This mechanistic understanding is highly important for the characterization of toxicological hazards, and for the risk assessment of chemicals and environmental samples such as surface waters and effluents. Prompted by this need, we developed and optimized a straightforward bioluminescent multiplexed assay which enables the measurement of four bioactivities, selected for their relevance from a toxicological perspective, in bioluminescent microtissues. The assay was developed to monitor inflammatory, antioxidant, and toxic activity, and the presence of heavy metals, and was successfully applied to the analysis of river water samples, showing potential applicability for environmental analyses. The assay, which does not require advanced equipment, can be easily implemented in general laboratories equipped with basic cell culture facilities and a luminometer.The development of predictive  Cell-based sensing represents a promising alternative to other biosensing techniques as it enables the acquisition of highly valuable information concerning bioactivity, toxicity, and bioavailability. Living cells possess the innate ability to respond to external stimuli via the expression of target genes ,2. This Owing to their superior predictivity, three-dimensional (3D) cell models are increasingly replacing conventional 2D cell cultures. 3D cell models, including spheroids, organoids, and microtissues, enable reconstitution of the extracellular matrix and cell–cell interactions, creating an architecture that faithfully reflects the native morphology of organs and tumors ,7. In reBioluminescent (BL) reporter assays represent the gold standard for the high-throughput screening assays employed in drug discovery ,11,12,13A number of cell-based assays have been reported as suitable to monitor protein–protein interactions within cells or living animals, spanning from reporter gene technology to bioluminescence resonance energy transfer (BRET) techniques . More reWhilst several 3D cell models have been reported in the literature, few efforts have been devoted to standardizing BL cell-based assays in 3D formats. Here, we report the development of a new multiplexed assay relying on 3D BL models that enables the acquisition of multiple pieces of information on four different activities via a single assay, without requiring sophisticated instrumentation . The met® HD transfection reagent; and D-luciferin potassium salt were all sourced from Promega . ZnSO4·7H2O, tert-butylhydroquinone (t-BHQ), Tumor Necrosis Factor-α , dimethyl sulfoxide (DMSO), MicroTissues® 3D Petri Dish® micro-mold spheroids, and all other chemicals were purchased from Sigma-Aldrich . River water samples from the Emilia-Romagna area, including transitional (delta and ria), and surface waters, were provided by ARPA Emilia-Romagna .Human embryonic kidney HEK293T cells were obtained from the American Type Culture Collection . Penicillin, streptomycin, and cell culture reagents were sourced from Carlo Erba Reagents . Charcoal stripped fetal bovine serum (FBS), was purchased from ThermoFisher Scientific . The mammalian expression plasmids pGL4.32[luc2P/NF-κB-RE/Hygro], pGL4.37[luc2P/ARE/Hygro], and pGL4.40[luc2P/MRE/Hygro], carrying Luc2P luciferase under the regulation of NF-kB, antioxidant, and metal transcriptional regulations respectively; plasmid pCDNA3Luc2P; PureYieldTM Plasmid Miniprep System kit; FuGENE−1 penicillin, 50 µg mL−1 streptomycin, and 2 mM L-glutamine. The day before transfection, the cells were plated into a 96-well optical-bottom black plate at a density of 2.0 × 104 cells/well with 100 µL of complete growth medium, and incubated under standard conditions for 24 h at 37 °C and 5% CO2. Afterwards, HEK293T cells were transiently transfected with 0.10 µg of the pGL4.32[luc2P/NF-κB-RE/Hygro], pGL4.37[luc2P/ARE/Hygro], pGL4.40[luc2P/MRE/Hygro] and pCDNA3Luc2P plasmids using the FUGENE® HD transfection reagent at a ratio of 1:3, and incubated for 48 h under the standard conditions. The 2D cell cultures were monitored every 24 h for a period of 3 days using Thermo Scientific Invitrogen Evos M5000 Imaging Systems with a 4× objective.The HEK293T cell line was cultured using DMEM supplemented with 10% (v/v) FBS, 50 U mLInflammation assay. HEK293T cells were transiently transfected into a 96-well plate format with a pGL4.32[luc2P/NF-κB-RE/Hygro] construct to monitor inflammatory pathway activation using TNFα as a model compound. After 48 h post transfection in the 2D format, the cell culture medium was replaced with 100 µL of fresh medium containing TNFα (concentration range from 0.1 to 20.0 ng/mL), and incubated for 5 and 24 h at 37 °C with 5% CO2. BL measurements (20 min with 1000 ms integration time) were obtained from the 96-well plates using a Tecan Microplate Reader Spark®  after injection of 100 μL of 1.0 mM D-luciferin citrate solution, pH 5.0.Antioxidant assay. HEK293T cells transiently transfected with a pGL4.37[luc2P/ARE/Hygro] construct were used to monitor antioxidant pathway activation with t-BHQ, which was selected as a model compound to induce nuclear factor erythroid 2–related factor 2 (Nrf2). After 48 h post transfection in the 96-well plate (2D format), the cell culture medium was replaced with 90 µL of DMEM with 0.5% v/v of charcoal stripped FBS. After 24 h, the cells were treated in triplicate with t-BHQ (concentration range from 0.1 µM to 1.0 mM), by adding 10 µL t-BHQ per well and incubating for 5 and 24 h at 37 °C with 5% CO2. BL measurements were obtained using a Tecan Microplate Reader Spark® after injection of 100 μL of 1.0 mM D-luciferin citrate solution, pH 5.0.Metal activation. pGL4.40[luc2P/MRE/Hygro] plasmid was used to transiently transfect HEK293T cells to monitor metal pathway activation using ZnSO4·7H2O. After 48 h post transfection in the 2D format, the HEK293T cell media in the 96-well plate was replaced with 90 µL of DMEM containing 0.5% v/v charcoal stripped FBS, and the cells were incubated for 24 h. Then, the cells were exposed in triplicate to increasing final concentrations (from 0.01 to 100 µM) of the metal inducer, by adding a volume of 10 µL ZnSO4 solution (concentration range from 0.1 to 1000 µM) per well, and subsequently incubating for 5 and 24 h at 37 °C with 5% CO2. BL measurements were obtained using a Tecan Microplate Reader Spark® after injection of 100 μL of 1.0 mM D-luciferin citrate solution, pH 5.0.Toxicity assessment. HEK293T cells were transiently transfected with pCDNA_Luc2P for toxicity assessment using TNFα, t-BHQ, and ZnSO4 as analytes. After 48 h post transfection in the 2D format, the cells were treated with TNFα (concentration range from 0.1 to 20.0 ng/mL), t-BHQ (concentration range from 0.01 µM to 100 µM), and ZnSO4 solution (concentration range from 0.1 to 100 µM), and incubated for 5 and 24 h at 37 °C with 5% CO2. BL measurements (20 min with 1000 ms integration time) were obtained from the 96-well plates using a Tecan Microplate Reader Spark®  after injection of 100 μL of 1.0 mM D-luciferin citrate solution, pH 5.0.−1 penicillin, 50 µg mL−1 streptomycin, and 2 mM L-glutamine. The day before transfection, cells were plated in flat-bottom, clear, 24-well plates at a density of 8.0 × 104 cells/well with 500 µL of complete growth medium, and incubated under standard conditions for 24 h at 37 °C and 5% CO2. Then, the HEK293T cells were transiently transfected with 0.50 µg of either pGL4.32[luc2P/NF-κB-RE/Hygro], pGL4.37[luc2P/ARE/Hygro], pGL4.40[luc2P/MRE/Hygro], or pCDNA3Luc2P plasmids according to the manufacturer’s instructions, using a FuGENE HD/DNA ratio of 3:1, and subsequently incubated at 37 °C with 5% CO2. The day after transfection, 3D spherical microtissues were generated through self-assembly in a non-adhesive agarose gel, with cylindrical microwells created using a 5 × 7 array micro-mold with 800 μm diameter rounded pegs fitting 24 well-plates . Briefly, a 330 µL-volume of a 2% w/v agarose solution in 0.9% w/v NaCl was pipetted into the micro-mold . After the agarose had gelled, the agarose gel was separated from the micro-mold and transferred to a single well of a clear 24-well plate. To equilibrate the 3D Petri Dish®, 1.0 mL of fresh cell culture medium was added per well, and then after 15 min incubation, replaced with fresh cell culture medium. This procedure was repeated twice, using 10% v/v FBS to monitor inflammatory pathway activation, and 1.0 mL of fresh medium containing 0.5% v/v charcoal stripped FBS to monitor antioxidant and metal pathway activations. Both 10% v/v FBS and 0.5% v/v charcoal stripped FBS were used for the general toxicity assay. Cells previously transfected in the 24-well plate were detached with trypsin, centrifuged for 5 min at 1200 rpm, resuspended in fresh medium, counted to a density of 3.5 × 104 in 75 µL, and transferred to micro-molds placed in the 24-well clear plate. After 10 min, to allow the cells to settle into the micro-mold, 1.0 mL of complete medium was added to each well. Spheroid formation was attained by incubating the plate at 37 °C and 5% CO2. Spherical microtissues were monitored every 24 h for a period of 7 days using the Thermo Scientific Invitrogen Evos M5000 Imaging Systems with a 4× objective.The HEK293T cell line was cultured using DMEM supplemented with 10% v/v FBS, 50 U mL® already transfected with the corresponding plasmids (as described in 4 (concentration range from 0.01 µM to 100 µM). For the antioxidant 3D format assay, the same procedure was used with 0.5% v/v of charcoal stripped FBS, and 120 µL t-BHQ solution in H2O (concentration range from 0.1 µM to 1.0 mM) was added to each well. For the metal pathway activation, 120 µL ZnSO4 solution (0.1 to 1000 µM) was added to the medium.For the 3D bioactivity multiplexed assay, we used one-day-old HEK293T spheroids from the 3D Petri Dish2, 1 mL and 50 μL aliquots of the media was removed from outside and inside the mold, respectively. A 50 μL volume of 1.0 mM D-luciferin citrate solution, pH 5.0, was added to the inside of the mold, and BL kinetics , t-BHQ (10 µM), and TNFα (5 ng/mL) were tested . Each sample was tested three times and in triplicate. A 120 µL volume of spiked river water sample was added per well, and the plates incubated for 5 h at 37 °C with 5% CO2; then 1 mL and 50 μL aliquots of the media was removed from outside and inside the mold, respectively. A 50 μL volume of 1.0 mM D-luciferin citrate solution, pH 5.0, was added to the inside of the mold, and the BL kinetics (20 min with 1000 ms integration time) were measured. BL responses were expressed as fold-changes relative to the control by dividing the BL signal of cells treated with spiked river sample  by the corresponding control .The suitability of the 3D BL high-content sensing technique to provide information about pro-inflammatory activity, antioxidant activity, general toxicity, and presence of heavy metals in environmental samples was investigated. To this end, river water samples from the Emilia-Romagna area were used to simulate a complex matrix with multiple bioactivities. Samples were spiked with ZnSO2O). TNFα, t-BHQ, and ZnSO4 dose-response curves in 2D and 3D formats were generated using GraphPad Prism v8.3.0 software , and the data were plotted normalizing the BL signal in respect to the control. The limit of detection (LOD) was calculated as the inducer that corresponded to the blank plus three times the standard deviation (s.d.).The BL bioassay responses were expressed as fold-changes relative to the control by dividing the BL signal of the treated cells by the corresponding control (either cell culture medium or ddH50), which is the concentration of the inducer which produces 50% of the maximum possible response, was calculated using the following equation:The half maximal effective concentration (EC50), which is the concentration of the inhibitor needed to inhibit a biological process or response by 50%, was calculated using the following equation:The half maximal inhibitory concentration .All measurements were performed in triplicate and repeated at least three times. A one-tailed In order to develop a multiplexed assay for monitoring four different activities in the same sample, we first optimized the conditions to obtain