Herein, the as-prepared VO2(A) hollow spheres via a feasible hydrothermal reaction exhibit superior zinc ion storage performance, large reversible capacity of 357 mA h g−1 at 0.1 A g−1, high rate capability of 165 mA h g−1 at 10 A g−1 and good cycling stability with a capacity retention of 76% over 500 cycles at 5 A g−1. Our study not only provides the possibility of the practical application of ZIBs, but also brings a new prospect of designing high-performance cathode materials.Rechargeable aqueous zinc ion batteries (ZIBs), owing to their low-cost zinc metal, high safety and nontoxic aqueous electrolyte, have the potential to accelerate the development of large-scale energy storage applications. However, the desired development is significantly restricted by cathode materials, which are hampered by the intense charge repulsion of bivalent Zn 2(A) hollow spheres exhibit superior zinc ion storage performance, large reversible capacity of 357 mA h g−1 at 0.1 A g−1, and good cycling stability with a capacity retention of 76% over 500 cycles at 5 A g−1VO In the process of exploring alternative systems, rechargeable multivalent metal ion batteries, such as Al-ion battery, Ca-ion battery and Zn-ion battery, have shown potential application due to their abundant sources and environmental friendliness.8–13 In particular, rechargeable Zn-ion batteries will play a role in energy storage devices owing to the following advantages: (a) the high theoretical Zn metal anode capacity (820 mA h g−1), and the low oxidation–reduction potential of Zn2+/Zn (−0.76 V vs. standard 2H+/H2); (b) the low-cost (USD $2 kg−1) and abundant Zn sources, as well as the nontoxic and safe aqueous electrolyte; and (c) transfer of more charges during electrochemical reactions to provide more energy storage than the univalent batteries.14–21Considering the increasingly serious energy shortage, climate change and other force majeure environmental factors, green renewable energy has become an active area of research. Secondary battery technologies afford a feasible way to store and convert the renewable energy sources; particularly, lithium-ion batteries account for half of the economic markets.et al. using MnO2 as the Zn-storage cathode with zinc sulfate or nitrate aqueous electrolyte.22–26 Afterwards, open-framework materials were extensively investigated. To date, some other layered or tunnel-type vanadium-based compounds, such as V2O5·H2O,27,28 H2V3O8,29 Na3V2(PO4)3 2·2H2O nanowires, showing an initial discharge capacity of 148 mA h g−1 at the current density of 200 mA g−1 and the capacity retention is 68% after 300 cycles.29 Liang et al. reported Na1.1V3O7.9 nanoribbon/graphene, showing a capacity of 220 mA h g−1 at the current density of 300 mA g−1 with a capacity retention of 77% after 100 cycles.32The first aqueous ZIB was reported by Kang V2(PO4)3  and VS2,2(A) hollow spheres have been synthesized and tested as a cathode for rechargeable aqueous zinc ion battery. The as-prepared VO2(A) hollow spheres delivered superior electrochemical performance including large specific capacity (357 mA h g−1 at 0.1 A g−1), high rate capability (165 mA h g−1 at 10 A g−1) and good capacity retention (76% over 500 cycles at 5 A g−1).Herein, novel VO2.2.14VO3 was added to 45 mL distilled water and stirred at 80 °C for 10 min. After cooling to room temperature, 1 mmol HCl solution was added dropwise to the above solution and stirred slowly for 2 min. Then, 3 mL N2H4·H2O was added dropwise into the solution with continuous stirring until its color gradually changed from yellow to gray. The above suspension was poured in a 50 ml Teflon-lined autoclave and kept at 120 °C for 4 h. After the reaction, the samples were washed with distilled water and ethanol three times to remove any possible residues and dried in vacuum. Finally, the resultant products were heated under Ar gas in a tube furnace at the temperatures of 300 °C, 350 °C and 400 °C for 2 h with an increasing rate of 5 °C min−1, respectively.Initially, 2 mmol NH2.2λ = 1.5406 Å) was used to investigate the structure of the materials. The morphology of the materials was studied by a Hitachi-4800 scanning electron microscope (SEM). High resolution transmission electron microscopy (HRTEM) was performed on a JEM-2100 PLUS high resolution transmission electron micro-scope. X-ray photoelectron spectroscopy (XPS) was conducted on an ESCALAB250 spectrometer using an Al Kα source. Total pore volumes of the materials were determined using the adsorbed volume at a relative pressure of 0.99. The multi-point Brunauer–Emmett–Teller (BET) surface area was estimated in the relative pressure range from 0.05 to 0.2.An X-ray diffractometer  with Cu Kα radiation (2.3−1 zinc trifluoromethanesulfonate (Zn(CF3SO3)2). Galvanostatic charge–discharge experiments were performed on a Land-2100 automatic battery tester. Cyclic voltammetry (CV) and galvanostatic intermittent titration technique (GITT) measurements were studied on a Bio-Logic VSP-300 multichannel.Electrochemical tests were performed by using 2032-type coin cells, where a metallic zinc foil was used as the anode electrode. The working electrode was prepared as follows. First, 70 wt% active material and 20 wt% Super P conductive additive were mixed with alcohol for 1 h. Then, 10 wt% polytetrafluoroethylene (PTFE) was added into the above mixture and rolled to a film on a carbon paper. Lastly, the working electrode was dried overnight under air at 60 °C. The electrode mass loading on the carbon paper was about 1 mg. The electrolyte was 3 mol L3.2(A) hollow spheres were characterized by XRD, as shown in 2(A) annealed at 350 °C, five broad major reflections are located at 2θ = 14.9°, 25.5°, 29.9°, 33.3°, and 45.5°, which correspond to the (110), (102), (220), (212) and (330) crystal planes . No diffraction peaks of other V–O impurities were detected, indicating the successful preparation of VO2(A) with purity. In addition, we prepared vanadium dioxide samples at the temperatures of 300 °C and 400 °C, as shown in 2(M) (JCPDS #44-0252). 3/2 in the VO2(A) could be resolved into two peaks at 516.2 and 517.5 eV, corresponding to V4+ and V5+.33,34 The intermediate oxidation peak between V5+ and V4+ is assigned to partial interaction with atmospheric oxygen because of the high surface area of the nanomaterials.35,36The crystal structures of VO2(A) hollow spheres can be described by the following reaction:In our experiment, the multi-step synthesis of VO2NH2 suspension can be formed by adding N2H4·H2O with the solution color changing from yellow to gray. After being sealed in the autoclave at 120 °C for 4 h, the VOOH hollow spheres were obtained.37,38 For the third step, VOOH was oxidized to VO2(A) hollow spheres, which could be due to the small amount of oxygen gas existing in the Ar gas.The reaction 2(A) sample, FESEM tests were performed, as shown in 2(A) submicro-spheres is between 300–500 nm. From some broken spheres, VO2(A) spheres can be clearly observed with the hollow interior, which was further confirmed by the TEM image, as displayed in 2(A) hollow spheres. The lattice fringe with an interplanar distance of 0.301 nm corresponds to the (220) plane of the VO2(A) hollow sphere. Furthermore, the specific surface area and porosity of the as-prepared VO2(A) hollow spheres were measured by nitrogen gas adsorption–desorption isotherm, as shown in P/P0, indicating the existence of mesoporous and a high BET surface area up to 55.5 m2 g−1, with the pore size of 7.5 nm and pore volume of 0.16 cm3 g−1. The high surface area of VO2(A) hollow spheres was favorable for fast ion transport during the de/intercalation process, further facilitating the electrolyte uptake.In order to observe the microscopic morphology of the VO−1 to investigate the redox reaction of VO2(A) hollow spheres annealed at 350 °C in the range of 0.2–1.4 V. The initial three cycles of the CV profiles were almost overlapping, indicating the high reversibility of VO2(A) hollow spheres. The three pairs of easily identifiable reduction/oxidation peaks were positioned at 0.90/1.02 V, 0.52/0.68 V, and 0.44/0.58 V, respectively, attributing to a three step reaction associated with Zn2+ ion intercalation and extraction of the VO2 lattice.392(A) hollow spheres at 300 °C, 350 °C and 400 °C at the current density of 100 mA g−1, respectively. It was worth noting that the large initial charge/discharge capacities of 370/357 mA h g−1 at 350 °C had been received at this low current density, finally achieving a high coulombic efficiency of 104%. The VO2(A) products synthesized at 300 °C and 400 °C delivered the initial charge/discharge capacities of 338/324 and 280/276 mA h g−1, respectively, which were inferior to those of the VO2 sample synthesized at 350 °C. The rate capabilities of the above three VO2(A) hollow spheres had been investigated in 2(A) hollow spheres synthesized at 350 °C exhibited average discharge capacities of 357, 350, 325, 306, 285, 246, and 165 mA h g−1 at current densities of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 A g−1, respectively. In addition, when the current density returns to 0.2 A g−1, the reversible capacities could generally recover to the corresponding original values under various current densities. It was also noted that when the current densities increased to 5 and 10 A g−1, VO2 materials at 300 and 400 °C had almost no available capacity at these high current rates, which were much worse than those of VO2(A) materials at 350 °C. This could be due to the low crystallinity of the sample at 300 °C and small layer spacing (d = 3.20 Å) of VO2(M) at 400 °C compared to that of VO2(A) (d = 5.95 Å), both of which inhibit the fast Zn-ion insertion/extraction process. The cycling stability of VO2(A) material was tested at the current density of 0.2 A g−1, as shown in 2(A) sample synthesized at 300 °C rapidly decreased after 50 cycles. In comparison, the VO2 sample synthesized at 350 °C and 400 °C was significantly improved with the retention of 94.1% and 92.5% of the maximum discharge capacity after 50 cycles. Lastly, the long-term cycle stability of the sample synthesized at 350 °C was investigated at the