in THMSNs was synthesized through PEI etching method following our previous report, , then lo1O2) was decisive process for PDT. RNO was employed to detect the singlet oxygen, due to its bleaching ability that the absorbance at 440 nm would be diminished triggered by 1O2 generated by 655 nm laser. As shown in 1O2 production of Ce6@THMSNs were still substantially increased from 34.8% to 50.1% of 1O2 generated by free Ce6 with the increase of irradiation time, although the fluorescence of Ce6 loaded in THMSNs were quenched at a certain degree, which contains only about 2% fluorescence of free Ce6. For Ce6@HMSNs, the 1O2 were substantially generated to 73.2% of that produced by free Ce6 after irradiation 10 min. The highly retained 1O2 production efficiency of Ce6 loaded on HMSNs or THMSNs and the slow release of 1O2 possess a promising chance to use Ce6@HMSNs or Ce6@THMSNs for PDT cancer treatment.The production of singlet oxygen (p < 0.001), even better than Ce6@HMSNs, could be efficiently internalized by cells, which was an effective nanoplatform for killing tumor cells with PDT.The efficacy of PDT depends on particle internalization. Therefore, we studied the cellular uptake profile of Ce6@THMSNs carefully. We firstly used CLSM to observe an obvious intra-cytoplasm fluorescence pattern of Ce6 in 4T1 cells after stimulated by Ce6@THMSNs and Ce6@HMSNs. In contrast, only weak fluorescence signals of Ce6 were noted in cells incubated with free Ce6 . Next, un = 3), which was lower than that in the cells incubated with Ce6@HMSNs (0.82 ± 0.02) (n = 3). In contrast, fluorescence staining with LysoTracker proved weak Ce6 fluorescence colocalization in free Ce6 group. These results indicate that Ce6@THMSNs promoted endosome/lysosome escape, possibly through incorporating of PEI-mediated “proton sponge effect” occurring in acidic lysosomes . When the incubation time was up to 6 h, the PDT efficacy of Ce6@HMSNs increased to higher level, but was still lower than Ce6@THMSNs (**p < 0.01). Based on these observations, our pretreatment of 4T1 cells with Ce6@THMSNs for 6 h showed excellent phototoxicities, which were much higher than that in Ce6@HMSNs (*p < 0.05) and free Ce6 group (***p < 0.001) under the same Ce6 concentration (1 μg/ml) after irradiation with 655 nm laser   cytotoxicity assay. As shown in , 1 min) . All ass, 1 min) . This de+/CD80+/CD86+ expression of CD11c+ DCs than HMSNs   than Ce6@HMSNs , with free Ce6  having no impact on DC maturation , can present antigens for the activation of naïve T cells . DC matu± 0.62%) , which w± 0.62%) . Similarturation . And no in vitro. The suspension was collected after various NPs incubated 4T1 cells followed by 655 nm laser irradiation , which were used to stimulate BMDCs  and free Ce6 . Taken together, Ce6@THMSNs-based PDT promoted emission of TAA from tumor debris after irradiation, and with the help of THMSNs’ immune adjuvant effect, could boost maturity of DC.TAA released after PDT treatment may augment immune responses by taking advantage of immunologic adjuvants . We condte BMDCs . After 2in vitro, we further evaluated in vivo immune responses and the therapeutic effect of Ce6@THMSNs on BALB/c mice bearing 4T1 tumors. Upon the growth of the 4T1 tumors reaching 80 mm3, we separated mice arbitrarily into 8 groups (n = 4): 1) PBS, 2) Ce6, 3) Ce6@HMSNs, 4) Ce6@THMSNs, 5) PBS + laser, 6) Ce6 + laser, 7) Ce6@HMSNs + laser, and 8) Ce6@THMSNs + laser. Tumor regions in groups 5, 6, 7, and 8 were irradiated by 655 nm laser  at 6 h post-intratumoral administration. The dose for the