spherical microtissues with a defined shape and dimensions using a reproducible protocol which can be easily implemented in other laboratories. −1), t-BHQ (concentration range of 0.01–100 µM), ZnSO4 (concentration range of 0.01–100 µM), or DMSO (concentration range of 0.01–20% v/v), for 5 and 24 h at 37 °C. The BL emission kinetics of the 3D cell cultures were obtained after the addition of 50 µL of D-luciferin substrate 1.0 mM, pH 5.0, and showed a BL signal maximum between 30 and 60 s   for 5 h and 24 h. Spheroids treated with 10 µM t-BHQ showed approximately a 1.9-fold increase in luciferase activity compared to the control (incubated with ddH2O) c. A thre2O) c. This i2O) . Figure 2O) .50 value of 0.6 ± 0.1 μM was observed in the 2D format after 24 h of treatment with t-BHQ. Despite the 2D cultures providing a higher dynamic range and sensitivity at 24 h, we observed a higher variability between replicates than that observed with the 3D spherical microtissues. This was confirmed by growth monitoring of 2D cell cultures in 10% v/v FBS and 0.5% v/v charcoal stripped FBS . After 5 h of incubation time, a 6.1-fold increase in BL was observed in the 3D spherical microtissue treated with 100 μM ZnSO4, compared to the 2D format , t-BHQ , and TNFα  for 5 and 24 h  and inflammatory (INF) bioactivities, the presence of heavy metals (HM), and toxicity (TOX). Once optimized, we employed the analytical procedure involving the multiplexed assay relying on 3D BL models , and incubated them at 37 °C for 5 h. Both the spiked superficial and transitional river samples showed a significant increase in inflammatory activity—approximately 5 times higher than the control treated with the same concentration of TNFα (5 ng/mL).To investigate using our BL 3D high-content sensing method for the predictive and cost-effective characterization of the bioactivity of unknown samples, river water samples were spiked with TNFα, t-BHQ, and ZnSOL models , to analin vitro sensing tools able to provide rapid information about various bioactivities of a sample is of pivotal importance, not only to monitor environmental toxicants, but also to understand their mechanisms of action on different molecular pathways. This mechanistic understanding is highly important when characterizing toxicological hazards, and for the risk assessment of chemicals and environmental samples such as surface waters and effluents  plasmid which expresses Luc2P under the regulation of the nuclear factor-kappa B (NF-κB) response element. NF-κB is a point of convergence of external and internal inflammation triggers, and thus represents a suitable transcription factor for monitoring pro-inflammatory stimuli. When comparing the dose-response curves for TNFα, which we used as a model analyte, we observed a higher NF-kB basal activation (3.2 ± 0.4-fold) in 3D spheroids than we did in 2D monolayer cultures a. This iFor the anti-oxidant activity experiments, we used pGL4.37[luc2P/ARE/Hygro] plasmid, which carries four copies of an Nrf2-sensitive antioxidant response element (ARE), driving transcription of the reporter Luc2P. The Nrf2-ARE signal transduction pathway contributes to the transcriptional regulation of stress response genes and is activated by cellular stresses . Nrf2 biThe metal-responsive transcription factor-1 (MTF-1)-MRE signal transduction pathway was selected because it plays a critical role in the homeostasis and detoxification of heavy metals. Moreover, it is interconnected with multiple cell stress response pathways, helping to protect the organism against hypoxia and oxidative stress. Upon zinc binding, MTF-1 shuttles to the nucleus where it binds to MRE and interacts with transcription factors  and coactivators , driving luciferase expression .To preliminarily investigate the suitability of this 3D platform, considering that both ATP and luciferin availability could be limiting factors for the BL reaction according to spheroid size, BL measurements were performed in non-lysing conditions using HEK293T transfected with a pCDNALuc2P construct expressing Luc2P under the regulation of CMV promoter. D-luciferin substrate in citrate buffer  was selected as a non-lysing substrate. We selected D-luciferin substrate because of the low cost per reaction in comparison to commercial substrates, and the possibility of performing non-destructive imaging.To assess toxicity, we decided to include a micro-mold for testing sample toxicity in both 10% v/v FBS and 0.5% v/v charcoal stripped FBS. This was motivated by the fact that we used both media for the reporter plasmids, as suggested by the producers, but in 0.5% v/v charcoal stripped FBS the toxicity was more pronounced, and this was confirmed by morphological studies . For exa4, t-BHQ, and TNFα. Lower antioxidant pathway activations were observed, probably due to dissolved organic matter photoreactions generating hydroxyl radicals in natural waters [To confirm the suitability of our BL multiplexed assay to measure four bioactivities, we simulated a real sample using superficial and transitional river waters spiked with ZnSOl waters . A highel waters . This isl waters ; accumulA multiplexed BL sensing assay based on 3D microtissues was reported. The feasibility of the assay was evaluated using HEK293T cells to assess inflammatory and antioxidant activities, the presence of metals, and the toxicity of a sample, using a unique protocol which enables the assessment of the four activities at once. To the best of our knowledge, this is the first bioluminescent assay of its kind, providing a new straightforward strategy to assess multiple bioactivities in a single sample. The method was applied to river water samples spiked with analytes with multiple bioactivities, and it could be helpful in situations in which mixtures of compounds with unknown bioactivities, toxicities, and different modes of action are present, such as in the aquatic environment, thus representing a useful tool for an initial screening.


Silicon (Si) is an alternative that is used to minimize these disturbances and maintain better growth under NH4+ toxicity. Nevertheless, the mitigatory effects of Si on NH4+-stressed salvia are unknown. Therefore, this study was carried out to determine how Si assists to alleviate the NH4+ toxicity degree in salvia. To this end, salvia plants were cultivated in a controlled environment supplied with a constant N (nitrogen) level (13 meq·L−1) in the form of three NH4+:NO3− ratios , each with (1.0 meq·L−1) or without Si. Physiological disorders and typical NH4+ toxicity symptoms, as well as interrupted photosynthesis, were observed in the 100% NH4+-treated plants. Furthermore, cation uptake inhibition and oxidative damage were also imposed by the 100% NH4+ supply. In contrast, in the presence of Si, the NH4+ toxicity degree was attenuated and plant growth was ensured. Accordingly, the NH4+ toxicity appearance ratio decreased significantly. Furthermore, Si-treated plants showed an ameliorated photosynthetic ability, elevated internal K and Ca levels, and enhanced antioxidative capacity, as reflected by improved major antioxidant enzyme activities, as well as diminished accumulation of ROS (reactive oxygen species) and MDA . Our findings enlightened the agronomic importance of additional Si to nutrient solutions, especially pertaining to bedding plants at risk of NH4+ toxicity.Ammonium (NH The absorption and assimilation of N by plants are predominately in the forms of ammonium (NHe NO3−) . The acqe (NO3−) . NH4+ as− . The a4+ level is unintentionally presented or as the exclusive N source [4+  for higher plants leads to the suppression of plant growth and development. Abundant ammonium assimilation causes acidic stress, which is considered the primary cause of NH4+ toxicity [+, Ca2+, Mg2+) and ion equilibrium, and interference with the overproduction of reactive oxygen species [4+ toxicity contribute to detrimental visual symptoms that are characterized by reduced plant growth, leaf chlorosis, and necrosis, as well as stunted roots [Paradoxically, it appears that most plants manifest poor growth performance when a high NHN source ,6. Indeetoxicity . This phtoxicity ,8,9. Con species . These sed roots ,11.Salvia splendens F. Sellow ex Roemer & J.A. Schultes) is a widely cultivated herbaceous bedding plant in the Salvia genus. It is extensively utilized for culinary purposes and essential oil extraction [4+ [4+ input. Thereafter, more optimal fertilization strategies regarding agronomic and agricultural attempts were warranted. Meanwhile, silicon supplementation benefits salvia growth in harsh environments [4+ toxicity stress degree [Salvia  [4+ toxicity, relevant work in salvia is highly insufficient.As the second-most abundant element after oxygen in the Earth’s crust, silicon (Si) was recognized to be a ‘quasi-essential’ inorganic constituent for higher plants . Outstan cations . On the  cations ,25. On t−, H2O2) ,26,27. R4+-stressed salvia plants, the growth attributes, photosynthetic ability, key ion accumulations, antioxidative capacity (major antioxidant enzymes activities), ROS content, and lipid peroxidation level in response to an increasing NH4+ nutrition supply were evaluated in the current study. To the best of the authors’ knowledge, this is the first attempt to unveil the mitigator role of Si against NH4+ toxicity in salvia.Therefore, to determine the alleviatory effects of Si supplementation on NHSalvia splendens ‘Vista Red’, provided by PanAmerica Seed company, West Chicago, IL, USA) were sown in 200-cell plug trays containing BVB medium  moistened with tap water. The seeds were germinated in a mist propagation bench under a naturally lighted greenhouse condition at Gyeongsang National University  from 20 October 2021 to 1 November 2021. Seedlings were cultivated with MNS (multipurpose nutrient solution) [−1): 6.0 Ca(NO3)2·4H2O, 2.0 MgSO4·7H2O, 5.0 KNO3, and 2.0 NH4H2PO4; and (micronutrients in μmol·L−1): 20 H3BO3, 0.5 CuSO4·5H2O, 10 Fe-EDTA, 10 MnSO4·4H2O, 0.5 H3MoO4, and 4.0 ZnSO4·7H2O. Furthermore, the seedlings were grown in a hydroponic substrate system by using a commercial BVB medium  consisting of peat, coconut, and perlite.Seeds of salvia (olution)  for anothttps://www.amazon.com/Seedling-Starter-Trays-Extra-Strength/dp/B01GBBZU2Y accessed on 26 November 2021), which were subjected to running tap water for three days (21 DAS) to leach all the nutrition and maintained in a controlled alternating diurnal regime with 10 h light , white LED) and 14 h darkness at an air-conditioned temperature of 23 °C/18 °C and 60 ± 10% relative humidity. Subsequently, three NH4+:NO3− ratios  at a constant N supply (13 me·L−1) were formulated based on the MNS, which corresponded with an optimized Si concentration (1.0 me·L−1) sourced from K2SiO3 according to our lab’s previous publication [2SiO3 in 0:100, 50:50, and 100:0 solutions were reduced using KNO3, K2SO4, and K2SO4, respectively, and the resultant losses of nitrate and sulfate were balanced with the addition of nitric and sulfuric acid. Therefore, a total of six solutions consisting of 0:100 NH4+:NO3− Si (−), 50:50 NH4+:NO3− Si (−), 100:0 NH4+:NO3− Si (−), 0:100 NH4+:NO3− Si (+), 50:50 NH4+:NO3− Si (+), and 100:0 NH4+:NO3− Si (+) were adopted for the treatments. For each treatment, three repetitions involving a total of 60 plants were laid out with a 2 × 3 factorial scheme in a completely randomized design. Moreover, blank columns of the plug trays between different treatments were intentionally kept.The seedlings with similar morphologies were screened out to new 200-cell plug trays (plug tray information can be found on lication  or withoThe treatment solutions were applied every two days for 15 days (36 DAS). Subsequently, the plants were demounted out of the plug tray and the medium was washed off (39 DAS). Whole-plant fresh weight (surface-blotted with absorbent paper) and dry weight (kept in an air-forced oven at 60 °C for 72 h) were measured with an electronic balance. Lengths of shoots and tap roots, as well as leaf lengths and widths, were measured with a metal ruler. The leaf area was determined with a leaf area analysis meter . The two topmost true leaves from each plant were