In an effort to improve in vivo potency of Vγ9Vδ2 T cells, we reduced FDPS expression in tumor cells using a lentivirus vector encoding a short-hairpin RNA that targets FDPS mRNA (LV-shFDPS). Prostate (PC3) or hepatocellular carcinoma (Huh-7) cells transduced with LV-shFDPS induced Vγ9Vδ2 T cell stimulation in vitro, resulting in increased cytokine expression and tumor cell cytotoxicity. Immune deficient mice implanted with LV-shFDPS transduced tumor cells showed dramatic responses to intraperitoneal injection of Vγ9Vδ2 T cells with strong suppression of tumor growth. In vivo potency was increased by transducing tumor cells with a vector expressing both shFDPS and human IL-2. Tumor suppression by Vγ9Vδ2 T cells was dose-dependent with greater effects observed in mice injected with 100% LV-shFDPS transduced cells compared to mice injected with a mixture of 50% LV-shFDPS transduced cells and 50% control (no vector) tumor cells. Delivery of LV-shFDPS by intratumoral injection was insufficient to knockdown FDPS in the majority of tumor cells, resulting in insignificant tumor suppression by Vγ9Vδ2 T cells. Thus, Vγ9Vδ2 T cells efficiently targeted and suppressed tumors expressing shFDPS in mouse xenotransplant models. This proof-of-concept study demonstrates the potential for suppression of genetically modified tumors by human Vγ9Vδ2 T cells and indicates that co-expression of cytokines may boost the anti-tumor effect.Human Vγ9Vδ2 T cells are attractive candidates for cancer immunotherapy due to their potent capacity for tumor recognition and cytolysis of many tumor cell types. However, efforts to deploy clinical strategies for Vγ9Vδ2 T cell cancer therapy are hampered by insufficient potency. We are pursuing an alternate strategy of modifying tumors to increase the capacity for Vγ9Vδ2 T cell activation, as a means for strengthening the anti-tumor response by resident or ex vivo manufactured Vγ9Vδ2 T cells. Vγ9Vδ2 T cells are activated  Human Vγ9Vδ2 (hereinafter referred to as Vδ2) T cells are a unique population in peripheral blood with frequencies ranging from 0.5-10% of peripheral blood mononuclear cells (PBMC). Due to unique features of their T cell receptor, these are often considered to have features representing both innate and adaptive immunity . Non-pepvia a cytoplasmic domain, resulting in a conformational change to the ectodomain that is recognized by Vδ2 T cells  have immunomodulatory properties including activation of Vδ2 T cells. NBPs are inhibitors of farnesyl diphosphate synthase (FDPS), an enzyme in the mevalonate pathway –34. NBPsor cells .Existing approaches for exploiting Vδ2 T cells in cancer therapy utilize soluble activators of Vδ2 T cells such as NBPs or PAg to increase the potency of Vδ2 T cells against tumor cell targets . Anotherin vivo. One is that soluble NBPs are absorbed by binding to hydroxyapatite on the bone surface, thus reducing bioavailability, and the second is that NBPs cannot be targeted to the tumors. Consequently, we constructed a lentiviral vector expressing shFDPS (LV-shFDPS) to determine if reducing FDPS mRNA and protein expression in prostate and hepatocellular tumors ex vivo or in vivo would lead to tumor suppression by Vδ2 T cells from healthy human donors. We also tested whether FDPS knockdown in addition to NBP treatment could increase the activation and cytolytic activity of Vδ2 T cells and whether this would be enhanced further with concurrent tumor cell expression of IL2.The combination of NBPs and Vδ2 T cell treatments used in xenograft mouse models were tested in several human tumor types including melanoma , glioma 7 cells/mL. The PBMC were placed at -80°C for overnight and then transferred to liquid nitrogen for long term storage.Leukopaks from anonymous donors were obtained from the New York Blood Center . PBMC were isolated using density gradient centrifugation using a Ficoll-Paque Plus gradient . The PBMC were transferred to a new tube and washed twice with PBS. The resulting cell pellet was resuspended in culture medium composed of RPMI 1640 complete medium: RPMI 1640 medium , 10% Fetal Bovine Serum (FBS) , and 10,000 U/mL Penicillin-Streptomycin (Thermo Fisher Scientific). PBMC were cryopreserved in 90% FBS + 10% DMS0 (Sigma-Aldrich) at a final concentration of 3 X 107 cryopreserved PBMC were thawed in a 37°C water bath and washed with RPMI 1640 medium. The PBMC were resuspended at a concentration of 1 X 107cells/mL in 10 mL RPMI 1640 medium supplemented with 1 μM zoledronic acid (ZA) (Sigma-Aldrich) and 0.1 mg/mL recombinant human IL2 (Thermo Fisher Scientific). The cells were expanded in T25 or T75 tissue culture flasks  for a minimum of two weeks prior to use for in vitro and in vivo experiments. During the first 10 days of expansion, the medium was supplemented with 0.1 mg/mL of recombinant human IL2 twice in the first week after which IL2 supplementation was continued at 0.01 mg/mL. After a minimum of two weeks in culture, the mixed cell population contained 20-90% Vδ2 T cells as determined by flow cytometry.3 X 102.PC3 prostate carcinoma cells were obtained from the American Type Culture Collection  and Huh-7 liver hepatocellular carcinoma cells were obtained from the Japanese Collection of Research Bioresources . The cells were thawed and passaged in DMEM complete medium: DMEM (Thermo Fisher Scientific), 10% FBS (Gemini), and 10,000 U/mL Penicillin-Streptomycin (Thermo Fisher Scientific). Cells were seeded into T-75 flasks  and cultured in a 37°C incubator with 5% CO6 cells/well in a 6-well plate followed by incubation overnight. The next day, when the cells had reached around 50% confluency, the cells were transduced with LV control, LV-shFDPS or LV-shFDPS-IL2 using a multiplicity of infection (MOI) of 5 or 10. Three days after transduction, the transduced PC3 or Huh-7 cells were collected, centrifuged, and resuspended in fresh, complete DMEM medium. The PC3 or Huh-7 cells were added at 5 X 105 cells/100 µL in wells of 96-well U-bottom plates. Next, 1 X 106/100 µL Vδ2 T cells were added to the wells and cytokine secretion was arrested by adding the GolgiPlug reagent per the manufacturer’s instructions . The coculture was incubated in a 37°C cell incubator for 4 h, whereupon cells were collected and cytokine expression was measured by flow cytometry.PC3 or Huh-7 cells were seeded at 0.5 X 105 cells in each well of a 6-well plate and cultured in 1.5 mL of complete DMEM medium in a 37°C incubator at 5% CO2 overnight. The next day, lentivirus vector was added to the cells at a MOI of 2.5, 5, 10 or 20 for 48 h. For PC3 cells, in addition to lentivirus, polybrene (Sigma-Aldrich) was added to the cell medium at a concentration of 2 μg/mL. Cells were lysed in 1% NP-40 lysis buffer containing a Pierce Protease Inhibitor Tablet (Thermo Fisher Scientific). Protein lysates were prepared in 1x NuPAGE LDS sample buffer (Thermo Fisher Scientific), samples were heated for 10 min at 70°C, and proteins were separated with 4-12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma) and probed with the antibodies anti-FDPS  and β-actin (MilliporeSigma). Anti-mouse or rabbit secondary antibody conjugated with horseradish peroxidase (HRP)  was visualized with the Immobilon Western HRP substrate (MilliporeSigma) and detected with the LI-COR C-DiGit Blot Scanner .293T cells were seeded at 8 X 10PC3 cells were transduced with lentivirus at 5 MOI in addition to 2 μg/mL of polybrene. The cell medium was changed after 6 h and then collected after 3 days whereupon IL2 expression was determined with a human IL2 ELISA kit (Thermo Fisher Scientific).Cells were collected and RNA was extracted with the RNeasy kit . The reverse transcription reaction was done using 1.4 μg of RNA and the Vilo SuperScript cDNA synthesis kit (Thermo Fisher Scientific) on a Veriti 96-well thermal cycler (Thermo Fisher Scientific). The PCR steps were according to the following: 25°C for 10 min, 42°C for 60 min, and 85°C for 5 min. For the Real-Time qPCR assays, 1.5 μL of the RT-PCR reaction was mixed with 1 μL of FDPS  and actin  primers and probes at 0.5 μM and the reaction components of the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). The qPCR reactions were performed on a QuantStudio3 Real-Time PCR system (Thermo Fisher Scientific) according to the following steps: 50°C for 2 min, 95°C for 20 secs, 40 cycles of 95°C for 1 sec and 60°C for 20 secs. Data were collected and analyzed with QuantStudio Design and Analysis software (Thermo Fisher Scientific).The 96-well U-bottom plate containing the co-cultured cells was centrifuged, followed by removal of the supernatant and resuspension in 50 µL of flow staining buffer  containing PE-labeled anti-human Vδ2 antibody . Next, the cells were treated with 50 µL of fixation buffer (BD Biosciences) before washing with 1X perm/wash solution (BD Biosciences). Then cells were resuspended in 50 μL of APC-labeled anti-human TNFα or IFNγ (BioLegend). The cells were incubated at 4°C for 20 min in the dark and washed 2 times with 250 μL of 1X perm/wash solution. Flow cytometry data were acquired on a FACS Calibur (BD Biosciences) and analyzed using FlowJo software.5 cells per well and incubated overnight. The next day, the cells were transduced with lentiviral vector stocks at a MOI of 5 or 10. For PC3 cells, in addition to lentivirus, polybrene was added to the cell medium at a concentration of 2 μg/mL. After 48 h, the medium was changed with or without 1 μM ZA for overnight incubation. The next day, 5 μM of Calcein AM (Thermo Fisher Scientifc) were added to the medium and cells were placed in a 37°C cell incubator for 20 min. Next, the cells were washed with culture medium. Finally, the labeled cells were resuspended to a concentration of 5 X 105 cells/mL.PC3 or Huh-7 cells were seeded in 6-well plates at 4 X 106/mL, 5 X 105/mL, 2.5 X 106/mL, and 1.625 X 106/mL cell concentrations. 