current density of 5 A g−1 with a pre-activation at 200 mA g−1 for the front five cycles, as shown in −1 is about 202 mA h g−1. After 500 cycles, VO2(A) hollow spheres demonstrated a capacity of 154 mA h g−1 with a good retention of 76%, corresponding to a capacity decay rate of 0.096% per cycle.2(A) electrode synthesized at 350 °C, CVs at the various scan rates from 0.1 to 1.0 mV s−1 were tested. Based on these scan data, the current (i) of CVs could comply with a power-law formula with the scan rate (ν) in the light of 40,41i is defined as the current density, ν is defined as the scan rate, and a and b are the adjustable parameters. When the b-value is 0.5, it suggests that the process is a diffusion-controlled process. When b-value is 1, it presents a capacitive-controlled process.42 The b values of the four redox peaks were calculated to be 0.74, 1.12, 0.75, and 1.03, respectively, as shown in 2+ diffusion kinetics and high-rate performance. Furthermore, the capacity can be separated into capacitive-controlling (k1ν) and diffusion-controlling (k2ν1/2) at a specific scan rate according to the following 43In order to assess the capacitive and diffusion contributions to the total capacity of the VO44As depicted in 2 hollow spheres, the Zn2+ diffusion coefficients (D2+Zn) of the VO2(A) cathode were further determined by GITT. The GITT data were collected at a current density of 100 mA g−1 for 10 min and a rest interval of 30 min in 2(A) hollow sphere electrode can be calculated as follows:45τ is the constant current pulse time, Vm, mB and MB are the molar volume, the mass loading and the molar mass of the inserted electrode material, respectively, S is the area interface between electrode material and electrolyte and ΔEτ and ΔES represent the steady-state voltage change by the current pulse and voltage change during the constant current pulse, respectively. 2+ diffusion coefficients of VO2(A) are in the range from 6.8 × 10−10 to 1.1 × 10−10 cm2 s−1, which is similar to that of VO2(D) hollow spheres.46 The high diffusion coefficient indicates the rapid Zn2+ movement in the VO2(A) hollow spheres.Additionally, to further understand the details of the electrochemical behaviors of VOEx situ XRD was performed to investigate the evolution of the crystal structure during the discharge and charge process in the VO2(A) hollow spheres. ex situ XRD patterns of the VO2(A) electrode at different charge and discharge states in the first cycle. No significant shift of any diffraction peaks could be observed, indicating the stability of the structure of the VO2(A) electrode during the discharge–charge process. This can be attributed to the high capacitive behavior of the VO2(A) electrode in the ZIBs. Moreover, HRTEM analysis was carried out after discharging at 0.2 V, as shown in 2(A) lattice expanded from 0.301 to 0.309 nm, corresponding to an increase in the c-parameter relative to the pristine VO2(A). The slight expansion of the lattice plane can be attributed to the large amounts of Zn2+ intercalation into the tunnels of VO2(A).2(A) cathode after the long-term cycling, XRD and SEM after 500 cycles at the current density of 5 A g−1 were performed, as displayed in 2(A) at 2θ = 15.4°, 25.5°, 30.2°, 33.2° and 45.4° can still be observed, indicating a stable structure of the sample in the zinc ion intercalation/de-intercalation process. Moreover, the morphology of the VO2(A) hollow spheres is well maintained after cycling, confirming the structure stability of the material during the long-term cycling.To provide further insight into the structural change of the VO2(A) electrode along with reported cathode materials for ZIBs. Compared to other cathode materials such as Zn3V2O7(OH)2·2H2O,47 Zn2V2O7,48 H2V3O8,35 Na2V6O16·3H2O49 and NH4V4O10,50 VO2(A) exhibited a competitive energy density and power density . When the power density was as high as 2736 W kg−1, the energy density remained at a high value of 137 W h kg−1. This demonstrates that VO2(A) hollow spheres are a promising cathode material for ZIBs.4.2(A) hollow spheres with a high rate capability and large capacity have been investigated in this study. Satisfactorily, the VO2(A) hollow spheres delivered large specific capacity of 357 mA h g−1 at the current density of 0.1 A g−1, high rate performance with high rate capability of 165 mA h g−1 at 10 A g−1 and good cycling stability with a capacity retention of 76% over 500 cycles at 5 A g−1. Furthermore, the zinc storage mechanism and electrochemical dynamic properties of the VO2(A) cathode have also been analyzed systematically through DSCV, GITT, ex situ XRD and HRTEM measurements. The highly reversible de/intercalation reaction of bivalent Zn2+ can be attributed to the stable structure of VO2(A) hollow spheres, high capacitive-controlled contribution and good diffusion coefficient.In conclusion, VOThere are no conflicts to declare.


Litopenaeus vannamei) using three different methods: 1) chronic toxicity after 28 days of feeding, 2) Effects against white spot syndrome virus (WSSV) challenged by oral route, and 3) transcriptional responses of immune-related genes  following WSSV infection. The results showed that the feed-AgNPs did not interfere with the growth and survival of shrimp. Also, mild lesions in the hepatopancreas were recorded, proportional to the frequency of the feed-AgNP supply. Challenge test versus WSSV showed that feeding every 7 days with feed-AgNPs reduced mortality, reaching a survival rate of 53%, compared to the survival rates observed in groups fed every 4 days, daily and control groups of feed-AgNPs for the 30%, 10%, and 7% groups, respectively. Feed-AgNPs negatively regulated the expression of PAP, ProPO, and Crustin genes after 28 days of treatment and altered the transcriptional responses of PAP, ProPO, CTL-3, and Crustin after WSSV exposure. The results showed that weekly feeding-AgNPs could partially prevent WSSV infection in shrimp culture. However, whether or not transcriptional responses against pathogens are advantageous remains to be elucidated.In recent years, the application of silver nanoparticles (AgNPs) as antibacterial compounds has been widely used in human and veterinary medicine. In this work, we investigated the effects of AgNPs (Argovit-4®) as feed additives (feed-AgNPs) on shrimp ( Litopenaeus vannamei) has grown from 2.6 million tonnes in 2010 to 4.9 million tonnes in 2018  is the primary mechanism of AgNPs toxicity . The antThe shrimp’s hepatopancreas (HP) is the principal organ that carries out the digestive process. It is the first organ to receive the nutrients and components of all the substances it ingests, including contaminating elements such as heavy metals that can cause cellular damage . TherefoThe crustacean immune system is based on innate immunity, mediated by hemocytes, through cellular processes such as phagocytosis, encapsulation, and nodule formation , synthesWe found that feeding shrimp once a week with AgNPs (Argovit-4®) in the feed did not affect shrimp growth and helped counteract the effects of WSSV infection.L. vannamei shrimp were donated by Inmobiliaria Osiba, SA de CV, Sinaloa, Mexico. Shrimp were selected and transported to Interdisciplinary Research Center for Integrated Regional Development facilities in Sinaloa, Mexico. Shrimp were acclimated for a week in 1,000 L tanks with filtered seawater at 30 practical salinity units (PSU), 25 ± 2 °C and constant aeration.Vibrio parahaemolyticus, which is the causal agent of acute hepatopancreatic necrosis disease (VpAHPND), following the protocols described by Ten shrimp were analyzed by PCR with specific primers to verify that they were free of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV) or According to a previous study , ArgovitThirty shrimp (15 ± 2 g each) were injected intramuscularly with 100 µl of the WSSV inoculum each, between the 3rd and 4th abdominal segments. The inoculum was prepared from experimentally infected shrimp as previously described . After iBefore the challenge test, the muscle tissue was thawed, weighed, and liquefied with seawater in a food processor (Nutribullet-600), filter through a 1.35 mm plastic mesh and add it immediately to the infection tank.AgNPs included in the feed (Feed-AgNPs) were supplied in three different cycles for 28 days to assess the chronic toxicity of shrimp (4 ± 0.5 g). The experimental system comprised 12 tanks (150 L each) in a recirculating aquaculture system (RAS) filled with seawater at 30 PSU, filtered and chlorinated. Twenty shrimps were placed in each tank (three tanks per treatment), 5% of the body weight was fed daily, and the diets were divided into two rations (9:00 and 16:00 h) for 28 days. Feed-AgNPs were supplied once in the morning (2.5% of the body weight) in three different cycles: shrimp fed feed-AgNPs every day (D1); shrimp fed feed-AgNPs every four days (D4); shrimp fed feed-AgNPs every 7 days (D7), and the control group was fed without AgNPs (Ctrol). Temperature, dissolved oxygen (DO), and mortality were recorded daily. Growth, percent weight gain (WG), specific growth rate (SGR), feed conversion factor (FCR), and survival were recorded weekly. Diet performance was assessed by calculating percentage of body weight gain WG = [/initial body weight] × 100; specific growth rate SGR = 100 /days in culture; feed conversion ratio FCR = total dry feed intake (g)/wet weight gain (g); percentage of survival =  × 100.ad libitum, organic waste was removed by siphoning daily, 50% water exchange was performed every two days, and mortality was recorded twice a day for 10 days. Dead or dying shrimp were removed from the aquarium and stored at −20 °C for later analysis. At the end of the experiment, three dead and three surviving shrimp from each treatment were analyzed by PCR to confirm WSSV status.At the end of bioassay 1, shrimp were orally challenged with WSSV by providing infected shrimp muscle. A total of 40 shrimp from each treatment were placed in cages (5 mm plastic mesh) into a 100 L tank containing 40 L of seawater (30 PSU) at 27 ± 1 °C, and constant aeration. Just before the challenge, 40 g of infective shrimp muscle was thawed and processed