intratumoral injection of Ce6 16 µg (0.8 mg/ml) in all Ce6 formulations was much lower than that used in other studies on photodynamic immunotherapy (in vivo (+ CD80+/CD11c+ CD86+) than those that received PDT with Ce6@HMSNs , free Ce6 , or NP injections without laser irradiation (+ cytotoxic T lymphocytes (CTLs)  and CD4+ T helper lymphocytes to produce antitumor immune responses. (+CD107a+ T lymphocytes (17.73% ± 2.16%) and CD4+ T lymphocytes (23.48% ± 1.94%) in tumors compared to the other treatment approaches  and Ce6@THMSNs (***p < 0.001) with laser irradiation showed better inhibition properties due to their superior PDT effect and PDT-induced TAA production, alongside proper adjuvant, resulting in enhanced therapeutic efficacy. Specially, Ce6@THMSNs + laser treatment drastically suppressed tumor development than Ce6@HMSNs + laser group (*p < 0.05), demonstrating the exceptional adjuvant efficacy of THMSNs. Additionally, tumor photography captured the smallest tumor volumes in the Ce6@THMSNs + laser group on day 12th, further establishing the superior antitumor property of Ce6@THMSNs  from all groups after treatment (in vivo.At the end of the above experiments (day 12th), hematoxylin and eosin (H&E) and Ki67 were employed to evaluate the antitumor efficacy of the various treatment options. As shown in reatment . Ce6@THM5 4T1 cells in the left flank (the primary tumor) of BALB/c mice, we injected the same amount of 4T1 cells into the right flank (the distant tumor) of the mice and partitioned them randomly into 6 groups (n = 4): 1) PBS, 2) PBS + surgery, 3) Ce6@THMSNs, 4) Ce6 + laser, 5) Ce6@HMSNs + laser, and 6) Ce6@THMSNs + laser. In the following day, the primary tumors of all the mice were subjected to the same parameters of the various NPs mentioned above, and 6 h later, the primary tumors from mice in groups 4, 5, and 6 were exposed to 655 nm laser , whereas, the distant tumors were monitored without treatment. All the mice received three rounds of treatment. On day 6th, the primary tumors in group 2 were removed through surgery after intratumoral injections of PBS (20 μl) for three rounds. Mice treated with Ce6 + laser or Ce6@HMSNs + laser later harbored untreated, distant tumors on day 8th and 10th, while distant tumors in groups without PDT treatment appeared as early as day 6  in the TDLNs of distant tumors were more significant than Ce6@THMSNs group  . Ce6@THMSNs-based PDT treatment prompted significant upregulation of CD80 (39.48% ± 2.36%) and CD86 (23.29% ± 2.25%) among DCs in the TDLNs of distant tumors compared to the other treatment groups . Meanwhi± 1.47%) , indicat± 1.47%) . CollectIn this study, we demonstrated an immunogenic Ce6@THMSNs nanotherapeutic system with efficient PDT performances for cancer immunotherapy. The Ce6@THMSNs act as both an effective vehicle for Ce6 and promising immunoadjuvant itself. Moreover, incorporating of PEI can not only enhance cellular internalization, promote endosome/lysosome escape to produce potent PDT efficacy, but also upregulate the maturity of DC to trigger antitumor immunity. Thus, Ce6@THMSNs-based PDT realized the synergy between Ce6-induced PDT and THMSNs-mediated adjuvant effect, strongly induced antitumor immune responses, thus enabling the forceful elimination of primary tumors and tumor metastasis with very small dose of Ce6. Therefore, we provided a powerful synergistic strategy of Ce6@THMSNs-based PDT for cancer immunotherapy. Their application provided a promising theoretical basis for future clinical development of immunotherapy strategies.