100 μL of each dilution of PBMC enriched for Vδ2 T cells were added to 5 X 104/100 μL of the Calcein AM-labeled PC3 or Huh-7 cells which resulted in effector to target cell ratios (E:T) of 20:1, 10:1, 5:1, 2.5:1, and 1.25:1; the ratio indicates numbers of enriched PBMC (Effector) and were not corrected for the percentage of Vδ2 T cells in each cell preparation. All co-cultures were performed in triplicate. The cocultures were incubated in a 96-well U-bottom plate in a 37°C cell incubator for 4 h. To account for spontaneous cell lysis, 5 X 104 of the cells were cultured without Vδ2 T cells. To determine maximum cell lysis, 5 X 104 cells cultured without Vδ2 T cells were completely lysed by the addition of 1% Triton-X-100 (Sigma-Aldrich) to the culture medium. After 4 h, 75 μL of supernatant from each well of the 96-well plate were transferred to a new 96-well black plate with a clear bottom and lid (VWR). Cell lysis was measured by the level of fluorescent Calcein AM detected in the supernatant using a fluorescent spectrophotometer . The percentage of specific lysis was calculated with the following formula: % specific lysis = (Co-culture lysis–spontaneous lysis)÷(Maximum lysis–spontaneous lysis) X 100.Serial dilutions of expanded Vδ2 T cells were prepared with 1 X 106 cells/0.1 mL PC3 or Huh-7 cell plus 0.05 mL Matrigel  into the right flank of each mouse. Mice were weighed and tumor volumes were measured twice a week using a caliper. Tumor volume was calculated using the following equation: Tumor volume (mm3)=d2 (d=the shortest diameter) X (D/2) (D=the longest diameter). Mice were euthanized when tumor volume reached approximately 2000 mm3, followed by excision and weighing of the tumor. 5-8 X 106 Vδ2 T cells expanded from PBMC were administered by intraperitoneal injection (IP) when the average tumor volume reached 200-300 mm3. Vδ2 T cells were injected by IP route every week for a total of 4 injections. ZA was administered to mice by IP injection with a dose of 100 mg/kg the day prior to each injection of Vδ2 T cells.PC3 or Huh-7 cells were seeded in a T75 flask. When the cells were 80% confluent, they were transferred to a T175 flask. PC3 or Huh-7 cells were transduced when the cells had reached 50% confluency by replacing the medium with lentiviral vector stocks diluted in medium to result in a MOI of 5 or 10. For PC3 cells, in addition to lentivirus, polybrene was added to the cell medium at a concentration of 2 μg/mL. Three days following cell seeding, the cells were collected by trypsinization and washed twice in complete medium for inoculation into mice. For each treatment, 5-8 NSG or NRG mice were inoculated subcutaneously with 3 X 10in vivo tumor studies was performed with the unpaired t test.The data was presented as the mean ± SEM where applicable and statistical analysis was performed with GraphPad Prism. Statistical analysis of the data from The lentiviral vector with a small hairpin (shFDPS) stem loop structure  for 48 h followed by ZA (1 μM) treatment for 24 h. The ZA dose of 1 μM was used because this is in the range of vivo bioavailability . The cel). In Huh-7 cells, the average percentage of cells positive for TNFα expression was 16.1± 15.9% (LV + ZA), 20 ± 10.8% (LV-shFDPS), and 36.9± 23.1% (LV-shFDPS + ZA) .In Huh-7 cells, the percent positive of IFNγ producing cells was 38.9 (LV + ZA), 49.3 (LV-shFDPS), and 76.3 (LV-shFDPS + ZA)  for 4 h. The supernatant from these co-cultures was evaluated for the presence of Calcein AM, which is indicative of tumor cell lysis. The results were analyzed, and the percent specific lysis values were calculated. At an E:T ratio of 2.5:1 for Vδ2 T cells to PC3 cells, the percent lysis was 22.2 ± 1.1% (LV), 45.1 ± 1.4% (LV + ZA), 38.8 ± 2.8% (LV-shFDPS), and 70.9 ± 3.1% (LV-shFDPS + ZA) (3. Expanded Vδ2 T cells (6-10 X 106) were injected weekly by intraperitoneal (IP) delivery. One group of mice additionally received ZA injections also given by IP. Mice were monitored for health and tumor volume was measured twice weekly until conclusion of the study. LV-shFDPS transduced tumors grew slower than the LV-Control (LV) tumors, but at the end of the study reached a similar average tumor volume which was not significantly different  . At the end of the study, 36 days post 1st Vδ2 injection, the average tumor volume for LV-shFDPS was 1680 ± 166 mm3 which was reduced to 795 ± 193 mm3 when combined with Vδ2 treatment   .Since the lentiviral vectors used in this mouse study also carried the firefly luciferase gene, bioluminescence imaging could be used to monitor tumor growth. Bioluminescence images of LV-shFDPS transduced PC3 tumors were captured on days 11 and 28 after the initial injection of Vδ2 T cells  in mice treated with Vδ2 T cells  .At the end of the study, all mice were sacrificed when PC3 tumors reached 2000 mmIL2 is used frequently to increase the activation and proliferation of Vδ2 T cells. We tested if adding an IL2 gene to LV-shFDPS and expressing both in a tumor cell line, would increase Vδ2 T cell activity in a coculture assay. PC3 cells were transduced with 5 MOI of LV-shFDPS-IL2 lentiviral vector and secreted IL2 was detected in cell culture medium by ELISA. The mean concentration of IL2 in culture medium was 52 ng/mL from 4 replicates as determined with a standard curve  was injected intraperitoneally with 6-10 X 106 Vδ2 T cells and the other group was injected with vehicle (N=8). One group of mice was injected with Vδ2 T cells once a week for a total of four weeks. Mouse body weights and tumor volumes were measured twice a week and the animals were euthanized when tumors reached 2000 mm3 or at the end of the study. At the end of the study, 39 days after the 1st Vδ2 T cell injection, the average tumor volume for LV-shFDPS and LV-shFDPS-IL2 was 1835 ± 289 and 1900 ± 210 mm3 which was reduced to 356 ± 140 mm3 and 116 ± 55 mm3 when combined with Vδ2 T cell treatment, respectively     (Expressing IL2 from LV-shFDPS transduced PC3 tumors impacted the growth of tumors in NSG mice treated with Vδ2 T cells. Mice were inoculated with 3 X 106 LV (N=10), 2 X 106 LV-shFDPS (N=10), or a 1:1 mixture of 1 X 106 LV + 1 X 106 LV-shFDPS . A week after injection, all mice were injected once a week with 7 X 106 Vδ2 T cells by IP delivery and half of the mice received ZA treatment for 5 weeks. There was a significant decrease in tumor volume in mice with 100% LV-shFDPS transduced cells and treated with ZA as compared with LV transduced cells    . Additionally, there was a significant survival advantage for mice with 100% versus 50% LV-shFDPS transduced tumors in the presence or absence of ZA. . The survival curve showed that mice with LV transduced tumors were sacrificed 21 days after the initial injection of Vδ2 T cells and mice with 100% and 50% LV-shFDPS transduced tumors remained alive at the end of the study on day 37    injection of LV-shFDPS in Huh-7 tumors in combination with Vδ2 T cells. NSG mouse were injected subcutaneously with 2 X 106 Huh-7 cells in the right flank. Three weeks after injection, the average tumor sizes ranged from 100-900 mm3; mice were placed into two groups with roughly equal distribution of starting tumor sizes. One group of tumors (N=8) were injected daily with 60 μL of LV and the other group (N=8) with 60 μL of LV-shFDPS for three consecutive days. Five days after IT injection of LV or LV-shFDPS, mice were injected with 7 X 106 Vδ2 T cells twice a week for a total of 4 weeks. Although the LV-shFDPS tumors (1934 ± 181 mm3) grew slower than LV tumors (2785 ± 447. mm3), the overall tumor volume showed no significant differences between LV and LV-shFDPS at the end of the study    and definitive conclusions about the relationship between vector dose and tumor suppression are not warranted at this time. Experiments with lentivirus vectors expressing both shFDPS and IL2 were limited in numbers and the precise cause of animal deaths was uncertain, although we note that the vector construct was not toxic as animal deaths occurred only after infusing Vδ2 T cells. In this context we did not test mixtures of individual vectors (LV-shFDPS + LV-shFDPS-IL2 in varying ratios) to see whether a lower proportion of IL2 producing tumor cells would support Vδ2 T cells effects without the associated toxicity. Finally, the intratumoral injection pilot studies are likely to require multiple repetitions and perhaps alternate delivery sites or methods.in vivo. Tumor suppression occurred in the absence of IL-2 treatment, although co-expressing IL-2 with shFDPS on the same vector appears to have increased potency. Dual targeting of FDPS activity, using LV-shFDPS plus ZA increased tumor suppression but the added benefit of adding ZA was modest compared to what was observed for dual targeting in vitro.Our key findings are that genetic manipulation of tumor cells increases the potential for Vδ2 T cell activation and tumor suppression Several published studies explored the effects of N-bisphosphonate (NBP) treatment in combination with Vδ2 T cells in mouse models. For example, Kabelitz et al. tested whether NBP alendronate plus IL2 suppressed the growth of the melanoma cell line MeWo in NSG mice treated with Vδ2 T cells . SantolaThe cytokine IL2 stimulates expansion and Vδ2 T cell cytotoxic activity. We observed that PC3 cells transduced with LV-shFDPS-IL2 as compared with LV-shFDPS stimulated more IFNγ production. Additionally the growth of PC3 tumors with LV-shFDPS-IL2 was decreased as compared with LV-FDPS in combination with Vδ2 T cells. However, we observed weight loss in some mice with LV-FDPS-IL2 transduced tumors, which may be caused by a supraphysiological level of circulating IL2 in the mice. In mice injected with a mixture of cells, 50% LV-FDPS-IL2 + 50% LV, there was no weight loss or deaths observed (data not shown). This suggests that the optimum level of IL2 can enhance the activity of Vδ2 T cell activity without causing toxicity. In the current vector, IL2 expression is being controlled by the strong CMV promoter, but weaker promoters or promoter regulatory units could be engineered into the vector for future studies.in vivo activation of these cells using PAg or bisphosphonate compounds, showed a significant event rate for Vδ2 T cell therapies and no significant association with severe adverse events (in vivo cell activation and expansion, can provide positive therapeutic benefit but overall have modest potency.Several groups have conducted exploratory clinical trials to evaluate the potential for activating Vδ2 T cells for cancer therapy. Zoledronic acid plus IL-2 therapy was tested in male patients with hormone-refractory prostate cancer . A simile events . These sOur approach was to improve upon these preclinical results by genetically targeting FDPS to further increase IPP levels in tumor cells and promote their subsequent destruction by Vδ2 T cells . Thus, win vivo stimulation of Vδ2 T cells is inefficient, possibly due to the unusual pharmacodynamics of these drugs which precipitate in bone (Several major obstacles must be overcome to realize the potential for gamma delta T cell-based cancer therapy. Use of bisphosphonates drugs for  in bone , 59 and  in bone , 61 and The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by University of Maryland School of Medicine-Institutional Review Board.in vivo studies and cellular assays, and wrote the initial version of the paper; TL is a shared first author of the manuscript, supervised the project, contributed to molecular and cellular assays, data analysis, and revision and preparation of the manuscript; HL contributed to study design and cellular assays; LX contributed to molecular assays; CDP supervised the project and contributed to revising the main text of the manuscript; TL is the corresponding author of the manuscript. All authors contributed to the article and approved the submitted version.M-LL is the first author of the manuscript, designed, performed, and analyzed the The authors M-LL, TL, and LX are employed by American Gene Technologies and HL and CDP are former employees of American Gene Technologies and currently employed by Viriom, Inc.