individually sampled, immediately frozen in liquid nitrogen, and preserved at −70 °C for further analysis.The toxicity ratio per replicate was determined using the following equation:The photosynthetic ability was estimated herein using the chlorophyll content and maximum photochemical quantum yield of PSII . Specifically, the total chlorophyll concentration (sum of chlorophyll a and b) was determined following a protocol proposed by Sims and Gamon . Fv/Fm wIon contents of Si, K, Ca, and Mg in the plants were determined with an inductively coupled plasma (ICP) spectrometer .Samples for the ICP analysis were prepared according to a slightly modified procedure : Fresh wFrozen leaf samples were finely ground in a pre-cooled mortar over an ice bath. Then, 100 mg of the fine powder was quickly weighed and homogenized in an extraction medium . The mixture was subjected to centrifugation  for the acquirement of the supernatant, which was used afterward for the total soluble protein quantification and enzymatic measurements.2O2 decomposition was employed for determining the catalase (CAT) activity [The total protein content was quantified using Bradford’s reagent . Analysiactivity . Guaiacoactivity . Dehydroactivity . Glutathactivity .2·− (superoxide) level was quantified following an approach proposed by Wu using hydroxylamine oxidization [2O2 (hydrogen peroxide) concentration was colorimetrically determined using a protocol according to Mukherjee [The Odization . The H2Oukherjee . The levukherjee . The detukherjee . The carukherjee  but the t-test to determine the significant differences at p = 0.05 using the SAS statistical software  and GraphPad Prism 8.0 , respectively. And the F-test was performed to find the significance between treatments using SAS statistical software 8.2. The PCA graph was plotted using Origin 2022 software  for the variability identification of antioxidative capacity results.All the data presented are the means ± standard deviations (SDs) of at least three biological replicates. They were subjected to the Duncan’s multiple range test or the two-sided Student’s 4+:NO3− nutrition, whereas plant growth was remarkably slowed in solely NH4+-treated plants  from 93.3 to 60% , the Fv/Fm value was significantly higher for the plants nourished with Si , CAT , APX (ascorbate peroxidase), GPX (guaiacol peroxidase), GR (glutathione reductase), and DHAR (dehydroascorbate reductase) were therefore quantified.4+ level increased from 50 to 100%, Si-deficient plants gradually improved the activity of antioxidant enzymes; similarly, the addition of 50% NH4+ to a solely NO3− solution to varying extents decreased the enzyme activities, except for APX (4+:NO3− ratios (4+:NO3− solution produced a 1.4-fold higher SOD activity relative to 100:0 NH4+:NO3− Si (−)   A. Concom− ratios B–F.2·− and H2O2) and lipid peroxidation  were evaluated.To better understand the responses of antioxidant capacity to the N form and Si application, the oxidative damage in terms of the ROS accumulation (O4+ stress rapidly increased the O2·− (17.9%), H2O2 (37.4%), and MDA (26.0%) contents, while reducing the carotenoids (11.9%) in contrast to the plants cultivated with 50:50 NH4+:NO3− , H2O2 (16.4%), and the production of MDA (22.7%); meanwhile, a significant increase was observed in the carotenoids content (6.3%).As expected, the supplementation of Si to varying extents decreased the ROS accumulation and lipid peroxidation, regardless of the NHnsidered . Outstan4+:NO3− ratios with and without Si on the antioxidant enzymes and oxidative damage, as well as the relationship between antioxidant capacity and Si supplementations, PCA  based on the antioxidant enzyme activities, ROS accumulation (O2·− and H2O2), and lipid peroxidation data set was performed.To visualize the influences of different NHThe computed model produced the related parameters along the first two principal components that captured 56.9%  of the total observed data variability . The maj4+-treated plants with Si supplementations displayed a higher activity of major antioxidant enzymes , which had strong negative correlations with H2O2  can be disturbed when plants are under abiotic stresses [4+ toxicity-induced osmotic changes can boost the excessive accumulation of O2·− and H2O2, accordingly resulting in higher lipid peroxidation and pigment degradation [2·− into H2O2, and subsequent rapid decomposition of H2O2 to water was aided by APX, CAT, and GPX [2O2 to water [2O2 into H2O by employing ASA (ascorbate) as an electron donor, while GPX decomposes the H2O2 to H2O using GSH (glutathione) [Generally, the balance between the production and detoxification of ROS  ,69.4+ supply increased from 50 to 100%, except for APX  and decreased photosynthetic ability . Similarly, NH4+-stressed salvia seedlings showed lower cation contents , together with increased synthesis of MDA and ROS. Conversely, the supplementation of Si significantly alleviated the NH4+ toxicity degree. This alleviation potential conferred by Si may be ascribed to the ameliorated photosynthesis, increased cation uptake , diminished lipid peroxidation (MDA), and enhanced performance of the antioxidative machinery to scavenge the over-production of ROS. It was accordingly concluded that the NH4+ toxicity could be mitigated by Si in Salvia splendens.To sum up, the damage of 4+ assimilation was elucidated as the primary cause of NH4+ toxicity. Therefore, further molecular study on the interaction between Si and H+ under high NH4+ conditions appears mandatory to interpret how Si assists in the alleviation of NH4+ toxicity.In addition, acidic stress caused by excessive NH


Inflammatory microenvironment promotes odontoblastic differentiation in human dental pulp stem cells (hDPSCs), but the regulatory mechanisms remain unclear. In this study, we aimed to explore the role of TAS2R in odontoblastic differentiation of hDPSCs in the inflammatory microenvironment.2+ were detected, respectively, by qPCR and Fluo-4AM Ca2+ fluorescent probe.Microarray analysis was performed to explore the differential mRNA profiles in inflammatory and healthy pulp tissues from the patients. hDPSCs isolated from the healthy pulp tissues were stimulated by LPS, TNFα and IL-6, respectively, to verify the effect of TAS2R. The expression markers related to odontoblastic differentiation of hDPSCs were observed by qPCR and chemical staining methods. TAS2R10 was overexpressed or silenced to observe the effect on odontoblastic differentiation of hDPSCs under LPS stimulation. The G protein and intracellular Ca2+ enhanced when TAS2R10 was overexpressed, but decreased when TAS2R10 was silenced.The expression of TAS2R was significantly upregulated in the inflammatory pulp tissues. In vitro, 5 subtypes of TAS2R mRNA expressions including TAS2R10, TAS2R14, TAS2R19, TAS2R30 and TAS2R31 in hDPSCs increased under the stimulation of LPS, TNFα or IL-6. In odontoblastic differentiation medium, we found LPS, TNFα or IL-6 stimulation promoted odontoblastic differentiation of hDPSCs. TAS2R10 overexpression in hDPSCs significantly increased the expression markers related to odontoblastic differentiation, whereas TAS2R10 silencing revealed the opposite effect. Furthermore, G protein was activated, and at the same time, intracellular Ca2+ via the G protein-coupled receptors (GPCR) conventional signaling pathway in inflammatory microenvironment, which may be a potential target for the development of effective conservative treatments for dental pulp repair.This study demonstrated that TAS2R was found to be expressed in hDPSCs, and TAS2R promoted odontoblastic differentiation of hDPSCs by mediating the increase in intracellular Ca Frequently, the inflammation and regeneration processes in dental pulp are often studied independently because they are antagonistic . HoweverIn this study, we investigated the differential expression profiles between inflammatory and healthy pulp tissues by microarray and validated the results using qPCR. Then, the effect of TAS2R on promoting odontoblastic differentiation of hDPSCs under the inflammatory microenvironment in vitro was detected. This study might reveal the mechanism of pulpitis repair and might provide a new idea for conservative treatment of pulpitis.The inflammatory samples diagnosed as irreversible pulpitis according to the endodontic diagnosis system of the American Association of Endodontist  were obtP value ≤ 0.05. Afterwards, Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to determine the roles of these differentially expressed mRNAs.Total RNA was extracted from the pulp tissues by RNeasy Kit  according to the manufacturer’s instructions. Microarray analysis  using inflammatory and healthy pulp tissues was performed. Differentially expressed RNAs were identified based on fold change ≥ 2.0 and 2.hDPSCs were isolated from the third molars of healthy adults aged 18–28 years by enzymatic digestion method . BrieflyThe third passage hDPSCs were identified via their surface antigen markers by flow cytometry. hDPSCs were resuspended and incubated in PBS  supplemented with 3% FBS for 45 min with primary antibodies against CD34, CD45, CD44 and CD146 . Flow cyAnd the third passage hDPSCs were used for subsequent experiments. hDPSCs were stimulated with 1 µg/mL LPS , 10 ng/m4 cells/dish) were seeded in 35 mm culture dishes in odontoblastic differentiation medium (ODI) containing DMEM  supplemented with 10% FBS, 1% Penicillin/Streptomycin, 50 µg/mL ascorbic acid , 10 mM β-glycerophosphate , 10 nM dexamethasone .The third passage hDPSCs (2 × 10Quinine (QUN) as a bitter compound can effectively activate TAS2R10 . 50 µM QSmall interfering RNA (siRNA) transfection was carried out using for TAS2R10 silencing studies. siRNA oligos were designed and synthesized by KeyGEN BioTECH Co. Ltd. . Non-specific siRNA-scramble were used as control. The sequences of siRNA-TAS2R10 were as follows:Sense, 5′-GCUUUAUUCUCACCGGCUUTT-3′Antisense, 5′-AAGCCGGUGAGAAUAAAGCCA-3′The sequences of siRNA-scramble were as follows:Sense, 5′-UUCUCCGAACGUGUCACGUdTdT-3′Antisense, 5′-ACGUGACACGUUCGGAGAAdTdT-3′hDPSCs were transferred to Opti-MEM medium . 50 nM siRNA and siRNA-scramble were transfected into hDPSCs via Lipofectamine 2000  according to the manufacturer’s instructions. After 24 h of transfection, the medium was changed to odontoblastic differentiation medium supplemented with 1 µg/mL LPS (ODI + LPS + siRNA).After 7 days or 14 days of odontoblastic differentiation, alkaline phosphatase (ALP) activity was evaluated by ALP staining . The num−ΔΔCT method.hDPSCs were harvested to extract total RNA using RNA Isolation Kit . Reverse transcription was performed with PrimeScript RT Master Mix  to synthesize first-strand cDNA. The mRNA expressions of ALP, runt-related transcription factor 2 (RUNX2), dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP-1), Gα and Gβ were measured by Viia 7 qPCR instrument  using SYBR Green Master Mix Reagent . Relative expression levels were determined by 22+ fluorescent probe  was added to each culture plate and incubated for 50 min at 37 °C with 5% CO2. Then, using Multi-Mode Microplate Reader  to detect the intracellular Ca2+ fluorescence intensity.hDPSCs were cultured in black-walled, clear-bottom 96-well plates  for 7 days. 2 µM Fluo-4 AM CaP < 0.05 was considered statistically significant.GraphPad Prism 7.0 software was used for One-way analysis of variance (ANOVA) and Student’s t-test. All experiments were repeated at least three times. The experimental data were expressed as mean ± standard deviation, and Microarray technology could reveal the differential expression profiles between inflammatory and healthy pulp tissues, which might help us discover the genes or pathways that played a key role in inflammation promoting differentiation of hDPSCs. Thus, we collected a total of 4 pulp samples, including 2 inflammatory samples and 2 healthy samples for microarray analysis. Two inflammatory samples were obtained from the mandibular first molar. The average age was 25, including 1 male and 1 female. Two healthy samples were taken from the mandibular third molar. The average age was 22, including 1 male and 1 female. By extracting total RNA and measuring the RNA