All synthesized samples crystallized in a monoclinic structure with similar thermal expansion coefficients. The phase transition temperature between the monoclinic and tetragonal structure increases with increasing praseodymium content from 500 °C for undoped LaNbO4+δ to 700 °C for PrNbO4+δ. Thermogravimetry, along with X-ray photoelectron spectroscopy, confirmed a mixed 3+/4+ oxidation state of praseodymium. All studied materials, in humid air, exhibited mixed protonic, oxygen ionic and hole conductivity. The highest total conductivity was measured in dry air at 700 °C for PrNbO4+δ, and its value was 1.4 × 10−3 S/cm.In this paper, the structural properties and the electrical conductivity of La These compounds are very interesting thanks to a wide possibility of modification of their chemical composition, which allows obtaining materials with different structures and properties. The first work focused on ABO4 type compounds was published by Keller in 1962 4 materiant (TEC) , Ba 20]4 materiant (TEC) , Pr (IIInt (TEC) , Nd and nt (TEC) , Tb (IIInt (TEC)  and Yb 4 materiant (TEC) , Cr, Mn,nt (TEC) ,29 and Tnt (TEC) , Co 30]4 materiant (TEC) ,32, Zr 4 materiant (TEC)  and W [3nt (TEC)  have bee2 and a proton transference number close to 1. The undoubted disadvantages of rare earth niobates are low, in relation to the results obtained for perovskite materials, total conductivities and the occurrence structural transformation at temperatures around 500 °C. As a result of the transformation from the monoclinic structure to the tetragonal structure, the thermal expansion coefficient (TEC) changes by over 31% (from 12 × 10−6 K−1 to 8.3 × 10−6 K−1). Such a large change in TEC increases the probability of mechanical failure of electrochemical devices consisting of anode, electrolyte and cathode. The chosen properties of various proton-conducting materials are presented in The advantages, compared to the other types of proton conducting ceramics, of the rare earth orthoniobates include high chemical stability in atmospheres containing impurities such as CO1−xWxO4+δ [0.9Pr0.1NbO4) [1−x−yCaxPryNbO4+δ, 0 < x < 0.02, 0 < y < 0.1) [1−xTbxNbO4+δ, 0 < x < 0.3) lanthanum orthoniobate [4+δ as a function of the temperature in wet air [+ and 4+ oxidation states.The proton conductivity in the lanthanum orthoniobate has been intensively examined; however, less effort has been devoted to oxygen ionic conductivity. In the lanthanum orthoniobate, the oxygen ionic conductivity may be enhanced by introducing donor substitutions, which may lead to oxygen interstitial formation. For example, several experiments were carried out concerning the use of tungsten substitution in the niobium sublattice of LaNb−xWxO4+δ ,52,53,54−xWxO4+δ . Additio0.1NbO4) , praseody < 0.1) ,22 and toniobate  were syn wet air . It shouIn this work, the structural and electronic properties of praseodymium-doped lanthanum orthoniobate and praseodymium orthoniobate are presented and discussed. An improved defect chemistry model, based on the obtained results, has been presented.1−xPrxNbO4+δ  were prepared with the use of solid-state synthesis. La2O3 , Pr6O11  and Nb2O5  were used. The stoichiometric amounts of the oxides were hand-milled in an agate mortar with the addition of isopropanol. The obtained powders were uniaxially pressed at 400 MPa into pellets characterized with 12 mm of diameter. The green bodies were calcined at 1000 °C for 12 h. After the calcination, the specimens were ground into powders, pressed again and sintered at 1400 °C for 12 h.Samples of La4+δ (ICSD: 01-073-6079) and PrNbO4+δ (ICSD: 01-074-6652) structure parameters were used during the analysis [The powder X-ray diffraction measurements were performed with a Philips X’Pert Pro MPD  with Cu Kα radiation. The data were analysed with the FullProf suite  . The monanalysis ,58. The The microstructure was characterized with FEI Quanta FEG 250  scanning electron microscope (SEM) equipped with EDAX Apollo-SD energy-dispersive X-ray spectroscopy  (EDS) detector. The microstructure imaging was performed in High Vacuum mode with an Everhart–Thornley detector working in secondary electrons (SE) and back-scattered electrons (BSE) mode.−8 mbar. The system incorporates a monochromatic Mg Kα X-ray source (hν = 1253.6 eV) and an Omicron Argus hemispherical analyser with a 128-channel detector. The X-ray source was operated at 20 mA emission current and 15 kV anode bias. Spectra were aligned by alignment of the C 1s core line at the binding energy of 284.8 eV. All data were analysed using the XPSPeak [The praseodymium orthoniobate was characterized using X-ray photoelectron spectroscopy (XPS). The spectra were recorded with an Omicron NanoTechnology photoelectron spectrometer system  operating at pressure below 1 × 10 XPSPeak .