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Est33) from an Antarctic bacterium. The protein was expressed and purified for biochemical characterizations. Site-mutation variants including S94A, D205A, and H233A were constructed to explore the structure–function relationship of the catalytic triad of Est33, and we found mutating Ser94, Asp205, and His233 residues lead to a complete loss of enzyme activity. In addition, the catalytic Ser94 located in a conserved pentapeptide motif GVSWG. Phylogenetic analysis showed that Est33 and its closely related homologs belonged to an independent group apart from other known family members, indicating that Est33 represented a new family of esterase. The Est33 enzyme was found to be a cold-active esterase retaining 25%–100% activity from 10°C to 30°C and to have optimal catalytic activity toward p-nitrophenol acetate (30°C and pH7.5). The serine modifying reagent phenylmethylsulfonyl fluoride inhibited the activity of Est33 by 77.34%, while thiol reagents such as dithiol threitol (DTT) activated the enzyme by 3-fold. Metal chelating reagents EDTA had no effects, indicating that Est33 is not a metalloenzyme. Collectively, these results indicate that Est33 constitutes the first member of a novel esterase family XXI that has been identified.Studies of microorganisms from extreme environments can sometimes reveal novel proteins with unique properties. Here, we identified a novel esterase gene ( Candida rugosa  families . SubsequLipolytic enzymes can also be found in organisms living in extreme environments, such as high-  and low-Pseudomonas sp. E5-12, which was isolated from Antarctic soil . The E. coli BL21 (DE3) cells were transformed with constructed pET22b-est33, pET22b-est33S94A, pET22b-est33D205A, or pET22b-est33H233A vectors and further verified by DNA sequencing.The primer synthesis and DNA sequencing were performed commercially at BioSune Company . The E. coli BL21 (DE3) containing recombinant plasmid was cultured in LB medium supplemented with 100 ug/ml ampicillin at 37°C. When the OD600 reached the range of 0.6–0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM to induce the expression of the target protein, and then, the cells were further cultivated at 17°C, 120 rpm for 48 h. The induced cells were collected by centrifugation at 10,000 × g for 10 min (4°C) and washed three times using Tris-buffered saline . After disrupting cells by sonication and centrifugation at 11,000 × g for 30 min at 4°C, a crude enzyme solution  was obtained. It was then passed over a Ni-Sepharose Fast Flow resin  against the C-terminal 6*His tag to purify the esterase protein. The protein was recovered with an elution buffer , and then was dialyzed using 50 mM Tris–HCl (pH 8.0) to remove NaCl and imidazole. Purity of the resulting protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-termini protein sequencing was performed by a commercial company .For protein expression, p-nitrophenyl ester substrates (pNPs), including acetate (C2), butyrate (C4), caproate (C6), caprylate (C8), caprate (C10), laurate (C12), and palmitate (C16), were prepared in 2-propanol to a concentration of 10 mM. The standard reaction system contained 0.02 ml of 10 mM pNP, 0.96 ml of 50 mM Tris–HCl buffer (pH 7.5), and 0.02 ml of enzyme. After incubation at 30°C for 10 min, 0.1 ml of 20% SDS (w/v) was added to stop the reaction. The esterase activity was calculated from the release of p-nitrophenol measured by the OD405. One unit of enzymatic activity was defined as 1 μmol of p-nitrophenol released per minute. The standard reaction system with 0.02 ml of 50 mM Tris–HCl buffer (pH 7.5) instead of enzyme was used as the blank control.Protein concentration of enzyme samples was determined by the Bradford method, with bovine serum albumin (BSA) as a standard . ActivitpNP esters. To evaluated the optimum temperature and pH, pNPC2 was as the substrate, with a temperature ranging from 0°C to 70°C  and a pH ranging from 4 to 10 using Britton–Robinson buffer at 30°C. To explore the enzyme thermal stability, Est33 esterase was incubated at 30°C, 40°C, 60°C, and 90°C for 120 min, and the residual activity was determined every 15 min.The substrate specificity of Est33 was determined with pNPC2 as the substrate at a final concentration of 0.01–4.0 mM in 50 mM Tris–HCl buffer (pH 7.5) at 30°C. The kinetic parameters including Km, Vmax, and kcat were calculated by curve fitting of the Michaelis–Menten equation, using GraphPad Prism . The catalytic efficiency of Est33 for pNPC2 was calculated as Km/kcat.The kinetic assays were performed using pNPC2 substrate. The potential inhibitors PMSF, EDTA, and DTT were tested at 1 mM and 10 mM final concentrations, while SDS and Tween-80 were tested at 1 and 10% (w/v), respectively. Various metal ions including Li+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ were tested at concentrations of 1 and 10 mM. The effects of different organic solvents were evaluated by addition of methanol, formaldehyde solution, ethanol, acetonitrile, acetone, isopropyl alcohol, and dimethyl sulfoxide to final concentrations of 20% and 40%. Enzyme activity without any additives was set at 100%. The experiment was performed at 30°C for 10 min.The effects of potential inhibitors, organic solvents, and metal ions on Est33 activity were evaluated using reaction mixtures described above (section Esterase Activity Assay) with the est33 gene contains an open reading frame of 825 bp, which encodes a protein of 274 amino acids. The theoretical molecular weight of the protein was 30.6 kDa. No signal sequence was detected in Est33. Protein BLAST analysis at NCBI revealed that the first 100 hits with highest sequence identity were all uncharacterized α/β hydrolases annotated from the bacterial genome. Among them, seven sequences with different identities (72.9%–94.5%) to Est33 were chosen for further alignment. Ser94, Asp205, and His233 residues were indicated as the catalytic triad (94 was found in a pentapeptide sequence (GVSWG) conforming to a conserved motif (GXSXG) of esterases.We found that the ic triad , among w94, Asp205, and His233, the corresponding codons were mutated. S94A, D205A, and H233A variants of Est33 were designed and expressed. These point substitutions all resulted in a complete loss of the enzyme activity (data not shown).To confirm the importance of SerSWISS-MODEL was used to predict the structure of Est33 and its homologs. The results indicated that Est33 and homologs possess a classical α/β hydrolase fold containing 9 β-sheet and 10 α-helices, with a highly conserved core structure and diverse loops . Est33 aE. coli BL21 (DE3) for expression. Nickel-affinity chromatography was used for purification against the 6*His tag contained in the recombinant protein. SDS-PAGE showed that the molecular weight of the recombinant enzyme Est33 was ~30 kDa, which was consistent with the theoretical molecular weight of 32.2 kDa  with different acyl chain-lengths were employed as substrates. The enzyme displayed a maximum activity toward pNPC2, and it showed high activity against other short-chain esters. The ability of Est33 to hydrolyze substrates with relatively long acyl-chains  was low  of 3.78 s−1 mM−1 . The first 100 hits with highest identity were revealed to be all uncharacterized α/β hydrolases via protein BLAST analysis. The phylogenetic analysis revealed that Est33 and its closely related homologs formed a separate branch from the known 20 families  higher catalytic efficiency at low temperature compared with their mesophilic analogues; (ii) lower optimum catalytic temperature; and (iii) lower thermal stability . With anA large number of cold-active enzymes are alkaline esterases and the optimum pH of approximately 8.0 . Est33, 2+, Ni2+, Cu2+, and Zn2+ had inhibitory effects while Mg2+ and Ca2+ enhanced activity slightly  International Cooperation Fund, grant number QLUTGJHZ2018002.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb. 2022.855658/full#supplementary-materialThe Supplementary Material for this article can be found online at: Click here for additional data file.


In squamous cell carcinoma, HPV18+ samples also showed increased median A3B expression when compared to HPV Alpha-9 species or only to HPV16+ samples. Our findings suggest that A3B expression is differentially upregulated in cervical cancer samples infected with HPV18. A3B could be potentially used as a biomarker for HPV infection and as a prognostic tool for clinical outcomes in the context of cervical cancer.The APOBEC3 (A3) proteins are cytidine deaminases that exhibit the ability to insert mutations in DNA and/or RNA sequences. APOBEC3B (A3B) has been evidenced as a DNA mutagen with consistent high expression in several cancer types. Data concerning the A3B influence on HPV infection and cervical cancer are limited and controversial. We investigated the role of A3B expression levels in cervical cancer in affected women positive for infection by different HPV types. Tumor biopsies from cancerous uterine cervix were collected from 216 women registered at Hospital do Câncer II of Instituto Nacional de Câncer, and infecting HPV was typed. A3B expression levels were quantified from RNA samples extracted from cervical biopsies using real-time quantitative PCR. Median A3B expression levels were higher among HPV18 The human papillomaviruses (HPV) are composed of non-enveloped particles containing a double-stranded DNA genome of approximately 8 kb in size . HPV is Cervical cancer is the fourth most incident cancer in women worldwide and in Brazil, responsible for over 8000 deaths in the latter in 2018 according to the last Globocan estimate ,10.The APOBEC family is a group of enzymes with cytosine-to-uracil deaminase activity grouped into five classes, namely AID, APOBEC1, APOBEC2, APOBEC3 and