concentration, the purity, the quality of the 4 samples was tested and could be used for the subsequent experiments.According to the analysis of differential mRNA profiles in inflammatory and healthy pulp tissues, the volcano plots showed 1727 differential genes between the two groups, of which 1128 were upregulated and 599 were downregulated   Fig. o.In the above experiments, we found that inflammatory microenvironment (such as LPS-induced inflammatory microenvironment) could promote the odontoblastic differentiation of hDPSCs. And the TAS2R expression (such as TAS2R10) was increased under LPS stimulation. To examine whether the LPS-induced differentiation of hDPSCs was dependent on TAS2R10, hDPSCs were co-treated with LPS and the TAS2R10 agonist QUN (50 µM) or siRNA-TAS2R10 transfection. Results showed that TAS2R10 overexpression in hDPSCs significantly enhanced the ALP activity and mineralized nodules formation, whereas TAS2R10 silencing revealed the opposite effect Fig. a–j. The Gα and Gβ are two subunits of G protein, which are downstream molecules of TAS2R.The mRNA expressions of Gα and Gβ were detected by qPCR. The results showed that the expression levels of Gα and Gβ in ODI group were similar to the Control group, but increased under LPS stimulation, indicating that TAS2R10 was activated. The expressions of Gα and Gβ were the highest in ODI + LPS + QUN group, but decreased in ODI + LPS + siRNA group Fig. o, p.2+ is a typical downstream effect mediated by TAS2R. Previous studies have reported that intracellular Ca2+ positively regulates the odontoblastic differentiation of hDPSCs [2+ fluorescent probe was used to evaluate the intracellular Ca2+ in this study. The results showed that the intracellular Ca2+ was increased in the ODI group compared with the Control group. After LPS stimulation, the intracellular Ca2+ further enhanced. The intracellular Ca2+ were the highest in ODI + LPS + QUN group, but decreased in ODI + LPS + siRNA group , but higher than that of ODI group. We speculate that inflammation promotes odontoblastic differentiation of hDPSCs may also involve other signal pathways like TLR4, ERK, P38 MAPK, AKT, or miRNA21/STAT3 , and inf2+ elevation is a downstream effect mediated by TAS2R that plays crucial roles in numerous physiological and pathological activities. Deshpande et al. [2+ concentration, evokes airway relaxation and reduces airway hyper-responsiveness. TAS2R negatively regulates the release of thyroid-stimulating hormone through increasing Ca2+ in thyrocytes [2+ positively regulates the odontoblastic differentiation of hDPSCs [2+ levels regulate dentin formation in vitro and in vivo [2+.TAS2R and its downstream signaling have been found in multiple extraterritorial tissues throughout the body such as digestive system , 39, repe et al.  have repyrocytes . Previouf hDPSCs  and Ca2+ in vivo . This is2+ in inflammatory microenvironment, and shed a new light on hDPSCs-based therapeutic strategies for regenerative endodontics.In summary, our study demonstrated that TAS2R was found to be expressed in hDPSCs, and TAS2R regulated odontoblastic differentiation of hDPSCs by mediating intracellular Ca


Mixed polyanion materials with a well-defined 3D framework channel can improve the electrochemical performance of sodium reversible insertion/extraction reactions, and can be especially beneficial for high rate performance and cycling capability. It approaches an initial reversible electrochemical capacity of 92.0 mA h g−1 with a high discharge potential over 3.85 V (vs. Na/Na+) and good cycling properties with a capacity retention of 81.4% after 300 cycles at a 0.5C rate in sodium systems. Taking into consideration the superior electrochemical characteristics, the phase-pure composite is considered to have a promising high rate capability as well as being a high capacity electrode material for advanced energy storage applications.A novel synthetic method has been investigated to fabricate a 1D nanostructure Na 7V4(P2O7)4PO4.A novel synthetic method has been investigated to fabricate a 1D nanostructure Na Therefore, there is an urgent desire to investigate large interstitial channel cathode electrode materials such as highly three-dimensional frameworks to cushion the volume expansion caused by rapid Na+ shuttling.9,10Lithium ion batteries (LIBs) are obviously distinguished from many other rechargeable batteries as power sources for applications of electrical vehicles due to their superior power and energy densities.11–14 for instance, NaxCoO2,15 Na0.44MnO2,16 NaFeO2,17 NaFePO4,18 Na3M2(PO4)3 19 and Na3M2(PO4)2F.20 Among the various cathode materials for SIBs, a vanadium-based ortho-diphosphate Na7V4(P2O7)4PO4 (NVPP) with a high energy density and stable cycling performance exhibits a single-valued voltage plateau at 3.88 V, where the remarkable single plateau and cycle life originate from an immediate phase , and has attracted comprehensive attention due to its unique 3D structure with a central tetrahedron [PO4] sharing corners with four [VO6] octahedra in the (VP2O7)4PO4 unit, and each diphosphate group [P2O7] bridging the two adjacent [VO6] octahedra by sharing the corners. The interconnected (VP2O7)4PO4 units form a 3D framework with well-defined ionic channels for Na (de)insertion.21 Na ions occupy three different crystallographic positions, Na1, Na2, and Na3, through the open 3D framework enabling reversible sodium de/intercalation which is favorable for surprising electrochemical behavior. Lim et al.21 reported a vanadium-based ortho-diphosphate NVPP that exhibited a single-valued voltage plateau at 3.88 V vs. Na/Na+ while retaining a substantial capacity (>78%) over 1000 cycles and proposed a theoretical scheme in which the reaction barrier arises from lattice mismatches suggesting that the presence of intermediate phases is beneficial for the cell kinetics by buffering the differences in lattice parameters between the initial and final phases. Deng et al.22 prepared a high-purity NVPP with a 1D nanostructure as a cathode material for rechargeable Na-ion batteries, facilitating its reversible sodium de/intercalation, which was beneficial to its high rate capability and cycling stability delivering 80% of the capacity (obtained at C/20) at the 10C rate and 95% of the initial capacity after 200 cycles. Deng et al.23 introduced a hydrothermal assisted strategy to prepare an NVPP/C nanorod and employed it as a novel high-property cathode material for aqueous rechargeable sodium-ion batteries. Favored by the open ion channel and 1D morphology, the composite exhibited superior high rate capability and 72% of the capacity remained at 1000 mA g−1. Zhang et al.24 designed a hierarchical NVPP/C nanorod–graphene composite as a sodium- and lithium-storage cathode material composed of a 1D rectangular NVPP/C nanorod, which was coated by in situ residual carbon and wrapped by a reduced graphene-oxide sheet. It approached initial reversible electrochemical capacities of 91.4 and 91.8 mA h g−1 with high discharge potentials over 3.8 V (vs. Na/Na+ or Li/Li+) and had good cycling properties with capacity retentions of 95% and 83% after 200 cycles at a 1C rate in sodium and lithium intercalation systems, respectively. Even at 10C, it still delivered 87.4% (for sodium) and 78.2% (for lithium) of the capacity and high cycling stability. In view of the reported coating modification, the low cost, easy synthesis, energy conservation and environmental friendliness will be promising themes in realizing the practical applications.In recent years, many considerable efforts have been made to develop novel cathode materials for rechargeable SIBs,7V4(P2O7)4PO4 nanorods by introducing Na3PO4 which acts as a melt-salt medium. Compared with other fabrication methods, the unique process not only yielded advanced synthetic materials in an environmentally benign system, but also enabled the control of the phase, morphology and crystallographic orientation of inorganic crystals. In addition, the 3D framework of NVPP nanorods with well-defined ionic channels can improve the electrochemical performance of sodium insertion/extraction reaction.In this paper, we report a novel facile sol–gel synthetic method to synthesise Na2.2.1.2CO3, Na3PO4, NH4H2PO4, and NH4VO3 and a desired amount of citric acid were used as starting materials. All of the reagents were dissolved in distilled water and the resulting solution was kept at 80 °C under magnetic stirring to evaporate the water until it turned into a wet gel. The wet gel was dried at 100 °C overnight under vacuum to obtain a dry gel which was ground and subjected to programmed heat treatment in an argon atmosphere. The furnace temperature was increased from 25 to 700 °C at 5 °C min−1, maintained at 700 °C for 12 h, cooled to 500 °C at a rate of 5 °C min−1 and quenched to 0 °C. The resulting intermediate product was soaked in hot water with argon bubbling. After filtering, washing, and drying, the final product, NVPP, was obtained.Stoichiometric amounts of Na2.2.Powder X-ray diffraction  using Cu Kα radiation was employed to identify the crystalline phase of the material. The experiment was performed in step mode with a fixed time of 3 s and a step size of 0.02°. The morphology was observed with a scanning electron microscope  and a transmission electron microscope . The carbon content in the NVPP/C composite was about 1.2 wt%, which was determined by an elemental analyzer .2.3.−1 NaClO4 dissolved in a mixture of ethyl carbonate (EC), diethylcarbonate (DEC) and dimethyl carbonate (DMC) (volume ratio 1 : 1 : 1) was used as an electrolyte. Cyclic voltammetry (CV) was conducted using an electrochemical work station (CHI660D). The scan rate was 0.5 mV s−1 and the voltage ranged between 2.0 V and 4.2 V. Galvanostatic charge–discharge tests were performed in a potential range of 2.0–4.2 V vs. Na/Na+ at ambient temperature on a land battery testing system . All of the specific capacities were calculated on the basis of Na7V4(P2O7)4(PO4) only.Coin cells were assembled to carry out the electrochemical measurements. The composite electrode was made from a mixture of the prepared sample, acetylene black, and polyvinylidene fluoride in a weight ratio of 70 : 20 : 10. A disk of sodium foil was used as a counter electrode and 1 mol L3.3V2(PO4)3 (NVP) phase slowly when the temperature exceeds 700 °C. It would be completely in the NVP phase when the temperature reached 900 °C. The diffraction peaks of the samples at 700 °C in P4̄21c. The 3D framework of [V4(P2O7)4(PO4)] facilitates sodium ion diffusion along the well-defined channels, which enables reversible sodium de/intercalation and thus is favorable for realizing a good electrochemical performance.The powder XRD patterns of the as-prepared NVPP sample are displayed in The morphology and microstructure of the hierarchical composite were investigated by SEM and HR-TEM. Irregular particles ranging from 2 to 5 μm are observed in the SEM image of the precursor multiphase product . As showx = 5 in NaxV4(P2O7)4PO4.17–19 Moreover, it is clearly seen that the capacity contribution mainly originates from the first split rather than the little second peak at a scan rate of 0.5 mV s−1. The high operating potential of NVPP makes it a promising cathode material for sodium ion batteries.i.e., 0.05C, 0.1C, 0.2C, 0.5C, 1C, 2C, 5C and 10C) were employed to evaluate the rate capability. With an increase in charge/discharge current density, the plateaus become shorter and vaguer, and the plateau voltage difference between the charge and discharge increases gradually for electrode polarization at high current densities.25,26 A discharge capacity as high as 92.0 mA h g−1 was obtained at a current density of 0.05C, which is very close to the theoretical capacity of Na7V4(P2O7)4(PO4) (92.8 mA h g−1). Although the discharge capacity declines with an increase in current density, 78% of the theoretical capacity (70.2 mA h g−1) is still realized at a current density of 10C. The superb rate capability of the NVPP nanorods can be attributed to their high purity and well-defined morphology. The nanorods of the material stack closely and crisscross with each other, forming pores and electron transport pathways which are available for short sodium-ion diffusion pathways as well as large electron transfer areas enabling rapid sodium-ion extraction and insertion. The long-term cycling performance at a current density of 0.5C is shown in −1). Therefore, the high cycling stability results from the excellent structural stability. The good electrochemical performance of the 1D nanostructured NVPP can be attributed to its favorable structural and morphological characteristics.The rate capability and cycling performance are summarized in 4.3PO4 as a melt-salt medium and carefully controlling the temperature ramp, which is favorable for rapid sodium intercalation. Based on the XRD, SEM, and TEM results, the nanorod architecture with rectangular sides is revealed which enables good electrochemical performance in terms of large reversible capacity, superb rate capability, and high cycling stability in sodium ion batteries. CV tests obviously indicate that the existence of Na5V4(P2O7)4(PO4) as an intermediate phase offers the main capacity contribution to the overall electrochemical behavior. Furthermore, the high operating potential together with the excellent electrochemical performance makes the NVPP nanorods promising cathode materials for advanced sodium ion batteries.We design an efficient and facile approach to prepare high-quality NVPP nanorods with a 1D nanostructure as high-performance cathode materials for sodium ion batteries by introducing NaThere are no conflicts to declare.RA-008-C8RA03682A-s001