® 449 F1 . The samples were heated to 900 °C with a heating/cooling rate of 2 °C/min. In the water uptake measurements, the as-prepared powders were heated to 800 °C under dry air to remove water and other impurities. The samples after dehydration were cooled to 300 °C in dry gas. After 2 h of stabilization, the dry gas was switched to the humidified gas (PH2O = 0.023 atm) and, after an additional 2 h, the gas was switched back to the dry gas. Proton defect concentration was calculated with Equation (1):The dilatometry was performed by Netzsch DIL 402 PC/4  dilatometer in argon in the temperature range of 50–1000 °C with a heating rate of 3 °C/min. Thermogravimetric measurements of mass change as a function of temperature were performed in technical air and nitrogen using a Netzsch Jupiter−30 atm) and water vapour (2.36 × 10−2 atm–6 × 10−5 atm) partial pressures. For the measurements performed in the air, the synthetic air mixture consisting of 20% O2 and 80% N2. The spectra were collected with Gamry Reference 3000  in a temperature range from 350 °C to 750 °C with 50 °C steps. The obtained data were analysed with ZView software. An equivalent circuit consisting of (RQ)(RQ)(RQ) elements has been used. The highest frequency semicircle is attributed to the grain interior conductivity process, the mid-frequency semicircle is related to grain boundaries and the third one, if distinguishable, describes processes related to the exchange of charge with the electrode. To identify the process for each semicircle, the capacitance was calculated with the equation:n are pseudocapacitance, resistance and the angle of misalignment, respectively. The total conductivity . The measurements were performed in a wide range of oxygen σ=σeAE, of the conductivity was calculated with the use of the Arrhenius Equation (7):The activation energy, 1−xPrxNbO4+δ powders are presented in I 2/a) LaNbO4+δ structure. The pattern of praseodymium orthoniobate has been indexed with the monoclinic (I 2/a) PrNbO4+δ. As can be seen, all samples were obtained as single-phase materials. The XRD patterns were analysed with the LeBail refinement method. An example of the fitted profile is presented in The XRD patterns of the LaThe calculated structure parameters  indicatedecrease , while t4+δ and PrNbO4+δ. The ceramics of intermediate compositions show higher porosity and vary in the microstructure. The microstructure of the La0.95Pr0.05NbO4+δ and La0.85Pr0.15NbO4+δ sample is quite dense, and a few large pores can be seen. On the other hand, the La0.9Pr0.1NbO4+δ, La0.8Pr0.2NbO4+δ and La0.7Pr0.3NbO4+δ are characterized by numerous pores with a size below 10 μm. The analysis with EDS and BSE detectors did not show the existence of any secondary phases, which is in accordance with the XRD results. The observed differences in porosity of the samples are consistent with the results of density measurements determined by the Archimedes method, which are presented in Scanning electron microscopy images of polished fractures of the sintered pellets are presented in 4 and PrAlO3 did the authors try to associate the presence of this signal with the presence of oxygen ions in interstitial positions; however, this thesis could not be confirmed in other experimental and theoretical studies [+ oxidation state of niobium [The XPS spectrum collected for the O 1s region is presented in  studies ,67. The  niobium ,68.5/2–3d3/2 doublets, with separation energy of 20.2 eV, and two single signals [94f3 and 3d94f2 states, respectively, and are observed for both 4+ (PrO2) and 3+ (Pr2O3) praseodymium oxidation states. Doublet I represents the 3d94f1 configuration and is observed regardless of the praseodymium oxidation state, but the intensity of this signal increases with an increase of Pr4+ content [4+δ. The spectra of PrNbO4+δ correspond to these obtained by Lütkehoff et al. for Pr6O11 [6O11, which is approximately 0.66.The 3d praseodymium band is presented in  signals ,69,70. Ar Pr6O11 . This me0.9Pr0.1NbO4+δ, La0.8Pr0.2NbO4+δ, La0.7Pr0.3NbO4+δ and PrNbO4+δ are presented in −4 mol/mol) was found in La0.8Pr0.2NbO4+δ, while the lowest was found in PrNbO4+δ (2.9 × 10−4 mol/mol).The thermogravimetric analysis of water uptake was performed to determine the concentration of proton defects forming in wet gases. The results obtained for LaC) and thermal expansion coefficient (TEC). The results of the measurements are presented in Dilatometry measurements were conducted to determine the phase transition temperature  at 700 °C in wet air was measured for La0.95Pr0.05NbO4+δ. The conductivity of materials with higher praseodymium content does not depend monotonically on the content of praseodymium. To analyse the influence of praseodymium content on the electrical properties of grains and grain boundaries, the components of total conductivity were determined. 