This method allows investigators to study gene expression changes within PPs in response to antigens, microbes, and oral vaccine delivery vehicles of interest that are sampled.Sampling and immune surveillance within gut-associated lymphoid tissues such as the intestinal Peyer’s patch (PP) occurs by an elegantly orchestrated effort that involves the epithelial barrier, B and T lymphocytes, and an extensive network of mononuclear phagocytes. Although we now understand more about the dynamics of antigen and microbial sampling within PPs, the gene expression changes that occur in individual cell subsets during sampling are not well characterized. This protocol describes the isolation of high-quality RNA from sorted PP, B and T-lymphocytes, and CD11c This ca and IFR . More re and IFR . These D and IFR ,11,12. A and IFR ,13. Recerophages . Bonnardrophages ,16.+ phagocytes. Total RNA isolated from sorted cells is then subjected to NanoString nCounter® technology that uses a novel digital color-coded molecular barcode technology to measure gene expression based on the counts of the target RNA [® PanCancer immune profiling array panel to gain insight into the gene expression levels of over 770 genes within PPs by interrogating B- and T-lymphocytes and CD11c+ phagocytes. To compare gene expression changes at baseline levels, we used Saccharomyces cerevisiae-derived β-glucan particles (GPs). Earlier studies using GPs demonstrated that these particles are taken up by both PP M-cells and CD11c+ phagocytes and are a good positive control as they are known to stimulate an immune response [+ phagocytes to determine gene expression changes using any antigen, microbe or delivery vehicle of interest in a murine model.To gain insight into the gene expression changes of specific PP cells at baseline and in response to vaccine delivery vehicles, we have developed a protocol that isolates high-quality RNA from specific PP cells such as B- and T-lymphocytes and CD11crget RNA . In thesresponse ,18,19. ISwiss Webster mice (8–12 weeks old) were obtained from Taconic Farms . Animals were housed under conventional, specific pathogen-free conditions and were treated in compliance with the Wadsworth Center’s Institutional Animal Care and Use Committee (IACUC) guidelines. We recommend usage of age-matched mice for consistency to reduce outlier variability.Experiments described in this study that involve mice were reviewed and approved by the Wadsworth Center’s Institutional Animal Care and Use Committee (IACUC) under protocol # 15-450. The Wadsworth Center complies with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and was issued assurance number A3183-01. Moreover, the Wadsworth Center is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Obtaining this voluntary accreditation status reflects that Wadsworth Center’s Animal Care and Use Program meets all of the standards required by law, and goes beyond the standards as it strives to achieve excellence in animal care and use.RNase Zap Decontamination Solution 70% Ethanol β-glucan particles (GPs) β-glucan particles with residual mannan content (GMPs) β-Mercaptoethanol 0.4% Trypan Blue RNeasy Mini Kit DNase RNase-Free Set (1500 Kunitz units) SUPERase·In RNase Inhibitor (20 U/µl) Agilent RNA 600 Pico Kit Agilent RNA 600 Nano Kit nCounter Low RNA input Amplification kit Target enrichment Primer pool ® PanCancer Immune Profiling Kit nCounterRat anti-mouse CD45R/B220 PerCp Clone RA3-6B2 Rat anti-mouse CD3 FITC-Clone 17A2 Rat anti-mouse Fc-Block CD16/CD32-Clone 2.4G2 Armenian Hamster anti-mouse CD11c-Clone N418 Luna Universal qPCR Master Mix ProtoScript II First Strand cDNA Synthesis Kit Fig. S1 for sequences)DNA Oglios Deoxynucleotide (dNTP) Solution Set 100 bp DNA Ladder EquipmentKleen Guard Gloves Straight Surgical Scissor Curved Surgical Scissor 70 µm nylon cell strainer 12 × 75 mm Polystyrene Tubes with Cell Strainer Cap Cell Strainer Pestle Countess Cell Counter Countess Cell Counting Chamber Slides Squared Petri Dishes 22 G × 1.5 in blunt-end feeding needle Fetal Bovine Serum Spin-X Centrifuge Tube 0.22 µm Filter 2% E-gel iBase Freeze Zone 4.5 Benchtop Lyophilizer 96 Well Thermal iCycler Chip Priming Station Vortex Mixer 16 pin bayonet electrode cartridges PCR Machine DNA Engine Dyad 2100 Expert Software )nCounter Prep Station Version 4.1.0.1nCounter digital analyzer Version 4.0.0.3nSolver analysis software 3.0FACSAria IIu Flow Cytometry Sorter BD FACSDiva Flow Cytometry Sort/Analysis Softer Ware Version 6.1.3 BD Falcon 5 ml Polystryrene Round-Bottom Tube with Cell-Strainer Cap Hanks’ balanced salt solution without sodium bicarbonate and phenol red pH 7.2 4, 0.048 g NaHPO4,1 g D-Glucose, 0.098 g MgSO4 (anhydrous), 0.14 g CaCl2 (anhydrous), Adjust the pH to 7.2 with HCl8 g NaCl, 0.4 g KCl, 0.06 g KH2PO1× phosphate buffered saline pH 7.4 (1 L)2HPO4, 0.24 g of KH2PO4, Adjust the pH to 7.4 with HCl8 g of NaCl, 0.2 g of KCl, 1.44 g of NaFlow buffer1× phosphate buffered saline pH 7.4, 2% fetal bovine serum1. Gavage mice with phosphate buffered saline (PBS) or with antigen of interest. In these experiments, we used 1 × 108 highly purified, Saccharomyces cerevisiae derived β-glucan particles (GPs) as a positive control. As a second positive control, 1 × 108 purified β-glucan particles with exposed mannans (GMPs) were used in 200 µl of 1× PBS. Delivery of particles was achieved using a 22 G × 1.5 in blunt-end feeding needle. Both GPs and GMPs are known to stimulate the immune response [response ,21.NOTE: Delivery volumes should not exceed 400 µl per mouse.2. Before euthanizing mice, prepare one tube containing 2 ml of Hanks’ balanced salt solution (HBSS) with 50 µl of SUPERase·In RNase inhibitor (20 U/µl), label as tube number 1, and place it on ice. Label one additional tube as number 2, add 2 ml of HBSS with 30 µl of SUPERase·In RNase Inhibitor (20 U/µl) and place on ice.NOTE: This step is critical, as addition of the SUPERase·In RNase inhibitor immediately stabilizes RNA when PPs are harvested. Without the SUPERase·In RNase inhibitor, RNA recovery will be poor. Keeping everything on ice is extremely important.CAUTION: DO NOT USE RNAlater RNA Stabilizer Reagent (Qiagen cat. #76104) specifically for PPs, as this reagent will significantly reduce the viability of PP cells to 40%. The RNA quality was also found to be very poor. We found that RNAlater made PPs float in solution during the harvesting step, suggesting that these tissues were damaged. PPs naturally sink to the bottom of the tube in solution.2.1. Euthanize mice by CO2 asphyxiation, according to institutional guidelines.2.2. Cleanse the mouse abdomen with 70% ethanol prior to necropsy. Perform a standard necropsy that involves a 1 cm incision using straight surgical grade scissors along the midline beginning about 1.5 cm from the base of the rib cage. Expose the peritoneal cavity and identify the cecum. Snip the terminal small intestine at the ileal-cecal junction, gently remove the small intestine in its entirety, and snip the stomach-ileal junction to release intact intestine.2.3. Identify individual PPs located on the anti-mesenteric side of the intestine. Mice typically contain 5 to 10 PPs spaced more or less evenly from the duodenum  to ileum .2.4. Using curved surgical scissors, gently excise individual PPs and place them immediately in cold HBSS that contains 50 µl of SUPERase·In RNase inhibitor (tube 1). The scissors should be placed curve-side up just above the PP and then gently applied to the tissue. Add RNAase Zap to surgical instruments and then rinse with ethanol between every mouse.NOTE: Two to three mice yield plenty of cells to be able to isolate RNA from all three PP cell types. To avoid cell clumping, isolate PPs from less than 5 mice per group.2.5. Decant HBSS in tube 1 and transfer PPs into tube 2 that contains 2 ml of HBSS with 30 µl of SUPERase·In RNase inhibitor. NOTE: Before moving on to this step, wipe petri dish, pestle and cell strainer with RNase Zap decontamination solution.3. To generate a cell suspension, decant tube 2 into a 70 μm nylon mesh cell strainer that is resting on a petri dish. This setup should be on ice. Grind PPs with pestle or with the back part of a syringe plunger. The 2 ml should yield approximately 1200 µl after grinding the PPs. Transfer the cell suspension from the petri dish into a microfuge tube [uge tube . If you 3.1. Save 10–15 µl of cells to count them in step 3.5.CRITICAL STEP: Prepare flow buffer that contains SUPERase· In RNase inhibitor, as this buffer will be used for all subsequent steps to stain and wash cells for cell sorting. It is recommended to prepare 20 ml of flow buffer at a 1:250 dilution.3.2. Using a 96-round bottom plate, add 50 µl of cells per well for single color controls. For B-cells, T-cells and DCs that will be sorted, fill ten wells per cell type, with 100 µl of cells per well. Spreading cells into multiple wells prevents cell clumping in subsequent steps.3.3. Spin the plate for 2 min at 2100 rpm (887× g), to pellet the cells and decant supernatant by inverting the plate.3.4. Prepare a (1:100) dilution of Rat anti-mouse Fc-block in flow buffer and resuspend cells in 100 µl of this buffer and incubate on ice for 15 min. Place the ice bucket on a rocker during incubation.3.5. During the incubation step with the Fc block, count viable cells in the suspension by preparing a 1:20 dilution of cells with trypan blue. Add 10 μl of the cell suspension to the countess chamber slides and count cells. 20 PPs should yield approximately 1.5 × 106 cells per ml with a viability range of 90%–98%.3.6. After the 15 min incubation, spin the plate for 2 min at 2100 rpm (887× g), and decant supernatant by inverting the plate.3.7. Resuspend the cells in 100 µl of fluorophore-conjugated antibodies added directly to cell suspensions (1:200 for B and T-cells and 1:40 for CD11c+ phagocytes) and incubate on ice for 30 min with continuous rocking.3.8. Spin the plate for 2 min at 2100 rpm (887× g), and decant supernatant by inverting the plate.3.9. Wash cells with 200 μl of flow buffer.3.10. Spin the plate for 2 min at 2100 rpm (887× g), and remove the supernatant by inverting the plate.3.11. Resuspend the cells from the plate into 3 ml of flow buffer; some of cells will clump. Pass the 3 ml cell suspension through a filtered cap flow tube that contains 1ml of flow buffer resulting in a total of 4 ml of cell suspension for cell sorting.4. Cells were analyzed and sorted on a FACS Aria IIu cell sorter. Prior to the collection of sorted cells, a cell purity test sort should be performed to check that the cell types of interest are free of debris and contamination by other cell types in the sample.NOTE: Magnetic bead isolation yields mixed populations of cells, making it difficult to interpret NanoString data. A combination of magnetic bead isolation with cell sorting also decreases RNA yield significantly.NOTE: RNA samples derived from PPs are sensitive to degradation, even with the addition of RNase inhibitors. The RNeasy Mini Kit calls for cell wall lysis by using a syringe, which leads to RNA degradation. We therefore developed an alternative method for cell lysis using the Spin-X filter column (step 4.1).4.1. Sort CD45R/B220+(CD3-/CD11c-) B-cells, CD3+(CD45R/B220-/CD11c-) T-cells and CD11c+(CD45R/B220-/CD3-) CD11c+ phagocytes onto a filtered Spin-X column that is moistened with 100 µl of flow buffer that containing SUPERase·In RNase inhibitor. For PP B and T-cells we sorted 300000 cells. Since CD11c+ phagocytes are the least abundant of the three cell types in PPs, we sorted a maximum of 50000–80000 cells.4.2. During sorting, centrifuge the Spin-X column every 100000 cells at 2000 rpm for 1 min at room temperature (20°C to 30°C) as this is the maximum volume that the tubes hold during cell sorting. Discard flow buffer through and continue sorting.4.3. When sorting is finished, centrifuge the Spin-X column at 2000 rpm for 2 min. Place the filter in a new 1.5 ml collection tube. Discard flow buffer through an old collection tube.4.4. Add 350 µl of Qiagen’s RLT buffer that contains β-Mercaptoethanol (BME) (10 µl of BME in 1 ml of RLT) to the filter, pipette 20–25 times to mix. Place tube on dry ice for 5 min.4.5. Incubate on rotary spinner for 10 min and vortex at the end of the incubation. The RNA extraction is performed at room temperature (20°C to 30°C).4.6. Centrifuge the Spin-X column for 2 min at 13400 rpm. Keep flow-through. To obtain a maximum yield of RNA, the flow through is passed a second time unto the filter.4.7. Transfer the flow-through back onto the filter, and centrifuge for 2 min at 13400 rpm. Keep flow-through.4.8. Add 100 µl of RNase free H2O to the filter and pipette 20–25 times, and centrifuge for 1 min at 13400 rpm. Keep flow through and discard the filter.4.9. Add 250 µl 100% ethanol to the tube.4.10. Transfer the liquid to the RNeasy spin column and pipette 20–25 times, incubate for 5 min on bench top. Centrifuge for 1 min at 13400 rpm. Discard flow-through. NOTE: The DNase RNase-Free Set was used according to manufacturer’s instructions.4.11. Add 350 µl buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 13400 rpm to wash the spin column membrane. Discard the flow-through.4.12. Add 10 µl DNase I stock solution to 70 µl buffer RDD. Vortex to mix and add 80 µl directly to each RNeasy spin column membrane, then incubate on the bench top for 15 min.NOTE: Be sure to add the DNase I incubation directly to the RNeasy spin column membrane. DNase digestion will be incomplete if part of the mix sticks to the walls of the spin column.4.13. Add 350 µl buffer RW1 directly onto the RNeasy spin column that contains the 80 µl of DNase I and spin for 15 s at ≥ 10000 rpm. Discard the flow-through.4.14. Add 500 µl buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s 13400 rpm to wash the spin column membrane. Discard the flow-through.4.15. Add 500 µl buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 13400 rpm to wash the spin column membrane. Discard the flow-through.4.16. Add another 500 µl buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 13400 rpm to wash the spin column membrane. Discard the flow-through.4.17. Place the RNeasy spin column in a new 2 ml collection tube and discard the old collection tube containing the flow-through. Spin at 13400 rpm for 1 min to eliminate any possible carryover of buffer RPE.4.18. Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30 µl of RNase-free water directly to the spin column membrane, incubate for 2 min, then spin at 13400 rpm for 2 min to elute the RNA. Keep flow-through.4.19. Add 10 µl of RNase-free water directly to the spin column membrane, incubate for 2 min, then spin at 13400 rpm for 2 min to elute the RNA. Keep flow-through. Vortex the 40 µl of collected RNA.4.20. Sample can be stored short-term at −20°C; for longer storage, store at −80°C.5. RNA stored at −80°C must be placed on dry ice and the lid of the tube covered with parafilm. Make small holes on the top of the tube using a flame-heated needle and place inside a lyophilization flask. Lyophilize for 3 h. To ensure that the flask remains cold and the RNA remains frozen, place the flask on dry ice while it is attached to the lyophilizer. Resuspend lyophilized RNA in 6 µl of RNase-free water.6. Before use, thaw sample and ladder on ice until fully resuspended. Take 1 µl of RNA sample and ladder and heat-denature at 70°C for 2 min.6.1. Agilent RNA 6000 Pico kit protocol was followed as per manufacturer’s instructions.NOTE: The RNA yield for B-cells was in the range of 3–20 ng. For T-cells, the yield was in the range of 1.5–14 ng, and for CD11c+ phagocytes, the yield was 0.06–0.6 ng, in a total volume of 5 µl each. Prior to hybridization with the reporter and capture probe set, we used the nCounter Low RNA Input Amplification kit to generate cDNA followed by the multiplexed target enrichment primer pool to amplify target genes in the codeset. Since the yield of RNA is low, it is important to check the desired amplification of targets in all cell types by reverse transcription polymerase chain reaction (PCR) and by reverse transcription quantitative PCR before proceeding with nCounter Low RNA Input amplification.7. Synthesize the cDNA using ProtoScript II first strand cDNA Synthesis Kit from NEB using 1 ng of RNA for each cell type with random hexamer primers.7.1. Use gene-specific primers to perform either reverse transcription-PCR (RT-PCR) or quantitative RT-PCR (RT-qPCR) on the cDNA. RT-PCR was performed on cDNA to ensure the amplification efficiency and specificity of the primers. Using both methods, we chose three housekeeping genes from the immune profiling codeset Hprt, Tubb5 and Sf3a3 .7.2. RT-PCR conditions: Initial denaturation 94°C for 3 min, 94°C for 20 s, 55°C for 20 s, 72°C for 20 s (30×) and 72°C for 5 min, followed by hold at 4°C. RT-qPCR conditions: Initial denaturation 95°C for 60 s, 95°C for 15 s and 60°C for 30 s (40×), followed by hold at 4°C.7.3. To estimate the minimum number of cycles needed for cDNA amplification, we used the RT-qPCR assay, using Luna Universal qPCR Master Mix and the primers for the housekeeping genes mentioned in 7.1.7.4. The estimated number of cycles from the RT-qPCR was used in the nCounter Low RNA Input Amplification assay.8. Add RNA (2 ng or less) with the provided reagents in the kit; 0.5 µl of 10× RT enzyme mix, 0.5 µl of 10× Primer Mix and bring up to 5 µl with RNase-free water.8.1. Synthesize cDNA in thermocycler using the following conditions: anneal primer at 25°C for 10 min, first strand cDNA synthesis at 42°C for 60 min, enzyme inactivation for 85°C for 5 min and hold at 4°C.8.2. Add the following to the cDNA: 1.5 µl 5× dT Amp Master Mix, 1 µl gene specific primers (500 nM per primer) corresponding to the codeset for multiplexed target enrichment. Gently, flick to mix and centrifuge.NOTE: Estimate the number of cycles to generate dsDNA for each cell type based on manufacturer’s instructions and the data collected during the RT-PCR/RT-qPCR in step 7.2.8.3. Set up the amplification reaction in thermocycler. Initial denaturation 95°C for 10 min, 95°C for 15 s and 60°C for 4 min, followed by hold at 4°C. Number of cycles needs to be estimated on cell type, as stated in the note.8.4. Proceed with NanoString hybridization or store the dsDNA at −80°C.9. Thaw the dsDNA if frozen or proceed with hybridization.9.1. Heat denatures the dsDNA at 95°C for 2 min. Immediately cool on ice.9.2. Pre-heat PCR thermocycler to 65°C with the lid set to 70°C.9.3. Thaw reporter code set and capture code set on ice. Mix by flicking or inverting tubes.NOTE: DO NOT VORTEX. Mix by flicking or inverting tubes, followed by a quick spin in picofuge. Make sure to change pipette tip every time.9.4. Prepare master mix for the 12 samples. The master mix includes 70 µl of sample hybridization buffer to the provided reporter code set.NOTE: DO NOT VORTEX. Mix by flicking or inverting tubes, followed by a quick spin in picofuge. Make sure to change pipette tip every time.9.5. Label strip tubes and cut the strip in the middle to yield 6 tubes each. Aliquot 8 µl of master mix into strip tubes.9.6. Adjust dsDNA input to 5 µl in dH2O.NOTE: It is important to empirically adjust the input amount so that the concentration of dsDNA is not too high or too low.9.7. Add dsDNA to the 8 µl of master mix into labeled strip tubes.NOTE: DO NOT VORTEX. Mix by flicking or inverting tubes, followed by a quick spin in picofuge. Make sure to change pipette tip every time.9.8. Add 2 µl of capture probe set to every tube.NOTE: DO NOT VORTEX, Mix by flicking or inverting tubes, followed by a quick spin in picofuge. Make sure to change pipette tip every time.9.9. Close the tubes and immediately transfer to the 65°C thermocycler set up in step 9.2.9.10. Incubate in 65°C for 12–30 h. 9.11. Warm sealed cartridge from −20°C to room temperature and sealed reagent plates from 4°C to room temperature (20°C–30°C).9.12. Centrifuge reagent plates 2000 g for 2 min.9.13. Follow prep station automated instructions until it requests sample loading. Immediately remove sample from 65°C, spin in Picofuge, carefully remove caps, and load in prep station. Immediately initiate protocol by pressing start.9.14. Remove cartridge and seal with adhesive tape to prevent evaporation. Tape is provided in kit.9.15. Take cartridge and place in digital analyzer for RNA copy counts. 9.16. Upload the Reporter Library file in the provided flash drive.9.17. Create a CDF file with sample name and description and upload into the digital analyzer.9.18. Insert cartridge with the seal (step 9.14) into the digital analyzer.9.19. Initiate counts by pressing start.9.20. When the digital analyzer is complete download your data for analysis.9.21. Follow instructions on nSolver or self-analyze.PanCancer Immune profiling kit. The results were analyzed using the raw count with DEseq2 [P-adjusted value ≤ 0.05.NanoString analysis was performed in duplicate per experimental condition with the nCounter Analysis System using the h DEseq2 ,23. A trFig. 1A). Each PP contain multiple follicles that are individually equipped with an epithelial barrier, an extensive network of mononuclear phagocytes, and T- and B-lymphocytes that play an important role in sampling luminal antigens, microbes, and oral vaccine delivery vehicles  [Fig. S2). We noted that PPs treated with SUPERase·In RNase inhibitor remained in the bottom of the tube, as expected, and single cell suspensions yielded 98% viability . Most importantly, we found that viability of PP single cell suspensions remained above 90% after 2–4 h on ice with SUPERase·In RNase inhibitor, a significant improvement from our previous protocol, which was done without inhibitors [PPs are aggregate lymphoid structures that can be easily seen with the naked eye and are located on the anti-mesenteric side of the small intestine  . To asseFig. 1B) . Additiohibitors .+ B-cells, CD3+ T-cells and CD11c+ phagocytes . Using this method, we sorted a single PP suspension of cells into three separate tubes that contained CD45R/B220+(CD3-/CD11c-) B-cells, CD3+(CD45R/B220- /CD11c) T-cells and CD11c+(CD45R/B220-/CD3-) phagocytes. During sorting, we carefully selected the gates denoted in green  such that there was no cross-contamination of each of the cell types of interest. Reanalysis of the sorted cells revealed that we accomplished 99% purity for B220+ B-cells , 99% purity for CD3+ T-cells , and 73% purity for CD11c+ phagocytes . Although we could not achieve a higher percentage of purity for CD11c+ phagocytes due to their complicated shape, we determined that there was no contamination by B220+ or CD3+ cells . We also found that using a combination of both magnetic bead cell isolation and cell sorting did not improve cell purity but, rather, it decreased RNA yield to 10%–15%, due to the increased experimental time and cell losses in each step (data not shown). Using both bioanalyzer nano and pico chips, we determined that RNA isolated using RNA-later was highly degraded . SUPERase·In RNase inhibitor alone yielded multiple bands. However, many of these bands were not clearly defined due to DNA contamination . RNA purified by SUPERase·In RNase inhibitor was of higher quality, although the preparation was contaminated with DNA. We therefore subjected the RNA preparation to DNase treatment, which resulted in DNA-free RNA. The bioanalyzer picochip results yielded reproducible bands that represent 28S (4.7 kb) and 18S (1.9 kb) rRNA . We also compared two RNA extraction methods by sorting cells directly unto Trizol reagent or Spin-X columns that contained a 0.22 µm membrane. Spin-X columns were subjected to dry ice and RNeasy reagents to extract RNA from cells. We determined that the Spin-X column RNA extraction method consistently yielded DNA-free RNA of high quality in the bioanalyzer data . We also determined the limits of detection of the RNA isolated per cell type and per single mouse. We found that PP RNA isolation for B- and T -lymphocytes can be achieved at an individual mouse level as both are abundant within PPs. For CD11c+ phagocytes, RNA amounts fall below the limits of detection; therefore, more than one mouse must be used for this cell type (data not shown).After determining which RNase inhibitor was optimal in prolonging cell viability and providing RNA stability, we used cell sorting to separate PP B220® mouse Pan Cancer Immune profiling kit that profiles over 770 murine immune system-related genes. To obtain both reproducible and biologically relevant results from PP RNA, it was critical to start with high quality RNA. To compare the differential expression of genes per cell type in comparison to the untreated control, we gavaged mice with β-glucan particles (GPs) derived from Saccharomyces cerevisiae cell walls. GPs are highly purified, spherical hollow shells that mainly consist of β-1, 3-D-glucans. Because GPs are recognized by Dectin-1 and complement 3 receptors, they are considered useful antigen presenting cell-targeted vaccine adjuvant/delivery vehicles. The inner hollow cavity of GPs can be loaded to deliver antigens to macrophages and dendritic cells [+ phagocytes . Since GPs and GMPs can serve as both antigen-presenting cell-targeted delivery systems and as adjuvants, we were particularly interested in differential gene expression of CD11c+ phagocytes [To assess the gene expression profiles of each cell type, we used the nCounteric cells . In addiic cells . Confocaagocytes .P-adjusted value to be ≤ 0.05. Mice were gavaged with GPs and GMPs, and PPs were harvested after 24 h. When comparing the differential expression within each cell type at baseline and with stimulation by GPs and GMPs, the results reveal that stimulation with GPs did not yield any significant differential gene expression in B-cells and T-cells. Only one gene, interleukin 1 receptor type 1, (IL-1r1), showed differential expression in CD11c+ phagocytes. In contrast, GMPs stimulated gene expression in CD11c+ phagocytes but not in B- and T-cells. The four genes that were most significantly upregulated in CD11c+ phagocytes by GMPs were interleukin-22 (IL-22), (21-fold), deleted in malignant brain tumors 1 (Dmbt1) 11-fold, IL-1r1 (6-fold), and Fibronectin-1 (Fn1) (6-fold). IL-22, is a key cytokine that has been demonstrated to induce