as described above. After that, tissue was added to the infection tank, and after 12 h, shrimp were transferred to 40 L aquariums containing 30 L of seawater (10 shrimp per aquarium = four aquariums per treatment). Shrimp were fed The fourth replicate of each treatment was used to assess transcriptional responses of immune-related genes.WSSV in shrimp was identified according to the method of 2, 0.3 µL 10 mM dNTPs, 0.5 µL 10 µM each primer (forward/reverse), 1.15 µL 2.0 µM TaqMan Probe, 0.1 µL Recombinant Invitrogen® DNA polymerase , 100 ng DNA (sample), and ultrapure water to a final volume of 15 µL.Each PCR reaction contains 1.5 µL 10X reaction buffer, 0.75 µL 50 mM MgClAmplification conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 20 s. Samples amplified after cycle 38 were considered negative.n = 4) at different times: pre-infection (0 hpi) and 6 and 12 h post-infection (hpi).The expression of five genes encoding shrimp immune-related proteins was assessed: phagocytosis-activating protein (PAP) [cellular defense]; and prophenoloxidase (ProPO), C-type lectin 3 (CTL3), Penaeidin 3 (PEN3), Penaeidin 4 (PEN4), and crustin ). Transcriptional responses were assessed in shrimp hemocytes  with 5% potassium oxalate w/v). Hemolymph was centrifuged at 800 × g for 10 min at 4 °C. The plasma was decanted, and the remaining plasma was removed with a micropipette. Finally, 200 µL of Trizol Reagent® was added to each sample and stored at −70 °C until RNA extraction.The hemolymph of individual shrimp (100–200 µl) was withdrawn from the ventral sinus of the first abdominal segment with a 1-mL syringe preloaded with 50 µl of anticoagulant . The RNA was treated with one unit of DNase I . cDNA synthesis was performed from 500 ng of total RNA using Improm II Reverse Transcriptase  and oligo dT20. The obtained cDNA was diluted with 80 µL of ultrapure water and stored at −70 °C until analysis. 5 µL of cDNA dilution was used as a template for qRT-PCR reactions.http://www.ciidirsinaloa.com.mx/RefFinder-master/?type=reference) (β-actin). Reactions were performed in a CFX96 real-time PCR thermal cycler  using 96-well plates.The stability of expression of the candidate housekeeping genes  was analference) . The exp2, 0.3 µL 10 mM dNTPs, 0.75 µL EvaGreen® 20X , 0.1 µL Invitrogen® DNA Polymerase Recombinant  and 5.9 µL ultrapure water to 9.3 µL.A qPCR master mix was prepared (2X concentration = 7.5 ul/reaction) in one batch for all PCR reactions and store in aliquots at −20 °C. Each 2X reaction comprised 1.5 µL 10X Reaction Buffer, 0.75 µL 50 mM MgClBefore PCR, each aliquot of the 2X master mix was thawed, and 0.35 µl of each 10 µM primer (forward/reverse) was added to reach 10 µl. Then 10 µl of mix were placed in each plate well and 5 µl of the corresponding cDNA were added (15 µl per reaction).After 28 days of treatment, three shrimp per treatment were collected and fixed in AFA Davidson’s solution for 48 h , 333 ml ethanol (96%), 233 ml filtered seawater, 100 glacial acetic acids) and treated according to post hoc Tukey’s comparison test were performed for expression, survival, WG, and FCR (p < 0.05). Histological damage data were analyzed using Kruskal-Wallis and post hoc Dunn’s test for multiple comparisons (p < 0.05).Statistical analysis was performed using the STATISTICA V6 program . Percentage values were normalized using the arcsine function before analysis. factorial-way ANOVA and There were no apparent signs of toxicity, and no mortality was recorded during treatment throughout the culture. Temperature, DO, and salinity values fluctuated between 25.3 and 27.5 °C, 5.5 and 6.7 mg/L, and 25 and 27 PSU, respectively. The shrimp performance values did not show significant differences between treatments .After culture, the experimental shrimp were challenged against WSSV by oral route.P = 0.0098 and P = 0.0007, respectively). Negative control had no mortality.Mortality of infected organisms was directly proportional to the frequency of feed-AgNPs supply . Shrimp The expression of immune-related genes was assessed after 28 days of treatment.PAP gene expression was down-regulated in control and D7 (6 and 12 hpi), and in D1, expression was up-regulated at 6 hpi . At 0 hpAfter infection (6 and 12 hpi), the expression of the ProPO gene was down-regulated in control and D7. In contrast, D1 expression was up-regulated at 6 hpi .Before infection, the control had the highest expression compared to the other treatments, and the expression of D7 was higher than D1 and D4. At 6 hpi, D1 reached higher expression than the other treatments. The control and D1 had higher expression than D4 and D7 at 12 hpi .At 6 hpi, CTL-3 expression was up-regulated in control, D1 and D4. Contrarily, in D4, expression was down-regulated at 12 hpi . Also, tThere were no significant differences between pre-challenge treatments. At 6 hpi, D4 and D7 reached higher PEN3 expression than control and D1. At 12 hpi, PEN3 was down-regulated in D7 compared to other treatments .PEN4 expression was up-regulated in D7 at 12 hpi . Also, tCrustin expression was up-regulated at 6 and 12 hpi in D1 and 6 hpi in D4. In contrast, crustin expression in D7 at 12 hpi was down-regulated . HoweverHistological analysis showed atrophy, tubular delamination, and hemocyte infiltration in the hepatopancreas of shrimp from D1 and D4 treatments .General damage was directly proportional to the feed-AgNP doses supplied, reaching 2.3 and 1.6 damage degrees in D1 and D4, respectively. However, only D1 was significantly higher than D7 and control (1.0 damage degree for both) . LikewisThe incidence of WSSV in shrimp farms worldwide has encouraged research into new technologies to reduce its impact. Nanotechnology is a promising tool to help shrimp fight this pathogen. In this sense, silver nanoparticles can modulate the immune system of human cells, trigger an inflammatory response, and help suppress microorganisms . Also, AAfter culture, the antiviral ability of AgNPs in the feed against WSSV was evaluated. The muscles from infected shrimp were liquefied and supplied to experimental shrimp, to simulate a natural infection. In this way, muscle tissue is divided into small fragments to reduce competition between organisms for larger particles. The results showed that protection against WSSV was provided by AgNPs and was directly proportional to the feed-AgNPs provided. This result suggests that the weekly application of feed-AgNPs suffices to protect shrimp from natural WSSV infection. However, an excess of silver can be counterproductive. This finding is similar to the other authors who reported that injection of 50, 200, and 2,000 ng AgNPs into 10 g shrimp prevented WSSV intramuscular infection. However, as shown in this study, mortality was higher with the highest dose of AgNPs, reaching 20%, 20% and 30% mortality, which differs from the 90% achieved in the control group without AgNPs .2O + O2  are produced as part of the phagocytic pathway . Superox2O + O2 . A retro2O + O2 . More sp2O + O2 . This ph2O + O2 . AgNPs i2O + O2 . Physioldiogenes . TherefoAssessing immune system gene expression by quantitative PCR is a tool for assessing the immunity of shrimp under specific conditions . The shrMytilus galloprovincialis) hemocytes to AgNPs did not affect their phagocytic capacity  after 6 hpi. capacity , and AgNcapacity . These fSilver also appears to inhibit basic defense mechanisms in shrimp in a long term. Besides, PAP and ProPO gene expression after culture were consistent with the mortality observed in the challenge bioassay .Lectins play essential roles in many biological processes, such as molecular effectors, cell signaling, and pathogen recognition . In thisDaphnia magna following 21-day exposure to AgNPs at concentrations above 1 µg/L, affecting feeding rates and reproduction. Antimicrobial peptides (AMPs) are components in many organisms’ pathogen defense mechanisms . The penL. vannamei treated with feed supplemented with unicellular microalgae Dunaliella sp. against WSSV, reaching mortality significantly lower in treated shrimp (20%) compared with the control group (44%). However, measurements were performed only once (on the sixth day after infection). Therefore, it is not clear what will happen to shrimp during the critical days 9–10. In this research, the application of AgNPs resulted in 53% mortality at day 10 post-infection , compared with 93% in the positive control group. The most important revelation, however, was that on day 10, the mortality curve plateaued, indicating the end of shrimp mortality  was more effective when supplied for a long time, possibly because, in this case, the silver exerted its antimicrobial effect while the shrimp also eliminated the excess metallic silver on their own.In conclusion, this study showed that supplementation of 1,000 µg AgNPs (Argovit-4®) per gram of feed did not interfere with shrimp growth and prevented WSSV infection when AgNPs were supplied once a week. Frequent feedings (every day or every four days) can lead to poor results. However, transcriptional responses of immune-related genes were affected. Whether this affectation makes the shrimp vulnerable to other pathogens is unknown and remains to be investigated. Also, further optimization of the AgNPs concentration in the feed and the feeding cycle could be more effective in preventing WSSV infection.10.7717/peerj.14231/supp-1File S1Mortality was recorded in each replicate from treatments every 12 hours for ten days. The cumulative mortality at the end of the experiment was normalized by arcsine function and analyzed by one-way ANOVA and a Tukey test using the Statistic program (STATISTICA 7.0).Click here for additional data file.10.7717/peerj.14231/supp-2File S2Transcriptional response of experimental shrimp after experimental infection with WSSV and at 9 and 12 hours post-infection. Data are represented as the relative expression of each gene in all treatments. The data were analyzed by two-way ANOVA using the time and treatment factors. Tukey test was interpreted as differences between treatment each time and the difference between before and after infection.Click here for additional data file.