0.85Pr0.15NbO4+δ.The total conductivity of La1−xPrxNbO4+δ for 0.05 ≤ x ≤ 0.3, the conductivity of grains is lower than that of the grain boundaries. At lower temperatures, the relation between them is opposite; the conductivity of grains is higher than the conductivity of the grain boundaries. Among all studied materials, at 700 °C, La0.9Pr0.1NbO4+δ and La0.95Pr0.05NbO4+δ exhibit the lowest and the highest conductivity of grain boundaries, respectively  determined in the oxygen partial pressure range above 1 × 10−2 atm are positive, and for the materials containing praseodymium they are in the range of 0.13–0.16. On the other hand, for praseodymium-doped and undoped LaNbO4+δ, an increase in the conductivity in hydrogen (pO2 = 1 × 10−30 atm) is observed, while for PrNbO4+δ a further decrease of the conductivity was observed. Measurements of total conductivity of PrNbO4+δ as a function of oxygen partial pressure, at different temperatures, are presented in 0.9Pr0.1NbO4+δ, where the slope factor value increases with increasing temperature  during phase transition, which occurs in the range of working temperatures of protonic devices [The observed decrease of unit cell parameters = 1.13 Å ) and Pr4= 0.96 Å ) compare= 1.16 Å ) and is = 1.16 Å . The det reports ,74. An itructure . The disperature . A similperature . What seperature  because  devices ,79,80.The influence of the introduction of the rare earth element on the electrical properties of lanthanum orthoniobate is related to two factors. First, the type and content of the rare earth element may influence the concentration and/or mobility of particular types of charge carriers under the given conditions. The second factor results from the microstructure of the material, which may also depend on the content of the rare earth element. The microstructure influences the overall conductivity of the material by affecting the conductivity of the grain boundaries and the porosity of the material.−3 atm. The presence of mixed conductivity in praseodymium-containing orthoniobates is related to the possible mixed valency of praseodymium. As the XPS results showed, in the analysed materials, praseodymium may occupy two oxidation levels, namely, 3+ and 4+, so that it may act either as an isovalent or a donor substituent. The change of mass of the samples recorded during thermogravimetric measurements  presents the formation of defects in La0.8Pr0.2NbO4+δ, with praseodymium being an isovalent (3+) substitution. In this case, oxygen nonstoichiometry, Below, we analyse possible defect processes which may occur in both cases. The equations represent defect chemistry for La4+, which is a donor dopant, two types of charge compensation processes may occur. The first one, which prevails in oxidizing conditions, is oxidation (Equation (9)) in which the excess positive charge of In the case of Pr4+ charge compensation is the introduction of additional electrons into the conduction band (Equation (11)), which may occur in the material with a narrow energy bandgap. Another factor to consider is the location of electron-holes. Depending on thermodynamic conditions, the holes may be localized on Pr4+ cations, forming small polarons, or at sufficiently high temperatures they may be excited to the valence band. The latter process is described by Equation (13). In this work, it was assumed that the analysed materials are similar to other oxides containing cations on various oxidation states, such as cerium oxide doped with gadolinium and cerium vanadate, in which the dominant charge carriers are small polarons [+ in the lanthanum sublattice-The results of thermogravimetric measurements  suggest polarons ,85,86,870.9Pr0.1NbO4+δ, the values of the slope factor increase with the temperature from 0.11 to 0.14. The temperature range in which an SL increase is observed (500–700 °C) corresponds to that in which the change of the mass recorded with TGA, related to oxygen incorporation ):eratures  indicateporation , occurs.urements  decreaseeratures , where t−4–4.9 × 10−4 mol/mol) are much lower than in classic proton conductors. For instance, the concentration of proton defects in BaZr0.8Y0.2O3−δ is 4 × 10−2 mol/mol [0.98Ca0.02Nb0.9Sb0.1O4−δ it is 1.7 × 10−3 mol/mol [1−xPrxNbO4+δ on the total conductivity (2O) (0.11–0.27) indicate different mechanisms of proton conductivity compared to that of the acceptor-doped lanthanum orthoniobate and other typical proton conducting oxides. In these materials, in which the slope coefficient is around 1/2 [1−xPrxNbO4+δ, oxygen ions in interstitial positions take part in proton defect formation. It should be noted that the thermogravimetry technique does not allow us to identify whether the dominant proton defect formation reaction involves oxygen vacancy or the incorporation of oxygen into an interstitial position. On the other hand, the possibility of interstitial oxygen presence in orthoniobates containing praseodymium has already been shown. Below we analyse the processes of proton defect formation in which, together with a proton defect, an oxygen interstitial ion is formed (Equation (17)). Such a mechanism was previously analysed by Xing et al. for TiNb2O7 and Fisher et al. for Ba2In2O5 [The substitution of lanthanum with praseodymium also influences proton conductivity. As was shown by the thermogravimetric studies , the mas mol/mol , while i mol/mol . The infuctivity  depends ound 1/2 , proton Ba2In2O5 ,92.