antimicrobial activity, stimulating tissue-damage protection and tissue repair and remodeling [Table 1). In addition, principal component analysis of the data show that gene expression profiles are clustered based on cell types regardless of treatment, indicating that there are larger changes in gene expression between cell types .To determine highly significant differential gene expression, we analyzed the RNA raw counts with DEseq2. Differential expression of transcripts was stringently defined, requiring transcripts to be up or downregulated at least 2-fold and the modeling . The Dmbmodeling . IL-1r1 modeling . Fibronemodeling  . We also found that 63 differentially expressed genes were only downregulated in B-cells, 70 genes that were only downregulated in T-cells, and 34 genes that were only downregulated in DCs . The data used in our representative heatmaps demonstrates the reproducibility of this method across all cell types under experimental conditions involving stimulation with GPs and GMPs.To determine whether we could find additional cell specific transcripts, we performed DEseq2 analysis between control B-cells, T-cells, and CD11c+ phagocytes which allowed the application of NanoString technology to determine changes in the transcriptional profiles of these cells. To achieve optimal results with this protocol, it is important to use an RNase inhibitor that is suited for PPs immediately after harvesting tissues. Careful cell sorting is also an important part of this method, as cells must be properly gated to ensure the highest cell purity during cell sorting. Lastly, the stringent data analysis that we describe will identify differentially expressed genes with high confidence. As an example of the utility of this method, we were able identify changes in genetic expression of GPs and GMPs β-glucan delivery vehicles that can target PP CD11c+ phagocytes. A major advantage of the presented method is that the investigator can perform intestinal immune profiling per cell type with any antigen or microbe of interest that is introduced into the intestinal mucosa.The reported method here provides a reproducible approach to isolating high-quality RNA from PP, B-cells, T-cells and CD11cTable 2.Possible problems and their troubleshooting solutions are listed in Supplementary informationFigure S1. Specific gene targets in all cell types are tested by RT-PCR.Figure S2. Percent viability of PP single cell suspensions.Figure S3. Stabilization of PP RNA is important for nanostring-based methods.Figure S4. Heatmap compares gene expression profiles of downregulated genes in B-cells, T-cells and DCs.Supplementary information of this article can be found online athttp://www.jbmethods.org/jbm/rt/suppFiles/246.


Cucumis sativus L.) is a typical monoecism vegetable with individual male and female flowers, which has been used as a plant model for sex determination. It is well known that light is one of the most important environmental stimuli, which control the timing of the transition from vegetative growth to reproductive development. However, whether light controls sex determination remains elusive. To unravel this problem, we performed high-throughput RNA-Seq analyses, which compared the transcriptomes of shoot apices between R2B1(Red light:Blue light = 2:1)-treated and R4B1(Red light:Blue light = 4:1)-treated cucumber seedlings. Results showed that the higher proportion of blue light in the R2B1 treatment significantly induced the formation of female flowers and accelerated female flowering time in this whole study. The genes related to flowering time, such as flowering locus T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), were up-regulated after R2B1 treatment. Furthermore, the transcriptome analysis showed that up-regulation and down-regulation of specific DEGs  were primarily the result of plant hormone signal transduction after treatments. The specific DEGs related with auxin formed the highest percentage of DEGs in the plant hormone signal transduction. In addition, the expression levels of transcription factors also changed after R2B1 treatment. Thus, sex differentiation affected by light quality might be induced by plant hormone signal transduction and transcription factors. These results provide a theoretical basis for further investigation of the regulatory mechanism of female flower formation under different light qualities in cucumber seedlings.Cucumber (The online version of this article (10.1186/s40529-018-0237-7) contains supplementary material, which is available to authorized users. Cucumis sativus L.) is a typical monoecious plant with distinct male and female flowers. It has been served as a model system for studying physiological and molecular aspects of sex determination and differentiation in plants . Organ-specific overexpression of CsACO2 driven by the organ-specific promoter P (AP3) significantly affected stamen but not carpel development, acting similarly to natural floral development of female cucumber flowers , a MADS-box gene, could be induced by ethylene and might be involved in formation of female flowers in cucumbers  application promoted the formation of male flowers in gynoecious plants (Pike and Peterson CsGAIP) is predominantly expressed in the male specific organs during cucumber flower development and belongs to the DELLA (the negative regulators of the GA action) family. CsGAIP inhibited stamen development through transcriptional repression of B-class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis  at all three stages under both treatments  mainly includes three categories: molecular function, cellular component and biological process. A total of 110 DEGs in pairwise R4B1-5-vs-R2B1-5 and 745 DEGs in pairwise R4B1-10-vs-R2B1-10 were divided into cell components, which were related to the cell membrane, cytoplasm and organelle Figs. . In pairTo further explore the biological pathways associated with sex differentiation development, DEGs in each comparison were mapped to the reference canonical pathways in the KEGG database. In pairwise R4B1-5-vs-R2B1-5, 108 DEGs were assigned into 52 KEGG pathways. They were significantly enriched in the following pathways: metabolic pathways, biosynthesis of secondary metabolites, starch and sucrose metabolism and plant hormone signal transduction  and CsGA2ox2 (Csa7G413380), involved in GA biosynthesis were identified. CsGA2ox2 (Csa7G413380) accumulated more transcripts at the R2B1-10 stage, while CsCPS1 (Csa6G410650) expressed more at the R4B1-15 stage  was used as the internal reference control to standardize the results  is responsible for turning the light transduction into a flowering signal. It showed a higher expression in 15 days samples after R4B1 treatment than after R2B1 treatment  is a key gene in regulating flowering time. FT was expressed more in 10 and 15 days samples after R2B1 treatment than after R4B1 treatment  and CRY2 (Csa3M88160). Only CRY1(Csa3M889670) was found in cucumber leaves. In the 10 days treatment samples, the expression of 0 and CRYCsa6G403570 (PHYA), Csa3G166340 (PHYB), Csa7G031720 (PHYC) and Csa1G461000 (PHYE)) responding to red light were identified from the shoot apices of cucumber seedlings. These genes showed a higher expression level after R4B1 treatment compared to R2B1 treatment  family transcription factors participate in multiple metabolic pathways. Six differently expressed WKRY transcription factors were identified in cucumber shoot apices, including 3  up-regulated genes and 2 (Csa1G547510 and Csa3G061560) down-regulated genes at R2B1-10 stage , 7  up-regulated genes and 2 (Csa4G338950 and XLOC_012211) down-regulated genes at the R2B1-10 stage  SOC1 belong to MADS-box gene, which both could regulate flowering time. Seven AGLs and two SOC1 s were identified from the shoot apices of cucumber seedlings. AGL (Csa5G156170) and SOC1  accumulated more transcripts at the R2B1-10 stage  also responds to inducing floral budding. Low red/far-red ratio promotes flowering in Arabidopsis through PHYA inhibiting PHYB  are flavo-proteins that direct a diverse array of developmental processes in response to blue light in plants  is a key transcription factor regulating flowering time. CO positively regulates two floral integrators. In Arabidopsis, CO activated SOC1 through FT to promote flowering  orthologous genes were found in cucumber shoot apices. SOC1 and AGL19 displayed the same expression pattern, with increased expression in R2B1-10 and R2B1-15 treatments  treatment, the first female flower node declined significantly and the number of female flowers within fifteen plant nodes increased markedly  was down-regulated in R2B1 samples  is also a key gene for GA synthesis pathway. It catalyzes catabolism and inactivation of bioactive GAs or their precursors   seeds were cultivated on a sponge block with a Yamasaki culture solution. When cotyledon appeared, we placed these seedlings under light conditions as follows: 300 μmol/m2/s, 12/12 (light dark) at 25 °C and a relative humidity of 70–80%. There were 2 treatments with ratios of red to blue being 4:1 (R4B1) and 2:1 (R21B), respectively. Each treatment lasted 20 days. As the flower primordium appeared 10 days after light treatment, the shoot apices and leaves after 5, 10 and 15 days of light treatments were collected immediately flash frozen in liquid nitrogen and stored at − 80 °C until use. Two biological replicates of shoot apices were used for RNA-seq analysis and real-time RT-PCR. After 20 days of treatment, 30–40 seedlings from each treatment were transplanted into the greenhouse of South China Agriculture University with a relative humidity ranging from 70 to 85% and 28 °C/20 °C (14 h/10 h) day/night temperature for further flowering time analysis.LED light produced by Kedao technology corporation  (Red light: 650–660 nm and Blue light: 450–460 nm) was chosen for this study. Cucumber . RNA isolation and RNA-Seq library preparation and sequencing were carried out by Guangzhou Gene Denovo Biological Technology Co., Ltd. . In brief, the workflow of library construction for transcriptome analysis was as follows: After RNA was collected, poly(A)-containing mRNA was purified using oligo(dT) magnetic beads. Following this, the mRNAs were fragmented and cDNA was synthesized using a random hexamer, DNA polymerase I and RNase H. The double-stranded cDNAs were purified and ligated to adaptors for Illumina paired-end sequencing. PCR amplication was done on the purified cDNAs with ligated adaptors. PCR products were separated by 2% agarose gel. The final PCR product chosen for sequencing is 400–500 bp band, which were cut and recovered from the gel. The quality and quantity of the library were verified using an Agilent 2100 Bioanalyzer and ABI real time RT-PCR, respectively  according to Kim et al. . The calculating methods for raw reads was conducted according to Cock . To obtam et al.  and Trapm et al. .The expression level of each gene was determined by the numbers of reads, which were uniquely mapped to the specific gene and the total number of uniquely mapped reads in the sample. Reads per kilobase of exon model per million mapped reads (RPKM) were calculated according to Mortazavi to estimate gene expression levels. The FDR was used to determine the threshold of the P-value in multiple tests. In our study, a FDR < 0.05 and an absolute value of |log2 (fold change)| > 1 were used as the threshold to determine the significant DEGs.To identify putative biological functions and pathways for the DEGs, the Gene ontology (GO) and Kyoto encyclopedia of gene and genomes (KEGG) database were searched for annotation. The methods for GO and KEGG enrichment analysis were according to Young and Mao respectively  according to the manufacturer’s instructions. The RNA quality and purity were verified by Nanodrop 2000 and electrophoresis on 1.0% agarose gels. The first-strand cDNA was synthesized from aliquots of 1 μg of total RNA using PrimeScriptTM RT Reagent Kit with gDNA Eraser  in a reaction volume of 20 μL. The synthesized cDNA was diluted 20 times with sterile water and used as the template for real-time PCR. The reactions were carried out in a Roche LightCycler 480 system with SYBR Premix Ex Taq II (Tli RNaseH Plus) . The reactions reagent mix was 5 μL SYBR Premix Ex Taq II, 1.5 μL cDNA template, 0.4 μL each primer (10 μmol/μL), and 2.7 μL nuclease-free water. The amplification program was 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Melting curve analyses were performed at the end of 40 cycles (95 °C for 5 s followed by a constant increase from 60 to 95 °C). In addition, TUA was used to normalize expression levels . The primer quality and efficiency were checked by qPCR on series of diluting cDNA. The expression of 26 genes chosen from RNA-seq were analyzed by real-time RT-PCR in shoot apex samples . Meanwhile, 9 genes were used for checking light signal transduction in leaves .Additional file 5: Table S2. Expression data of all the genes in R2B1 and R4B1 samples.Additional file 6: Table S3. List of primers used in the expression studies.Additional file 7: Table S4. Real-time quantitative PCR (qPCR) quality control (QC).