Bacillus thuringiensis, known to be one of the most important biocontrol microorganisms, contains three AA10 family lytic polysaccharide monooxygenases (LPMOs) in its genome. In previous reports, two of them, BtLPMO10A and BtLPMO10B, have been preliminarily characterized. However, some important biochemical features and substrate preference, as well as their potential applications in chitin degradation, still deserve further investigation. Results from present study showed that both BtLPMO10A and BtLPMO10B exhibit similar catalytic domains as well as highly conserved substrate-binding planes. However, unlike BtLPMO10A, which has comparable binding ability to both crystalline and amorphous form of chitins, BtLPMO10B exhibited much stronger binding ability to colloidal chitin, which mainly attribute to its carbohydrate-binding module-5 (CBM5). Interestingly, the relative high binding ability of BtLPMO10B to colloidal chitin does not lead to high catalytic activity of the enzyme. In contrast, the enzyme exhibited higher activity on β-chitin. Further experiments showed that the binding of BtLPMO10B to colloidal chitin was mainly non-productive, indicating a complicated role for CBM5 in LPMO activity. Furthermore, synergistic experiments demonstrated that both LPMOs boosted the activity of the chitinase, and the higher efficiency of BtLPMO10A can be overridden by BtLPMO10B. Chitin is responsible for providing important characteristics such as rigidity and strength to the cell wall, and it also adds to the defense of the cell against pathogens and various predators  and lytic polysaccharide monooxygenases (LPMOs) . GHs typBacillus thuringiensis is one of the most important biocontrol microorganisms, which has been used in agriculture as a biopesticide for a long time to control various invertebrate species  and colloidal chitin, and generates products with different patterns (BtLPMO10B (reported by BtLPMO10A-FL) is a multi-modular LPMO and the roles of individual domains in substrate  binding have been characterized. However, biochemical features such as the effect of substrate binding on H2O2 generation, as well as their synergistic activity with chitinase in chitin degradation still worth further investigation. In this study, the biochemical features and substrate preference of BtLPMO10A and BtLPMO10B were compared with the aim of identifying and understanding their functions in chitin degradation. The obtained results showed the significantly different substrate preferences of the two enzymes although they shared highly conserved substrate binding surfaces in their catalytic domain. The C-terminal domains of BtLPMO10B do enhance the substrate binding ability of the enzyme, especially on colloidal chitin, whereas it has little effect on the activity of BtLPMO10B. Synergetic assays indicated that the efficiency of chitinase can be significantly improved by both the BtLPMO10A and the BtLPMO10B, whereas the higher effect of the BtLPMO10A can be attenuated by BtLPMO10B. species . Besides insects . In the cterized . BtLPMO1patterns . In contBtLPMO10A (GenBank ID: AJP62637) and BtLPMO10B (GenBank ID: ALP73598) can be accessed in the Genbank database and the crystal structure of BtLPMO10A was obtained from the Protein Data Bank database with accession code 5WSZ (BtLPMO10B (BtLPMO10B-CD) was generated by homology modeling using Modeller 9.19 (BaLPMO10A from Bacillus amyloliquefaciens (PDB ID: 2YOX) (BtLPMO10A and BtLPMO10B was conducted using Mega 7.0 (The sequences of ode 5WSZ . The thrler 9.19  with theD: 2YOX)  as the tD: 2YOX) . After bMega 7.0  and ESPrMega 7.0 .B. thuringiensis kurstaki ACCC10066 in E. coli BL21 (DE3), including the previously reported BtLPMO10A which stored in the lab. The gene encoding BtLPMO10B was amplified from the genomic DNA of B. thuringiensis kurstaki ACCC10066 using a forward primer F1: 5′-GGAATTCCATATGCACGGTTTTGTTGAAAAGCCCGGTA-3′ encoding a restriction site for NdeI and a reverse primer R1: 5′-CCGCTCGAGCACTGTTTTCCATAATGATAATGCA- 3′ with a restriction site for XhoI. The amplified gene was then subcloned into the pET23b vector through double digestion with the two restriction enzymes. The catalytic domain of BtLPMO10B (BtLPMO10B-CD) was synthesized and cloned into the same vector by the Taihe Biotechnology Co., Ltd . After verification by sequencing, three recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells, respectively, for protein expression.We produced three recombinant LPMOs from E. coli BL21 (DE3) cells were cultivated in 1 L Luria-Bertani (LB) medium at 37 °C with constant shaking at the speed of 180 rpm. When the OD600 of the culture reached 0.6, a final concentration of 0.05 mM IPTG and 0.2 mM CuSO4 were added and the cultivation was continued for an additional 4 h at 30 °C. Afterword, the cells were harvest by centrifugation at 4 °C for 10 min with the speed of 8,000 × g, and then resuspended in 100 mL of hypertonic solution containing 100 mM Tris–HCl pH 8.0, 20% sucrose and 0.5 mM EDTA. This step was performed two times. Finally, the precipitated cells obtained by centrifugation were resuspended in 100 mL hypotonic solution (1 mM MgCl2) and incubated on ice for 10 min. After 10 min of centrifugation at 8,000 × g, the supernatant was collected for further purification.The recombinant BtLPMO10A, a chitin beads affinity chromatography method was performed as described previously  and 0.15M (NH4)2SO4, and the protein was eluted by 20 mM acetic acid. For the purification of BtLPMO10B-CD, an ion exchange chromatography with HiTrap Q column  was performed. The protein solution was loaded onto the column equilibrated with 20 mM Tris–HCl buffer (pH 7.5) and eluted with a linear salt gradient using 1 M NaCl (pH 7.5). The obtained fractions were pooled and concentrated using the Amicon 8400 stirred cell  installed with a 3kDa cut-off membrane. Samples purity was analyzed by SDS-PAGE and the protein concentrations were measured by Bradford, using bovine serum albumin as a standard.For the purification of eviously . For the−1α-chitin, β-chitin and colloidal chitin, respectively, prepared according to the procedure described previously (The reactions were conducted in 20 mM Tris–HCl (pH 8.0) buffer containing 1 µM enzyme and 5 mg mLeviously . The mix−1α-chitin, β-chitin and colloidal chitin, respectively. The mixture was incubated 2 h at 30 °C with constant shaking at 800 rpm using Thermo block . After been separated from the reaction mixture by filtration through a 0.22 µm membrane, the concentrations of H2O2 in the supernatant were measured using the Fluorimetric Hydrogen Peroxide Assay Kit . The reactions without substrate were set as the control.The reactions were conducted in 20 mM Tris–HCl (pH 8.0) buffer containing 1 µM enzyme, 1 mM ascorbic acid and 5 mg mL−1 substrate, 20 mM Tris–HCl (pH 8.0), 1 µM enzyme, and 1 mM ascorbic acid. For the reaction using both BtLPMO10A and BtLPMO10B, 0.5 µM BtLPMO10A and BtLPMO10B was added. The reaction was last for 16 h at 30 °C with constant shaking at the speed of 800 rpm. After been separated from the reaction mixture by filtration through a 0.22 µm membrane, the generated oligosaccharides were analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC) with protocols described previously , 1 µM SmChiB (anexo-type chitinase from Serratia marcescens), 1 µM LPMO (BtLPMO10A or BtLPMO10B) and 1 mM ascorbic acid mixed in 500 µL reaction. The mixtures were incubated at 30 °C for 4, 8, 12, 24, 48, 72 h with an 800 rpm shaking. After separated the products from the reaction mixture by filtration through a 0.22 µm membrane, an equal volume of acetonitrile was added into the product solution. For BtLPMO10A and BtLPMO10B synergy studies, 0.5 µM BtLPMO10A and BtLPMO10B was added into the mixture. The reaction without the presence of LPMO was used as the control. The (GlcNAc)2 released from the reactions was analyzed by HPLC equipped with an X-Amide column and a UV detector at 195 nm. The concentration of (GlcNAc)2 in samples was calculated using commercial (GlcNAc)2 as a standard. All experiments were performed in triplicates.The chitinase synergetic experiments were carried out in 20 mM PBS buffer (pH 6.0) containing 5 mg mLBtLPMO10A and BtLPMO10B shares a sequence identity of 61%, and both enzymes contain a typical active site of AA10 family LPMOs that are comprised of a type II copper ion coordinated with two fully conserved histidines  generated by BtLPMO10B-CD were similar as compared to BtLPMO10B. The combination of BtLPMO10A and BtLPMO10B led to similar product profiles on both α-chitin and colloidal chitin as compared to BtLPMO10B. In contrast, the product profile on β-chitin with the combined BtLPMO10A and BtLPMO10B was similar to that of BtLPMO10A. These results suggest that BtLPMO10B can affect the activity of BtLPMO10A on α-chitin and colloidal chitin.The enzymatic activity of  by HPLC , Fig. 3Dd DP6 ox . Moreoveh enzyme , the amo2O2 produced by BtLPMO10A was much higher than that produced by BtLPMO10B and BtLPMO10B-CD 2  after 12 h of reaction. In contrast, the additional supply of BtLPMO10A or BtLPMO10B can both significantly boost the accumulation of chitobiose, which has reached 1.464 mg ml−1 for BtLPMO10A and 1.232 mg ml−1 for BtLPMO10B, respectively, after 72 h of incubation. Moreover, when both BtLPMO10A and BtLPMO10B were provided, the curve of the concentration of GlcNAc2 over time was similar to that observed when only BtLPMO10B was provided.The synergetic effects of nt study . SmChiB GlcNAc)2 . The synBtLPMO10A and BtLPMO10B. It is well known that both BtLPMO10A and BtLPMO10B have a typical AA10 catalytic domain. Sequence and structure analysis indicated that both enzymes have a typical carbohydrate-binding surface and a distinct active site with a phenylalanine rather than tyrosine as the axial residual . In contrast, the multi-modular BtLPMO10B is more inclined to bind to the colloidal chitin. As for the crystalline chitins, BtLPMO10B favors the β-form rather than the α-form, which is contrary to the results reported by BtLPMO10B (named BtLPMO10A-FL) prefers to bind the α-form chitin. Furthermore, BtLPMO10B-CD only retained a small portion of the binding abilities of the full-length enzyme toward all three chitins, which are significantly lower than BtLPMO10A. Interestingly, it exhibited comparable binding ability to both α- and β-chitin which is different from the report by BtLPMO10B-CD showed no binding to α-chitin while retained almost half the binding ability to β-chitin. This suggests that the substrate-binding capacity of BtLPMO10B is mainly contributed by CBM5 which is in accordance to previous reports 2 by HPLC chromatogram.Click here for additional data file.