APOBEC4. APOBEC3 is further subclassfied into seven members , characterized by a cytidine deaminase activity on single-stranded DNA ,12. PhysA3B expression is consistently reported at low levels in normal human tissues . HoweverRNA Later , conditioned in liquid nitrogen gallons and transported to Banco Nacional de Tumores e DNA (BNT) at INCA. After 24 h, the RNA Later was removed from the cryotubes and samples were placed in a −80 °C freezer until nucleic acid extraction. This study was approved by the Ethical Committee of INCA (registration # 156/10 and CAAE 53398416.0.0000.5274). An Informed Consent Term was applied to all the patients enrolled in the project.The population studied included 216 women with cervical cancer whose samples were extracted from uterine cervix biopsies. The subjects were registered and followed-up at Hospital do Câncer II of Instituto Nacional de Câncer José de Alencar Gomes da Silva (INCA). Tumor biopsies were collected at surgery rooms before patients initiated any therapy, and were stored in cryotubes with 1 mL of All 216 subjects included in the study were previously investigated with respect to infecting HPV genotypes and HPV16/18 genetic variability ,21. All 2O, supplementing the final volume to 10 µL in order to degrade any DNA traces present in the samples. The mix was then incubated at 37 °C for 30 min and the DNase reaction was interrupted by adding 1 µL RQ1 DNase Stop Solution and proceeding to an incubation at 65 °C for 10 min. Continuing with the RNA treated with DNase, complementary DNA (cDNA) synthesis was performed as follows: 10 µL of input RNA after DNase treatment (1–500 ng/µL), 2 µL of 50 ng random primers (random hexameric primers—Thermo Fisher Scientific), 2 µL of 10 µM dNTPs (for each nucleotide) and ultrapure nuclease-free H2O complementing the final volume to 12 µL. The reaction was incubated at 65 °C for 5 min. After, 4 µL 5X First-Strand Buffer were added for each reaction, as well as 2 µL of 0.1 M DTT. The reaction was incubated at 25 °C for 1 min. For the last step, 2 µL of SuperScript™ II RT were added for each reaction following a cycling with 25 °C for 10 min, 42 °C for 50 min and 70 °C for 15 min.Total RNA extraction from the collected samples was performed using the All Prep DNA RNA kit , following the manufacturer’s protocol. After RNA isolation, samples were quantified using NanoDrop ND-1000 . RNA was kept on ice during all the procedures and stored at −80 °C. All RNA samples were treated with 1 µL RQ1 RNase-Free DNase , 1 µL RQ1 DNase 10X Reaction Buffer, 1–8 µL of input RNA (1–500 ng/µL) and ultrapure nuclease-free HAPOBEC3B mRNA was performed through real-time PCR (qPCR). TaqMan™ probes for GAPDH  and APOBEC3B  were used, and the reactions were carried out in a ViiA 7 Real-Time PCR System apparatus (Thermo Fisher Scientific). Reactions were performed in triplicates containing 5 µL TaqMan™ Gene Expression Master Mix, 0.5 µL TaqMan™ probe for APOBEC3B, 0.25 µL TaqMan™ probe for GAPDH, 2 µL of input cDNA and ultrapure nuclease-free H2O, supplementing the final volume to 10 µL for each reaction. The cycling conditions were as follows: polymerase activation at 50 °C for 2 min, denaturation at 95 °C for 10 min and 40 cycles of denaturation at 95 °C for 15 s, followed by annealing and extension for fluorescence detection at 60 °C for 1 min. The analytical method used for calculating gene expression differences was the 2-ΔCT [The relative quantification of he 2-ΔCT .Statistical Package for the Social Sciences (SPSS) was used for statistical analyses. The Mann–Whitney U test was used to analyze differences in APOBEC3B expression levels between the samples with distinct infecting HPV types and clinical variables . Results were considered significant when p-values < 0.05. A multivariate analysis was also performed in the SPSS software using the Poisson regression model.The software n = 3; 1.4%), HPV45  and HPV59 , and other members of the alpha-9 species: HPV31 and HPV35 , and HPV33, HPV52 and HPV58 . Moreover, we also found four samples positive for HPV73 (1.9%) and one positive for HPV56 (0.5%), belonging to the alpha-11 and alpha-6 species, respectively.In this study, we analyzed a total of 216 women with cervical cancer, all of them previously reported with single infection with different HPV types. The most frequent types observed were HPV16 (Alpha-9), present in 139 samples (64.4%) and HPV18 (Alpha-7), present in 30 samples (13.9%). Besides these, 10 additional HPV types were present in smaller frequencies. These included other members of the alpha-7 HPV species: HPV39 (+ and in 28 (93.3%) of 30 HPV18+ samples. Among the HPV16+ samples, ninety-two (66.2%) belonged to lineage A, twenty-eight (20.1%) to lineage D, six to lineage C (4.3%) and five to lineage B (3.6%). Among the HPV18+ patients, we also observed a higher frequency of lineage A in twenty-six samples (92.9%) and only two samples (7.1%) carried lineage B. HPV18 lineages C and D were not identified in this study. When we performed multiple intratype comparisons between samples carrying different lineages of HPV16 or HPV18 with respect to A3B expression levels, no significant differences were observed .HPV lineages were identified in 131 (94.2%) of 139 HPV16p = 0.753; Kruskal–Wallis). The most frequent histological type observed in our cohort was the squamous cell carcinoma (SCC) (84.8%). Women with HPV16 showed a significantly increased frequency of SCC (89.2%) when compared to those carrying HPV18 (56.7%) given the non-overlapping confidence intervals (respectively 84–94.4% and 38.2–75.2%). Conversely, HPV18-infected women exhibited a higher incidence of adenocarcinoma (ADC) (43.3%) in comparison to women with HPV16 (10.8%) without superposition between the confidence intervals (respectively 24.8–61.8% and 5.6–16%). Most patients presented at intermediary to late stages at diagnosis  and did not differ between HPV types in that regard.Demographic and clinical characteristics of the patients and their relation to HPV are listed in APOBEC3B expression levels in cervical cancer samples, we compared them among distinct HPV type groups. The median APOBEC3B expression level was higher among HPV18+ samples (n = 30) when compared to HPV16+ samples (n = 139) . As expected, HPV18+ samples continued to exhibit significantly increased levels of APOBEC3B expression compared to that group . On the other hand, HPV18+ samples continued to display an increase in the median APOBEC3B expression levels when compared to samples infected with the Alpha-9 (HPV16-related and included) species (n = 148)  species (n = 54)  showed an increased median APOBEC3B expression level when compared to Alpha-9 species (n = 148) . That prompted us to analyze the effect of each variable in the A3B expression levels. A final model was tested in a multivariate analysis in order to simultaneously consider all variables . After multivariate testing .With respect to the clinical variables under study, we did not find any significant association between A3B expression and tumor subtypes or clinical staging using univariate analyses. However, when analyzing age at diagnosis, we found that women above the median age (49 years) displayed higher A3B expression levels than those under the median  showed the uterine cervix to be one of the cancer sites with the greatest A3B expression and APOBEC-related mutational signatures [+ cervical cancer samples exhibited an increased A3B mRNA expression when compared to HPV16+ samples (p = 0.027). These results not only reinforce the association between HPV and A3B expression in cervical cancer, but also suggest that distinct HPV types can modulate A3B expression to different extents.In this study, we describe that eoplasia ,24. Moregnatures ,14. In tOur efforts were concentrated in A3B, and not in other members of the APOBEC3 subfamily, for a number of reasons. In addition to the abovementioned evidence of the importance of A3B in multiple cancers , Burns et al.  showed tHigh-risk persistent HPV infection is well established as the main risk factor for the development of premalignant intraepithelial lesions and cervical cancer progression ,26,27. A+ samples [The data presented herein are consistent with those from previous studies that showed an increase in A3B mRNA levels upon HPV infection ,30. Viei samples . In norm samples .MTA1 mRNA levels and HPV18 E6 could induce its expression. It was also evidenced that transfection of the HPV18 genome induced an increase in DNA deaminase activity, which was proportional to A3B upregulation and required MTA1 participation [A recent report evaluated the potential mechanistic link between metastatin-associated protein 1 (MTA1), whose levels are tightly associated with tumor progression in cervical cancer, and A3B in cervical cancer cells. It was observed that HPV18 infection was significantly associated with cipation .TP53 mutated breast cancer samples [TP53 loss or E6-mediated degradation of p53 prevents the inhibition pathway and possibly leads to A3B upregulation [APOBEC3B [Although a mechanistic model linking HPV to A3B upregulation is notfully established, recent reports suggest promising pathways. Higher A3B mRNA levels were observed among  samples . The p53gulation . MoreoveAPOBEC3B . In thisAPOBEC3B .Another possible link between HPV and A3B overexpression could be through the antiviral responses elicited by the innate immunity. During HPV infection of human keratinocyte cell lines, the viral E5 protein induces the expression of IFN-β . Either + oropharyngeal squamous cell carcinoma [Taken together, different reports indicate A3B upregulation upon HPV infection. However, APOBEC3 deaminase activity can also target the HPV genome, induce hypermutation and limit its infectivity, contributing to virus clearance ,40,42. Iarcinoma . Therefoarcinoma . Interesarcinoma .Our study is limited in the sense that it could not unveil the molecular determinants that govern the differential effect of HPV18 towards A3B upregulation when compared to HPV16 or other high-risk HPV types. We have previously shown that HPV18 infection of human immortalized keratinocytes caused higher levels of A3B induction (1.5-fold) in comparison to HPV16 . In the In conclusion, we found that high APOBEC3B expression was associated with HPV18 infection in women with cervical cancer. Our results suggest that A3B expression is an important biomarker of HPV infection, and potentially a prognostic biomarker of cervical cancer clinical outcomes such as recurrence, response to treatment and overall survival, issues that will require further investigation.