Herein, α-Fe2O3 and CdS were in situ synthesized on fluorine-doped tin oxide (FTO) glass by hydrothermal and SILAR (successive ionic layer adsorption and reaction) methods, respectively. The direct Z-scheme charge transfer path between Fe2O3 and CdS and the effective electron migration toward the PdAg mainly contributed to the excellent photocatalytic CO2 reduction performance. The controllable work function based on Pd (5.12) and Ag (4.26) constructed an appropriate band alignment with α-Fe2O3/CdS and displayed favorable production for CH4 rather than CO. The optimum ratio of PdAg 1:2 performed a 48% enhancement than pure Pd for photoreduction of CO2. Meanwhile, the enhanced charge separation improved the photoelectrochemical performance and photocurrent generation, and reduced the electrical resistance between components. This work provided insights into the dual-metallic co-catalyst for boosting the activity and selectivity of photocatalytic CO2 reduction.Metal nanoparticles have been extensively used as co-catalysts in photocatalytic systems in order to pursue improvements in both reaction kinetics and selectivity. In this work, PdAg dual-metallic nanoparticles synthesized by the co-reduction method were decorated on a well-established α-Fe However% light) . In ordeparation . For ins and CdS . Based onditions . This isnditions . In compnditions . Furthernditions .2 into valuable solar fuels was regarded as an efficient means to address the energy supply and CO2 emission (2O3) to achieve this conversion goal had attracted extensive attention, since they were an environmentally friendly low-cost technology , cadmium acetate dihydrate (Cd(Ac)2·2H2O, Macklin, AR), ethanol , sodium sulfide nonahydrate , dimethyldioctadecylammonium chloride , silver nitrate , palladium chloride , hydrochloric acid , polyvinylpyrrolidone n, Aladdin), ascorbic acid , and sodium sulfite  were used. All the raw materials were used without further separation and purification.Iron chloride hexahydrate (FeCl2O3 nanorod arrays were prepared following a modified literature report (3 (0.15 M) aqueous solution and heated to 100°C for 6 h. After the hydrothermal reaction, the samples were collected and rinsed with deionized water and N2 stream drying. The sample was then annealed in a muffle furnace at 550°C for 2 h (10°C/min) and collected after cooling down to room temperature.The Fee report . The cle2O3 nanorods deposited by CdS was fabricated via the SILAR (successive ionic layer adsorption and reaction) method (2O3 film was first soaked in Cd(Ac)2 (25 mM) ethanol solution for 1 min, dried by N2 stream. Next, it was immersed in Na2S (25 mM) aqueous solution for 1 min and then washed with deionized water, dried by N2 stream. The deposition of CdS was accomplished by following chemical reactions for 10 SILAR cycles. Then, the electrode was finally annealed under Ar stream at 400°C for 30 min (1°C/min).The electrode of Fe) method . The as-3 (10 mM) and H2PdCl4  were added to DODAC (2 mM) which was dissolved in deionized water. In the next step, PVP (10 mg/ml) was added for better dispersion. During the co-reduction process of Pd and Ag, ascorbic acid (0.3 M) aqueous solution was added with the color change (slight yellow to black) under 60°C for 30 min.The preparation of PdAg was more complicated through the co-reduction of Pd and Ag using DODAC as a structure-directing agent . AgNO3 . It was noted that the Pd:Ag ratio was controlled by the molar amount of each precursor.The dispersant absorption method was employed for the deposition of PdAg on Fe2 was conducted in a home-made 100-ml Pyrex reactor with an optical window. The reactor was sealed using a rubber septum. A Xe lamp with 300 W and an AM 1.5-G filter were used as the simulated solar light. The samples were scraped from the FTO slides and sonicated for 1 h before the reaction. During the reaction, 1 ml of gas was acquired from the reactor at a specified interval (1 h), and subsequent gas concentration analysis was performed in a GC-2014C Shimazu equipped with a flame-ionization detector. The photoelectrochemical (PEC) performances were measured in an alkaline solution  using a standard three-electrode PEC cell, under the simulated sunlight (with AM 1.5-G filter). The sample, Ag/AgCl, and Pt wire were employed as the working electrode, reference electrode, and counter electrode, respectively. The potential vs. RHE was determined using the equation, from which the E(RHE) = E(Ag/AgCl) + 0.964 V  and a transmission electron microscope , respectively. X-ray photoelectron spectroscopy (VG ESCALAB XPS System with a monochromatized Al Kα X-ray source) was carried out to investigate the chemical state. UV-vis diffuse reflectance spectroscopy and absorption spectroscopy equipped with a DH-2000-BAL lamp (deuterium/helium) of wavelength ranging from 200 to 1,100 nm were used to characterize the optical properties and bandgap. The photoreduction of CO 0.964 V  E(RHE)=2O3 film was first grown on the FTO glass substrate, which exhibited the nanorod morphology in SEM images  and (200) planes, respectively. Meanwhile, it certified the weak diffraction peak of PdAg NPs in XRD patterns. From UV-vis diffusion reflectance spectroscopy (DRS), the light absorption of α-Fe2O3/CdS basically showed no differences after coupling with PdAg  confirmed the main products (CH4 and CO) from the samples decorated with different Pd:Ag ratios. As shown in −1 h−1), PdAg 1:2 (9.80 μmol g−1 h−1) presented a 48% enhancement in the total conversion rate. More importantly, as the modulating research predicted, by controlling the Pd:Ag ratio, the selectivity toward CH4 was optimized from 58.7 to 83.2%.The photoelectrochemical properties of α-Fes report , an imprwith CdS . Next, tneration , due to neration  comparedvia an α-Fe2O3/CdS heterojunction, and a CO2 reduction reaction occurred on the PdAg sites. In detail, the adsorbed CO2 reacted with electrons accumulated on the PdAg sites to form CO and CH4, respectively . On the other hand, the selectivity was attributed to the dual-metallic active sites supplied by PdAg NPs, which conducted continuous protonation of C atoms in M1-C-O-M2 intermediates. As a result, the sample with a ratio of PdAg 1:2 was in favor of producing CH4 instead of CO, which was consistent with the best photocurrent and smallest radii.Overall, α-Fe


GO was synthesized by a modified Hummers method, however GO was reduced using the plant extract to produce rGO. The as-synthesized rGO/Fe3O4 NCs were characterized by UV-vis spectrophotometer, Fourier transform infrared (FT-IR) spectroscopy, FT-Raman spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy supported with energy dispersed X-ray spectroscopy (FESEM-EDX), transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). The synthesis of magnetic rGO/Fe3O4 NCs was confirmed from characterization results of FT-Raman, TEM and VSM. The FT-Raman results showed the D and G bands at 1306.92 cm−1 and 1591 cm−1 due to rGO and a peak at around 589 cm−1 due to Fe3O4 NPs that were anchored on rGO sheets; TEM results showed the synthesis of Fe3O4 with an average particle size of 8.86 nm anchored on the surface of rGO sheets. The VSM result confirmed the superparamagnetic properties of the rGO/Fe3O4 NCs with a saturation magnetization of 42 emu g−1. The adsorption capacity of rGO/Fe3O4 NCs towards crystal violet (CV) dye was calculated to be 62 mg g−1. The dye removal behavior fitted well with the Freundlich isotherm and the pseudo-second-order kinetic model implies possible chemisorption. Besides, rGO/Fe3O4 NCs showed antifungal activities against Trichophyton mentagrophytes and Candida albicans by agar-well diffusion method with a zone inhibition of 24 mm and 21 mm, respectively. Therefore, rGO/Fe3O4 NCs can be used as an excellent adsorbent to remove organic dye pollutants and kill pathogens.This work reports the synthesis and application of magnetic rGO/Fe 3O4 NCs using pod extract of Dolichos lablab L. as a reducing agent and its applications.This work reports synthesis of magnetic rGO/Fe The combination of GO or rGO with magnetite produces Fe3O4–graphene hybrid which has a planar geometry, high conductivity, fascinating carrier transport properties, large surface area, strong magnetism, low cost and environmentally benign nature which has opened an opportunity for various applications.5–8 Therefore, rGO/Fe3O4 hybrids have reported for catalysis, water purification, sensors, toxic heavy metal removal, capacitors, bio-medical diagnosis therapy, microwave absorption, water desalination, photocatalysis and antimicrobial.7,9–15 The hybrid combination of magnetic Fe3O4 NPs and graphene sheets best produces rGO/Fe3O4 NCs that have recognized properties such as great dispersibility, large surface area, superparamagnetism and fabulous extraction capacity.16 The π–π stacking, hydrogen bonding and electrostatic interaction between rGO/Fe3O4 NCs and dye molecules are responsible for the adsorptive removal of dye molecules from aqueous solution.17 Moreover, preceding reports indicated that rGO/Fe3O4 NCs can efficiently remove toxic heavy metals,7,18 fluoroquinolones,19 organic dyes16,20 and MB dye via degradation.17Graphite oxide, also known as graphitic oxide or graphitic acid, is a compound of carbon, oxygen and hydrogen in variable ratios, obtained by treating graphite with strong oxidizers.21 even