(17)If this hydration reaction occurs in the atmospheres with high oxygen and water vapour partial pressure, the concentration of proton defects, small polarons, 1−xPrxNbO4+δ with As can be seen, the predicted dependencies of proton defect concentration on the water vapour partial pressure  are consistent with the values of slope coefficients  close to1−xPrxNbO4+δ  versus log (pH2O) is negative and close to −1/6. The decrease of total conductivity in wet atmospheres is also visible in the measurements as a function of temperature  and PrNbO4+δ materials were synthesized with the solid-state reaction method. All samples were single-phase, dense ceramics showing similar thermal expansion coefficients. The volume of the unit cell decreases with an increase of praseodymium content. The addition of praseodymium stabilizes the monoclinic structure, which leads to an increase of phase transition temperature from 500 °C for undoped LaNbO4+δ to 700 °C for PrNbO4+δ. The XPS and thermogravimetric analyses indicated that praseodymium occupies the mixed 3+/4+ oxidation states.A series of praseodymium-substituted lanthanum orthoniobate La1−xPrxNbO4+δ have changed. In atmospheres characterized with both high oxygen and water vapour partial pressures, these materials conduct three types of charges: proton defects, oxygen ions and electron-holes. Analysis of the conductivity of the materials as a function of water partial pressure indicates that the incorporation of proton defects is accompanied by oxygen interstitial formation. The highest conductivity in dry air at 700 °C was 1.4 × 10−3 S/cm for PrNbO4+δ. A non-monotonic dependence of the total conductivity on praseodymium content was observed. The influence of higher porosity and oxidation of grain boundaries on the electrical properties of materials has been discussed. In comparison to praseodymium orthoniobate, the major type of defects are proton defects, while for the PrNbO4+δ it is electron holes/small polarons, Owing to the presence of praseodymium with a mixed oxidation state, the electrical properties of La


Almost all of the iron sand found in Indonesia, from Sumatra to Papua, is sandy deposit. Despite its variety of minerals, iron sand is commonly mined for low economic uses such as building material. As iron sands from different localities might have different characteristics , such characteristics might in turn be used for provenance study or for selective mining. This study aims to examine iron sand deposits from two geographically close but geologically different regions, Lampanah and Anoi Itam, and to test the grain size distributions and their relationship with magnetic susceptibility as well as Fe content. In both Lampanah and Anoi Itam, the sizes of iron sands were predominantly medium sand (MS) and fine sand (FS), but they differed in mass percentage (M%). Generally, magnetic susceptibility increases as grain size decreases. Fe content is also grain size dependent, with higher Fe content in finer sizes. The results imply that the combination of grain size distribution and magnetic mineral composition might be used not only as a provenance indicator for iron sand deposits, but also as a criterion for selective mining. Iron sand; Magnetic susceptibility; Magnetic mineralogy; Grain size distribution; Provenance study; Aceh; Indonesia. This study is a maiden attempt to test whether different iron sand deposits have different grain size distributions and to verify whether different grain sizes indeed have different mineral compositions. If successful, the combination of grain size distribution and magnetic mineral composition might be used not only as a provenance indicator for iron sand deposits but also as a criterion for selective mining.2Samples were taken from 2 sites, Lampanah and Anoi Itam beaches. Lampanah is located in the Northern tip of Sumatra  to the east of Banda Aceh , and Anoi Itam is located on Weh Island  across a narrow strait to the north of Banda Aceh see . Both LaEach sample was then sieved through a 10-mesh sieve (2 mm opening) to eliminate particles larger than sand size. A small amount was set aside and is referred to as the bulk subsample. The remaining samples were then sieved through a series