The experimental conditions for the preparation single-phase material with small particle size were optimized. Single-phase spinel was formed by low-temperature annealing at 700 °C. For discharge rate 0.2 C, the reversible capacities 109 and 112 mAh g−1 were obtained for LiMn1.95Co0.025Ni0.025O4 and Li1.1Mn1.95Co0.025Ni0.025O4, respectively. A good cycle performance and capacity retention about 90% after 30 cycles at discharge rate 0.2–4 C were observed for the materials cycled from 3 to 4.6 V vs. Li/Li+. Under the same conditions pure LiMn2O4 cathode materials represent a reversible capacity 94 mAh g−1 and a capacity retention about 80%. Two independent experimental techniques  were used in order to investigate the diffusion kinetics of lithium. This study shows that the partial substitution of Mn in LiMn2O4 with small amounts of Ni and Co allows the cyclability and the performance of LiMn2O4-based cathode materials to be improved.A new route for the preparation of nickel and cobalt substituted spinel cathode materials (LiMn In spite of emerging power sources, Li-ion batteries still remain the most attractive ones, since their first commercial application in 1991. The cathode materials and their synthesis procedure play a tremendous role in the electrochemical performance of Li-ion batteries [2O4 spinel was firstly proposed as cathode materials by Thackeray in 1983 [+), high safety, low cost and toxicity compared to LiCoO2 and LiNiO2 [3+ ions content during lithium intercalation at low potentials (~3 V). Upon cycling, these ions cause a significant variation of unit cell volume due to Jahn-Teller effect which leads to break the interparticle contacts [LiMnd LiNiO2 . One of contacts .3+ cannot resolve all the factors which cause a capacity loss [3+ to improve the cycling life of LiMn2O4 cathode materials [This problem can be overcome by the partial substitution of Mn by other 3d-metals  ,8,9,10 wity loss . A syneraterials ,13.2O4 can be divided into conventional solid-state route [chimie douce methods [2O4 materials.The synthesis methods of single- or multi-cation doped LiMnte route ,15,16 an methods ,23,24,25 methods . These f0.1Ni0.1Mn1.8O4 materials by sol–gel method and found that the phase transitions were significantly suppressed during charging and discharging. This allows the cathode materials with discharge capacity 118 mAh g−1 and capacity fade rate less than 10% after 40 cycles to be obtained, while the undoped LiMn2O4 phase demonstrates the capacity decrease of about 44% under the same conditions (0.3 C discharge rate) [0.25Ni0.25Mn1.5O4 spinel materials using three different chelating agents and tested their charge–discharge properties in the 3–5 V range. The utilization of oxalic acid as a chelating agent yields a LiCo0.25Ni0.25Mn1.5O4 spinel with discharge capacity of 110 mAh g−1 and capacity fading lower than 3% during 15 cycles [2O4 spinel allows to decrease the quantity of d-metal dopant, which is not active during cycling at potential range near 4 V and to decrease the Mn3+ concentration for the retention of high discharge capacity. Li1.1Mn2−2xCoxNixO4  spinel powders were successfully synthesized using a liquid stirred tank reactor method by Liu et. al. 119 mAh g−1 reversible capacity and capacity retention about 94% after 80 cycles were obtained for Li1.1Mn1.85Co0.075Ni0.075O4 and potential range from 3 to 4.3 V [1.95Ni0.025Co0.025O4 by sol-gel mediated solid-state route at 650 °C, using highly dispersed ultra-fine Mn3O4 particles as a Mn source [Wet-chemical preparation techniques including sol-gel ,21 or coge rate) . Rajakum5 cycles . Simultato 4.3 V . Fang etn source . As-obta4 [2  [1+yMn1.95Ni0.025Co0.025O4  cathode materials. Extra lithium was added to the material for overcoming a well-known capacity loss during the first charge of lithium-ion battery [2O4-based cathode material and a partial substitution of Mn with Co and Ni allows to improve the electrochemical performance of cathode materials due to the improvement of lithium diffusion.Another well-known technique which allows to obtain highly homogeneous precursor mixtures is the cryochemical (freeze drying) synthesis. A simple combination of solution freezing with the following sublimation allows to retain a uniform salt distribution obtained in the starting solution and to prepare precursors mixed at the atomic level for the synthesis of complex oxide powders. This method has been successfully used for the complex oxide preparation  for a long time ,32. More4 , LiMO2  ,35,36,37 battery . The mor3COO)2·4H2O, Co(CH3COO)2·4H2O, Mn(CH3COO)2·4H2O , lithium acetate solution was prepared by dissolving Li2CO3  in the acetic acid.The aqueous solutions of the following salts were used for the preparation of freeze dried precursors: Ni.Two solutions were prepared containing Li, Mn, Ni and Co in molar ratio 1.00:1.95:0.025:0.025; 1.10:1.95:0.025:0.025, respectively. The quantitative composition of all the solutions was confirmed by ICP/MS analysis . For the preparation of pristine LiMn2 via pneumatic nozzle under continuous stirring. As-obtained cryogranules were placed in a freeze dryer chamber ) and subjected to freeze drying at a pressure of 0.2–0.5 mbar for 72 h. Upon freeze-drying, the temperature in chamber was gradually increased from −40 to +30 °C for efficient ice removal.Then the solutions were sprayed into liquid N−1, then dwelling at required temperature for 4 h. The mass of the sample per run of treatment was equal to 2 g.The thermal treatment of freeze dried (FD) precursors was performed in air in a Nabertherm tube furnace at a heating rate of 5 °C minθ range from 10 to 90° . XRD data analysis and processing were performed in WinXPow software. Rietveld refinement technique was used for the determination of cell volume and cell parameter.X-ray diffraction patterns for the materials obtained were registered using Rigaku D/MAX 2500 diffractometer  in the reflection mode (Bragg-Brentano geometry) with CuKα radiation and graphite monochromator in the 2The morphology study was performed by scanning electron microscopy  at an accelerating voltage of 21 kV and magnification ranging from ×1000 to ×100,000. The particle size distribution was determined by statistical analyses of several SEM images using the software ImageJ with a treatment of more than 200 nanoparticles.−1 heating rate.The thermal analysis accompanied by mass-spectrometry (MS) analysis of evolved gases was performed by STA 209 PC Luxx thermal analyzer equipped with QMS 403C Aëolos mass spectrometer  in air by heating to 800 °C at a 10 °C minaqua regia. Multi element standards Perkin Elmer N9300234 and N0691579 were used for the mass spectrometer calibration.The chemical composition of the obtained cathode materials was determined by ICP mass spectrometry . Before analysis, the samples were dissolved in 1+yMn1.95Ni0.025Co0.025O4 with 10 wt. % of acetylene black, followed by adding 5 wt. % of polyvinylidene fluoride . After that, the cathode mass was placed on the stainless-steel mesh (0.05 mm thick), pressed and dried in vacuum . The cathode mass load was varied from 9 to 12 mg cm−2 and the geometric area of the cathode was equal to 1 cm2.The cathode mass was prepared by mixing 85 wt. % of Li6 in solution of dimethyl carbonate, diethyl carbonate, and ethylene carbonate (1:1:1 by volume)). According to the Fischer titration data, water content in the electrolyte did not exceed 25 ppm.The electrochemical measurements were carried out in the three-electrode Teflon cells containing an active electrode, Li counter and Li reference electrodes separated by a porous polypropylene membrane soaked with electrolyte (1M LiPF+ at room temperature. The current density during cycling was varied in the range from 20 to 400 mA g−1. Cyclic voltammograms (CV) were registered using Biologic VMP-3  potentiostat in the potential range 3–4.7 V, the potential scan rate was varied from 100 to 2000 µV s−1. Electrochemical impedance spectroscopy (EIS) measurements were carried out using Solartron 1255B , the frequency range was varied from 10 MHz to 1 MHz and the AC signal amplitude was 5 mV. The obtained hodographs were treated using ZView-impedance software.Galvanostatic curves were registered using the multichannel setup manufactured by the OJSC “Buster”  in the potential range 3–4.6 V vs. Li/Li2 and H2O evolution (confirmed by MS), and well-pronounced exothermic effect. The porous structure of FD precursor facilitates both the oxygen diffusion which is necessary to transform Co(II) to Co(III) and the gas removal from the reaction zone; hence, mass loss almost finishes at 350 °C.TG (Thermogravimetry) curve of FD precursor obtained from the solution containing Li, Mn, Ni and Co acetates in molar ratio 1.1:1.95:0.025:0.025 is shown in 2O4 spinel, is formed just at 400 °C (2MnO3 and MnO2) were also detected. Hence, the heat treatment at higher temperatures was necessary to achieve the crystallographic ordering and the formation of single-phase Li1+yMn1.95Ni0.025Co0.025O4. The increase in the annealing temperature to 600 °C and 700 °C resulted in narrowing the reflections in XRD pattern  and subsequent natural cooling with the furnace.XRD analysis revealed that the target phase, substituted LiMnt 400 °C . However pattern  caused b1+yMn1.95Ni0.025Co0.025O4  spinel with 8a positions while transition metal cations occupy octahedral 16d positions. Doping of LiM2O4 spinel with Ni and Co cations led to decrease in the cell parameter a and in the cell volume V , β is the peak width at a half maximum intensity, θ is Bragg angle, the coherent size domain for the materials obtained at 700 °C was estimated taking account of instrumental errors, it was equal to 160 nm which coincided well with the SEM results.According to Scherrer equation:1.95Co0.025Ni0.025O4 and Li1.1Mn1.95Co0.025Ni0.025O4 were studied by cyclic voltammetry at a potential scan rate of 0.1 mV s−1  and capacitance of surface layer (Cf). The second semicircle in the medium frequency region corresponds to the lithium charge transfer resistance (Rct) and the double layer capacitance (Cct). Linear part in the low frequency region corresponded to the diffusion controlled Warburg impedance W. The Nyquist plots obtained for LiMn1.95Co0.025Ni0.025O4 and for Li1.1Mn1.95Co0.025Ni0.025O4 at different potentials during charge and discharge are shown in The lithium diffusion processes during intercalation and deintercalation were also studied by the electrochemical impedance spectroscopy. The Nyquist plots of all the samples consisted of a linear part in low frequency region, of the first semicircle at medium to high frequency region and of the second semicircle in high frequency region . The firσ is Warburg coefficient, Ohm s−0.5, F—Faraday constant, s—surface area, cm2, mV—specific molar volume, cm3·mol−1, dE/dx—potential derivative of the amount of intercalated/deintercalated lithium. The calculated diffusion coefficients for charge and discharge potential 3.9–4.2 V are listed in The Warburg impedance regions in Nyquist plots were used for the calculation of apparent diffusion coefficients of lithium ions:2O4-based cathode material, where Mn is partly substituted by Ni [−12 to 10−13 cm2 s−1 and from 10−13 to 