though these applications are time-consuming and require extra cost.22 Compared with traditional centrifugation and filtration methods, magnetic separation method is an efficient, fast and economic method for the separation of magnetic adsorbents from the medium after the adsorption treatment of pollutants is completed.23 Hence, it is important to synthesize magnetically separable and high surface area rGO/Fe3O4 NCs for removal of water pollutants and treat pathogens. Diverse methods have been used to synthesize rGO/Fe3O4 NCs, such as chemical co-precipitation,13,14 solvothermal reduction,11,24 chemical reduction,7 and green synthesis.12,15,25 Different types of adsorbents have been used as way of removal of organic compounds and metals from aqueous solution. For example, Harijan and Chandra26 have stated facile fabrication of magnetite graphene composite through thermal reduction of graphene oxide to graphene in alkali medium with extreme sorption capacity for Cr6+ (5.5 mg g−1) at pH = 6.6. The graphene sheets prohibited agglomeration of the Fe3O4 NPs while enabling a good dispersion of the Fe3O4 NPs and at the same time the specific surface area of the composite is considerably enhanced. Similarly, Sun et al.11 have reported one step solvothermal synthetic route of rGO/Fe3O4 NCs for excellent removal of toxic dyes, i.e., rhodamine B and malachite green with removal efficiency of 91% and 94%, respectively. He et al. have reported waste biofilms biosorbents to treat Cd2+ from aqueous solutions. The maximum adsorption capacity of dry waste biofilms for Cd2+ is 42 mg g−1 when the initial concentration of Cd2+ is 50 mg L−1.27 Bionanomaterials such as Saccharomyces cerevisiae and nano Fe3O4 encapsulated in a sodium alginate–polyvinyl alcohol matrix and Penicillium doped with nano Fe3O4 entrapped in polyvinyl alcohol–sodium alginate gel beads have been reported to remove atrazine from aqueous solution by biodegradation.28,29 Moreover, synthesis of rGO/Fe3O4 NCs through a facile and environmental benign approach is a prime concern for practical application. Plant extracts currently attract a tremendous research interest for synthesis of rGO/Fe3O4 NCs owing to environmentally friendly and ability to reduce GO into rGO. Gurunathan et al.30 have recorded reduction of GO into rGO with spinach leaf extract as a simultaneous reducing and stabilizing agent. The plant extracts of Solanum trilobatum17 and Averrhoa carambola12 were previously reported as reducing agent for synthesis of rGO/Fe3O4 NCs. To the best of our information, Dolichos lablab L. has not been reported to synthesize rGO or rGO/Fe3O4 NCs. Dolichos lablab L. is a plant that is extensively scattered in India that contains lectins sugar in the form of mannose/glucose specific,31 galactose specific,32 crude lipid, crude protein, insoluble dietary fibre, soluble dietary fibre, carbohydrate, and amino acids.33 For this reason, the existence of many alternative phytoconstituents of the plant motivated the researchers to prepare rGO/Fe3O4 NCs for different applications.In general, centrifugation and filtration methods are used to separate the adsorbent material from aqueous solution3O4 NCs using a facile co-precipitation method of Fe3O4 onto rGO sheet in the presence of extract of Dolichos lablab L. for the adsorptive removal of crystal violet (CV) and antifungal activity against pathogens. The pod extract of Dolichos lablab L. was used as reducing and capping agent during preparation of rGO and rGO/Fe3O4 NCs. The crystal structure, electronic property and surface morphology of the as-synthesized materials were investigated by UV-vis, FT-IR, FT-Raman, powder XRD, FESEM-EDX, TEM and VSM techniques. The CV dye was selected as a model water pollutant to investigate the adsorption capacity of rGO/Fe3O4 NCs. Besides, Trichophyton mentagrophytes and Candida albicans pathogens were chosen to examine the antifungal activities.Herein, in this study, the researchers have synthesized rGO/Fe2.2.1.ii) sulfate heptahydrate (FeSO4·7H2O), hydrogen peroxide , sodium nitrate , concentrated sulfuric acid , potassium permanganate (KMnO4), crystal violet dye (C25H30ClN3) and sodium hydroxide (NaOH) were from Merck, India; iron(iii) chloride hexahydrate (FeCl3·6H2O) from HiMedia, India; and Graphite flake with +100 mesh from Sigma-Aldrich, India. The standard strains of Trichophyton mentagrophytes and Candida albicans (fungus) were obtained from Adhya Biosciences Pvt. Ltd., Visakhapatnam. Milli-Q water was used in the experiment. Pocket-sized pH meter acquired from Hanna instruments was used to regulate the pH of the solutions. Hot Air Oven, Kemi was used to dry washed samples. Dolichos lablab L. pods were collected from market near Andhra University, Vishakhapatnam, India.All the chemicals in this paper were analytical grade acquired from Merck, HiMedia and Sigma-Aldrich and used without further purification. Iron mixer grinder. The optimization of percent aqueous plant extract was taken based on our previous work.2.3.1 To prepare rGO from GO, pod extract of Dolichos lablab L. was used as a reducing agent. 100 mg of GO was added into round bottomed flask containing 50 mL Milli-Q water and sonicated for 30 min. Meanwhile 10 mL of 1% pod extract of Dolichos lablab L. was added and refluxed at 80 °C while stirring for 12 h to produce a black precipitate. Finally, rGO was collected by centrifugation at 12 000 rpm and dried at 100 °C in vacuum oven.GO was processed by modified Hummers strategy.2.4.3O4 NCs were fabricated through co-precipitation using FeSO4·7H2O and FeCl3·6H2O in the presence of GO in alkaline medium according to the procedure in −1 dispersed GO was exfoliated into 100 mL of iron source solution containing both FeSO4·7H2O and FeCl3·6H2O salts (1 : 2 molar ratio) and it was sonicated for 1 h. Ten (10) mL of 1% pod extract of Dolichos lablab L. solution was added slowly to the above suspension of GO at 30 °C with vigorous stirring under nitrogen atmosphere for 1 h. In short period, 10 mL of ammonia solution was added drop wise to modify the mixture pH to a value of 10. Moreover, reducing agents in the plant extract may reduce Fe3+ as well. Thereupon, the mixture was heated to 80 °C for 12 h to form dark colored solution indicating formation of rGO/Fe3O4 NCs. Finally, black colored rGO/Fe3O4 NCs was produced, separated, washed with Milli-Q water and ethanol, and then dried in hot air oven at 60 °C for 12 h.The rGO/Fe2.5.−1 using a SHIMADZU-IR PRESTIGE-2 spectrometer. Raman spectrum was obtained using FT-Raman spectroscopy  with laser source Nd (YAG 1064 nm) at 2 cm−1 resolution. 16+ mW laser power was irradiated on rGO/Fe3O4 NCs to collect Raman spectrum over the wide range 4000–50 cm−1. XRD patterns were recorded by PANalytical X'pert pro diffractometer at 0.02 degree per sec scan rate using Cu Kα1 radiation . TEM images were acquired through (TEM model FEI TECNAI G2 S-Twin) at an accelerating voltage of 200 kV. The morphology of the sample was characterized using FE-SEM  equipped with EDX. VSM was used to examine the magnetic property of rGO/Fe3O4 NCs .The UV-visible absorption spectra were recorded using a  spectrophotometer in the range of 200–800 nm. FT-IR spectrum was recorded over the range of 4000–400 cm2.6.3O4 NCs took place with different initial concentrations of CV dye at different pH values at room temperature. 100 mL  of CV dye solution was placed into 250 mL beaker and the pH (4–12) of the solution was maintained by using 0.1 M HCl/0.1 M NaOH solution. Afterward, 20 mg rGO/Fe3O4 NCs was added to the beaker and kept agitated in an incubator for 220 min until equilibrium was established. The initial concentration of the dye was reported as Co and in the consecutive 20 min time interval, concentration of the dye was recorded as Ce. The amount of CV dye adsorbed per gram of adsorbent at time, t, qt (mg g−1) and equilibrium, qe (mg g−1) were determined using , ‘Ct’ is concentration of CV dye at time, t (mg L−1), ‘Ce’ is concentration of CV dye at equilibrium (mg L−1), ‘V’ is the volume of solution (L), ‘m’ is the mass of rGO/Fe3O4 NCs adsorbent (g) and ‘η’ is the dye removal percent (%). The adsorption isotherm was studied using Langmuir and Freundlich isotherm models, while, the kinetics was studied using pseudo-first and pseudo-second-order kinetic models.In addition, CV dye removal efficiency was calculated using .3where, 2.7.3O4 NCs, the adsorption isotherm was analyzed by Langmuir36 and Freundlich37 isotherm models. The Langmuir adsorption isotherm argues consistent adsorbent surface activity and unilayer adsorption, with partial adsorbent active sites, is revealed as in (Ce’ is the equilibrium concentration of CV in the aqueous solution (mg L−1), ‘KL’ is the Langmuir adsorption constant (L mg−1) correlated to heat of adsorption, ‘qmax’ is the maximal adsorption capacity of the adsorbent (mg g−1), and ‘qe’ is the amount of CV adsorbed per mass of adsorbent at equilibrium (mg g−1).To determine the maximum adsorption capacity of rGO/Feed as in .4where, qe’ is the quantity of dye adsorbed per unit weight of adsorbent (mg g−1), ‘Ce’ is the equilibrium concentration of dye in the bulk solution (mg L−1), ‘KF’ is the Freundlich constant indicative of the relative adsorption capacity of the adsorbent (mg g−1) (L mg−1)n1/ and ‘n’ is adsorption intensity in the Freundlich equation.The Freundlich isotherm signifies