of 18-mesh, 35-mesh, 60-mesh, and 120-mesh sieves. Through this series of sieves, iron sand samples could be divided into 5 subsamples depending on their grain size, i.e., very coarse sand , coarse sand , medium sand , fine sand , and very fine sand  see . The sub3 was set aside and placed inside 3 cylindrical sample holders  for magnetic susceptibility measurements. These samples in cylindrical holders were weighed using an Ohaus analytical balance before they were measured using a Bartington MS2 magnetic susceptibility system  with MS2B dual-frequency sensor. The results of these measurements were low-frequency (460 Hz) and high-frequency (4.6 kHz) mass-specific magnetic susceptibilities, denoted respectively as χLF and χHF. The third parameter, termed frequency dependent susceptibility or susceptibility χFD (%), could be derived as χFD(%) = 100% × (χLF − χHF)/χLF.For each subsample (including the bulk samples), about 30 cmSelected subsamples were also subjected to x-ray fluorescence (XRF) analysis using a Supermini 200 x-ray fluorescence spectrometer  to identify their major and trace elements. Selected samples were also subjected to x-ray diffraction (XRD) analysis using a SmartLab x-ray diffractometer  equipped with a Cu target to identify the minerals contained in the subsamples. For these analyses, a few grams of subsamples were pulverized so that they could pass through a 200-mesh sieve.3Results of M% and magnetic susceptibility (χLF) for all subsamples from Lampanah and Anoi Itam are summarized in CU) and the coefficient of curvature (CC) of each subsample could easily be calculated from the curves in CU = D60/D10 and CC = (D30 × D30)/(D60 × D10) where D10 is the particle size at which 10% of the particles are finer and 90% of the particles are coarser than D10 size, D30 is the particle size at which 30% of the particles are finer and 70% of the particles are coarser than D30 size and D60 is the particle size at which 60% of the particles are finer and 40% of the particles are coarser than D60 size (CU and CC for Lampanah varies from 0.1833 and 0.970 (for subsample L-8) to 2.050 and 1.098 (for subsample L-4). For Anoi Itam subsamples, the values of CU and CC varies from 1.727 and 0.938 (for subsample S-3) to 2.384 and 0.993 (for subsample S-6). Despite slight differences in their values of CU and CC, both Lampanah and Anoi Itam samples could be classified as poorly graded as expected for beach sand.D60 size . The valLF value varied for each sand grain size. Based on the average, the χLF values for Lampanah sand grains increase from coarser to finest grain size, aligned as VCS = 719.2 ± 4.3 (× 10−8 m3/kg), CS = 1313.3 ± 1.1 (× 10−8 m3/kg), MS = 2582.8 ± 2.0 (× 10−8 m3/kg), FS = 2531.0 ± 2.0 (× 10−8 m3/kg), and VFS = 3933.9 ± 2.8 (× 10−8 m3/kg), with the highest χLF value being VFS size. Similar to Lampanah sand grain size, the χLF values for Anoi Itam sand grains also increase from coarser to finest grain size, aligned as VCS = 966.9 ± 3.7 (× 10−8 m3/kg), CS = 1897.3 ± 2.8 (× 10−8 m3/kg), MS = 3571.8 ± 3.4 (× 10−8 m3/kg), FS = 3033.7 ± 4.2 (× 10−8 m3/kg), and VFS = 3087.1 ± 5.1 (× 10−8 m3/kg), with the highest χLF value being MS size.Samples from L-7 and S1 were selected for XRF analysis, and the results are shown in 2GeO4) was also found in the VFS sample of S-1.Figures 4In densely populated countries such as Indonesia, massive exploitation of iron sand deposits for low economic use is a controversial issue, as it affects not only the environment but also the livelihoods of coastal communities. Therefore, selective and low-quantity exploitation might be preferable compared to the more common method of strip mining. As shown in this study, the Fe content varies greatly between localities as well as between different grain sizes, so combined grain-size distribution and magnetic susceptibility tests might be useful for preliminary investigation for selective mining. In Japan, iron sand is still being used  in the making of classical weapons .In this study, although Fe content correlates linearly with particle size (finer particles tend to have higher Fe content), it does not always correlate linearly with magnetic susceptibility as some iron-bearing minerals could be paramagnetic see . SimilarThus, if the objective of exploration is to find magnetic minerals or iron-bearing minerals then according to Tables LF) of bulk samples from Lampanah and Anoi Itam are comparable to those of iron sand from Papua, Indonesia  so that their grain size distributions are known. Second, bulk and subsamples are measured for their magnetic susceptibility. Third, bulk and subsamples are measured for Fe, Si, and Ca content. Measurements of other elements and other types of analyses are optional.2GeO4) in the VFS sample of S-1 is also interesting. This mineral is one of the compounds containing an oxyanion of germanium, collectively called germanates .This work was supported by Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