Schizosaccharomyces pombe (sp) uses two transcription factors to regulate intracellular iron levels: spFep1 under iron-rich conditions and spPhp4 under iron-deficient conditions, which are controlled by spGrx4. However, bioinformatics analysis to understand the role of the spGrx4/spFep1/spPhp4 axis in maintaining iron homeostasis in S. pombe is still lacking. Our study aimed to perform bioinformatics analysis on S. pombe proteins and their sequence homologs in Aspergillus flavus (af), Saccharomyces cerevisiae (sc), and Homo sapiens (hs) to understand the role of spGrx4, spFep1, and spPhp4 in maintaining iron homeostasis. The three genes’ expression patterns were also examined at various iron concentrations. A multiple sequence alignment analysis of spGrx4 and its sequence homologs revealed a conserved cysteine residue in each PF00085 domain. Blast results showed that hsGLRX3 is most similar to spGrx4. In addition, spFep1 is most closely related in sequence to scDal80, whereas scHap4 is most similar to spFep1. We also found two highly conserved motifs in spFep1 and its sequence homologs that are significant for iron transport systems because they contain residues involved in iron homeostasis. The scHap4 is most similar to spPhp4. Using STRING to analyze protein-protein interactions, we found that spGrx4 interacts strongly with spPhp4 and spFep1. Furthermore, spGrx4, spPhp4, and spFep1 interact with spPhp2, spPhp3, and spPhp5, indicating that the three proteins play cooperative roles in iron homeostasis. At the highest level of Fe, spgrx4 had the highest expression, followed by spfep1, while spphp4 had the lowest expression; a contrast occurred at the lowest level of Fe, where spgrx4 expression remained constant. Our findings support the notion that organisms develop diverse strategies to maintain iron homeostasis.Due to iron’s essential role in cellular metabolism, most organisms must maintain their homeostasis. In this regard, the fission yeast  Schizosaccharomyces pombe (sp) uses two transcription factors, spFep1 and spPhp4, to regulate iron levels within cells , Saccharomyces cerevisiae (sc), and Homo sapiens (hs) to understand the role of spGrx4, spFep1, and spPhp4 in maintaining iron homeostasis. Under various ferrous concentrations, expression analysis was also conducted to gain a deeper understanding of the critical roles of spGrx4, spPhp4, and spFep1 in iron homeostasis. In light of this, we proposed a model in which spFep1 and spPhp4 interact to regulate the expression of each other, thus controlling the strict regulation of intracellular iron concentrations  to determine whether the candidate proteins contained the conserved active site. Using SMART, Simple Modular Analysis Research Tool, the presence of conserved domains in Te candidate proteins was determined . We utilMultiple amino acid sequences were aligned to construct a phylogenetic tree using the MUSCLE algorithm incorporated into MEGA X software; version 10.0.5 . The physbg.bio.ic.ac.uk/phyre2/) was used for protein prediction, modeling, and analysis in the intensive mode. The STRING database (https://string-db.org) was used to analyze protein-protein interactions using the amino acid sequences.According to S. pombe cells were cultured overnight in YES medium at 30°C before diluting to an OD600 of 0.2 and re-culturing for the same period. The cells were then assembled and assigned an OD600value of 3.0. A tenfold dilution series was prepared, and each dilution was spotted on plates containing 3 μl YES media containing 0.25, 0.5, 3, or 6.0 mM Fe2 (SO4)3, or was left untreated.S. pombe cells using the High Pure RNA Isolation kit (Roche). Next, quantitative RT-PCR was conducted (Stratagene) using a FastStart SYBR Green Master kit  and the Mx3005P system. Four specific primers were employed for the following genes: act1, grx4, fep1, and php4, as follows (act1: Forward (F) 5′-ACTATGTATCCCGGTATTGCC-3′, Reverse (R) 5′-GACAGAGTATTTACGCTCAGG-3′; grx4: (F) 5′-GCGTCGCCTATTGTGCAAGG-3′, (R) 5′-GTTGGTTGTCGGCAGGTTCG-3′; fep1: (F) 5′-CCCGAAGAGCCACCCTCAAA-3′, (R) 5′-CCGGCGAGGTAGAGGATTGG-3′; php4: (F) 5′-CAAGACCTGGGCCGACTTCA-3′, (R) 5′-AGCCTGCAGCATCTCCAACT-3′). We constructed the first-strand cDNA template in accordance with the manufacturer’s guidelines. Next, 50 µl of ultra-pure nuclease-free H2O was added to a mixture of 25 ml of FSSGM, 0.2 µM of each F and R primer, and 0.1 µg of cDNA. PCR was configured as follows: Pre-incubation at 95°C for 10 min, then 40 cycles of 95°C for 10 s, 55°C for 10 s, and 72°C for 20 s. The final step consisted of gradually increasing the temperature from 55°C to 95°C at a rate of 1°C/10 s to collect melting curve data , three from A. flavus , and three from humans. We found that hsGLRX5 was the most closely related by sequence homology to spGrx4 with a 50% identification rate .As part of our study, we used the protein sequences of spGrx4, spFep1, and spPhp4 as query sequences, and performed BLAST searches against the human and fungal genome databases to determine the functional and structural diversity of these proteins. For further examination of spGrx4, eight spGrx4 homologs were chosen: two from ion rate , while hion rate . Our resproteins . Our mulS. cerevisiae , two from A. flavus (afSreA and afSreP), and six from humans (hsGATA1 to hsGATA6). According to the NCBI basic sequence blast results and the two major ortholog predictors (OrthoFinder and PHYLome) tools, scDal80 is most similar to spFep1, 60.9% identity  domain, which is depicted in Ten spFep1 homologs were chosen : two froidentity . To deteidentity . Our stu domains . AccordiS. cerevisiae (scHap4), one from A. flavus (afHapX), and three from humans . The scHap4 is more similar to spPhp4 than others  domain, as depicted in We chose 5 spPhp4 homologous: one from n others . It sharn others  demonstrn others . Based oThe spGrx4 protein contains 244 amino acids, with 28 negatively charged residues (Asp and Glu) and 19 positively charged residues (Arg and Lys). At its N-terminus, spGrx4 has an additional domain containing a WAAPCK motif, similar to the WCGPCK motif in thioredoxin’s active site . spFep1 spgrx4 expressed itself more than spfep1, while spphp4 expressed itself the least. By decreasing the iron concentration, spgrx4 continued to exhibit the highest expression level, while fep1 expression decreased and spphp4 expression increased . The highly conserved residues C172GFS, which are required for Grx4’s cellular functions, are located in the C-terminal GRX-like domain of the protein  and GRX (PF00462) domains  domain  in spFep1 and its homologs. A region containing four conserved Cys residues that regulate iron homeostasis in r motifs . We also) domain . This do) domain . When ce) domain . MoreoveA. flavus, S. cerevisiae, and humans all contain the Hap4_Hap_bind (PF10297) domain. This domain is essential for scHap4 to form the scHap complex by allowing it to bind to scHap2, scHap3, and scHap5 , and in mitochondria, respectively (Our study also revealed that spGrx4 interacts strongly with spPhp4 and spFep1, and that spGrx4, spPhp4, and spFep1 interact with spPhp2, spPhp3, and spPhp5, indicating that our three proteins play cooperative roles in iron homeostasis . spGrx3,S. pombe . They arectively . At the ectively . Earlierectively . The spGectively .spgrx4 expressed itself more than spfep1, while spphp4 expressed itself the least. By decreasing the Fe concentration, grx1 continued to exhibit the highest expression level, while spfep1 expression decreased and spPhp4 expression increased (S. pombe cells inhibit the transcription of many genes that encode iron-utilizing proteins (S. pombe uses spFep1 and spPhp4 in iron-rich and iron-deficient conditions, respectively (spfep1 and spphp4 mutually regulate one another’s expression using iron-dependent mechanisms (S. pombe, spPhp2, spPhp3, and spPhp5 operate as heterotrimers to bind CCAAT cis-acting elements and activate gene expression (At the highest level of Fe, ncreased . When irproteins . Interacproteins . spPhp4 proteins . S. pombectively  to regulchanisms . Moreovechanisms . Under ichanisms . In S. ppression . This copression . spPhp4 pression . Therefopression .via its N- and C-termini TRX and GRX domains (The activities of spFep1 and spPhp4 are also controlled post-translationally by the cytosolic CGFS glutaredoxin spGrx4 . spGrx4  domains . In cont domains . Cys221  domains .S. pombe and their sequence homologs in A. flavus, S. cerevisiae, and H. sapiens, and an expression analysis with different iron concentrations to gain a deeper understanding of the critical roles of spGrx4, spPhp4, and spFep1 in iron homeostasis. Our analyses of Grx4 revealed a conserved cysteine residue in each TRX domain, and that human hsGLRX3 is most similar to spGrx4. Moreover, spFep1 is highly correlated with scDal80, whereas scHap4 is most similar to spFep1 and to spPhp4. Our analysis also showed that spGrx4 interacts strongly with spPhp4 and spFep1, and that spGrx4, spPhp4, and spFep1 interact with spPhp2, spPhp3, and spPhp5, indicating that our three proteins play cooperative roles in iron homeostasis. At the highest level of Fe, spgrx4 had the highest expression, followed by spfep1, while spphp4 had the lowest expression; a contrast occurred at the lowest level of Fe, where spgrx4 expression remained constant. Our analysis improves our understanding of the structure and function of spGrx4, spFep1, and spPhp4.Here, we performed a bioinformatics analysis on