surface heterogeneity of the adsorbent as well as multilayer coverage on the surface. The linear equation of Freundlich isotherm is expressed as in .5where, 2.8.3O4 NCs, the mechanism of the adsorption process was studied by fitting pseudo-first-order and pseudo-second-order reactions to the experimental data. The adsorption equilibrium time of CV by rGO/Fe3O4 NCs was 200 min. The Lagergren pseudo first-order kinetic model38 is known as in , ‘k1’ is rate constant of pseudo-first-order kinetic model (min) and ‘t’ is time (min). Likewise, the pseudo-second-order kinetic model39 is expressed as in (k2’ is the rate constant of pseudo-second-order (g mg−1 min−1), ‘qt’ is the amount of CV dye adsorbed on surface of rGO/Fe3O4 NCs at time, t (mg g−1), ‘qe’ is the equilibrium sorption capacity (mg g−1).The physical and chemical properties of the adsorbent as well as mass transfer mechanisms are among the several prominent parameters to determine the adsorption mechanism. To revise the effect of adsorption time on CV dye removal by rGO/Fewn as in .6ln. About 20 mL of the nutrient agar medium/potato dextrose agar seeded with the respective fungal strains were transferred aseptically into each sterilized Petri plate. The plates were left at room temperature for solidification. Each plate, a single well of 6 mm diameter was made using a sterile borer. The rGO/Fe3O4 NCs were freshly reconstituted with suitable solvent (DMSO) and tested at various concentrations . The samples (50 μL) and the control along with standard (clotrimazole (5 μg mL−1)) were placed in 6 mm diameter well. Then the fungal plates were incubated at 28 ± 2 °C. Activity diameter of the zone of inhibition was measured using Himedia antibiotic zone scale.The antifungal activity was carried out using agar-well diffusion method by employing 24 h cultures with given rGO/Fe3.3.1.3.1.1.3O4 NCs using iron salts (Fe3+/Fe2+) in 2 : 1 molar ratio, GO and pod extract of Dolichos lablab L. A systematic schematic representation of synthesis of rGO/Fe3O4 NCs for the possible application of removal of CV dye is represented in 3O4 NCs by using UV-vis spectrophotometer. The absorption peak of GO at 228 nm is due to π to π* transition of the aromatic C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bond,413O4 NCs at around 267 nm, In this study, one pot co-precipitation green method was used to synthesize rGO/Fe3.1.2.3O4 NPs and rGO/Fe3O4 NCs. Pod extract of Dolichos lablab L. showed absorption peaks at 3324, 2925, 1645, 1051 and 660 cm−1 due to O–H stretching, sp3 C–H stretching, CO of amide or CC stretching, C–O stretching and C–H bending, respectively  and 1591 cm−1 (G band) had ratio of ID/IG = 1.19. The D band is assigned to the breathing mode of the k-point phonons with A1g symmetry of disordered graphite structure, whereas the G band introduces the E2g vibration mode between two sp2 carbon atoms.46 The three peaks at 269, 589, and 767 cm−1 were related to Fe–O vibration of Eg and A1g modes in Fe3O4. Hence, this result also proved magnetite nanoparticles were decorated on surfaces of rGO sheets.47 Similarly, two peaks were absorbed at 1591 and 1307 cm−1 for magnetite–rGO composites synthesized by using hydrazine hydrate as a reducing agent.7 The G band of rGO  is red shifted by 10 cm−1 to 1591 cm−1 in rGO/Fe3O4 NCs.FT-Raman spectroscopy is used to characterize carbon containing materials and to identify amorphous and crystalline carbon structures.3.1.4.3O4 NPs and rGO/Fe3O4 NCs were characterized through powder XRD as indicated in θ = 10° with a lattice reflection of (001), θ = 21° with a lattice reflection of (002) in 48 The XRD pattern of Fe3O4 NPs and rGO/Fe3O4 NCs in θ values as 33.09°, 35.64°, 43.44°, 53.91°, 57.41° and 62.9° with diffraction lines matching to (220), (311), (400), (422), (511) and (440), respectively, are consistent with the standard XRD data for the face centered cubic (fcc) Fe3O4 structure (JCPDS card no. 19-0629). The peak at 2θ = 21° (002) which is seen in the XRD pattern of rGO/Fe3O4 NCs , ‘k’ is Scherrer constant (0.94), ‘λ’ is the X-ray wavelength, ‘β’ is the half width of XRD diffraction line, and ‘θ’ is the Bragg's angle in degrees.The phase structures of GO, rGO, Fee3O4 NCs , confirmequation .8where, 3.1.5.3O4 NCs were studied by using FESEM-EDX. 3O4 NPs that were consistently distributed on the surface of rGO sheets. The size of the Fe3O4 NPs from the image sources range from 9.75 to 14.85 nm. The EDX spectrum in 3O4 NCs. 3O4 NPs are homogeneously anchored at the surface of disorderly distributed rGO sheets. The SAED patterns of rGO/Fe3O4 NCs in 3O4 and disorderly oriented nature of rGO sheets. The average particle size of Fe3O4 in rGO/Fe3O4 NCs was calculated to be 8.86 nm using (ImageJ) software, 3O4 NPs observed on the surface of rGO sheets. Hence, the large surface area to volume ratio of the NCs causes the rGO/Fe3O4 NCs to have an efficient adsorption property towards CV dye pollutant.The morphological looks and surface characteristics of rGO/Fe3.1.6.3O4 NCs was measured by VSM at room temperature and confirmed its superparamagnetic property. 3O4 NCs with a magnetic saturation (Ms) 42 emu g−1 which was lower than that of l-Met capped Fe3O4 NPs50 indicated Fe3O4 NPs were anchored on the surface of rGO sheets.The magnetic property of rGO/Fe3.2.3.2.1.3O4 NCs as a function of time, while 3O4 NCs surface is positively charged. Similarly, Duman et al.21 have reported adsorption of CV dye onto magnetic OMWCNT–Fe3O4 NPs is lowest at pH = 2 . This is due to the electrostatic repulsion between positively charged adsorbent surfaces and positively charged CV dye. However, at the higher pH solution (pH = 12), the surface of the rGO/Fe3O4 NCs became negatively charged which resulted to electrostatic forces of attraction between CV dye molecules and the adsorbent that increased adsorption efficiency to 95%. 3O4 NCs using different initial CV dye concentration at pH = 10. Dye adsorption increased with increasing initial concentration of organic dye pollutant. 3O4 NCs. The correlation coefficients (r2) and other parameters from the models are presented in r2 = 0.998) than Freundlich (r2 = 0.992). Hence, the adsorption mechanism was monolayer, i.e., physical intermolecular attraction between the adsorbent and adsorbate predominated. Similar results were reported for the adsorption isotherms of various pollutant-adsorbent systems.51,52 The monolayer adsorption maximum capacity (qmax) of CV on rGO/Fe3O4 NCs was found to be 62 mg g−1. Hence, current study shows rGO/Fe3O4 NCs has better adsorption capacity than Fe3O4 NPs alone towards CV dye53 due to increased number of sites of functional groups in rGO/Fe3O4 NCs.3.2.2.3O4 NCs are shown in 3O4 NCs follows pseudo-second-order kinetics implies that the rate determining step is chemisorption.54 Similar results were reported for the adsorption kinetics of various pollutants onto activated carbon cloth.55–57 Therefore, adsorption was facilitated due to the interaction of cationic dye (CV) and rGO sheets through the oxygen functional groups and vacancy defects, by either electrostatic or π–π interactions.58 The maximum adsorption capacity of Fe3O4–rGO NCs synthesized by solvothermal method against methyl violet dye has been reported 196 mg g−1 by Cherukutty et al.59 Similarly, the maximum adsorption capacity of Fe3O4/GO hybrid to remove methylene blue dye from aqueous solution at 35 °C has been reported 96.05 mg g−1 by Liao et al.60 In addition, successful photocatalytic degradation of malachite green using Ag3PO4@MWCNTs@Cr : SrTiO3 composite from aqueous solution under sunlight or visible light irradiation has also been reported by Lin et al.61The linear plots of pseudo-first-order and pseudo-second-order kinetic models for adsorption of CV dye on rGO/Fe3.3.3O4 NCs showed antifungal activity against Trichophyton mentagrophytes and Candida albicans with maximum zone of inhibition of 24 mm and 21 mm, respectively. See both et al.62 reported biosynthesized AgNPs using aqueous leaf extract of Erythrina suberosa (Roxb.) showed antifungal activity against Trichophyton mentagrophytes with inhibition zone of 16 ± 0.8 mm, but no zone of inhibition was observed against Candida albicans. In addition, green synthesized ZnS NPs using Phyllanthus niruri plant extract and thorn-like ZnO NPs synthesized by sol–gel method both showed antifungal activity against Candida albicans with maximum inhibition zone of 32 mm and 20 ± 1.5 mm, respectively.63,64 Hence, the as-synthesized rGO/Fe3O4 NCs have showed better antifungal activity against the above studied fungus and therefore can be used as antimicrobial applications.Finally, the product rGO/Fe4.3O4 NCs using pod extract of Dolichos lablab L. as reducing agent was reported. The result of TEM analysis showed the average particle size of Fe3O4 NPs in the as-synthesized rGO/Fe3O4 NCs was 8.86 nm. The maximum CV dye removal efficiency of the adsorbent was calculated to be 95% at pH = 12. The adsorption mechanism well fitted to Langmuir than Freundlich isotherm and followed pseudo-second-order kinetics. Dolichos lablab L. mediated rGO/Fe3O4 NCs can be used as efficient removal of toxic dyes and metals from contaminated water and antifungal against microorganism pathogens.Eco-friendly, cost-effective and single-step green co-precipitation synthesis of magnetically separable rGO/FeThere are no conflicts to declare.


Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.Increased risk of intestinal dysfunction has been reported in patients after  Clostridioides difficile (C. difficile) is an anaerobic, gram-positive, spore-forming bacterium which is transmitted by oral ingestion of spores. Once the microbiota is disrupted in the gut, these spores can germinate and grow into vegetative cells, which in turn secrete toxins (C. difficile toxins A (TcdA) and B (TcdB) are the main virulence factors involved in C. difficile infection (CDI), promoting gut cell death and inflammation  , 7. CDI The pathogenesis of CDI involves several pathways, including adenosine receptors A2B and A2A signaling. Deletion of A2B during CDI in a pre-clinical model in mice showed to be protective, for both intestinal damage and disease severity, while deletion of A2A demonstrated to promote worsened CDI .So far, four isoforms of G-protein coupled adenosine receptors were described: A1, A2A, A2B, and A3, which are activated by adenosine, an endogenous, purine-based nucleoside that can be released by all eukaryotic cells . A1 and C. difficile toxins on EGCs. Our findings showed that EGCs expressed all four subtypes of adenosine receptors, which differentially modulate IL-6 and S100B expression and cell death induced by TcdA and TcdB. These findings reveal new insight to understanding the role of these receptors during CDI and aid in the development of therapies able to decrease inflammation during infection, as well as decrease the damage to ENS components that can contribute to future intestinal dysfunction associated with post-CDI sequelae.Given that adenosine receptors have been shown to play an important role in CDI, we investigated how the modulation of adenosine receptors could contribute to the deleterious effects of in vitro assays using an immortalized rat enteric glial cell (EGC) line PK060399egfr . This cell exhibits similar morphology and functional properties to primary enteric glial cells from humans and mice .EGCs (PK060399egfr) were cultured in Dulbecco’s Modified Eagle’s Medium  and supplemented with 10% fetal bovine serum, 1% antibiotics  and 1 mM sodium pyruvate (Gibco) at 37°C in a humidified incubator under 5% CO2 for no more than 20 passages. For all experiments, EGCs were released using 0.05% trypsin-EDTA for 5 min. Cells were incubated with 300 µM ATL313 (an A2a-selective adenosine receptor agonist), 3 µM SCH58261 (an A2a-selective adenosine receptor antagonist), 1 µM BAY60-6583 (an A2b-selective adenosine receptor agonist), 100 µM ATL801 (an A2b-selective adenosine receptor antagonist), 30 µM CI-IB-MECA , 0.01 µM MRS1220 , 0.5 µM 666-15 , and 10 µM H89 dihydrochloride  added 1 hour prior to incubation with TcdA (50 ng/mL) or TcdB (1 ng/mL). In some experiments, EGCs were incubated with recombinant IL-6 . All drug concentrations used were based on MTT assay results (5 cells/well) were seeded in 6-well plates and treated with TcdA or TcdB and pharmacologic modulators . After incubation, total RNA was extracted with a RNeasy Plus Mini Kit  using QIAcube (Qiagen). RNA was quantified with a Qubit 3.0 fluorometer (Life Technologies) using a Qubit RNA BR Assay Kit . After DNA contamination was removed by RNA treatment with Dnase I , a total of 1000ng of RNA was then reverse transcribed using an iScript cDNA Synthesis Kit  according to the manufacturer’s protocol.EGCs  with the following conditions: 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 30 s, and melt curve analysis from 60-95°C in 0.5°C increments for 2 s each. All PCRs were performed with iTaq Universal SYBR Green Supermix . The primer sets are listed in qPCR amplification of 4 cells/well) plated on 4-chamber glass tissue culture slides in a polystyrene vessel and treated with TcdA or TcdB for 18 h were fixed in 4% PFA solution (Alfa Aesar) in PBS for 30 min at room temperature and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) and 3% bovine serum albumin  in PBS for 10 min at 4°C. After blocking with 5% normal donkey serum  in PBS for 30 min at room temperature, the cells were incubated with anti-A2A , anti-A2B , anti-A3  or anti-IL-6  overnight at 4°C. After three washes with washing buffer (0.01% Tween 20 in PBS), the cells were incubated for 2 h with Alexa fluor 594-conjugated donkey anti-rabbit , Alexa fluor 488-conjugated donkey anti-rabbit  or Alexa fluor 488-conjugated donkey anti-goat  antibodies, washed with PBS and mounted with ProLong Gold antifade reagent containing DAPI . The samples were visualized by fluorescence microscopy . The intensity of fluorescence was measured using Image J software.EGCs (4x104 cells/well) were seeded in white tissue culture-treated 96-well plates  and treated with TcdA or TcdB for 18 h. Then, after removal of the supernatant, cells were incubated with 20 µL of cAMP-Glo lysis buffer with shaking at room temperature for 20 min. After this, 40 µL of cAMP-Glo detection solution (2.5 µL of PKA in 1000 µL cAMP-Glo reaction buffer) were added to each well and the plates were shaken for 60s and incubated at room temperature for 20 min. Then, 80 µL of kinase-Glo reagent were added to each well, and the plates were shaken for 60 s and incubated at room temperature for 10 min. The luminescence was recorded using a multi-mode reader , and the intrinsic reagent luminescence  was subtracted from the luminescence signals in the sample to obtain the relative luminescent units (RLUs).To measure the levels of cyclic adenosine monophosphate (cAMP) in EGCs treated with TcdA or TcdB for 18 h, the cAMP-Glo assay  was performed following the manufacturer’s instructions. The assay is based on the principle that cAMP stimulates protein kinase A (PKA) holoenzyme activity, decreasing available ATP and leading to decreased light production in a coupled luciferase reaction. Briefly, EGCs (103 cells/well) were seeded in 96-well plates and treated with several concentrations of adenosine receptors modulators , inhibitors of CREB (666–15), and PKA (H89) for 18 h. Cells treated with DMSO (100%) was used as a positive control of cell death. Then, the cells were incubated with thiazolyl blue tetrazolium bromide  for 2 h at 37°C in a humidified incubator under 5% CO2. After removal of the MTT solution, 150 µL of 100% dimethyl sulfoxide was added to each well. The plates were then shaken for 2 min at room temperature, and the absorbance of the reaction at 570 nm was measured using an ELISA reader.To determine the viability of the EGCs, cells (5x104 cells/well) were seeded in white tissue culture-treated 96-well plates  and treated with TcdA or TcdB for 18 h in the presence or absence of 300 µM ATL313 (an A2a-selective adenosine receptor agonist), 3 µM SCH58261 (an A2a-selective adenosine receptor antagonist), 1 µM BAY60-6583 (an A2b-selective adenosine receptor agonist), 100 µM ATL801 (an A2b-selective adenosine receptor antagonist), 30 µM CI-IBMECA (an A3AR-selective adenosine receptor agonist), 0.01 µM MRS1220 (an A3AR-selective adenosine receptor antagonist), 0.5 µM 666-15 (CREB inhibitor), and 10 µM H89 (PKA inhibitor) added one hour prior to C. difficile toxin challenge. Then, 200 µL of 2x detection reagent  were added to each well, and the cells were incubated at 37°C in a humidified incubator under 5% CO2. The luminescence was recorded using a multi-mode reader , and the intrinsic reagent luminescence  was subtracted from the luminescence signals in the sample, then divided by the mean of control cells to obtain the relative luminescent units (RLUs).Cell death was evaluated with a live cell real-time assay , following the manufacturer’s instructions. EGCs (104 cells/well) were seeded in white tissue culture-treated 96-well plates  and treated with TcdA or TcdB for 18 h in the presence or absence 300 µM ATL313 (an A2a-selective adenosine receptor agonist), 100 µM ATL801 (an A2b-selective adenosine receptor antagonist) or 30 µM CI-IBMECA (an A3AR-selective adenosine receptor agonist) added one hour prior to C. difficile toxin challenge. Then, the plates containing the cells were removed from the incubator for 15 min. A volume of 100 µL of Caspase-Glo reagent was added to each well, and the wells were mixed with a plate shaker at 500 rpm for 30 s. The plates were incubated for 2 h at room temperature. Then, the luminescence of each sample was acquired in a multi-mode reader  to obtain the relative luminescent units (RLUs).Caspase 3/7 activity was evaluated with a Caspase-Glo assay kit  following the manufacturer’s instructions. EGCs  and A2B sgRNA CRISPR/Cas9 . Plasmids were transformed into NEB® Stable Competent Escherichia coli (High Efficiency) following the manufacturer’s protocol. Clones were confirmed by Sanger sequencing.To ensure successful knockdown of the respective genes, we performed a lentiviral-mediated CRISPR/Cas9 genome editing using three different targets for each