Abrus precatorious seeds, which comprises a catalytically active A chain and a lectin-like B chain linked by a disulfide bond. Four isotoxins of abrin have been reported with similar amino-acid composition but different cytotoxicity, of which abrin-a is the most potent toxin. High lethality and easy availability make abrin a potential bioterrorism agent. However, there are no antidotes available for managing abrin poisoning, and treatment is only symptomatic. Currently, neutralizing antibodies remain the most effective therapy against biotoxin poisoning. In this study, we prepared, identified, and acquired a high-affinity neutralizing monoclonal antibody (mAb) 10D8 with a potent pre- and post-exposure protective effect against cytotoxicity and animal toxicity induced by abrin-a or abrin crude extract. The mAb 10D8 could rescue the mouse injected intraperitoneally with a 25 × LD50 dose of abrin-a from lethality and prevent tissue damages. Results indicated that 10D8 does not prevent the binding and internalization of abrin-a to cells but inhibits the enzymatic activity of abrin-a and reduces protein synthesis inhibition of cells. The high affinity, good specificity, and potent antitoxic efficiency of 10D8 make it a promising candidate for therapeutic antibodies against abrin.Abrin is a types II ribosome-inactivating protein (RIP) isolated from  Abrus precatorious seeds, which inhibits protein synthesis in eukaryotic cells and consequently triggers apoptosis . Ta. TaRicinIt has been reported that abrin induces apoptosis in HeLa cells and three leukemic cell lines through caspase activation ,21. To pSDS-PAGE and Western blot under reducing or nonreducing conditions were performed to explore the epitopes recognized by 10D8. Abrin-a, abrin-b, and AAG displayed different profiles in reducing and nonreducing SDS-PAGE, while the abrin crude extract showed characteristics of all three toxins A. The reTo examine whether 10D8 inhibits the internalization of abrin into cells, cells treated with abrin-a and 10D8 were analyzed by a high-content cell analysis system. The extent of abrin-a internalization into cells was determined by measuring the fluorescence intensity of Alexafluor-488 labeled abrin-a in the cells. There was no significant difference in the fluorescence intensity in the presence of 10D8, suggesting that 10D8 does not prevent the attachment of abrin-a to cell surface and the further internalization of abrin-a into cells D,E. Thes50 of abrin-a or a lethal dose of abrin crude extract. The mAb 10D8 was administered via the same route 1 h before or 1, 3, 6, 9, or 12 h after abrin-a challenge. All of the mice became very ill and showed weight loss, diarrhea, drowsiness, and weakness, dying within 48 h of abrin-a intoxication without 10D8 treatment. The mAb 10D8 rescued 100% of mice from lethality via either prophylactic or 1, 3, or 6 h post-exposure treatment, and all of the mice remained healthy and lived up to 21 days  and treated with 100 μg of antibody at 6 h after intoxication. All of the mice without antibody treatment died by 7 days after intoxication [50 of abrin-a, and all of the mice died within 48 h of intoxication , China .Purified abrin-a protein dissolved in PBS at a concentration of 2 mg/mL was dialyzed using a Slide-A-Lyzer dialysis cassette  against 4% paraformaldehyde solution at 47 °C for 16–18 h. Then, the cassette was transferred to PBS solution (pH 7.4) to remove the remaining paraformaldehyde through dialysis, and the procedure was repeated every 12 h for a total dialysis time of 72 h. At last, the abrin-a solution in the cassette was pipetted and collected into a new tube. The concentration of purified inactivated abrin-a protein was determined by BCA assay.Six week old BALB/c mice were immunized intraperitoneally with 33 μg of inactivated abrin-a mixed with Freund’s complete adjuvant  four times at 2 week intervals. Hybridomas were produced by fusion of spleen cells from the immunized mice with myeloma cells 4 days after the last immunization, according to a published procedure . Brieflym/v) BSA for 2 h at 37 °C. After the blocking steps, 100 µL of supernatant from the hybridoma cultures serially diluted in PBST containing 0.5% BSA was added to the wells to incubate for 1 h at room temperature. After the wells were washed three times with PBST, bound antibodies were detected with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody . Following washing, the colorimetric reaction was measured using 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate after a 10 min development period. The reaction was then terminated with 2 M H2SO4 , and the absorbance at 450 nm was measured using a