Thyroid hormone (T3) and high glucose concentrations are critical components of β-cell maturation and function. In the present study, we asked whether T3 and glucose signaling pathways coordinately regulate transcription of genes important for β-cell function and proliferation.RNA-seq analysis was performed on cadaveric human islets from five different donors in response to low and high glucose concentrations and in the presence or absence of T3. Gene expression was also studies in sorted human β-cells, mouse islets and Ins-1 cells by RT-qPCR. Silencing of the thyroid hormone receptors (THR) was conducted using lentiviruses. Proliferation was assessed by ki67 immunostaining in primary human/mouse islets. Chromatin immunoprecipitation and proximity ligation assay were preformed to validate interactions of ChREBP and THR.We found glucose-mediated expression of carbohydrate response element binding protein alpha and beta (ChREBPα and ChREBPβ) mRNAs and their target genes are highly dependent on T3 concentrations in rodent and human β-cells. In β-cells, T3 and glucose coordinately regulate the expression of ChREBPβ and PCK1 (phosphoenolpyruvate carboxykinase-1) among other important genes for β-cell maturation. Additionally, we show the thyroid hormone receptor (THR) and ChREBP interact, and their relative response elements are located near to each other on mutually responsive genes. In FACS-sorted adult human β-cells, we found that high concentrations of glucose and T3 induced the expression of PCK1. Next, we show that overexpression of Pck1 together with dimethyl malate (DMM), a substrate precursor, significantly increased β-cell proliferation in human islets. Finally, using a Cre-Lox approach, we demonstrated that ChREBPβ contributes to Pck1-dependent β-cell proliferation in mouse β-cells.We conclude that T3 and glucose act together to regulate ChREBPβ, leading to increased expression and activity of Pck1, and ultimately increased β-cell proliferation.  •T3 is necessary for and glucose-mediated transcription on rodent and human β-cells.•T3 and glucose coordinately regulate the expression of ChREBPβ and PCK1 among other important genes for β-cell maturation.•Fine-tuning of glucose and T3 signals can regulate gene expression and proliferation of rodent and human pancreatic β-cells.•THR and ChREBP are interacting have common target genes and interact in close proximity in β-cells.•PCK1 activity is sufficient to drive β-cell proliferation. ThyroidOne way that T3 affects glucose homeostasis is through its influence on β-cell mass. Thyroid hormone (T3) is required for islet development and function . T3 promUcp1, Glut4 and Fasn, resulting in increased thermogenesis, decreased body weight, and improved glycemic levels. Recently, T3 was shown to promote lipogenesis in hepatocytes [Pklr), which encodes an enzyme that catalyzes the conversion of phosphoenolpyruvate to pyruvate, the last step of glycolysis [Txnip) [In mouse brown adipose tissue (BAT) we demonstrated that T3 and glucose synergistically regulate ChREBP, which in turnupregulates atocytes . Similaratocytes . In isleatocytes , are expatocytes . The expatocytes ,32. In βatocytes . Furtheratocytes ,32. In pycolysis . ChREBP  [Txnip)  which is [Txnip) ,36. Othe [Txnip) PCK1) is a main control point for the regulation of gluconeogenesis. PEPCK-C converts oxaloacetate and GTP into phosphoenolpyruvate, GDP and CO2. PEPCK promotes cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. This effect is mediated at least partially by mTORC1 [PCK1 was demonstrated by Shalev et al. to be the second most glucose responsive gene in pancreatic human islets after Txnip [Since ChREBP was shown to play a key role in glucose stimulated β-cell proliferation ,40, we ty mTORC1 ,42. PCK1er Txnip . In the er Txnip ,44, and er Txnip ,46. HowePCK1 [PCK1 [While it is now established that human and murine α-cells express PCK1 , it is wK1 [PCK1 . In thisK1 [PCK1 , at a tiK1 [PCK1 ,55. We h22.1l-glutamine, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin and further supplemented with 11 mM glucose, at 37 °C in a 5% CO2 incubator. To specifically study the effect T3, 10% resin-stripped FCS, was used to deplete thyroid hormones as described in Cao et al. [INS-1–derived 832/13 rat insulinoma cells were maintained in RPMI 1640 medium with 10% FBS, 10 mM HEPES, 2 mM o et al. .2.2Total RNA from ∼100 islets per condition, from five different human donors was isolated using the RNAeasy micro kit (Qiagen) according to the manufacturer's protocol. RNA integrity was assessed using Ribogreen to determine total mass and Fragment Analyzer. All samples passed QC. The RQN  scores ranged from 7.7 to 10. Samples were submitted to the New York Genome Center and RNA was amplified via the NuGEN Ovation RNA-Seq System V2 prior to RNA sequencing. 35–40 million 2 × 50 bp paired-end reads were sequenced per sample on the HiSeq2500 instrument (Illumina). Raw count data was pre-filtered to keep genes with CPM >1.0 for at least 60% of the samples. After filtering, count data was normalized via the weighted trimmed mean of M-values  and normComparisons between groups (log-fold-changes) were obtained as contrasts of the fitted linear modes generated using weighted least squares (lmFit) and empirical Bayes method ,61. A fa2.3Carbohydrate response elements (ChoREs) binding motifs were downloaded from the Schmidt et al. paper , which a2.4https://remap.univ-amu.fr) [Coordinates of the binding sites for the human THRB and RXRA transcriptional regulators were downloaded from the ReMap2022 database (available at: -amu.fr) . For eacMurine Thrb and Rxra transcriptional regulator binding sites were downloaded from, respectively, the Mendoza et al.  paper an2.514597658) ClueGO (v2.5.7 PMID: 19237447) and CluePEDIA (v1.5.7 PMID: 23325622) apps. Pathways were reported with Benjamini–Hochberg (BH) multiple test correction >0.05. Gene sets were tested for transcription factor target enrichment using the GTRD , Reactome (downloaded 17-02-2020) and Gene Ontology (downloaded: 03-04-2020) pathway databases using the Cytoscape . Islet cells were either uninfected or transduced with a multiplicity of infection (MOI) of 150 of the adenoviruses indicated. Thereafter, cells were incubated overnight in fresh medium with 10% strip FBS containing indicated glucose and T3 concentrations. Then, cells were rinsed with PBS and fixed in 4% paraformaldehyde, and β-cell proliferation by staining for ki67 (Thermo Scientific) and Insulin (Dako). At least 2000 β-cells were blindly counted per human donor/mouse. Cells were imaged on a Zeiss 510 NLO/Meta system , using a Plan-Apochromat 20× objective.2.7Total RNA was extracted using the Qiagen RNeasy micro kit, reverse transcription was performed using the MMLV reverse transcriptase (Promega), following by real-time PCR with the SYBER-green reagent (BioRad). The sequences of primers used are shown in 2.86 Ins1 cells. DNA was obtained with the Active Motif  chromatin shearing kit. Chromatin was precipitated by incubation with 3 μg of ChREBP antibody  or 3 μg thyroid hormone receptor antibody which recognizes both THRA and THRB genes  assays were performed with 100 mg of cell chromatin extracts from 20 × 10clone C3 ) 1:10,002.9PLA was used to determine endogenous protein–protein interactions . ChREBP 2.10Human cadaveric islets received from the Integrated Islet Distribution Program were dispersed by trypsinization as described previously . To obta2.11One-way or Two-way Anova was used to compare sets of data obtained from independent groups of samples. All data were analyzed using Prism version 9 . Statistical significance was considered at P < 0.05.33.1To explore the relationship between T3 and glucose, we measured the expression of numerous genes following 48 h h of exposure to various concentrations of either glucose or T3 or a combination of both agents in INS-1 832/13 rat insulinoma cells [  henceforWe next studied the effect of T3 and glucose concentrations on the expression of ChREBPα and β and the same target genes in human islets. Remarkably, we obtained very similar effects on mRNA expression in both model systems I–L. In t3.2Thra and Thrb) would alter the expression of ChREBP. In rats, the two genes of Thr are expressed at different amounts during development. Thra is the predominate form just after birth in rodents. Thra and Thrb are expressed at equal levels from postnatal day 9–15, and after 15 days, Thrb becomes the predominant isoform in islets [Thrb is expressed at much higher levels than Thra [as can be appreciated by the respective mRNA levels compared to actin  and in the presence or absence of T3 (10 nM). All donors were between the age of 24–61 and with body mass indexes (BMIs) ranging from between 18 and 26 . CovariaPathway enrichment analyses of the DEGs associated with T3 and high glucose treatment are summarized in 3.5As a first approach to investigate cooperativity between T3 and glucose signaling, we concentrated on genes that are co-upregulated by both T3 and glucose-namely ChREBPβ, PCK1, SLC9A4, RGS16, ABHD17C OXGR1, KLF10 . We idenWe identified conserved thyroid response element (TRE) and ChREBP binding sites in the promoter of the ChREBPβ isoform . We vali3.6https://www.ncbi.nlm.nih.gov/sites/GDSbrowser), we are clearly able to demonstrate that Pck1 is expressed in rodent and human islets as well as in purified β-cells  rats and adult rats, found that Pck1 is expressed 1000-fold higher in young rat islets compared with adults . Similar β-cells .Figure 6To test if PCK1 and its activity can control proliferation in β-cells, we overexpressed PCK1 in human islets cultured with non-stripped FCS and found that overexpression of PCK1 increases proliferation of adult human β-cells A. Furthe4In this paper, we demonstrate that thyroid hormone and glucose co-regulate ChREBP transcription and together the fine-tuning between the two signals can regulate gene expression and proliferation of rodent and human pancreatic β-cells. Our data indicate that the expression of ChREBPα is potentiated by T3, and that the expression of ChREBPβ and other downstream targets require and are augmented by T3. By stripping the serum in the growth media using resin we manage to eliminate thyroid hormone . HoweverOther genes that we found to be upregulated by T3 in both low and high glucose are Chodl, involved with carbohydrate sensing, enforcing the notion that T3 regulates glucose metabolism. Recently Ackerman et al. found Chodl (chondrolectin) to be one of the genes that is exclusively expressed in β-cells and not alpha cells  indicatiCytosolic PCK1 is best known as a gluconeogenic enzyme, essential for the production of glucose in the liver in the fasted state ,112. PCKIn summary, T3 is necessary for glucose-mediated transcription in rodent and human βcells. T3 and glucose together upregulate Pck1, which is sufficient to drive βcell proliferation. Finding a mechanism and link between thyroid disorders and diabetes could help predict, prevent, and possibly treat diabetes. In the long term, ChREBP may be a target for therapeutic regulation of β-cell function, proliferation and survival. Additionally, a T3 analog with islet-selective activity could be designed, similar to the liver-specific thyroid hormone analog developed for the treatment of hyperlipidemia , and thu


The chemical composition, crystal structure and morphology have been characterized by FT-IR spectra, XRD, Raman spectra, XPS, SEM and TEM in detail. Reflection loss (RL) values of NG/SnO2 hollow sphere hybrids less than −10 dB and −20 dB are found in the wide frequency range of 4.5–18 GHz and 5–16.2 GHz within 1.3–3.5 mm, and a minimum RL of −50.3 dB is achieved at 8.6 GHz with the matching thickness of only 2.3 mm. The results indicate that the NG/SnO2 hollow sphere hybrids with high-performance microwave absorption properties have a promising future in decreasing electromagnetic wave irradiation and interference.Nitrogen-doped graphene (NG)/SnO 2 hollow sphere hybrids were synthesized and show high-performance microwave absorption properties.Nitrogen-doped graphene/SnO It had been found that coupling nanostructured materials with graphene sheets could improve the EM wave absorption properties significantly in recent studies.19–28 For graphene-based composites, the real part of permittivity exhibits a sharp decrease, while the imaginary part shows only a slight decrease compared with graphene.19 The combination of a binary dielectric or magnetic material with graphene not only benefits its attenuation ability, but also obtain a good impedance matching.20–28Electromagnetic (EM) wave absorbing materials with the properties of broad bandwidth, strong absorption, light weight and thin-thickness have attracted much attention due to increasing EM interference and radiation.29–31 Zhao et al. prepared the hollow three-dimensional CuS hierarchical microspheres and the effective bandwidth was 3.0 GHz with absorber thickness of 1.1 mm.32 Considering the outstanding properties of graphene as well as hollow structures, hollow structures/graphene hybrids would be very attractive for practical applications. Han et al. synthesized the graphene-wrapped ZnO hollow spheres and a maximum absorption of −45.05 dB at 9.7 GHz with the thickness of 2.2 mm.33 Fu et al. prepared CoFe2O4 hollow spheres/graphene composites and a minimum reflection loss of −18.5 dB was observed at 12.9 GHz with a thickness of 2 mm and the effective absorption frequency range from 11.3 to 15.0 GHz.34Hollow structures exhibit special physical and chemical properties due to their low density and large surface area, having attracted attention as candidates for microwave absorption in recent years.2 as one of the most important semi-conductor transition-metal (band gap 3.6 