Cryptococcus gattii. Host innate and adaptive immune responses can be subverted by C. gattii, that blocks the differentiation of T helper (Th) 1 and Th17 cells, which are involved in the protection against cryptococcosis. Moreover, the macrophage polarization is modulated by C. gattii infection that requires a balance in the macrophage subsets to control the C. gattii infection. Toll-like receptor (TLR) 2 agonists are important immunomodulators favoring a pro-inflammatory response with potential fungicidal activity, and TLR2 agonists have been used as adjuvants in vaccines against infections caused by bacteria or viruses. Therefore, this work aimed to evaluate the immunomodulatory effect of the tripalmitoyl lipopeptide S-glycerol cysteine (Pam3CSK4 or P3C4), a TLR2 agonist, as an adjuvant in the vaccination against C. gattii infection.Cryptococcosis is a relevant invasive fungal infection that affects immunocompromised and immunocompetent individuals when caused by 7 inactivated yeasts of C. gattii via intranasal route on day 1, 14 and 28 (Immunized group). Immunization was associated with 1µg or 10µg of adjuvant P3C4 (Immunized+P3C4-1µg or Immunized+P3C4-10 µg), followed by C. gattii infection on day 42 after the immunization protocol. Immunized+P3C4-1 µg group had reduced levels of IgG1, IgG2a and IgA and no significant difference in the IgG and IgM anti-GXM antibody titer was detected, compared to the Immunized group. High levels of IL-17 and IL-1β in lung tissue of mice from the Immunized+P3C4-1µg group did not promote a predominance of Th17 cells, in contrast, the frequency of TLR2+ cells was increased in immunized mice that received 1 µg of P3C4. The reduction in the relative expression of T-bet and high levels of Foxp3 detected in the lungs of the Immunized+P3C4-1µg group suggest a prevalence of regulatory T cells in the tissue, which did not contribute to the control of C. gattii infection. The immunization protocol associated with 10 µg of adjuvant P3C4 induced high levels of IL-17 in the lung tissue, whereas the levels of pro-inflammatory cytokines were downregulated. To evaluate the effect of adjuvant P3C4 in the control of C. gattii infection, quantification of the fungal burden in the lungs was performed by the CFU assay, and the groups with adjuvant P3C4 showed a pulmonary C. gattii burden that was not significantly altered when compared with the immunized group. The mice that received 1 µg of adjuvant P3C4 had a lower percentage of inflammatory infiltrate in the lungs.C57BL/6 mice were immunized with 2 × 10C. gattii infection, which is related to the immune response characterized by a suppressive/regulatory profile in the pulmonary microenvironment after C. gattii infection.The immunomodulatory effect of P3C4, associated with the immunization protocol, plays an imbalance between pro- and anti-inflammatory response in the lungs that did not favor a protection against  Cryptococcus gattii is a relevant fungal species that causes disseminated infection, namely cryptococcosis, in immunocompromised patients as well as in healthy individuals   .γ, interleukin (IL)-2, and IL-12, unlike the Th2 subset, which is associated with an anti-inflammatory response , which leads to increased expression of this complex on the cell surface. Furthermore, dendritic cell (DC) presentation of epitopes via MHC-II to specific T cells increases in the presence of the TLR2 agonist P3C4 (Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that are composed of 12 receptors in mice. These receptors are produced in the endoplasmic reticulum and recruited to the cell membrane or intracellular compartments, such as endosomes . After tP kinase . TLR2 foP kinase , zymosanP kinase , phosphoalbicans , and glytophilum . Synthettophilum . Cell acist P3C4 . Therefoist P3C4 .Cryptococcus neoformans could partially protect against C. neoformans infection (C. gattii associated with the adoptive transfer of dendritic cells (DC) pulsed with an acapsular strain of C. gattii (ΔCAP60) , associated with the culture filtrate antigen (CneF) of nfection . There w(ΔCAP60) . The pro. gattii . In addihe lungs . To datehe lungs . The TLR B cells .C. gattii. Doses of 1 µg and 10 µg of adjuvant P3C4 were tested, and it was demonstrated that 10 µg of P3C4 reduced the levels of pro-inflammatory cytokines in the lung tissue and did not alter the subtypes of CD4+ T cells. The administration of 1 µg of P3C4 induced the predominance of Th17-associated cytokines in the lung tissue and a prevalence of Foxp3 transcription factor that characterizes regulatory CD4+ T cells (Treg). However, this microenvironment in the lung tissue is not sufficient to control C. gattii infection.The development of vaccines to combat cryptococcosis is an advancement aimed at mitigating drug-resistant organisms and antifungal side effects. The present study evaluated the immunomodulatory effects of P3C4, a TLR2 agonist, as an adjuvant in an immunization strategy against C. gattii infection.Male C57BL/6 mice (6–8 weeks old) were acquired from the animal facility at the Campus of Ribeirão Preto of the University of São Paulo, Brazil. The mice were housed in the animal facility of the Department of Cell and Molecular Biology and Pathogenic Bioagents of Ribeirão Preto Medical School, and all animal experiments were performed according to the Ethical Principles Guide for the Care and Use of Laboratory Animals adopted by the Brazilian College of Animal Experimentation. The protocols were approved by the Committee on Ethics in Animal Research of Ribeirão Preto Medical School (protocol 072/2019). Mice were anesthetized with ketamine hydrochloride  and xylazine hydrochloride  as previously reported , and theC. gattii strain  was recovered from 25% glycerol stocks stored at −80 °C and cultured on Sabouraud dextrose medium at 30 °C with constant agitation (150 rpm) for 16–18 h. C. gattii yeast with a thin capsule (C. gattii-thin) were obtained after inoculation in YPD medium containing 0.5 M NaCl, as described by C. gattii-thin was heat-inactivated at 70 °C for 1 h (HK-C. gattii-thin). The cell concentration was determined using a Neubauer chamber with Chinese ink. A fraction of the HK-C. gattii-thin yeast was spread on Sabouraud dextrose agar to confirm the heat inactivation.R265 C. gattii that were cultured on Sabouraud dextrose medium at 30 °C with constant agitation (150 rpm) for 16–18 h. Yeast cells were harvested by centrifugation at 7,600 × g for 10 min at 25 °C and washed three times with sterile PBS (Thermo Fisher Scientific). Cell concentration was determined using a Neubauer chamber with Chinese ink.C57BL/6 mice were infected with viable yeast of 7 H.K.-C. gattii with or without P3C4 (1 or 10 µg/mouse) intranasally, and the control group received PBS alone (n = 5 in each group). Immunization was performed three times with a two-week interval among them . The animals were separated into the following groups: PBS, Immunized, Immunized +P3C4 (1 µg), and Immunized +P3C4 (10 µg). On day 35, the blood was collected to measure the levels of IgM and IgG anti-GXM. On day 42, mice were anesthetized and infected with 1 × 105C. gattii yeast cells intranasally. Fourteen days after infection (day 56), the animals were euthanized to collect blood samples and lungs.Mice were anesthetized with ketamine hydrochloride  and xylazine hydrochloride . Animals were immunized with a cell suspension of 2 × 10C. gattii infection (day 56), and the tissue was stored in one mL cold 1x sterile PBS buffer (pH 7.2) and immediately weighed and homogenized . Suspensions of homogenized lung tissue were diluted and plated on Sabouraud dextrose (Oxoid) agar medium containing 100 µg/mL ampicillin. The plates were incubated at 30 °C for 48 h, and the C. gattii colonies were counted to perform the colony forming unit (CFU) assay, which is expressed in CFU/g.The lungs of the animals were removed 14 days after C. gattii infection (day 56), the serum was obtained from all mice to quantify the levels of the immunoglobulins isotyping  using the Easy-Titer™ Mouse IgG Assay Kit (Thermo Fischer Scientific) as previously reported  for 2 h at 25 °C  for blocking. The plate was washed five times and mouse serum was added and incubated for 2 h at RT, furthermore, the plate was washed five times. Then, goat anti-mouse IgM (μ-chain specific)-peroxidase antibody  or rabbit anti-mouse IgG (whole molecule)–peroxidase antibody  was added in the plate and incubated for 1 h. The plates were washed seven times and 50 µL 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added. After 30 min of incubation at RT, 30 µL of stop solution (2N H2SO4) was added. Absorbance was recorded at 450 nm using a spectrophotometer .The levels of IgG and IgM anti-GXM in the serum of the animals were measured 7 days after the immunization protocol (day 35) and it was performed in 96-well microplates coated with 20 µg/mL GXM isolated from d Levitz , and incC. gattii infection (day 56). The tissue was stored in one mL cold 1x sterile PBS buffer (pH 7.2), immediately weighed, and homogenized . The supernatant was collected after centrifugation  and a protease inhibitor was added . Cytokine levels were quantified by ELISA using the OptEIA™ Kit  according to the manufacturer’s instructions. IFN-γ, IL-12(p70), IL-10, IL-4, IL-17A, TNF-α, IL-1β, IL-2, and IL-6 levels were measured in the lungs, and IFN-γ, IL-17, and IL-4 levels were quantified in the serum. The levels of cytokines in the tissues were normalized relative to the organ mass (mg) and expressed in pg/mL.Lung homogenates of the animals were obtained 14 days after 6 cells/mL from each sample were stained with antibodies conjugated to fluorophores. Cells were incubated for 30 min at 4 °C with 0.5 µg of anti-CD16/CD32 monoclonal antibody . Subsequently, 5 µg/mL of the following antibodies (obtained from BD Pharmingen) were used: anti-CD3 , anti-F4/80 , anti-CD11c  and anti-TLR2-PE . The cells were incubated for 45 min with the antibodies, and washed twice with PBS buffer and fixed with PBS-formaldehyde (1%). After these steps, the percentage of cells positive for CD3, F4/80, or TLR2 was measured using flow cytometry (Guava easyCyteTM Mini System).Pulmonary leukocytes were obtained as previously described by + T cell differentiation was determined by the relative expression of T-bet, ROR γt, GATA-3, Foxp3, and TGF-β transcripts, and macrophage polarization was evaluated by the relative transcript expression of Arginase-1, iNOS, and IL-23. Relative transcript expression was quantified using the ΔΔCt method and normalized to β-actin expression. The primers used for PCR reaction were: β-actin (F: 5′-CCTAAGGCCAACCGTGAAAA-3′/R: 5′-GAGGCATACAGGGACAGCACA-3′), T-bet (F: ′-CACTAAGCAAGGACGGCGAA-3′/R: 5′-CCACCAAGACCACATCCAC-3′), ROR γT (F: 5′-TGGAAGATGTGGACTTCGTT-3′/R: 5′-TGGTTCCCCCAAGTTCAGGAT-3′), GATA-3 (F: 5′-AAGAAAGGCATGAAGGACGC-3′/R: 5′-GTGTGCCCATTTGG ACATCA-3′), Arginase-1 (F: 5′-GTTCCCAGATGTACCAGGATTC-3′/R: 5′-CGATGTCTT TGGCAGATATGC-3′), iNOS2 (F:5′-CCGAAGCAAACATCACATTCA-3′/R: 5′-GGTCTAAAGGCTCCGGGCT-3′), IL-23 (F: 5′TCCGTTCCAAGATCCTTCGA3′/R: 5′TGTTGGCACTAAGGGCT CAG3′), Foxp3 (F: 5′-ATTGAGGGTGGGTGTCAGGA-3′/R: 5′-TCCAAGTCTCGTCTGAAGGCA-3′), and TGF-β (F: 5′-GACTCTCCACCT GCAAGACC-3′/R: 5′-GGGACTGGCGAGCCTTAGTT-3′).Total RNA was isolated from the lung cells using TRIzol reagent  according to the manufacturer’s instructions. Total RNA was converted to cDNA using an iScript cDNA Synthesis Kit . qRT-PCR was performed in a final volume of 10 µL using the EvaGreen Supermix (Bio-Rad). The CFX96 Touch System  was used under the following reaction conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 91.5 °C -−95 °C for 5 s. CD4 2) in a total area of one mm2 per animal.The procedures to obtain the lungs and the preparation of tissue samples for histology were performed as reported by . Moreovep <0.05), one-way ANOVA followed by Tukey’s multiple comparisons test or Kruskal–Wallis test followed by Dunn’s multiple comparisons test was performed.All