one: 6 HEK 293T/17 cells in 10 cm dishes. These cells were maintained with Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% bovine calf serum (BCS) and 0.1% of gentamicin. Twenty-four hours later, cell medium was changed to serum-free IMDM. One hour later, cells were co-transfected with 15 ug of each different construct, 12.84 ug of psPAX2, and 2.58 ug of pMD2.G (VSV-g) using a 1:3 ratio of 1 mg/mL of polyethylenimine (PEI) pH 7.0. Seventy-two hours after transfection, cell culture supernatants were collected, centrifuged to remove cell debris, aliquoted, and frozen at -80° C.In order to produce lentiviral particles for viral transduction, integration and consequently constitutive expression, we packaged our lentiviral sgRNAs seeding a total of 3.5x10To establish an EGC line PK060399egfr knockdown of A2A and A2B, cells were infected with lentiviral particles containing the plasmids for the target genes overnight. The medium was removed and a supplemented DMEM containing puromycin (0.2 µg/mL) was added and changed every 48h for 7 days. The knockdown of A2A and A2B was confirmed by immunofluorescence (5 cells/well) were seeded in six-well plates and treated with TcdA or TcdB for 1.5, 3.5, and 4h. After incubation, the supernatant was removed, the cells were lysed using RIPA lysis buffer  and centrifuged , and the supernatant was collected. Protein concentrations were determined through the bicinchoninic acid assay according to the manufacturer’s protocol (Thermo Fisher Scientific). Reduced 40 µg protein samples (prepared with sample reducing agent- Invitrogen- and protein loading buffer-LI-COR) were denatured at 99°C for 5 min, separated on NuPAGE 4-12% BIS-Tris gel (Invitrogen), and transferred to nitrocellulose membranes (Life Technologies) for 2 h. The membranes were then immersed in iBind fluorescent detection solution (Life technologies) and placed in a iBIND automated Western device (Life Technologies) overnight at 4°C for blocking, incubating with primary antibodies  and secondary antibodies . Then, the membranes were immersed in ultrapure water, and fluorescent signal was detected using the Typhoon system . Densitometric quantification of bands was performed using ImageJ software .To evaluate the protein levels of pCREB and PKA, EGC lines (6×105 cells/well), which were plated in 6-well plates and treated with TcdA or TcdB for 4 h, using radioimmunoprecipitation assay (RIPA) buffer  containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13000 rpm for 15 min, and the supernatant was used to perform the protein assay using the bicinchoninic acid assay (Thermo Fisher Scientific). The transcription factor pCREB was measured using a commercial ELISA kit  according to the manufacturer’s instructions. The absorbance (450 nm) was determined using an Epoch plate reader (BioTek). Levels of these cytokines were measured as picograms per milligram of protein.Protein lysates were extracted from seeded EGC lines .Male C57BL/6 mice WT, A2A, and A2B Knockout  were housed in temperature-controlled rooms under 12 h light-dark cycles. The animals received water and food C. difficile colonization or infection, wild type (WT) or Knockout (A2A or A2BKO) C57BL/6 mice (n=8-9 for each group) received an antibiotic treatment  in drinking water for 3 days. After 1 day off antibiotics, an intraperitoneal injection of clindamycin (32 mg per kg) was given 1 day before C. difficile challenge. Then, 105 CFU of the vegetative C. difficile strain VPI10463  was administered by oral gavage. Control mice received the same vehicle. Mouse weights and the development of disease symptoms were monitored daily. Animals that became moribund or lost >20% of their body weight were euthanized.Our murine CDI model was established as previously described , 23, 24.A2AKO  were then stained with hematoxylin and eosin staining (H&E) and examined using light microscopy. Histopathological scores were performed by a blinded investigator, using a previously described method  µm were . Histopa2O2 for 10 min to reduce nonspecific binding. The sections were then incubated with the primary antibodies A2A , A2B , A3 , HuC/D , or IL-6 , overnight. The sections were then incubated for 30 min with polymer . The antibody binding sites were visualized by incubating the samples with diaminobenzidine–H2O2  solution. Sections incubated with antibody diluent without a primary antibody were used as negative controls. Antibody specificity was evaluated using positive controls for each antibody in the mouse large intestine (data not shown).The immunostaining for A2A, A2B, A3, HuC/D and IL-6 in cecum samples was performed at the UVA Biorepository and Tissue Research Facility. Briefly, Sections (4 µm thick) were prepared from paraffin-embedded mouse cecum tissues. After deparaffinization, antigens were recovered by incubating the slides in citrate buffer (pH 6.0) for 20 min at 95°C. Endogenous peroxidase was blocked with 3% HThe amounts of DAB products after immunostaining were estimated from at least 6-8 and from different areas of each section (from 8-9 specimens per group) for mouse samples, at 200X magnification using Image J software.EGC death in cecum tissues of infected WT, A2AKO, and A2BKO mice were investigated using a TdT-mediated dUTP nick end-labeling (TUNEL) assay and S100B immunoassaying (a marker for EGCs) according to the manufacturer’s protocol . After TUNEL staining was performed according to the manufacturer’s protocol, tissues were blocked using 5% donkey serum in PBS for 30 min at room temperature followed by incubation with S100B primary antibody  overnight at 4°C. Then, the cells were washed twice in PBS solution with 0.1% Tween 20 and incubated with secondary antibody  for 2 h at room temperature. After this time, cells were washed, stained with 4’,6-diamidino-2-phenylindole, dihydrochloride , washed in PBS, and mounted with antifade mounting medium (Vector). Cells were acquired using Leica DFC900 GTC VSC-11976 system (Leica) of at least three-eight distinct fields of each well per group from 2-3 different experiments. Cells were counted using ImageJ software . Fluorescence intensity of S100B was also measured by using ImageJ software .P< 0.05 was considered to indicate significance.The data are presented as the mean ± standard error of the mean (SEM). Student’s t test or one- or two-way analysis of variance (ANOVA) followed by the Turkey test was used to compare means. To identify the expression profile of adenosine receptors in EGCs challenged by TcdA and TcdB, we performed a qPCR to A1, A2A, A2B, and A3. We found that TcdA and TcdB upregulated A2A and A3 and downregulated A2B at 12 h incubation , but not A2A antagonist (SCH58261), decreased the levels of annexin and phosphatidylserine binding induced by TcdA and TcdB in EGCs , A2B antagonist (ATL801), and A3 agonist (CI-IBMECA). We found that A2A agonist (ATL313), A2B antagonist (ATL801), and A3 agonist (CI-IBMECA) decreased the levels of caspase 3/7 induced by both toxins , cell infiltrate (p<0.0001) and edema (p<0.0001) compared to WT infected mice (A2A knockout infected mice showed higher cell infiltrate (p<0.0001) and edema (p=0.002) score compared to WT infected mice  decreased the IL-6 expression induced by TcdA and TcdB in EGCs  and downregulated the proinflammatory-related receptor (A2B). Increased A2A expression was also found in murine macrophages, bone marrow-derived macrophages challenged by lipopolysaccharides (LPS)  , 30, andes (LPS) , 31. We ll lines . Decreasll lines .in vitro and in a murine model of CDI, respectively. These findings indicate the critical role of A2B in C. difficile toxin-induced EGC death. A2A deletion did not increase the EGCs death induced by C. difficile toxins which we speculate to be due to the presence of A3 receptors, whose activation promotes an antiapoptotic effect. As previously reported, deletion of A2B was protective against CDI, decreasing the intestinal epithelial damage and release of pro-inflammatory mediators in vivo , caspases 3, 6 and 7 (effector caspases) are part of the apoptotic caspases. Caspases 3 and 7 are found in the cytoplasm of cells in an inactive homodimers form that became proteolytic active once the initiators and activated executioner caspases cleave the linker that separates their large and small catalytic subunits, culminating in apoptosis  can be found in the article/The animal study was reviewed and approved by Committee on the Ethics of Animal Experiments of the University of Virginia (Protocol number: 4096).DC wrote the manuscript, performed all the experiments, analyzed and organized the data. JS, SG, DB, FM, AL and MJ helped in the acquisition of data. GB reviewed the manuscript. CW contributed to the organization of the manuscript, reviewed, and approved the final version. All authors contributed to the article and approved the submitted version.