Hence soil samples were taken from a vineyard free-air CO2 enrichment (VineyardFACE) study in Geisenheim and examined for possible changes in the soil active bacterial composition (cDNA of 16S rRNA) using a metabarcoding approach. Soil samples were taken from the areas between the rows of vines with and without cover cropping from plots exposed to either eCO2 or ambient CO2 (aCO2).Elevated carbon dioxide concentrations . In contrast, the bacterial composition in bare soil was unaffected. In addition, the microbial soil respiration  and the ammonium concentration  were significantly different in the samples where cover crops were present and exposed to eCO2. Moreover, under eCO2 conditions, qPCR results showed a significant decrease in 16S rRNA copy numbers and transcripts for enzymes involved in N2 fixation and NO2− reduction were observed using qPCR. Co-occurrence analysis revealed a shift in the number, strength, and patterns of microbial interactions under eCO2 conditions, mainly represented by a reduction in the number of interacting ASVs and the number of interactions.Diversity indices and redundancy analysis (RDA) demonstrated that eCO2 concentrations changed the active soil bacterial composition, which could have future influence on both soil properties and wine quality.The results of this study demonstrate that eCOThe online version contains supplementary material available at 10.1186/s12866-023-02781-5. Vitis vinifera L.) grow in a complex and dynamic ecosystem, where climate, soil, microorganisms, and management practices are key factors for plant health, productivity, and wine quality. These complex interactions in the local growing area, together with the viticulture and enological techniques, lead to the wine's unique taste (the terroir). Therefore, altering factors in this balance may alter the terroir and lead to changes in consumer acceptance and profitability. The increasing CO2 concentrations and the resulting climate change could both conceivably influence plant physiology and microbial ecosystems in vineyards.Vineyards are important economic and agricultural ecosystems. According to the “Deutsche Wein Statistik” in 2017, the total land used for vineyards worldwide are 7.654 million hectares of which 3.312 million are in the European Union and 0.102 million in Germany. As a perennial culture, grapevines . A FACE study facility on vines was established in the wine-growing region Rheingau by the Geisenheim University in 2014. Since then, several studies have been conducted in this facility examining the effects of expected future eCO2 concentrations on different aspects of grapevine physiology, yield efficiency, grape composition, and ecology  sodium dodecyl sulfate) were added to the resulting soil pellet and the disruption process was performed again as described above. Both supernatants from the lysis processes were pooled in one reaction tube. The pooled supernatant was immediately extracted, initially with the addition of 500 µl of phenol/chloroform/isoamyl alcohol (25:24:1) and subsequently with chloroform/isoamyl alcohol (24:1). Afterwards, each time sample was centrifuged for 5 min at 17,000 g and 4 °C. The resulting upper aqueous phase was transferred to a new reaction tube, 800 µl of PEG solution was added (30% [wt/vol] polyethylene glycol 6000 and 1.6 M NaCl), incubated in ice for 30 min, and centrifuged for 30 min at 17,000 g and 4 °C. Subsequently, the DNA/RNA pellet was washed with 800 µl of ice-cold 75% ethanol, dried out and dissolved in 50 µl of nuclease-free water.RNA extraction was performed following a modified protocol of Mettel et al. . For theAfter extraction, samples were treated for DNA digestion with RNase-Free DNase Set (QIAGEN GmbH—Germany) according to the manufacturer’s instructions; DNase reaction was stopped with 10 µl of 50 mM EDTA. With the DNA-free RNA, a PCR was carried out using the universal 16S rRNA gene primers 27F (5’-AGAGTTTGATCMTGGATCMTGGCTCAG-3’) and 1492R (5’- GGTTACCTTGTTACGACTT-3’) , 77 and The 16S rRNA gene hypervariable regions (V4&V5) were PCR amplified using the set of primers 520F (5’-AYTGGGYDTAAAGNG-3’)  and 907R2 conditions and soil habitats using the Wilcoxon test [2 conditions, and ring plus soil habitats were assessed by a Permutational Multivariate