eV) with temperature and environment stable dielectric properties, has been studied intensively by some groups acting as microwave absorbing materials.35–41 Zhao et al. synthesized the order honeycomb-like SnO2 foams and reflection loss values less than −10 dB reaches 5.6 GHz with a thin thickness of 2.0 mm.36 The SnO2 act as shells of Fe3O4, Ni, Fe etc. also have been studied and obtained good microwave absorption properties.37–39 The composites with SnO2 decorated on graphene sheets have been used for supercapacitor application and microbial fuel cells.42,43 So far, the EM absorption properties of N-doped graphene/SnO2 hollow spheres hybrids have not been studied.SnO2 hollow spheres hybrids were fabricated using a hydrothermal method with SiO2 act as sacrificial template, urea act as nitrogen source and reducing agent in this work. The microwave absorption performance of NG/SnO2 hollow spheres hybrids was investigated in the frequency range of 2–18 GHz. The high performance EM absorbing mechanism of the NG/SnO2 hollow spheres hybrids is further discussed.Herein, NG/SnO2.44 The silica nanospheres with particle size of about 200 nm were prepared via a Stöber method.45All the chemicals and reagents were used without further purification. Graphene oxide (GO) was synthesized from natural graphite by using a modified Hummers' method.2.1.2 hollow spheres were prepared by according to previous method with some modification.45,46 Typically, 100 mg of as-prepared SiO2 nanospheres were dispersed into 60 mL of deionized water and ultrasound for 2 h. Then, 1.2 g of glucose was added into the suspension and stirred for 10 min. After that, 0.2 g SnCl2·2H2O was added into the suspension under stirring. After stirring for 30 min, 0.044 g of NH4F was added into the mixed suspension and stirred another 30 min. After that, the mixed suspension was transferred into a Teflon-lined stainless steel autoclave, and then placed in an oil bath at 160 °C with magnetic stirring for 10 h. After cooled down to room temperature, the hollow SnO2@polysaccharides nanospheres were collected by centrifugation, washed with deionized water and ethanol thoroughly, and dried in an oven at 60 °C. Finally, the SnO2 hollow spheres composite was obtained by heat treatment at a temperature of 500 °C for 3 h with a ramping rate of 4 °C min−1 in air.The SnO2.2.2 hollow spheres (amino-SnO2) were achieved via a post-grafting strategy. Typically, 100 mL of anhydrous toluene and 200 mg of SnO2 hollow spheres was mixed under ultrasound and mechanical stirring for 1 h. Then 0.5 mL of APTES was added dropwise to the suspension and kept at 110 °C with reflux for 6 h. The amino-SnO2 were collected by centrifugation and washed for several times with ethanol and deionized water and dried in an oven at 60 °C. The NG/SnO2 hollow spheres hybrids were prepared by using a hydrothermal method. Briefly, 100 mg GO was dispersed into 60 mL deionized water and ultrasound for 2 h, 100 mg amino-SnO2 hollow spheres were added into the suspension with ultrasonic and stirred for 1 h. Then 2 g urea was added into the suspension and stirred for another 30 min. The suspension was transferred into a stainless-steel autoclave and kept at 180 °C for 6 h. The product was collected by centrifugation and then washed with ethanol and deionized water and dried at 60 °C. The NG was also prepared by using the same method without amino-SnO2.Amino-function SnO2.3.via Raman microscope. X-ray photoelectron spectroscopy (XPS) measurements were obtained with an AXIS-ULTRA DLD-600W electron spectrometer from Kratos. The morphology was examined by field emission scanning electron microscope (Gemini SEM 300) and transmission electron microscope . The measurements of FT-IR were performed with spectroscopy (VERTEX 70) using the KBr pellet method at room temperature. The electromagnetic parameters of the samples were measured by a vector network analyzer (Agilent E5071C) within 2–18 GHz. The toroidal shape samples with an inner diameter of 3.04 mm, out diameter of 7 mm and thickness of 2 mm were prepared by mixing the paraffin and samples with a mass ratio of 6 : 4.The crystal phase was investigated with X-ray powder diffraction . Raman spectroscopy was carried out on a LabRAM HR800 in 3.2 hollow spheres hybrids is schematically illustrated in 2 hollow spheres were prepared by facile strategy of one-pot hydrothermal following a subsequent heat treatment with SiO2 act as sacrificial template. Second, amino-functional SnO2 hollow spheres were obtained via a post-grafting strategy with reflux. Third, amino-function SnO2 hollow spheres anchored on the GO sheets surface through electrostatic interaction and the NG/SnO2 hollow spheres hybrids were prepared by using a hydrothermal method with urea as the nitrogen source and reducing agent.The formation mechanism of NG/SnO PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching vibration at 1723 cm−1, CC deformation vibration at 1613 cm−1, C–OH stretching at 1226 cm−1 and C–O stretching at 1053 cm−1, indicating that abundant oxygen functional groups  were introduced during the exfoliation of the graphene sheet.2 Compared the FT-IR spectrum of amino-function SnO2 hollow spheres with SnO2 hollow spheres, two new bonds were found at 3373 and 1570 cm−1 correspond to the coupling of N–H stretching vibration, suggesting that NH2 group have adhered to the surface of SnO2 hollow spheres.47 All samples except for GO show strong absorption at 500–700 cm−1 corresponding to characteristic vibration of O–Sn–O in the SnO2.48 For NG/SnO2 hollow spheres hybrids, the peaks at 1723, 1226 and 1503 cm−1 almost disappear, indicating that most oxygen function groups have been removed.2 There are three peaks at 1555, 1185 and 623 cm−1 can be assigned to the CC, C–N(C–O) and Sn–O stretching vibration in NG/SnO2 hollow spheres hybrids, indicating that the SnO2 hollow spheres have been successful anchored on the NG surface.2 hollow spheres and NG/SnO2 hollow spheres hybrids, as shown in θ = 25.3° attributed to the (002) reflection of graphitic carbon.49 The XRD pattern with the (110), (101), (200), (211), (222), (310), (112), and (321) planes confirmed the formation of SnO2 (JCPDS no. 21-1250). No apparent diffraction peak could be found at around 25° for NG/SnO2 hollow spheres hybrids, indicating that SnO2 hollow spheres have been decorated on the NG sheets surface, suppressing the stacking of graphene layers. 2 at 472, 632 and 775 cm−1 corresponding to Eg, A1g and B2g modes of crystalline SnO2, respectively.50 For GO, NG and NG/SnO2 hollow spheres hybrids, two apparent peaks centered at 1350 and 1590 cm−1 are attributed to the D bands associated with structure defects and G bands for the E2g vibration mode of sp2 carbon domains of carbon materials, respectively. The ratio of the intensity of D-band to G-band (ID/IG) is a measure of the degree of disorder in the graphene or GO.51 The D and G bands cannot be found in SnO2 hollow spheres, indicating that there is no carbon on the surface of SnO2 hollow spheres. There are three additional peaks corresponding to the SnO2 in the NG/SnO2 hollow spheres hybrids indicating that the SnO2 hollow spheres were efficiently deposited on the NG surface. The ID/IG increases from 0.97 for GO to 1.1 for NG/SnO2 hollow spheres hybrids, which suggest that more defects are formed during the SnO2 hollow spheres anchored on the NG surface and the reduction of GO.52 Both XRD and Raman measurement confirmed the successful integration of NG and SnO2 hollow spheres.The XRD was utilized to investigate the phase structure of GO, NG, SnO2 hollow spheres and NG/SnO2 hollow spheres hybrids were further investigated by FESEM and TEM. As showed in 1 High resolution SEM image clearly shows that the SnO2 hollow spheres with an average diameter of 220 nm, as displayed in 2 hollow spheres are uniformly anchored on the NG surface, forming sandwich-structure NG/SnO2 hollow spheres hybrids. 2 hollow spheres hybrids, the NG is nearly transparent with lots of wrinkles on its surface and the hollow feature of SnO2 nanospheres is further confirmed by TEM image , indicating most of the C atoms in the N-doped graphene are arranged in a conjugated honeycomb lattice. The peaks at 285.8 and 287.5 eV reflect different bonding structure of C–N bonds, N-sp2 C(–CN) and N-sp3 C(–C–N) bonds, respectively, which originate from substitution of N atom, defects or the edge of the graphene sheets.53 Small peaks at 286.9 eV reflect different bonding structure of C–O bonds, indicating that there are also some oxygen functional groups on NG surface. Deconvolution of the high-resolution N 1s spectra shows distinct N-containing functional group coexisted, identified by their unique bonding states in the hybrids.54 The peaks locate at 398.6, 400 and 401.7 eV can be attributed to pyridinic-N, pyrrolic-N and graphitic-N respectively.54 Two symmetrical peaks at 487.4 eV and 495.8 eV are attributable to Sn 3d5/2 and Sn 3d3/2 are showed in 2.55The surface chemical composition of the hybrids were characterized by XPS technology. 2 hollow spheres, NG and NG/SnO2 hollow spheres hybrids were are shown in μ′) and imaginary part (μ′′) of the complex permeability of the samples are close to 1 and 0, respectively, due to the SnO2 and NG is nonmagnetic at room-temperature. The real part (ε′) and imaginary part (ε′′) for SnO2 hollow spheres are nearly constant within 2–18 GHz, with an inconspicuous undulation (ε′ ≈ 3.2 and ε′′ ≈ 0.28) as shown in ε′ and ε′′ for NG decrease gradually from 34.51 to 19.03 and 34.93 to 9.69, respectively, with a resonance peak at about 13.5 GHz. 2 hollow spheres hybrids. The ε′ values gradually decrease from 17.8 to 12.7 with the increase of the frequency and the ε′′ values are varied in the range of 3.78–7.5 over 2–18 GHz. The complex permittivity of NG/SnO2 hollow spheres hybrids is enhanced in the whole frequency range after the NG introduced compared with SnO2 hollow spheres, which should be ascribed to the increased electrical conductivity in the presence of NG. The obvious improvement of complex permittivity for NG/SnO2 hollow spheres hybrids is possibly due to relaxation polarization of the residual defect and groups of the NG surface which are generated by the reduction and N doping process of GO during the formation of NG/SnO2 hollow spheres hybrids.The frequency dependence of the complex permittivity and complex permeability for the SnO13,36εs and ε∞ are the static dielectric constant and the dielectric constant at infinite frequency, respectively.2 It is clear that the plot of ε′ versus ε′′ would be a single semicircle, which is denoted as a Cole–Cole semicircle. Each semicircle is linked to one Debye relaxation process.1,2ε′′ versus ε′ for NG and present two semicircles corresponding to two relaxation processes. Nevertheless, four semicircles are obviously obtained in the curve of the NG/SnO2 hollow spheres hybrids .11 Nevertheless, the effective bandwidth is narrow due to the poor impedance matching of NG. The RL of the NG/SnO2 hollow spheres hybrids with different thickness are shown in 2 hollow spheres hybrids, the RL values less than −10 dB and −20 dB are found in the wide frequency range of 4.5–18 GHz and 5–16.2 GHz range with a variation in absorber thickness from 1.3 mm to 3.5 mm, and a minimum RL of −50.3 dB is achieved at 8.6 GHz with the thickness of 2.3 mm. The effective absorption bandwidth (RL < −10 dB) is 4.2 GHz for NG/SnO2 hollow spheres hybrids with the thickness of only 1.3 mm, which is thin enough as an absorber.Based on the generalized transmission line theory, the reflection loss (RL) curve of the absorber can be calculated based on the complex permittivity and permeability for the given absorber thickness and frequency by the following equation:tm) and the peak frequency (fm) can be described by the following equation:1,6,57,581,6 The minimum RL shifts toward the lower frequency as the absorber thickness increase also can be explained by the quarter-wavelength cancellation model. The black squares in texpm), almost locating on the λ/4 curve, which demonstrating that the good EM wave absorption properties of NG/SnO2 hollow spheres hybrids can be explained by the quarter-wavelength cancellation model. Apart from dielectric loss and magnetic loss, the RL is highly relevant to the impedance matching characteristic. When Z = |Zin/Z0| = |εr/μr|1/2|tanh{j(2πfd/c)(εrμr)1/2}| is close to 1, resulting in zero reflection at the air–absorber interface, most of EM wave can enter into the absorber. Z–f curves of the NG/SnO2 hollow spheres hybrids, in which the relationship between fm and Z at the matching thickness is indicated by the dash lines. The minimum RL value can be obtained and the corresponding Z is close to 1 when the matching frequency is 8.6 GHz, and the corresponding matching thickness (2.3 mm) is almost on the λ/4 curve. Such high performance EM wave absorption properties of NG/SnO2 hollow spheres hybrids are ascribed to the combined contribution from quarter-wavelength interference cancellation and impedance matching characteristic.The quarter-wavelength cancellation model and zero reflection have been applied to analyze the enhanced microwave absorption. In the quarter-wavelength cancellation model, the relationship between the absorber thickness  is 4.2 GHz for NG/SnO2 hollow spheres hybrids with the thickness of only 1.3 mm. The enhancement of microwave absorption for NG/SnO2 hollow spheres hybrids is attributed to the synergistic effect of dielectric loss, better impedance matching, multiple internal reflection and quarter-wavelength interference performance. These results indicate that the NG/SnO2 hollow spheres hybrids are ideal candidate as microwave absorbing materials.In summary, the NG/SnOThere are no conflicts of interest to declare.