microplate reader .Indirect ELISAs were employed to screen the established hybridomas. Briefly, microwell plates were coated with 100 µL of 5 µg/mL abrin protein in 0.05 M bicarbonate buffer (pH 9.6) at 4 °C overnight and blocked with 300 µL of 1% (m/v) BSA for 2 h at 37 °C. Then, 100 µL of 10 μg/mL screened mAbs were added and incubated for 1 h at 37 °C. Wells were washed with PBST, before adding 100 µL of 1:1000 dilution of HRP-labeled anti-mouse antibody and then incubating for 40 min at 37 °C. Wells were washed with PBST, before adding 100 µL of TMB substrate solution and incubating for 10 min. The reaction was terminated by 50 µL of 2 M H2SO4. Absorbance at 450 nm was measured using a microplate reader .Microplates were coated with 100 µL 10 μg/mL of either abrin-a, abrin-b, AAG, ricin, or RCA120 in 0.05 M bicarbonate buffer (pH 9.6) at 4 °C overnight and blocked with 300 µL of 1%  supplemented with 10% fetal bovine serum , 100 units/mL penicillin, and 100 μg/mL streptomycin , and incubated at 37 °C in the presence of 5% CO4 cells/well. For the cell death study, HeLa cells were pretreated with 50 μM Z-VAD-FMK , 50 μM Nec-1 , or 100 nM wortmannin  1 h prior to abrin-a intoxication. Then, abrin-a was diluted with serum-free RPMI-1640 medium at 10 or 100 ng/mL and added into each well. Toxins were diluted with serum-free RPMI-1640 medium at various concentrations and preincubated with or without 10 μg/mL monoclonal antibodies at 37 °C for 1 h. Alternatively, antibody 10D8 was added to cells 1, 3, or 6 h after intoxication. Cells were incubated at 37 °C for 24 h. A10 μL aliquot of CCK-8 reagent  was added into each well and incubated at 37 °C for 1 h. Absorbance at 450 nm was measured using a microplate reader . The survival rate of cells was calculated as follows: survival rate % = (A450 sample/A450 control) × 100%.HeLa cells were seeded in 96-well cell culture plates at a density of 1 × 10® T7 coupled reticulocyte lysate systems  containing total amino acids, RNase inhibitor, and luciferase mRNA and incubated for 1.5 h at 37 °C. After the incubation, 1 μL of the reaction mixture containing synthesized luciferase was mixed with 50 μL of luciferase assay reagent pre-equilibrated to room temperature, and the extent of the synthesized luciferase was evaluated by measuring the luminescence.Abrin-a (10 ng/mL) alone or along with 10D8 (10 μg/mL) was added to TNT4 cells/well. The cells were treated with abrin-a (100 ng/mL) or abrin-a preincubated with 10D8 (20 μg/mL) for 3 h at 37 °C. The cells were washed three times with PBS and fixed with 4% paraformaldehyde. The fixed cells were incubated with 0.1% Triton X-100 for 30 min for penetration and blocked for 30 min with 5% BSA at room temperature. The cells were treated with 10D8 for 2 h at 37 °C and washed three times, before incubating with Alexa-488 labeled anti-mouse IgG  for 1 h and washing three times. The nuclei were stained with Hoechst-33342. The plate was visualized and analyzed using a high-content cell analysis system .HeLa cells were seeded in a 96-well black cell culture plate at a density of 1 × 10Firstly, 4 μg of abrin-a, abrin-b, and AAG and 20 μg of abrin crude extract were loaded and separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions, stained with Coomassie Blue solution .Firstly, 100 ng of abrin-a was loaded and separated on 10% SDS polyacrylamide gels under reducing or nonreducing conditions, before transferring onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% BSA, the membranes were incubated with 10D8 antibody diluted to 1:1000 and HRP-labeled anti-mouse secondary antibody  diluted to 1:5000. The immunostaining signal was visualized using ECL Plus reagent  and imaged with FluorChem E .50 of abrin-a (25 μg/kg) [Adult BALB/c mice (eight mice per group) were injected intraperitoneally with 25 × LD5 μg/kg) ,32,33 orp < 0.05, ** p < 0.01, *** p < 0.001).Data were expressed as the means ± standard deviation (SD) of three independent experiments. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test with GraphPad Prism 8 software (*


Various studies have reported that altered expression of METTL3 is associated with an aggressive malignant phenotype and favors migration and invasiveness, but its role in Thymic Tumors remains unknown.Thymic epithelial tumors (TETs) are rare neoplasms, originating from epithelial thymic cells. The oncogenic potential of these rare neoplasms is still largely undefined, and a deeper molecular characterization could result in a relevant advance in their management, greatly improving diagnosis, prognosis and treatment choice. Deregulation of N6-methyladenosine (mIn this study, we characterized that METTL3 contributes to Thymic Epithelial Tumor phenotype. We evidenced that METTL3 is overexpressed in tumor tissue compared to normal counterpart. Silencing