data were analyzed using Prism 7.0 , and the results were expressed as mean ± standard deviation (SD). Homogeneity of variance was analyzed using the Kolmogorov–Smirnov test. For all data with Gaussian distribution, an ANOVA test followed by Bartlett’s test was applied to the three groups of experiments . In all data with a non-normal distribution, the Kruskal–Wallis test was applied to the three groups of experiments. To determine the differences between the means of groups  at a two-week interval . The animals were infected with live C. gattii on day 42, as shown in C57BL/6 mice were immunized with three inoculums of heat-killed C. gattii infection (day 56). The supernatant of the mouse lung homogenate was used to quantify the levels of IFN-γ, IL-12p70, TNF-α, IL-1β, IL-2, IL-17, IL-6, IL-10, and IL-4 , alveolar macrophages and dendritic cells are unaltered.Since alterations in cytokine levels were observed in the lungs of immunized mice that received adjuvant P3C4, the frequency of alveolar macrophages, T cells, and TLR2+ cells could indicate immunomodulatory activity induced by 1 and 10 µg of P3C4. Therefore, a suspension of pulmonary leukocytes obtained from each group was incubated with anti-CD3, anti-F4/80 or anti-CD11c antibodies, and the frequency of T cells, alveolar macrophages and dendritic cells was measured by flow cytometry –3C. On dµg group . The levµg group . These d+ T cells was investigated by qRT-PCR using molecular markers that characterize M1/M2 subsets  and Th1/Th2/Th17/Treg profiles . The relative expression of iNOS and Arginase-1 quantified in all groups did not differ  to measure the fungal burden by the CFU assay. The groups with 1 µg or 10 µg of adjuvant P3C4 showed a pulmonary C. gattii burden that was not significantly altered when compared with the immunized group  in vitro to produce high levels of cytokines related to Th1/Th17 responses, with a likely contribution of TLR4 and TLR9, but not TLR2. C. neoformans with a minor role of CD14 and TLR2 . In contrast, C. neoformans, and C. gattii induced a TLR2-mediated response and high production of NO by macrophages . Nevertheless, immunomodulation of TLR2 agonists can be a relevant strategy to assemble a robust inflammatory response, although a balance between pro- and anti-inflammatory responses is necessary. A previous study by our group revealed that TLR2 agonists can polarize peritoneal macrophages to the M1 subset, producing robust immunity; however, it did not result in a fungistatic effect on the growth of C. gattii (C. gattii. Alveolar macrophages represent more than 90% of the cells in the alveolar space (The administration of synthetic TLR2 agonists can be relevant because of the induction of a pro-inflammatory immune response that is beneficial to combat cryptococcosis. Although soluble GXM, a major component of the and CD14 , the rol. gattii , and prear space , and theC. gattii, and the animals in the immunized +P3C4-1 µg group had reduced levels of IgG1, IgG2a, and IgA. The levels of these immunoglobulins indicate that immunization with P3C4-1 µg did not favor the production of effector isotypes against C. gattii infection, since IgG1 is the most abundant isotype after vaccination, and the IgG2a isotype is important for the recognition of carbohydrates.Previous studies have shown that specific antibodies against GXM during infection can be produced at low titers; therefore, the administration of monoclonal antibodies specific to GXM has shown promising results in the control of cryptococcosis in murine models and clinical studies . We evalβ in the lung tissue of animals that received 1 µg of adjuvant; however, the relative expression of the transcription factor ROR γt did not change significantly among the groups. These data demonstrate that P3C4 induced the production of IL-17 in the lung tissue even in the absence of a predominance of the transcription factor ROR γt that is a specific marker of Th17 cells. Previous studies have revealed that C. neoformans infection causes elevated levels of IL-17 in the lungs in the absence of Th17 cells (β were also detected in PBMC incubated with C. gattii (C. gattii yeast containing a mutation in E3 ligase led to the induction of IL-17-producing CD4+ T cells that did not play an essential role in protecting mice against C. gattii infection (We observed high levels of IL-17 and IL-117 cells , and inc. gattii . Furthernfection .+ T cells does not contribute in combating C. gattii infection. These data are justified by other vaccination strategies that have been successful in controlling cryptococcosis through the induction of Th1 cells, along with high levels of IFN- γ (C. gattii infection. In addition, the capacity of TLR2 activation to induce the production of IL-10 by APCs and T cells associated with the presence of pro-inflammatory cytokines can maintain the suppressive function of Treg cells, and this regulation can be modified as verified in the current work that showed a discrepancy in the balance of immune response between the adjuvant P3C4 at different concentrations. An interesting finding is the ability of Treg cells to suppress the Th2 response through the suppression of IRF-4 in the lungs, which promotes the growth of C. neoformans (C. gattii infection progression must be well studied. In addition, evaluation of animals that received 10 µg of adjuvant P3C4 demonstrated an absence of high levels of relative expression of Foxp3, T-bet, and ROR γt. Administration of 10 µg of P3C4, associated with immunization, maintained elevated levels of IL-17 in the lungs after C. gattii infection; however, the cytokine profile in the lungs was characterized by a reduction in the levels of IFN-γ, IL-12p70, IL-6, and IL-10. These findings suggest an immunosuppressive effect in the lungs through high concentrations of P3C4 associated with inactivated yeasts of C. gattii. Finally, the high IL-4 serum levels in the animals of the immunized group in association with 10 µg of P3C4 and infected with C. gattii reinforce the suppressive effect of the TLR2 agonist in the induction of cytokines and Th1 cells. Therefore, the immunomodulatory effect of adjuvant P3C4 is related to an immune response with suppressive/regulatory characteristics after C. gattii infection, which impairs the control of cryptococcosis.The animals in the immunized +P3C4-1 µg group revealed a reduction in the relative expression of T-bet and increased relative expression of Foxp3, suggesting that immunization associated with P3C4-1 µg promotes the prevalence of regulatory T cells in the lung tissue along with a low frequency of Th1 cells, and this balance of differentiated CD4f IFN- γ . There ioformans . HoweverC. gattii associated with different doses of TLR2 agonist that should induce a predominant pro-inflammatory response, which is considered beneficial to combat cryptococcosis. We demonstrated that the immunomodulatory effect of adjuvant P3C4 occurs in the lungs of immunized mice, but it did not culminate in protection against C. gattii infection, and suppressive/regulatory characteristics in the microenvironment of pulmonary tissue were remarkable after C. gattii infection.In this study, mice were immunized with heat-killed yeast of 10.7717/peerj.14778/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14778/supp-2Supplemental Information 2Click here for additional data file.


Preliminary study results demonstrated parenchyma-sparing surgery (PSS) as an effective approach which allowed to remove colorectal cancer (CRC) metastatic lesions within the central liver cites and increased the probability of the liver re-resections.The prospective analysis re-evaluation of the 185 CRC patients surgical treatment has been performed.CI: 44.4–66.1) months. The 5-year OS for CRC patients whose metastatic lesions were predominantly located within peripheral and central liver segments was 56 ± 8% and 27 ± 9%, respectively (p = 0.08). A 5-year disease-free survival (DFS) rates of patients with peripheral and central liver cites metastatic lesions were 31 ± 7 % and 15 ± 7%, p = 0,12. And the DFS median was 34.2 and 46.5 months for R1v and R0 cohorts, respectively, p = 0.62.An overall 5-year survival (OS) of the 185 enrolled patients was 43 ± 7%, and the mean and median value for OS was 48.7 ± 1.9% and 55.2 ± 5 (95% Parenchyma-sparing surgery should be a priority pathway for complex treatment of patients with deeply located lesions of the right liver lobe.https://www.researchregistry.com/browse-the-registry#home/registrationdetails/5ed9f60863e9bf0016624456/, no. 5679.The study is registered in  CRC is ranged 3rd in the structure of the most common malignant neoplasms in Ukraine . Proper approach . Howeverapproach . PrelimiThe aim of this article was to show the long-term oncological results of PSS strategy for CRC liver surgery.p = 0.05 was taken as the critical level of significance when testing statistical hypotheses. Patients’ overall (OS) and disease-free survival (DFS) have been analyzed according to the Kaplan–Meier method. The multivariate Cox regression analysis of factors contributing to overall survival of CRC patients has been performed.The prospective analysis re-evaluation of the 185 CRC patients surgical treatment has been performed. The description of the liver surgery aspects, study design, inclusion and exclusion criteria, and endpoints has previously been published . StatistCI: 44.4–66.1) months, while CRC patients OS whose metastatic lesions were predominantly located within peripheral and central liver cites was 56 ± 8% and 27 ± 9%, respectively (p = 0.08)  and 46.5 ± 6.5 (95% CI: 33.7–59.3) months, respectively  of the 185 enrolled patients was 43 ± 7%, and the mean and median value for OS was 48.7 ± 1.9% and 55.2 ± 5  of CRC patients with R1v margin did not reach a 5-year follow-up, the DFS median was 34.2 and 46.5 months for R1v and R0 cohorts, respectively, p = 0.62  had no impact on survival rate in either univariate analysis or MVA. During 5 years, no local recurrence was detected on the radiological follow-ups in the cohort of 27 patients who underwent intraparenchymal preservation of 1–2 order vascular structures. Consequently, we believe that the adaptation of PSS in CRC patients has the potential of liver surgery personification.The modern concept of CRC is based on the perception of the metastatic process as a chronic illness and not as a lethal disease . That isstrategy . This stParenchyma-sparing surgery has shown appropriate surgical and oncological outcomes of treatment of the colorectal cancer patients with bilobar liver metastases. Parenchyma-sparing surgery should be a priority pathway for complex treatment of patients with deeply located lesions of the right liver lobe.