10−16 cm2·s−1 for the materials, where Mn is substituted by Ni and Cu, respectively [D values are equal to 2.28 × 10−14 and 2.60 × 10−14 cm2·s−1 for the potentials 3.9 and 4.1 V, respectively. So, we can conclude that the simultaneous substitution of Mn with Ni and Co using freeze-drying acetate precursors followed by low temperature heat-treatment allows to enhance the Li+ diffusion in comparison with substitution with only by one element  using a classical solid-state preparation technique. The enhancement of lithium diffusion coefficient in materials obtained by freeze-drying can be also explained by reducing particles size in comparison with the particles obtained via solid state or sol-gel technique. The values of lithium diffusion coefficients calculated from the electrochemical impedance spectroscopy data are in good agreement with the ones calculated from cyclic voltammetry.According to the literature data, the apparent lithium diffusion coefficient for LiMned by Ni  is varieectively . In our 1.95Co0.025Ni0.025O4, Li1.1Mn1.95Co0.025Ni0.025O4 and LiMn2O4 at the first cycle measured at С/5 charge-discharge rate are represented in 2O4 spinels. The highest discharge capacity obtained for the sample Li1.1Mn1.95Co0.025Ni0.025O4 at C/5 discharge rate was 112 mAh·g−1. This value is in good accordance with the one obtained from cyclic voltammetry at a similar rate capability. The Coulombic efficiency measured at the first cycle for all the samples is about 90%, excess charge could be attributed to the decomposition of electrolyte and formation of SEI.Charge-discharge curves for LiMn1.1Mn1.95Co0.025Ni0.025O4 are represented in −1 was obtained for C/5 discharge rate. The increase in discharge current density led to the fal of the discharge capacity to 95, 75, 55 and 17 mAh·g−1 for discharge rates C/2.5, C, 2 C and 4 C, respectively.Galvanostatic curves at different discharge rates for Li1.95Co0.025Ni0.025O4, Li1.1Mn1.95Co0.025Ni0.025O4 and LiMn2O4 during galvanostatic cycling at different discharge rates. During the first 5 cycles at C/5, the discharge capacity of Li1.1Mn1.95Co0.025Ni0.025O4 was stable and equal to 112 mAh·g−1. The increase in discharge current density to 4 C led to the drastic fall of the discharge capacity to 17 mAh·g−1. However, the following decrease in the discharge rate down to C/5 after cycling at 4 C led to the recovery of discharge capacity up to 102 mAh·g−1. This means that the discharge capacity loss after cycling was about 10%, while for the pristine LiMn2O4 the discharge capacity loss is about 20% under the same conditions. Moreover, the excess of lithium in doped spinel allows to increase the capacity of the final material: the capacity of LiMn1.95Co0.025Ni0.025O4 exceed the capacity of Li1.0Mn1.95Co0.025Ni0.025O4 for all discharge rates. The doping of spinel with excess lithium undertakes the domination of Mn4+ on the particles surface, which suppresses the formation of Mn2+ on the electrode surface and improve cyclability of cathode material [1.95Co0.025Ni0.025O4 cathode materials prepared by sol-gel mediated solid-state route are better in comparison with ours, however the preparation procedure described in ref. [material . Also, wmaterial  and multmaterial  spinel. material . For thematerial . So, our in ref.  is quite1.95Co0.025Ni0.025O4 and Li1.1Mn1.95Co0.025Ni0.025O4 cathode materials were obtained via freeze drying of acetate precursors followed by thermal treatment. Well-formed single-phase spinel was obtained at 700 °C. For 0.2 C discharge rate, the reversible capacities 109 and 112 mAh·g−1 were obtained for LiMn1.95Co0.025Ni0.025O4 and Li1.1Mn1.95Co0.025Ni0.025O4, respectively, which is higher than the ones obtained for pure LiMn2O4 prepared under the same conditions (94 mAh·g−1). The diffusion kinetics of lithium during charge-discharge processes was determined via two independent techniques: cyclic voltammetry at different scan rates and electrochemical impedance spectroscopy. The obtained value of diffusion coefficients shows that the partial substitution of Mn simultaneously with Co and Ni allows to improve the electrochemical performance of cathode materials due to the improvement of lithium diffusion kinetics. A good cycle performance and capacity retention about 90% after 30 cycles at discharge rate 0.2–4 C were obtained when the cathode materials were cycled from 3 to 4.6 V. The study shows that the partial substitution of Mn with a small amount of Ni and Co in LiMn2O4 structure allows to improve the electrochemical properties and LiMn1.95Co0.025Ni0.025O4 and Li1.1Mn1.95Co0.025Ni0.025O4 prepared by freeze-drying technique will be promising materials for lithium ion batteries.LiMn


The morphologies, phase crystals, elemental composition, and magnetic properties of the composites were also analyzed. In addition, microwave absorption of the as-prepared samples was investigated and it revealed that the impedance mismatching could be responsible for inferior microwave absorption; higher conductivity can lead to skin effect that inhibits the further incidence of microwaves into the absorbers. Furthermore, the optimum reflection loss (RL) of GNPs@NixSy@MoS2-2 can reach −43.3 dB at a thickness of 2.2 mm and the corresponding bandwidth with effective attenuation (RL < −10 dB) of up to 3.6 GHz (from 7.0 to 10.6 GHz). Compared with the GNPs, the enhanced microwave absorption can be assigned to the synergistic effects of conductive and dielectric losses.Graphene-based powder absorbers have been used to attain excellent microwave absorption. However, it is not clear if inferior microwave absorption by pure graphene materials can be attributed to impedance mismatching or inadequate attenuation capability. In this comparative study, we focus on these aspects. Graphene nanoplatelets (GNPs) multi-component composites (GNPs@Ni Electromagnetic (EM) pollution by the modern industry has become inevitable because EM radiation harms health, military devices performance, and information transmission. To alleviate this harm, various EM absorbing materials have been developed. Amongst them, magnetic ferrites  were the2 [2 hybrids such as MoS2/Ni nanoparticles [2/carbon nanotubes [2@MoS2 nanosphere [2 nanosheets (CS/MoS2) [2 composites are still effective high-performance EM absorbers [The absorber’s structure is one of the factors that affects the EM wave attenuation ,8. Mater2 ,15,16,172 ,19,20,21articles , MoS2/caanotubes , core-shnosphere , and selCS/MoS2)  are faci2@MoS2, physically combined by NiS2 and MoS2, can achieve an excellent microwave absorption ability of −41.05 dB at 12.08 GHz with a thickness of 2.2 mm [2S3 foam reportedly has a wide range of effective attenuation bandwidth (12.75–18.0 GHz) and an intense reflection loss (−50.7 dB) with a thickness of 3.6 mm [Impedance matching is presumably another key factor for materials’ MAP; this explains why semiconductor materials are preferred over insulators for high MAP absorbers. Metal sulfides and their composites are associated with excellent EM absorption due to their higher dielectric loss than metal oxides ,26. Hierf 2.2 mm . Ni@Ni2Sf 3.6 mm . Interesf 3.6 mm .xSy@MoS2) with hierarchical structures. Firstly, we deposited nickel particles on GNPs through a simple electroless nickel-plating method. Through the sulfurization process, we used GNPs@Ni, sodium molybdate, and thioacetamide as the precursors for nickel, molybdenum, and sulfur source, respectively, to obtain the GNPs@NixSy@MoS2 composites with hierarchical structures from a single-step procedure of facile hydrothermal reaction. For ideal absorbers, different hierarchical structures were obtained by controlling S/Mo molar ratios. Studies on morphologies and EM absorption properties suggest that the as-prepared GNPs@NixSy@MoS2 with hierarchical nanostructures have optimized morphology and impedance matching, strong dielectric loss, and interface polarization, thus resulting in excellent MAP.Considering nano-structural engineering and impedance matching ,34,35,362MoO4·2H2O, AR) and thioacetamide (C2H5NS) were bought from Janus New-Materials Co., Ltd. . Oxidized graphite (OG) was supplied by Qingdao Yanxin Graphite Products Co., Ltd. . Sodium hydroxide , stannous chloride , hydrochloric acid , palladium dichloride , sodium hypophosphite , nickel sulphate hexahydrate , ammonia solution , and malic acid  were all obtained from Sinopharm Chemical Reagent Co., Ltd. . Ethanol  was provided by Fuyu Fine Chemical Reagent Co., Ltd. . All reagents were used without further purification.Sodium molybdate /HCl (10 g/L) mixed solution for another hour at room temperature (RT). Finally, the as-treated GNPs were transferred into a Ni-plating solution containing nickel sulphate (25 g/L), sodium hypophosphite (30 g/L), malic acid (30 g/L), and an excess NH3·H2O (28%). The Ni-plating reaction was kept at 60 °C for 1 h. The products were collected and a robust yield calculated by mass showed that the nickel mass ratio (mass of nickel in GNPs@Ni composite) was approximately 38%.Graphene nanoplatelets (GNPs) were prepared as in a former report  and GNPs for 1 h . Thirdly3·H2O at 60 °C for 1 h.For comparison, the Ni nanoparticles were prepared by mixing the solution of nickel sulphate (25 g/L), sodium hypophosphite (30 g/L), malic acid (30 g/L), and an excess NH2MoO4 ·2H2O, 1 mmol), and a certain amount of thioacetamide (C2H5NS) were dissolved in 120 mL of deionized water under sonication for 30 min. The resulting solution was then mixed and transferred into a 200 mL Teflon-lined stainless-steel autoclave and kept at a temperature of 220 °C for 10 h. It was then cooled to room temperature. After the products were collected by centrifugation they were washed with pure water and ethanol several times. Freeze-drying resulted in GNPs@NixSy@MoS2 composites with three different amounts of C2H5NS .In a typical procedure, 0.1 g of as-prepared GNPs@Ni, 0.242 g of sodium molybdate  were displayed by the FE-SEM Hitachi S4800 microscope . The TEM images of GNPs@NixSy@MoS2-3 were carried out by the JEOL JSM-2010 microscope . Meanwhile, crystal structures of GNPs@NixSy and GNPs@NixSy@MoS2 composites were analyzed by the Bruker D8 Advanced X-ray  in the range of 2θ = 5–80°. On the other hand, elementary compositions of the composites were surveyed by an XPS spectra with the PHI 5000 Versa Probe . The electrical conductivities (σ) of all the samples were obtained with RTS-8 . Thermogravimetric analysis (TGA) was performed on Netzsch STA-449F3 Thermal Analyzer  at air atmosphere with the heating rate of 10 °C min−1 in air atmosphere. The magnetic property was tested with a physical property measurement system . The research on EM parameters were carried out with a vector network analyzer  using the coaxial wire method within the range of 2.0–18 GHz. The samples used for EM parameter measurement were prepared by mixing the GNPs@NixSy@MoS2 composites with paraffin with a mass ration of 70%. The mixtures were then cold-pressed into a toroid  at a proper pressure.The morphologies of the as-prepared samples (GNPs@Ni and GNPs@NixSy composites and the pattern shows two phases in the sample. The diffraction peaks located at 2θ = 16.2° (111), 26.6° (220), 31.3° (311), 37.9° (400), 