Analysis of Variance (PERMANOVA) using the Adonis method and employing 999 permutations with the R package Vegan version 2.4–6 [Alpha and Beta diversity analyses were performed using R studio software 1.1.419, R packages Phyloseq 1.28.0 , and Vegxon test  with thexon test , 92, usixon test . Later, xon test , 92 and xon test . Statiston 2.4–6 . Additioon 2.4–6 .Core microbiome ASVs of green and open inter-row soils were calculated by transforming the ASV counts to relative abundance with Microbiome version 1.8.0 . Later, Differential abundance of ASVs and genera from green inter-row soils was assessed by comparing the core bacterial compositions of each one utilizing the R package ALDEx2 1.22.0 . First, 2 and eCO2 green inter-row soils, which showed an absolute ALDEx2 effect size > 0.5. Later, ASVs were analyzed utilizing a co-occurrence network with the R package Spiec-easi 1.1.1 [–2 and the Stability Approach to Regularization Selection (StARS) using its default settings. Subsequently, the network visualization was performed on Cytoscape 3.8.2 [Network analysis was performed using the core ASVs from aCOsi 1.1.1 , using tsi 1.1.1 , a lambdpe 3.8.2 .2 and eCO2 green inter-rows samples were assessed with Spiec-easi 1.1.1 [–1 and the Stability Approach to Regularization Selection (StARS). Additionally, prior to SPRING partial correlation analysis, a modified central log ratio (mclr) transformation of the genera counts was performed.Similarly, Core genera co-occurrence from aCOsi 1.1.1  and SPRIsi 1.1.1  using gesi 1.1.1 , a lambdCorrelation analysis between green inter-rows ASVs and genera with environmental parameters was performed using ALDEx2 1.22.0  and its nifH), ammonia oxidation (amoA), nitrite reduction  and nitrous oxide reduction (nosZ) were performed using primers and amplification protocols described on Table The quantification of the 16S rRNA gene to estimate total bacterial abundance was performed following the protocol described by Kaplan et al. , but insAdditional file 1. Elevated atmospheric CO2 concentrations caused a shift of themetabolically active microbiome in vineyard soil. Table S1, Figure S1-S10.Additional file 2: Table S2. Chemical and soil respiration results from the Geisenheim VineyardFACEAdditional file 3:Table S3.1. Correlation of green inter-rows ASVs with environmental parameters using ALDEx2 with Pearson and Sperman correlation test and False Discovery Rate (FDR) for p-values correction. r = Pearson correlation coefficient.


Bacillus cereus (S6D1–105) strain was selected for in vitro and in vivo studies. B. cereus was able to solubilize potassium, and phosphate as well as produce protease and siderophores during plate essays. Moreover, we observed the response of hydroponically grown rice plants, inoculated with B. cereus which was able to promote plant growth, by increasing plant biomass, chlorophyll contents, relative water content, different antioxidant enzymatic activity such as catalase, superoxide dismutase, ascorbate peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase and reducing malondialdehyde content in both roots and leaves of rice plants under Cd stress. Our results showed that the B. cereus can be used as a biofertilizer which might be beneficial for rice cultivation in Cd contaminated soils.Heavy metal resistant bacteria are of great importance because they play a crucial role in bioremediation. In the present study, 11 bacterial strains isolated from industrial waste were screened under different concentrations of cadmium (Cd) (100 µM and 200 µM). Among 11 strains, the Cd tolerant  In the present era, heavy metal stress is growing at a frightening level, and it has become a universal concern. Abiotic stresses such as drought, salinity, and metals are among the most deteriorating factors that hinder crop growth and yield . Althougin situ fixation , excavation , and soil washing. Still, these procedures are costly and less proficient  media: NaCl (5 mg), Tryptone (5 g), Agar (7.5 g), and Yeast (2.5 g) in 500 ml distilled water. Streaking of bacterial strains was performed on each plate containing 20 ml agar media. After streaking, the plates were secured with parafilm to avoid any contamination. Petri plates were then placed in an incubator at 30 °C for approximately 