Thus, many efforts have been made by researchers, employing a doping strategy, in order to further improve its ionic conductivity, but with little success. In this work, a series of pure phase Ca12Alx14−GaxOδ33+ (0 ≤ x ≤ 1.2) materials were synthesized by a traditional solid state reaction method. Scanning electron microscopy (SEM) combined with energy dispersion spectrum (EDS) analyses disclosed well-sintered ceramics with uniform Ga distributions. The defect formation energies for Ga replacing the two distinguishable Al1 and Al2 sites in Ca12Al14O33 calculated by static lattice atomistic simulation are nearly identical, ∼3.03 and ∼3.04 eV, respectively, consistent with the results of Rietveld refinements based on the XRD data, from which no preferred distribution of Ga on Al1 or Al2 site was observed. The electrical properties investigated by alternating current (AC) impedance spectroscopy show increased bulk conductivities for 0 ≤ x ≤ 0.4. Thus, here we present the first work that successfully improves the bulk oxide ion conductivity of Ca12Al14O33 by Ga-doping.Although mayenite Ca 12Al14O33 can be apparently enhanced by Ga-doping on the Al sites.The bulk conductivity of Ca Thus, the development of new oxide ion conductors with considerably high conductivity for use in intermediate temperature (500–750 °C) SOFCs is an urgent and pressing need.9–12Solid oxide fuel cells (SOFCs) have attracted great attention over the last few decades, due to the clean energy conversion technology associated with high efficiency and fuel flexibility.12Al14O33, a stable ceramic material, was first identified by M. Lacerda et al. in 1988.13 M Lacerda et al. reported that the oxide ion conduction (∼ 1.5 × 10−3 S cm−1) of Ca12Al14O33 materials was only slightly lower than that of YSZ, making Ca12Al14O33 a competitive candidate for SOFC electrolyte, if the oxide ion conductivity could be further improved through metal cationic doping strategy. The parent Ca12Al14O33 adopts a cubic structure with the I4̄3d space group and a lattice constant of ∼11.99 Å. There are two Ca12Al14O33 molecules in the unit cell which is composed of a positively charged framework built from 12 cages and two free O ions randomly occupying 2 different cages. Thus, it can also be represented by the chemical formula [Ca24Al28O64]4+·2O2−. The cages, composed of framework Ca, Al (6-coordinated Al1 and 4-coordinated Al2) and O atoms, are approximately 6 Å wide and connected to 8 other cages via ∼3.7 Å wide windows  and an increasing in activation energy. Similar electrical behaviors were also found for the zinc and phosphorus co-doped compositions. Later, G. Ebbinghaus et al. studied iron-doped single crystal mayenite in which they observed slight decreases in conductivity  for the doped samples.21 Other works have included copper,22,23 nickel,24 gallium,25 manganese,26 and iridium27 incorporation into mayenite, but did not examine the effect of these dopants on the conductivity. Some other cations doped materials, such as Bi3+ and Ln3+(Tb3+/Sm3+/Er3+/Nd3+/Yb3+/Ho3+/Pr3+) substituting for Ca ions, were also reported and focused mainly on the luminescent properties,28,29 without oxide ion conductivities being investigated.The oxide ion conductivity of aluminate Ca windows . The fre−, OH−, O2−, H−, O−, and so on,30–36 had also been reported with some interesting properties. In addition, electrons can occupy the empty space inside each cage in a similar manner, forming electride materials. However, although these anions or just electrons substituted Ca12Al14O33-based materials may have much higher conductivity, such as the H− introduced and photo-activated material had an electrical conductivity as high as 0.3 S cm−1 (mainly n-type electronic conduction) at room temperature, the oxide ion conduction was not improved, and therefore did not benefit its application in SOFCs as electrolyte. These anions or electrons substituted Ca12Al14O33-based materials are thus out of the scope of our study interesting.Besides metal cations doping, substituting the free oxygen ions with other anions such as F12Al14O33 materials have been previously reported by Luis Palacios et al. with the structures and reduction behaviors being studied.25 Through the Rietveld refinements based on the combined neutron and X-ray powder diffraction data, unit cell expansions were observed for these doped materials and the Ga ions were reported to mainly occupy the 4-coordinated Al2 sites. In order to get Ga-doped Ca12Al14O33 electrides, they fired these doped materials under a strong reducing condition, but resulted finally in decompositions and forming a mixture of Ca12Al14O33, Ca3Al2O6, and amorphous Ga metal. In this study we focused on investigating the effects of Ga doping on the phase, defect formation energy, structure and electrical properties of Ca12Al14O33 by X-ray diffraction, SEM/EDS, static lattice atomistic simulation, and AC impedance spectroscopy techniques. The results show that Ga ions can be substituted for up to about ten percent of Al ions in the crystal structure, similar with that reported in Luis Palacios et al.’s work. However, we found that both the Rietveld refinements based on the high-resolution XRD data and the defect formation energy calculations, did not suggest a preferred occupation for Ga ions replacing the two distinguishable Al1 and Al2 sites. This is different from that reported by Luis Palacios et al. The bulk electrical conduction of Ca12Alx14−GaxOδ33+ was increased for Ga content in the range of 0 ≤ x ≤ 0.4, after which a decrease in the conductivity was observed for x > 0.4.As mentioned above, the gallium doped Ca2.12Alx14−GaxOδ33+ were prepared by traditional solid-state reaction method using stoichiometric amounts of CaCO3 , Al2O3  and Ga2O3  as the starting raw materials. First, the well-mixed and ground raw materials were fired at 1000 °C for 12 h. After regrinding, the calcined powders were then uniaxially pressed into pellets and sintered at 1300 °C for 24 hours in an air atmosphere. The densities of these prepared ceramics were estimated from the samples' weight and geometry.The samples of Caθ range of 5−120°. The variable temperature XRD measurements were performed over a temperature range 25–900 °C with the 2θ range of 10−80 °C. The Rietveld refinements of the XRD data were carried out using Topas-Academic software.37 The microstructure and EDS analyses were performed on a Hitachi  S4800 scanning electron microscope (SEM). Before the SEM/EDS measurements, all these as-made ceramic pellets were well polished, followed with thermal etched. AC impedance spectroscopy (IS) measurements were performed with a Solartron 1260 frequency response analyzer over a 107 to 10−1 Hz frequency range. Prior to the IS measurements, electrodes were formed by coating platinum paste on opposite faces of the pellets and fired at 750 °C for 3 h to remove any organic components. Before the impedance measurements, the temperature was equilibrated at each set point for 1 hour.The XRD data were collected on a Panalytical X'pert Pro X-ray diffractometer with Cu Kα radiation over a 23+ ions substituting for Al3+ ions were investigated through atomistic-static-lattice simulation, using the General Utility Lattice Program (GULP)38,39 based on interatomic potential approach. In this work, the Buckingham potential function40 was used to model the interaction between ions with the shell model41 to describe the electronic polarizability for the structural modeling. The interatomic potential parameters used for the atomistic simulation are listed in The energies of Ga3.3.1.12Alx14−GaxOδ33+ (0 ≤ x ≤ 1.4). We can see clearly that pure phase can be obtained for compositions x ≤ 1.2, and all the reflection peaks match well with the mayenite structure Ca12Al14O33 (PDF#70-2144). For x = 1.4, reflections from the impurity Ca5Al6O14 (PDF#11-0357) appear. This solid solution limit is close to that (x = 1.0) reported by Luis Palacios et al.25 The refined cell parameters of different composition were plotted in x ≤ 1.2, obeying the Vegard's law; similar cell lattice expansion for these Ga-doped Ca12Al14O33 materials was also observed in Luis Palacios et al.’s work. The cell parameters of x = 1.4 are nearly the same as that of x = 1.2, indicating the solid solution limit being close to x = 1.2. The linear increase for x ≤ 1.2 can be explained by the larger effective ionic radius of Ga3+ ions  than that of Al3+ ions .42 Rietveld structure refinements were subsequently carried out on these doped materials. For these Rietveld refinements, a parent cubic Ca12Al14O33 structure (space group I4̄3d) containing two Ca sites (24d), one 3-linked and one 4-linked Al site  and three oxygen sites (one 16c and two 48e) was used as a model. Here, a typical Rietveld refinement was described for the x = 1.2 composition which has the highest nominal Ga content, while refinements for other single phase compositions were provided in the ESI of this paper .et al. that the Ga ions shown a preferred occupation on the 4-coordinated position. The refined chemical formula of Ca12Al12.784(2)Ga1.216(2)Oδ33+ agrees well with the nominal composition of Ca12Al12.8Ga1.2O33.x = 1.2, confirming the dense structure of this prepared ceramic. The grain size ranges from 2 μm to 5.0 μm, with irregular morphology. †3.2.i.e.,E(X) is the calculated total lattice energy or point defect energy of the species of interest after geometry optimization.As stated previously, no more than 10 percent of the Al ions can be replaced by Ga ions. This narrow solid solution usually corresponds with the relatively high defect formation energy for substitution. To confirm this, static lattice simulation technology was used to calculate defect formation energies based on the appropriate combination of dopant and vacancy defect energies and lattice energies of the binary oxides, and can be determined using the following defect equation:12Al14O33, Ga2O3, and Al2O3. Using the interatomic potentials presented in The starting point of the study was to reproduce the experimentally observed crystal structures of Ca2O3 and Al2O3, the defect formation energies for Ga ions replacing Al ions can be deduced from Next, point defect energies for Ga ions replacing both Al1 and Al2 site ions were calculated. Combining the total lattice energies of the interested binary oxides Ga3.3.12Alx14−GaxOδ33+ ceramic samples were investigated by AC impedance spectroscopy. Compared to four electrode technique that commonly used in the direct current (DC) conductivity measurements, the alternating current (AC) impedance spectroscopy measurements have the advantage to separate the resistance related to grains (bulk) and grain boundaries due to their different relaxation times, leading to separate semicircles in the complex impedance spectrum. Therefore, the bulk conductivity (σb), grain boundary conductivity (σb), and the total conductivity (σt) can be well calculated