46.9° (422), 49.9° (511), 54.7° (440), 64.4° (533), 68.5° (444), 74.9° (642), 77.3° (731) are assigned to polydymite Ni3S4 (JCPDS Card no. 47–1739). Other peaks at 2θ = 35.3° (210), 38.8° (211), 45.3° (220), 53.6° (311), 56.3° (222), 58.8° (230), 61.2° (321) can be perfectly indexed to the vaesite NiS2 (JCPDS Card no. 11–0099). After the sulfurization process, the three main diffraction peaks of Ni disappeared at 2θ = 44.5°, 51.8°, and 76.4°. NiS2 and Ni3S4 peaks, however, appeared, thus indicating that metallic Ni was completely transformed into NiS2 and Ni3S4 composites. The strong and sharp diffraction peaks confirmed that the obtained products were properly crystallized [Phase crystals investigation of the products is performed by XRD. tallized .xSy@MoS2 composites , (101), (103), respectively, (JCPDS Card no. 37–1492) [xSy are polydymite Ni3S4 and vaesite NiS2 during the hydrothermal reaction and the existence of Ni3S4, NiS2, and MoS2 was confirmed in GNPs@NixSy@MoS2 composites.For GNPs@Nimposites b, after 37–1492) . Thus, txSy@MoS2 composites were further confirmed by XPS. xSy and GNPs@NixSy@MoS2. As expected, Mo, S, C, and Ni signals were observed in the survey spectra. The peaks at approximately 227–240 eV and 400–410 eV can be ascribed to Mo 3d and Mo 3p. Other 2 peaks at 229.2 and 232.5 eV in 5/2 and Mo 3d3/2 of MoS2. The peak located at 226.3 eV corresponds to S 2s. In the spectrum of S 2p , the as-synthesized NixSy@MoS2 nanosheets increased in size to such an extent that the space was insufficient for the nanosheets to assemble into nanospheres  and the (002) planes of polycrystallinity of MoS2 in the sample. To search the elementary compositions of GNPs@NixSy@MoS2-3, elemental mappings of GNPs@NixSy@MoS2-3 are displayed in xSy@MoS2-3 frame except for Ni whose mapping is distributed in a corner. This indicates that the Ni nanoparticles only existed in that corner of the GNPs region. These results coincide with the analysis of XRD and XPS patterns.The microstructures of the samples were further investigated via TEM analyses. As seen in  of GNPs b. The sexSy@MoS2 composites were successfully synthesized. Furthermore, different hierarchical structures could be obtained by controlling the ratios of S/Mo, which may be significantly correlated to the MAP.All the morphological, crystalline, and elementary characterizations above have demonstrated that the hierarchical GNPs@NisM) of GNPs@Ni composite is 13 emu/g, which is lower than that of pure Ni nanoparticles (35 emu/g). The decrease in Ms values is mainly attributed to the demagnetizing field caused by GNPs. According to Equation (1), the weight percentage of Ni over GNPs@Ni is 37.14%, which corresponds to the calculated values obtained from the experiment.As presented in iμ). According to the Equation (2), iμ values of GNPs@Ni are higher than those of pure Ni. Thus, we can conclude that GNPs with Ni particles can enhance that magnetic loss more than pure Ni.Coating magnetic nanoparticles is one of the effective ways to enhance the material’s MAP because of the role of magnetic loss. Normally, the magnetic loss can be evaluated by initial permeability −(5).material ,58.xSy@MoS2 composites display excellent microwave absorption abilities . The 3D surface plots , dielectric loss behaviors depend on xSy@MoS2 composites. As a result, conductivity loss, dipole orientation polarization, and interfacial polarization are the possible candidate mechanisms to attenuate EM waves [Dielectric loss is the unique pathway to attenuate EM wave in GNPs@NiEM waves ,63,64.σ) of the composite samples are listed in σ values for GNPs@NixSy@MoS2-1, GNPs@NixSy@MoS2-2, and GNPs@NixSy@MoS2-3 are 3.3433 S/cm, 1.7177 S/cm, and 0.5258 S/cm. The higher ε″ of GNPs@NixSy@MoS2-1 nanocomposite mainly originates from the higher The electrical conductivities ( EM wave .ε = ε″/ε′) and attenuation constant (α) are displayed in In addition, the important parameters of dielectric loss tangent . The contour maps of Z and RL of GNPs and as-prepared GNPs-based composites are shown in xSy@MoS2 composites covers broader frequency and the area is bigger than that of GNPs and GNPs@Ni composites. This shows that NixSy and MoS2 can significantly optimize the impedance matching of GNPs@NixSy@MoS2 composites. Compared to xSy@MoS2-2 > GNPs@NixSy@MoS2-3 > GNPs@NixSy@MoS2-1, which coincides with the corresponding MAP order. For GNPs@NixSy@MoS2-1, the minimum RL can be obtained at 6.2 GHz, where α reaches only 100 and Z is equal to 1. For GNPs@NixSy@MoS2-2, Z is close to 1 in the range of 8.5–10.0 GHz with the thickness increase from 1.8 mm to 2.5 mm. The minimum RL value of −43.3 dB can be achieved at 9.1 GHz and 2.2 mm coating thickness, but the α value only reaches 110. While α value reaches a maximum of 750 at 16.0 GHz, Z is too small (~0.2). In other words, the minimum RL are not obtained at 16.0 GHz due to the impedance mismatch, which is consistent with xSy@MoS2-3, the minimum RL values of −39.5 dB and −28.3 dB are achieved at 8.1 GHz (2.4 mm thickness) and 4.8 GHz (4 mm thickness), respectively. Most importantly, the corresponding Z values are much close to 1 at 4.8 GHz and 8.1 GHz. Therefore, this clearly infers that impedance mismatching is the limiting factor of GNPs and GNPs@NixSy@MoS2 composites’ MAP. In addition, GNPs@NixSy@MoS2 composites with different hierarchical structures obtained by sulfurization process have optimized the impedance matching.Concerning impedance matching properties  can be contributed to the excellent MAP of GNPs@Niadiating ,66. TherxSy@MoS2 composites with different microstructures, that were analyzed via SEM, TEM, XRD, XPS and TGA. With respect to the MAP of GNPs@NixSy@MoS2 composites, the optimum RL is −43.3 dB at a thickness of 2.2 mm and the corresponding bandwidth with effective attenuation up to 3.6 GHz. In addition, the absorption mechanism could be responsible for the conductive loss, multi-interface polarization, and dipole polarization. Therefore, this study has not only scientifically revealed the key limiting factor for the inferior MAP of GNPs, but also a new method to optimize impedance matching. In addition, materials with moderate conductivity are still promising to deal with the pollution of EM wave; single component, especially, dielectric materials, are not enough to attenuate EM wave by dielectric loss. Concerning the cost, rational design GNPs-based materials with first-rank component are still a great challenge in the future.The key limiting factor of MAP of GNPs and GNPs@Ni composites is impedance mismatching owing to high conductivity leading to skin effect, that inhibits the EM wave permeation as revealed in this comparative study. The conductivities were adjusted by simple sulfurization process, resulting in a series of GNPs@Ni


Arthrobacter were among the most efficient GA-degrading strains.Potato juice, a by-product of starch processing, is a potential high-value food ingredient due to its high protein content. However, conversion from feed to human protein requires the removal of the toxic antinutritional glycoalkaloids (GAs) α-chaconine and α-solanine. Detoxification by enzymatic removal could potentially provide an effective and environmentally friendly process for potato-derived food protein production. While degradation of GAs by microorganisms has been documented, there exists limited knowledge on the enzymes involved and in particular how bacteria degrade and metabolize GAs. Here we describe a series of methods for the isolation, screening, and selection of GA-degrading bacteria. Bacterial cultures from soils surrounding greened potatoes, including the potato peels, were established and select bacterial isolates were studied. Screening of bacterial crude extracts for the ability to hydrolyze GAs was performed using a combination of thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and liquid chromatography mass spectrometry (LC-MS). Analysis of the 16S rRNA sequences revealed that bacteria within the genus  Periplaneta americana ] and high-tech [liquid chromatography mass spectrometry (LC-MS)] techniques we successfully isolated and screened bacterial strains capable of degrading GAs demonstrating the utility of this pipeline as a starting point for enzyme discovery. To the best of our knowledge, this is the first report describing bacteria capable of degrading potato GAs.Crude extraction of (GAs) from potato protein supplied by Kartoffelmelcentralen (KMC), Amba was performed as follows: 0.5 g of potato protein was added to 5 ml methanol-water-acetic acid (at a ratio of 80:9.5:0.5) in a 50 ml tube and shaken vigorously for 10 min. The tube was centrifuged 10 min at 4°C and the upper phase gently decanted into a fresh 50 ml tube. Extractions were pooled and dried in a vacuum centrifuge before resuspension in 1.5 ml filter-sterilized potassium phosphate buffer at pH 7.5. To improve the solubility of the GAs, 1.5 μl acetic acid was added. The crude extract was filter-sterilized using a 0.45 μm filter after which the purified GAs were detected and identified by HPLC analysis (see below).Pure α-chaconine was obtained from PhytoLabs  and α-solanine was purchased from either PhytoLabs or Sigma-Aldrich .Bacteria were isolated on potato dextrose agar at fifth strength (PDA 1/5) by inoculation of material from greened potatoes and the surrounding soil. Alternatively, minimal media as described by To determine whether the bacterial isolates could use the GAs as a sole carbon source, the strains were grown overnight in LB at 20°C and 5 μl culture plated on Petri dishes containing minimal media as described above enriched with either α-chaconine or α-solanine as a sole carbon source. Strains that scored positive for growth were selected for further characterization.g after which the supernatant removed. For extraction of DNA, the pellet was resuspended in 100 μl sterile water and boiled 10 min and immediately placed on ice before adding 900 μl ice-cold sterile water. Cell debris was pelleted by centrifugation for 5 min at 10,000 × g. The supernatant containing DNA was transferred to a clean tube ready for PCR analysis or storage at −20°C. The 16S rRNA gene sequence was determined by PCR using the universal bacterial 16S rRNA primers: 16S-27F (5′- AGA GTT TGA TCM TGG CTC AG-3′) and 16S-1492R (5′- CGG TTA CCT TGT TAC GAC TT-3′)  buffer in a sterile Eppendorf tube. The cells were concentrated by centrifugation for 5 min at 10,000 × C TT-3′) . Sanger −1 final concentration of α-chaconine or α-solanine and incubated 20 h under the same conditions. To obtain crude extracellular extracts, cultures were centrifuged at 20°C for 30 min at 4000 × g and either used directly or frozen at −20°C. Enzyme reactions consisting of 10 μl 50 mM sodium acetate buffer pH 5.5, 40 μl substrate (0.2 mM GA in 100 mM sodium acetate buffer pH 5.0) and 45 μl of crude extract was set up and incubated 24 h at 20°C. Enzyme reactions were terminated by heating for 10 min at 90°C. After centrifugation or 10 min at 17,000 × g, the supernatant was transferred to a fresh 1.5 ml tube and dried in a vacuum for further analysis. Pellets were resuspended in 20 μl of methanol and 8 μl were spotted on TLC plates for analysis or 10 μl were