24 h. Afterward separate bacterial colonies were obtained on plates and stocked at 4 °C for further use.2) tolerance of bacterial strains was assessed by growing bacteria in nutrient agar media with different concentrations of CdCl2 (100 µM and 200 µM). Control plates were also maintained. Eleven different strains i.e. S3D1–1, S3D1–4, S3D4–6, S1D2–33, S1D2–34, S6D1–42, S1–89, S6D1–105, S2D2–111, R1, and R2 were used. Streaking of these strains was performed afterwards on agar plates and incubated for 48 h at 30 °C. Growth of the tested strains was observed under both the concentrations of CdCl2 to screen tolerant bacterial strains.Cadmium  along with 1% polyvinylpyrrolidone (PVP). Afterwards, samples were incubated at 65 °C for 2 min. The blend was then incubated in a water bath at 65 °C for 10 min. The samples were then centrifuged at 16,000×g for 10 min along with the extraction of supernatant twice with 500 μl chloroform. The upper layer was collected and incubated for 1 h at room temperature, thereafter, merging it with the twofold volume of CTAB solution. The samples were further centrifuged at 16,000×g for 5 min. The pellet was mixed in the mixture of 350 μl chloroform and 1.2 M NaCl, and centrifuged for 10 min at 16,000×g. Again, the upper layer was shifted to a new test tube, blended with isopropanol, and further centrifuged for 10 min at 16,000×g. Subsequently, supernatant was discarded, and pellet was washed with 500 μl of 70% ethanol. Following centrifugation, the supernatant was thoroughly disposed, and the pellet was dried up and was dissolved in 100 μl ddH2O.For genomic DNA extraction, Cetyltrimethylammonium Bromide (CTAB) based procedure was used. Samples were merged with 500 μl of CTAB buffer, 300 μl sterile dH2, 10× Taq buffer, Taq polymerase 5 U/µl, reverse primer 10 µM, forward primer 10 µM, dNTPs 10 mM, and DNA template ≥1,000 ng. The PCR was carried out in a thermal cycler manipulating cycling condition, that contained 1 cycle of initial denaturation at 95 °C for 5 min, then 35 cycles of denaturation for 45 s at 95 °C, annealing at 54–65 °C for 45 s and extension for 1 min 30 s at 72 °C for 16S rRNA gene and final extension was executed for 10 min at 72 °C. The PCR products were assessed by 1% agarose gel electrophoresis. The bacterial 16S rRNA gene sequence was identified using the Basic Local Alignment Search Tool (BLAST) algorithm at the National Centre for Biotechnology Information (NCBI) and accession number (OK310861) of the most potent bacterial sequence submitted to NCBI Gene Bank was obtained, accordingly.The 16S rRNA gene of bacteria was amplified using universal primers as, F-primer MP1 (5’-CGGGATCCAGAGTTTGATCCTGGTCAGAACGAACGCT-3’) and R-primer MP6 (5’-CGGGATCCTACGGCTACCTTGTTACGACTTCACCCC-3’). Amplification was carried out by PCR with a reaction mixture of 25 µl, that consisted of PCR water, MgClPlant growth promoting (PGP) traits such as: phosphate solubilization, potassium solubilization, as well as protease and siderophores production by Cd tolerant bacterial strain were examined.The bacterial strain was tested for phosphate solubilization by an agar assay method using Pikovaskaya’s media. The bacterial strain was streaked on Pikovaskaya’s plates, and then placed in an incubator for 7 days at 30 °C. After incubation period, plates were observed for the presence of clear halo-zones surrounding the culture spot which points towards the isolate’s phosphate solubilization ability. The solubilization index was accessed by the following formula:The bacterial strain was tested for potassium solubilization using Aleksandrov media. The media was dispensed into petri plates. After the media was solidified, the bacterial strain was streaked on the media. Plates were incubated for 3 days at 30 °C. After incubation, the presence of clear halo-zones surrounding the culture spot, indicated the potassium solubilization potential of the bacteria. Potassium solubilization index was noted by the following formula:The protease production test was performed according to the method mentioned by Bacterial isolate was observed for siderophores production by nutrient agar medium added with an indicator dye. The nutrient agar media containing the bacterial culture was incubated for 7 days at 30 °C. The