from the bulk resistance (Rb), grain boundary resistance (Rg), and total resistance (Rt = Rb + Rg). The densities of these prepared ceramics, estimated from the samples' weight and geometry, were all higher than 92% of the theoretical prediction. The AC impedance measurements were performed within a temperature range of 400−900 °C. The results showed that the conductivity of Ca12Alx14−GaxOδ33+ ceramic samples did not vary linearly with the doped Ga content. In fact, the conductivity increased within the range 0 ≤ x ≤ 0.4, as shown in x ≥ 0.6. For x ≥ 0.6 the values are plotted separately in x = 0.4, with a value of ∼ 2.76 × 10−3 S cm−1 at 800 °C. For the composition x = 0.6, although the conductivity was lower than that of x = 0.4 but still higher than that of the parent material Ca12Al14O33. For x = 0.8, the measured conductivity was almost the same as that of the un-doped material. Conductivity lower than the un-doped Ca12Al14O33 was observed for samples with x = 1.0, and the lowest measured conductivity of ∼6.96 × 10−4 S cm−1 occurred for the x = 1.2 sample. This value, measured at 800 °C, is about four times lower than the sample with composition x = 0.4. A more direct visual comparison of the conductivities of the Ca12Alx14−GaxOδ33+ ceramic samples is seen in x at a temperature of 800 °C. In x values can be clearly seen.The electrical properties of the Cain situ VT-XRD measurements  materials were prepared by a conventional solid state method. Pure mayenite phases were obtained for 0 ≤ x ≤ 1.2, as was confirmed by Rietveld refinements based on the XRD data, and also by combined SEM and EDS element distribution map and element concentrations analyses. Static lattice atomistic simulation was used to calculate the defect formation energies of Ga ions replacing Al ions. The simulation values of 3.03 eV and 3.04 eV for the defects on Al1 and Al2 sites, respectively, were consistent with the non-preferred occupation of Ga on Al1 or Al2 sites, as derived from Rietveld refinements. The bulk conductivities increased with Ga content for 0 ≤ x ≤ 0.4. In summary, we believe that this paper presents the first example of successful improvement of the bulk oxide ion conductivity of Ca12Al14O33 through Ga-doping.In this work, a series of Ga-doped CaThere are no conflicts of interest to declare.RA-009-C8RA08254E-s001


Raphanus sativus) methanol (RME) and aqueous extracts (RAE) was tested against the second stage (J2) root knot nematode Meloidogyne incognita model. The EC50 of RME after 3 d of J2 immersion in test solutions was 312 ± 65 μg/ml. However, no activity was noted for RAE . The chemical composition analysis of the methanol extract carried out by the GC–MS technique showed that 4-methylthio-3-butenyl isothiocyanate (raphasatin) was the most abundant compound at 20%. This pure compound strongly induced J2 paralysis with an EC50 of 1.3 ± 0.2 μg/ml after 24 hr. Comparison with other 11 selected isothiocyanates with structure similarity showed that the double bond at position 3 of the alkyl side chain is important for nematicidal activity, followed by the sulfur group at position 5 and the benzyl group at position 2. In addition, raphasatin showed the highest nematicidal activity with the corresponding lowest antioxidant activity of about 92 ± 18 μg/ml. In conclusion, the results of this investigation reveal that R. sativus and its major compound raphasatin can be integrated into the pest management system.The nematicidal activity of radish bulb ( Meloidogyne represent a serious problem in crop production (Root-knot nematodes (RKN) of the genus Sambucus nigra and Melia azedarach methanol extracts were reported to have nematicidal activity against Meloidogyne incognita , belonging to the Brassicaceae family, is used in indigenous Asian herbal medications  after tissue damage , (ii) the study of the in vitro biological activity (EC50) of RME and its major compound having a nematicidal effect against M. incognita second-stage juveniles (J2), (iii) preliminary structure–activity relationship with other 11 selected isothiocyanates, and (iv) the correlation between nematicidal analysis and antioxidant power of the tested compounds.In the present investigation, we report for the first time: (i) the GC–MS chemical characterization of the bulb of R. sativus was obtained from a local market in Beja-Tunisia  in January, 2017. It was identified according to the flora of Tunisia by Professor Hichem Sebai. Bulbs were air-dried for 7 d at room temperature (20 ± 2°C), ground into a fine powder using a Retsch blender mill, sifted through a 0.5 mm mesh screen to obtain a uniform particle size, stored in the Laboratory of Functional Physiology and Valorization of Bioresources, High Institute of Biotechnology of Beja, Tunisia, with the voucher specimen number # RS-BL-20-01-17, and subsequently used for extraction.Fresh Then, 100 g of ground bulb was extracted using 400 ml of water or methanol (HPLC grade), sonicated for 15 min, and macerated for 24 hr at room temperature. The extracts were centrifuged for 15 min at 13,000 rpm after being filtered through a Whatman No. 40 filter paper. We didn’t use a rotary evaporator to dry the extracts to avoid the loss of volatile compounds such as isothiocyanates. The extracted samples were kept at 4°C until further analysis. The plant material was also dried at 105°C for 24 h, and the moisture content was found to be 62.51%.R. sativus were performed on a gas chromatograph Varian model 3800  equipped with a Varian 7800 autosampler, a split/splitless injector Varian 1079, and an ion trap mass detector (ITMS) 2000. The analytical column was a Varian VF5m s (30 m × 0.25 mm i.d. × 0.25 mm film thickness). Helium was the carrier gas at 1 ml/min. Then, a 1-μl sample, previously dehydrated with sodium sulfate and filtrated, was injected in the splitless mode with a purge valve on for 1 min. The injector temperature was set at 200°C. The mass spectrometer detector was operated in the electron ionization positive mode. Trap, manifold, and transfer line temperatures were at 200°C, 80°C, and 200°C, respectively. The oven was programmed as follows: 50°C (1 min), raised to 100°C (5°C/min), held for 1 min, then raised to 180°C (20°C/min), and held for 4 min. Qualitative analysis was performed in the scan mode (50–550 amu). Peak identification was made by comparing full mass spectra and retention times from authentic standards and the NIST MS Spectra Library .Chromatographic separation and identification of components of methanol extract of M. incognita originally reared from tomato roots (Solanum lycopersicum L.) cv. Belladonna, a cultivar that is very susceptible to RKN, was collected from a greenhouse in Cagliari . All plants were maintained in a growth chamber at 25°C to 28°C, with 60% relative humidity and a 16-h photoperiod, in plastic pots (18-cm diameter). Plants used for inoculations were 7 wk old, at the five-leaf stage. After 40 d, the plants were uprooted, and the roots were washed to be free of soil and cut into 2 cm pieces. Eggs were extracted according to the sodium hypochlorite procedure, and J2 were allowed to hatch in modified Baermann funnels at 28°C. All J2 hatching in the first 3 d were discarded, and thereafter, J2 collected after 24 h were used in the experiments (A population of Raphanus sativus methanol extract and Raphanus sativus aqueous extracts (RAE) were tested against RKN at a dose range of 10−5,000 μg/ml and assessed for toxicity at 72 h using fosthiazate as a chemical control according to 2 at a dose ranging from 1 μg/ml to 1,000 μg/ml using the organophosphorus fosthiazate (Sigma Aldrich) as a chemical control  at ×10 and ×20 after 72 h for extracts and 24 h for pure compounds. J2 were ranked in two distinct categories: motile or paralyzed/dead. Paralysis experiments were performed in three replicates.Chemically, the more a compound is oxidant, the more it will generate free radicals and consequently the more it has nematicidal activity. The antioxidant capacity of the tested compounds was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, as described previously by 50) value was determined as the concentration of the compound required to scavenge 50% of the DPPH radicals.Ascorbic acid (Sigma Aldrich) was used as a reference molecule in the same concentrations as the tested extract. All the analyses were carried out in triplicates. The efficacy concentration 50 . In the regression equation, the test compound concentration (% w/v) was the independent variable (x), and the paralyzed J2 was the dependent variable (y). The mean value of the six replicates per compound concentration and immersion period was used to calculate the EC50 value.where C is the lower limit, D is the upper limit, b is the slope at the ECM. incognita, a clear dose−response relationship was established and significant paralysis/death of J2 was evident after 3 d of exposure with a calculated EC50/3 d value of 312 ± 65 μg/ml , at 20%, while the identification, by the NIST library of other constituents of the extract, was unsuccessful.2, 4-methylthio-3-butenyl was the most active followed by benzyliosothiocyante, 4-methylthiobutyl isothiocyanate (erucin), allylisothiocynate, and methylisothiocynate with an EC50/1 d of 1.3 ± 0.2 μg/ml, 1.9 ± 0.3 μg/ml, 3.2 ± 1.7 μg/ml, 6.6 ± 2.5 μg/ml, and 7.9 ± 3.1 μg/ml, respectively (Compared with the selected isothiocyanates previously tested against Jectively .(12) followed by isobutylisothiocyanate (11) and 3-methylsulfinylpropyl isothiocyanate (iberin) (10), with EC50/24 h values of 2.6 ± 0.7 μg/ml, 5.3 ± 0.9 μg/ml, and 6.55 ± 1.1 μg/ml, respectively. The least antioxidant compound was raphasatin (1) followed by benzylisothiocyanate (2), and erucin (3), with EC50 values of 92 ± 18 μg/ml, 90 ± 17 μg/ ml, and 43 ± 12 μg/ml, respectively  and raphasatin (1). Besides, the addition of sulfur group at position 5 of the side alkyl chain increased the nematicidal activity more than thrice in the case of erucin (3) and hexylisothiocyanate (7). Moreover, when the double bond in position 2 was substituted by a benzene moiety as in the case of allyisothiocyanate (4) benzyl isothiocyanate (2), the compound increased thrice from an EC50/24 h of 6.6 ± 3.4 μg/ml to 1.9 ± 1.1 μg/ml  . IsothioM. incognita was treated with V-ATPase inhibitors such as maleimide and arylhydrazones (A previous study reported the depletion of monoacylglycerol levels when drazones . We specM. incognita. Radish is rich in 4-methylthio-3-butenyl isothiocyanate, which has nematicidal activity against this RKN and allows us to use this plant for crop protection against pests. Comparison with other isothiocyanates showed that double bounds at position 3 are very important for nematicidal activity. This gives insights that would enable the development of new pesticides with high nematicidal activity.This article deals with the study of the nematicidal activity of the radish methanol extract against