characterized by HPLC analysis.For preliminary screening, strains were grown overnight in 5 ml LB shaking at 200 rpm at 20°C and then diluted 1:100 into LB medium either water (control) or 10 μg ml600nm of 1.0. To 100 ml shake flask, 1 ml of bacterial culture  was added to 9 ml LB supplemented with acidic phosphate buffer (control) or a glycoalkaloid mix  and grown shaking 200 rpm 20°C for 20 h. To obtain crude extracellular extracts, cultures were centrifuged at 20°C for 20 min at 4000 × g and used directly for enzyme activity analysis. Enzyme assays consisting of 10 μl 50 mM sodium acetate buffer pH 5.5, 40 μl substrate (0.2 mM of α-chaconine and 0.2 mM of α-solanine) in 100 mM sodium acetate buffer pH 5.0 and 45 μl of crude extract were set up and incubated at 20°C for 35 h. Enzyme reactions were terminated by heating for 10 min at 90°C and subsequently centrifuged for 10 min at 17,000 × g before transferring the supernatant to either an HPLC vial (60 μl) for semi-quantitative analysis or to a fresh 1.5 ml tube (30 μl) for qualitative TLC analysis. For TLC analysis, 30 μl extract was dried 1 h in a vacuum centrifuge and resuspended in 7 μl cold methanol. Samples were briefly centrifuged and 5 μl spotted on TLC plate. TLC plates were run twice in n-butanol-acetic acid-water (2:1:1) solvent system and sprayed with 50% vv−1 H2SO4 and heated for 10 min at 85°C. For HPLC analysis, a 25 μl sample was injected and conditions for analysis were as described below. For analysis of intermediate metabolites produced during degradation or detection of solanidine, 30 μl of sample was analyzed by LC-MS.For selecting strains for further characterization, strains were grown overnight in 5 ml LB shaking 200 rpm 20°C and then washed with 0.9% NaCl and standardized to an ODn-butanol-acetic acid-water (2:1:1) as a running solvent and plates were developed on a hot plate after spraying with 50% vv−1 H2SO4.TLC was performed on silica gels 60 F254 (Merck) using 18 with 4 μm particle size, operated at 40°C. Solvent were 60% acetonitrile and 40% phosphate buffer at pH 7.6  at a flow rate of 1.5 ml/min.Quantification and identification of α-chaconine and α-solanine were performed using a Waters 2695 Alliance system with Waters 2487 Dual absorbance detector at 202 nm. The column was a Waters Nova-Pak CQuantification and identification of α-chaconine and α-solanine was based on the ion intensity of the single charged monoisotopic mass . The presence of solanidine was monitored at m/z 398.34. Ion intensities were normalized to the level in untreated LB media. Samples were analyzed in an LC-MS setup with an eksigent nanoLC 415 system (Sciex) connected to a TripleTOF 6600 mass spectrometer (Sciex) equipped with a NanoSpray III source (AB Sciex). Ion-intensities were extracted in Pekaview 1.2 (Sciex).A previous study demonstrated a rapid turnover of potato (GAs) in top soils of Danish potato fields, presumably due to microbial metabolism . TherefoOf the 14 GA degrading bacterial strains isolated from a series of enrichment cultures, four strains S37, S39, S40, and S41, were the best at degrading potato GAs. Unsurprisingly, the four strains were all isolated from minimal media solidified with agar containing a mixture of pure α-chaconine and α-solanine as a sole carbon source. Enrichment cultures containing GAs as a sole carbon source was the best method to enable the isolation of GA degrading bacteria.Clavibacter michiganensis and Streptomyces scabies are bacteria that can degrade the tomato GA α-tomatine, however, to the best of our knowledge no studies report the bacterial degradation of potato GAs  showed 98% sequence identity to species of the genus Serratia. All strains were capable of degrading both α-chaconine and α-solanine. Published information on GA-degrading bacteria is sparse, but it has previously been reported that an α-chaconinase enzyme produced by Gibberella pulicaris is induced by α-chaconine, α-solanine and the tomato glycoalkaloid α-tomatine (Identity of the four strains that produced GA-degrading extracts was done by analysis of 16S rRNA gene sequences (Table tomatine . All fouArthrobacter and Serratia. Functional genomics of the identified strains is now required to identify the genes and metabolic pathways related to the biodegradation of potato GAs.The isolation method here described identified four strains, which demonstrated the ability to metabolize and degrade the potato-derived GAs α-chaconine and α-solanine. The 16S rRNA gene sequences of these strains showed highest similarity to strains belonging to genera We successfully developed a screening method to isolate GA-degrading bacteria from potato soil extracts. This will enable the future identification and characterization of enzymes involved in the degradation of GAs to upgrade industrial waste products into valuable food ingredients.RH and PS conceived the study. RH, NJ, and CS performed the experiments. All authors analyzed and interpreted the data, drafted and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oxidation–stabilization using pressurized air flows of 0.5 and 1.0 MPa could successfully shorten the total stabilization time to less than 60 min for manufacturing mesophase pitch-based carbon fibers without deteriorating mechanical performance. Notably, the carbonized fiber heat-treated at 1000 °C for 30 min, which was oxidative–stabilized at 260 °C without soaking time with a heating rate of 2.0 °C/min using 100 mL/min of pressurized air flow of 0.5 MPa , showed excellent tensile strength and Young′s modulus of 3.4 and 177 GPa, respectively, which were higher than those of carbonized fiber oxidation–stabilized at 270 °C without soaking time with a heating rate of 0.5 °C/min using 100 mL/min of atmospheric air flow . Activation energies for oxidation reactions in stabilization using pressurized air flows were much lower than those of oxidation reactions using atmospheric air flow because of the higher oxidation diffusion from the outer surface into the center part of pitch fibers for the use of the pressurized air flows of 0.5 and 1.0 MPa than the atmospheric one. The higher oxygen diffusivities resulted in a more homogeneous distribution of oxygen weight uptake across the transverse section of mesophase pitch fibers, and allowed the improvement of the mechanical properties. Carbon fibers (CFs) have attracted substantial attention again owing to their novel applications in the automotive, windmill, and other energy-saving industries ,2. The pCFs are produced through a manufacturing process that includes several important stages, such as selection and purification of raw material, preparation of CF precursor (PAN or pitch), spinning, oxidative stabilization, carbonization and further heat treatment of graphitization, and surface treatment ,18,19. TIn the manufacturing process, development of the carbonization and graphitization processes has almost been completed. However, there is still much room for improvement in the oxidation–stabilization process, because this process remains the least efficient, most time-consuming, and costliest ,24. In tMany studies have been conducted to optimize the mechanical properties of CFs in terms of air oxidation conditions in the stabilization process ,25,26. FDespite the accumulation of such studies during the last three decades, the appropriate tensile strength of the carbonized MPCF of over 2.5 GPa through oxidation–stabilization with a total oxidation–stabilization time of less than 1 h and successive carbonization at a relatively low temperature of 1000 °C has not been achieved, because the slow diffusion rate of oxygen molecules into the center part of pitch fiber and its low oxidation reactions always prevent optimized homogeneous stabilization across the transverse section of pitch fibers within a short time. The main reason for such a slow diffusion rate of oxygen may be the compact alignment of structural units, that is, stacking clusters and domains, of pitch materials ,28. YangSome pioneering studies of the oxidation–stabilization of various types of mesophase pitch including fibrous form under a mild pressure of oxygen or air flow have been carried out ,31,32,33In this study, we aimed to reduce the total stabilization time to less than 1 h without causing deterioration of the mechanical performance of the resultant MPCFs. We aimed for the target mechanical performance of tensile strength and Young′s modulus of the carbonized fiber to be equal to the mechanical performance of the carbonized fiber, which could be prepared using the same precursor pitch fiber, oxidative–stabilized at 270 °C with the heating temperature of 0.5 °C/min using atmospheric 100 mL/min of air flow, and carbonized at 1000 °C for 30 min. To achieve this goal, we attempted oxidation–stabilization of mesophase pitch fibers at the temperature range of 250–270 °C using the pressurized air flows of 0.5–1.0 MPa. For comparison, we also performed oxidation–stabilization of the same mesophase pitch fibers using atmospheric air flow. We carried out thermo-gravimetric analysis (TGA) to analyze the oxidation reactions in the stabilization kinetically.AR mesophase pitch (ARMP) was supplied by Mitsubishi Gas Chemical Company, Tsukuba, Japan, and used as a model mesophase pitch precursor in this study without further treatment. ARMP has a softening point at 240 °C and carbon aromaticity of 0.84 and 100 vol. %, in terms of the mesophase texture. ARMP was melt spun into as-spun AR mesophase pitch fiber (ARMP-F) using a single-hole spinneret at 340 °C with a laboratory-type mono-hole melt spinning apparatus, which, as a spinneret, has a stainless-steel die hole with a diameter and length of 0.3 and 0.6 mm (L/D = 2), respectively ,36. FiguOxidation–stabilization of spun mesophase pitch fibers was carried out through thermal oxidation under dried air flow of 0.1–1.0 MPa with a flow rate of 100 mL/min. After stabilization of the ARMP-Fs under various conditions, ARMP-SF14s were successively heat-treated at 800 °C for 5 min with a heating rate of 5.0 °C/min in a nitrogen atmosphere for carbonization, and further heat-treated at 2400 °C for 10 min with a heating rate of 15 °C/min under an argon atmosphere for graphitization. ARMP-SF10s were heat-treated at 1000 °C for 30 min with a heating rate of 20 °C/min in a vacuum atmosphere, and some of the carbonized fibers were also further graphitized at 2800 °C for 10 min with a heating rate of 15 °C/min in an Ar atmosphere to evaluate the mechanical performance. The carbonized and graphitized fibers of ARMP-SF10 and ARMP-SF14 are designated ARMP-CF10 and ARMP-CF14, and ARMP-GF10 and ARMP-GF14, respectively.Tmax, q, and Ea designate peak temperature, heating rate, and activation energy, respectively.Spun mesophase pitch fibers were subjected to TGA to track the amount of oxygen uptake under atmospheric and air flow pressure conditions using a magnetic suspension balance . To evaluate the distribution of the amounts of oxygen uptake in the transverse sections of stabilized fibers, ARMP-SF14s were analyzed using a scanning electron microscope (SEM) equipped with an electron probe micro-analyzer (EPMA)  . Images Elemental analysis was used to determine the total amount