positive or negative reaction of bacterial strain to the assay was recorded. After incubation, plates were detected for halo-zones formation and the following formula for accessing the solubilization index was used:2O2 (3%) and were washed with distilled water. These seeds were grown in the germination box on wet double-layer filter paper. Seeds were germinated under different levels of Cd concentration . Seeds were kept in germination boxes in the dark until the plumule (shoots) and radical (roots) of the seeds emerged. After 7 days of germination fresh and dry weights, shoot, and root lengths were measured.Seeds of three rice genotypes were acquired from Rice Research Institute , District Shaikhupura Punjab, Pakistan. Fifty seeds of each genotype were soaked for 3 min in H4)2SO4 48.2, K2SO4 15.9, MgSO4.7H2O 154.88, KNO3 37, KH2PO4 49.6, Ca (NO3).2H2O 172.36, MnCl2.4H2O 1.8, ZnSO4.7H2O 0.22, Fe-Citrate 14, CuSO4.5H2O 0.08, H3BO3 5.8, H2MnO4 0.02 and prepared according to 6D1–105 was pre inoculated with 2 weeks old rice seedlings. After 5 days of inoculation, rice plants were exposed to four treatments i.e., (1) Control, (2) 100 µM CdCl2 stress, (3) bacterial strain-105, (4) 100 µM CdCl2 + bacterial strain-105 with three replicates.After screening, test seeds of Super Basmati rice with high germination percentage were selected for further experimental work. After 7 days of germination, seeds of Super Basmati were grown hydroponically in Hoagland nutrient solution in plastic pots containing foam-plugged holes placed in a greenhouse. Nutrient solution composition at its full strength was mg/L  plants were gathered separately and rinsed three times with deionized water. Roots taken from each treatment sample were spread carefully in a transparent tray containing distilled water and scanned with Epson Expression 16801.0 scanner for analysis.Fresh leaves of rice (0.2 g) were homogenized with mortar and pestle in 20 ml (80%) acetone and samples were placed in dark for 24 h. After that, samples were centrifuged for about 10 min and Chlorophyll contents were measured by taking supernatant. Chlorophyll-a, chlorophyll-b, and carotenoids were checked by recording the corresponding absorbance at 470 nm, 647 nm, and 664 nm respectively. The quantity of photosynthetic pigments was expressed in mg/g of the fresh weight (FW) .Fresh rice leaves (0.5 g) were homogenized in 5 ml (0.1%) trichloroacetic acid (TCA). Malondialdehyde (MDA) determination was carried by measuring the absorbance of supernatant at 532 nm and 600 nm and it was calculated according to its extinction coefficient of 155 nm/cm .Superoxide dismutase (SOD) activity was measured by its ability to inhibit photochemical reduction of NBT on blue formazan, and then monitored by the absorbance of assay mixture spectrophotometrically at 560 nm .2O2 decomposition at 240 nm. The CAT activity was measured in accordance with protocol followed by Activity of catalase (CAT) in the leaves and roots of rice was assessed with spectrophotometer at 30 °C by monitoring the absorbance resulting from H2O2. Reaction mixture was composed of 2.5 ml of 50 mmol/L potassium phosphate buffer (pH 6.1), 1 ml (1%) hydrogen peroxide, 1 ml (1%) guaiacol and 10–20 μl enzyme extract (Peroxidase (POD) activity was determined by analysis of POD ability for oxidizing guaiacol using H extract .2O2 (0.1 mM), ascorbate (0.5 mM), and enzyme extract (20 μl) was used. The APX activity was observed at 290 nm, using the extinction coefficient (2.8 mM/cm).Ascorbate peroxidase (APX) activity was measured according to Determination of polyphenol oxidase (PPO) activity was carried out by adding 100 μL of the enzymatic extract into 3 mL catechol (0.07 M) in sodium phosphate buffer (0.05 M) having pH 6.5. Assay mixture absorbance was measured in the kinetic mode at 420 nm (25 °C for 10 min) using the UV-visible spectrophotometer .PAL activity was determined according to Relative water content of rice leaves was determined according to FW, fresh weight; DW, dry weight; TW, turgid weight.P value ≤ 0.05 . Variability in data was expressed as standard error and the assessment of level of significance among different treatments was carried out by Least Significance Difference (LSD) at a significance level of e ≤ 0.05 . All