of oxygen uptake of the stabilized fibers for evaluating the mechanical performance of the obtained carbonized and graphitized fibers. The mechanical properties of the carbonized and graphitized fibers were measured using a universal tensile tester , in accordance with the JIS R 7606:2000 method (a method of single filament test) . Twenty-2 before the main decomposition of aromatic molecules of mesophase pitch fibers. In these results, the temperature at which the maximum oxygen uptake in oxidation–stabilization occurred was shifted to a lower position with increasing air pressure. This proved that the main oxygen uptake could start at a lower temperature under pressurized air flow conditions than under atmospheric conditions, and the application of pressure in oxidation–stabilization had a similar effect to decreasing the heating rate. Such an earlier start of oxidation and higher amounts of oxygen uptake are well matched with the previous reports [ reports ,31.Regarding the maximum amount of oxygen uptake in the oxidation–stabilization with applied pressure, different effects of decreasing the heating rate were observed. Specifically, the stabilized fiber of ARMP-SF14 at 0.5 MPa had a higher amount of oxygen uptake at its maximum point of oxygen uptake than the stabilized fiber under atmospheric conditions. However, the maximum amount of oxygen uptake was higher at 0.5 MPa, but slightly lower at 1.0 MPa compared with those under atmospheric pressure conditions. As is clearly shown in In the oxidation–stabilization under atmospheric conditions, the amounts of oxygen uptakes of the stabilized fibers with a soaking time of 0 min at temperatures of 250, 260, and 270 °C with a heating rate of 2.0 °C/min were evaluated as being 5.2, 6.2 and 8.5 wt. %, respectively. The amounts of oxygen uptakes with a heating rate of 3.0 °C/min after soaking for 0, 30, and 60 min at 270 °C were 6.5, 7.8 and 8.5 wt. %, respectively. The increase of the amounts of oxygen uptake for soaking times between 30 and 60 min was very small because the ARMP-F10 almost reached a fully oxidized state under atmospheric air flow pressure with a soaking time of 60 min at 270 °C and a heating rate of 3.0 °C/min. Compared with this, the amounts of oxygen uptake with a heating rate of 0.5 °C/min after soaking for 0 min at 270 °C was higher with 11.7 wt. %. This higher amount of oxygen uptake at 0.5 °C/min demonstrates that a more stabilized state of ARMP-F10 could be obtained with a decreased heating rate of 0.5 °C/min in oxidation–stabilization under atmospheric air flow pressure.In the oxidation–stabilization under air flow pressure of 0.5 MPa, the amounts of oxygen uptake of the stabilized fibers with a soaking time of 0 min at temperatures of 250, 260, and 270 °C and a heating rate of 2.0 °C/min were 6.3, 11.4 and 11.9 wt. %, respectively. The amount of oxygen uptake after soaking for 15 min at 260 °C with a heating rate of 3.0 °C/min was 11.1 wt. % and those with soaking times of 0 and 10 min at 270 °C with the same heating rate were 10.8 and 11.8 wt. %, respectively. The increase of oxygen uptake between 260 and 270 °C with 0 min of soaking was very small and the oxygen uptake after soaking for 10 min at 270 °C with a heating rate of 3.0 °C/min was also 11.8 wt. %, which indicates that ARMP-F10 was almost fully stabilized upon soaking for 10 min at 270 °C with a heating rate of 3.0 °C/min under air flow pressure of 0.5 MPa.In the oxidation–stabilization under air flow pressure of 1.0 MPa, the amounts of oxygen uptakes of the stabilized fibers at soaking temperatures of 250, 260, and 270 °C with a heating rate of 2.0 °C/min were 7.0, 11.5 and 11.2 wt. %, respectively. The amount of oxygen uptake after soaking for 0 min at 260 °C with a heating rate of 3.0 °C/min was 11.5 wt. %, and those after soaking for 0 and 5 min at 270 °C with the same heating rate were 11.2 and 11.1 wt. %, respectively. The amounts of oxygen uptakes of the stabilized fiber soaked at 270 °C were slightly lower than those of the stabilized fiber at 260 °C, which indicated that the oxidation decomposition occurred at 270 °C with a heating rate of 2.0 °C/min through excess oxidation reactions of mainly the exterior of the stabilized fiber. The decrease in oxygen uptakes with soaking times of 0 and 5 min at 270 °C with a heating rate of 3.0 °C/min were also the result of oxidation decomposition. The amount of oxygen uptake after soaking for 0 min at 270 °C with a heating rate of 3.0 °C/min was 11.2 wt. %. From these results, ARMP-SF10 was fully or excessively stabilized with soaking for 0 min at 270 °C with a heating rate of 3.0 °C/min under air flow pressure of 1.0 MPa.The carbonization yields of ARMP-CFs stabilized with soaking for 0 min at 250, 260, and 270 °C with a heating rate of 2.0 °C/min under atmospheric pressure were 81.0, 85.2, and 83.2 wt. %, respectively. Moreover, the yields of ARMP-CFs stabilized with soaking for 0, 30, and 60 min at 270 °C with a heating rate of 3.0 °C/min under atmospheric pressure were 83.2, 85.6 and 87.0 wt. %, respectively. The carbonization yield of ARMP-CF stabilized with soaking for 0 min at 270 °C with a heating rate of 0.5 °C/min under atmospheric pressure was 88.3 wt. %. Furthermore, the carbonization yields of ARMP-CFs stabilized with soaking for 0 min at 250, 260, and 270 °C with a heating rate of 2.0 °C/min under air flow pressure of 0.5 MPa were 90.0, 90.9, and 89.1 wt. %, respectively. The yields of ARMP-CFs stabilized with soaking for 15 min at 260 °C and soaking for 0 and 10 min at 270 °C with a heating rate of 3.0 °C/min under air flow pressure of 0.5 MPa were 88.8, 89.0, and 89.5 wt. %, respectively. The analyses also revealed that the carbonization yields of ARMP-CFs stabilized with soaking for 0 min at 250, 260, and 270 °C with a heating rate of 2.0 °C/min under air flow pressure of 1.0 MPa were 85.3, 85.4, and 85.3 wt. %, respectively. The yields of ARMP-CFs stabilized with soaking for 0 min at 260 °C and soaking for 0 and 5 min at 270 °C with a heating rate of 3.0 °C/min under air flow pressure of 1.0 MPa were 89.6, 85.2, and 84.3 wt. %, respectively. From these results, if the stabilization under air flow pressures of 0.5 and 1.0 MPa could be carried out on optimal conditions (soaking time and temperature), the carbonized fibers that were stabilized under air flow pressure of 0.5 and 1.0 MPa had higher carbonization yields than those stabilized under air flow pressures of the atmospheric level. Generally, the stabilized fibers with oxygen uptakes of less than 7.0 wt. % and more than 13.0 wt. % had lower carbonization yields than the stabilized fibers with oxygen uptakes of around 11.0–12.0 wt. %. These results suggest that oxygen uptakes of 11.0–12.0 wt. % might be close to the optimal oxidation state for the oxidation–stabilization of ARMP-F10. The deficiency of oxygen uptake in the fibers stabilized at 270 °C with heating rates of 2.0 and 3.0 °C/min under the atmospheric air flow pressure was ascribed to the insufficient delivery of oxygen into the center of the pitch fibers under these conditions, conferring excess volatility of light aromatic molecules in the carbonization. In contrast, excessive oxygen uptake in the stabilized fibers usually occurred under an air flow pressure of 1.0 MPa in our study, indicating that the excess oxidation mainly occurred in the exterior of pitch fibers, which might be the principal reason for the oxidation decomposition of pitch and stabilized fibers in the stabilization and carbonization processes.The graphitization yields revealed a trend very similar to those of carbonization. The average weight loss in the graphitization from the carbonization was estimated to be approximately 1.0–2.0 wt. %. The graphitized fibers stabilized under an air flow pressure of 0.5 MPa had higher graphitization yields than those stabilized under air flow pressure conditions of the atmospheric level and 1.0 MPa.With regards to the mechanical performance of the carbonized and graphitized fibers stabilized with soaking for 0 min at 270 °C with a heating rate of 2.0 °C/min under atmospheric pressure, they had tensile strength of 2.4 and 3.5 GPa, elongation ratios of 1.5% and 0.6%, and Young′s moduli of 159 and 508 GPa, respectively. The carbonized and graphitized fibers stabilized with soaking for 0 min at 270 °C with a heating rate of 0.5 °C/min under atmospheric pressure had markedly improved values of tensile strengths of 2.9 and 4.0 GPa, elongation ratios of 1.7% and 0.6%, and Young′s moduli of 171 and 663 GPa, respectively. The carbonized and graphitized fibers stabilized with soaking for 0 min at 260 °C with a heating rate of 2.0 °C/min under air flow pressure of 0.5 MPa had the best mechanical performance of tensile strengths of 3.4 and 4.6 GPa, elongation ratios of 1.7% and 0.6%, and Young′s moduli of 177 and 765 GPa, respectively. These results indicate that tensile strength, elongation ratio, and Young’s modulus of the carbonized fibers of higher than 1.7 GPa, 1.7%, and 170 GPa, respectively, after carbonization at 1000 °C for 30 min, were successfully obtained within a total stabilization time of less than 60 min. From these results, stabilization under air pressures of 0.5 MPa cause homogeneous oxidation and increases mechanical properties more than other graphitized fibers. In contrast, the mechanical properties of ARMP-GF10 stabilized under air pressures of the atmospheric level and 1.0 MPa may decrease owing to the deficient and excess oxygen uptake in the center and outer parts of pitch fibers, respectively.It usually takes more than 4 h to carry out the optimal oxidation–stabilization of mesophase pitch fiber to obtain the maximum mechanical performances, especially the tensile strength. The oxidation–stabilization under mild air flow pressures of 0.5 and 1.0 MPa could greatly shorten the total stabilization time to less than 60 min to obtain mesophase pitch-based carbonized and graphitized fibers without deteriorating the mechanical performance. Notably, carbonized fibers with high tensile strengths and Young′s moduli of over 3.0 and 170 GPa, respectively, which had only been heat-treated at 1000 °C for 30 min, were successfully obtained with a total stabilization time of less than 60 min through oxidation–stabilization under air flow pressures of 0.5 MPa.Activation energies for oxidation reactions in stabilization under air flow pressure are much lower than those of oxidation reactions under atmospheric air flow pressure because of the higher diffusivity of oxygen into the center and a more rapid oxidation reaction on the molecules of mesophase pitch fibers under mild air flow pressures of 0.5 and 1.0 MPa. The higher oxygen diffusivities resulted in a more homogeneous distribution of oxygen uptake across the transverse section of mesophase pitch fibers and allowed higher yields of carbonization and graphitization, which are directly related to the improvement of the mechanical properties. Additionally, excess oxidation can bring about the oxidation decomposition of pitch molecules with oxidation–stabilization under air flow pressures of 0.1 and 1.0 MPa, which decreases the carbonization yield and tensile strength of the obtained carbon fibers.