dati.e. S3D1–1, S3D1–4, S3D4–6, S1D2–33, S1D2–34, S6D1–42, S1–89, S6D1–105, S2D2–111, R1, and R2 were used. Only three strains S6D1–42, S6D1–105 and S2D2–111 showed growth at 100 µM and 200 µM Cd concentrations  showed maximum similarity with B. cereus (97.30%).For screening, 11 strains of bacteria trations . We sele opacity . Based o6D1–105 was able to grow up to 1,000 µM CdCl2 concentration which was determined by steadily increasing CdCl2 concentration on growth media till the colony failed to propagate on agar plates  were exposed to 100 µM, 200 µM, and 300 µM CdClr 6 days . On the 2 stress  and 4B. After 7 days of exposure to Cd stress, plants inoculated with bacterial strain showed enhanced growth as compared to uninoculated seedlings . CadmiumPlants grown with bacterial strain exhibited a significant increase in the total root volume, root length, root area, and average diameter as compared to the Cd-treated plants . An 11% Chlorophyll a, b, and carotenoid content was increased in bacteria treated plants by 6%, 39%, and 26% respectively, while Cd treated plants showed a decrease of 50%, 58%, and 44%, respectively. In Cd + bacteria inoculated plants, a decrease of 44%, 32%, and 43% was observed as compared to the control plants –7C. An i6D1–105 was observed on the PPO, PAL, and SOD activity  activities were increased in stress-treated plants compared to the control –9F. CAT Heavy metal contamination, resulting from naturally occurring processes or anthropogenic activities, has polluted the environment, and has become a serious cause of concern among majority of the countries around the world. Cadmium is ranked as 7th among the top 20 toxic metals  and is aNumerous procedures, such as ion exchange, adsorption, or coagulation-flocculation, are available to remove Cd from Cd-contaminated regions. However, they are expensive and require high amounts of energy consumption as they rely on transforming the soil into different phases . Bioreme105, was selected for further experimentation as it had the highest MIC value . The assessment is important not just because of the need to measure resistance trend, but more importantly, the MIC value is correlated to the dosage required for effective therapy . Only three bacterial strains grew under Cd stress. These three isolates were further screened by determining the MIC of Cd after which one isolate, S therapy .6D1–105 strain of B. cereus was able to solubilize potassium and phosphate and synthesize protease and siderophore. Pseudomonas aeruginosa the enzyme recombinase regulates the zinc-induced production of siderophore. Zinc and Cd are both divalent cations thus it is predicted that Cd-induced siderophore production is controlled by an analogous mediator  which is further broken down into water and oxygen by CAT and glutathione peroxidases (Rice inoculated with bacteria showed improved growth and biomass under Cd stress. This improvement was determined by comparing the length, fresh weight, and dry weight of the rice leaves and roots under different conditions. Rice plants that were not inoculated with the bacteria showed a decrease in these parameters when exposed to Cd stress, as compared to the control. Previously, it was reported that bacterial inoculation by S8 strain could enhance the agronomic traits under Cd stress . Monocotc stress . Thus, sc stress . The chlc stress . Photosyc stress  as Cd isc stress . In the c stress . Cadmiumc stress . Bacteric stress  and S8 (c stress  in rice c stress . Anotherc stress . Reactivc stress . Althougc stress . To counc stress . SOD catoxidases . Howeveroxidases . Severaloxidases . In the B. cereus strain S6D1–105 were able to show improved plant growth, increased plant biomass, chlorophyll contents, relative water content, different antioxidant enzymatic activity such as catalase, superoxide dismutase, ascorbate peroxidase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase, and reduced MDA content in both roots and leaves of rice plant under Cd stress. Our results showed that the B. cereus S6D1–105 can be used as a biofertilizer which might be beneficial for rice cultivation in Cd polluted soils.Hydroponically grown rice plants, inoculated with 10.7717/peerj.13131/supp-1Supplemental Information 1Click here for additional data file.