of METTL3 expression in thymic carcinoma cells results in reduced cell proliferation and overall translation rate. Of note, METTL3 is responsible for the induction of c-MYC expression in TET cells. Specifically, high expression of c-MYC protein is enabled by lncRNA MALAT1, which is methylated and delocalized by METTL3. Interestingly, blocking of c-MYC by using JQ1 inhibitor cooperates with METTL3 depletion in the inhibition of proliferation and induction of cell death.This study highlighted METTL3 as a tumor promoter in Thymic tumors and c-MYC as a promising target to be exploited for the treatment of TET.The online version contains supplementary material available at 10.1186/s13148-021-01159-6. Thymic epithelial tumors (TETs) are very rare neoplasms comprising thymoma (THY) and thymic carcinoma (TC) that establish a unique group of cancers arising from the epithelial cell of the Thymus . On the The malignant potential of thymomas, ranging from low to very aggressive locally, is in relation to the stage of these tumors , 5. The Complete surgical resection is the gold standard when feasible; chemotherapy like the combination of cisplatin, doxorubicin and cyclophosphamide (PAC) and/or radiotherapy can be administered as an induction at advanced stages or prior to surgery as adjuvant treatment in case of advanced pathological stages , 8.The management of TET is a paradigm of synergistic strategy to guarantee an advance in clinical and translational research in these tumors .Significant advances have recently been made in the characterization of the molecular landscape at the basis of TET underlined by The Cancer Genome Atlas (TGCA)  data pub6A), one of the most abundant internal modification of mRNA and noncoding RNA (ncRNA), is an important modification involved in RNA splicing [N6-methyladenosine  and METTL14 (or KIAA1627) [6A modification, like WTAP, required for complex localization into nuclear speckles [mIAA1627) . The firIAA1627) , 25. Othspeckles , VIRMA  methylation of RNAs to thymic cancer, we first took advantage of the thymoma GEPIA dataset to evaluate whether any modulation of these components was present between cancer and normal tissues. As shown in Fig. N = 22). Interestingly, analysis of METTL3 protein staining intensity in epithelial cancer cells revealed an increase of intensity from types A/AB (score of intensity = 1) to types B1-B3 (score of intensity = 2), and reached a maximum of intensity (score = 3) in thymic carcinoma cases (C) , its substrate ribosomal protein S6 (S6), and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), revealed that METTL3 depletion causes a marked and significant decrease of the phosphorylated forms of these upstream regulators, compared to control cells  , 45, 46.6A immunoprecipitation confirmed that c-MYC is a target of METTL3 methyltransferase activity, as silencing of METTL3 led to a 40% reduction of c-MYC mRNA methylation . We observed that CDDP treatment or METTL3 depletion significantly induced cell death and that the combination of CDDP and METTL3 silencing determined an additive effect, compared to individual treatments, leading to reduced proliferation Fig. A, left a μg/mL. WOn the basis of METTL3-dependent c-MYC protein induction, we evaluated the effect of the combination of METTL3 depletion and c-MYC inhibition on cell death and proliferation. c-MYC was inhibited using the small molecule JQ1, a selective bromodomain inhibitor able to block the c-MYC inducer BRD4 –52. 24 hThis result suggests that a block of c-MYC cooperates with METTL3 depletion, impinging on cell viability and proliferation, and highlighting this axis as a promising target to be exploited for the treatment of TET.6A methyltransferase complex, plays a crucial role in many biological processes especially in tumorigenesis, either dependently on or independently from its m6A RNA methyltransferase activity[This study focused on the study of a specific enzyme, METTL3, and its role in Thyimic epithelial tumors (TETs). METTL3, the main component of the m activity, 54.As described in a variety of studies , 56, MET6A modification of its mRNA in several tumors as for example in thyroid cancer [One of the most well-known oncogenes and key regulator of cell proliferation is c-MYC. Several reports have demonstrated that abnormal expression of c-MYC may be related to increased md cancer , or in od cancer . Consistd cancer .6A modifications can also regulate the biogenesis and function of noncoding RNAs, such as miRNAs, lncRNAs, and circRNAs [Recently, different studies have reported that mcircRNAs . In this6A modification of c-MYC mRNA (data not shown), suggesting that it is not required for METTL3 enzymatic activity.Similarly, to METTL3-depleted cells, MALAT1-depleted TC1889 also showed decreased c-MYC protein, suggesting that METTL3 could regulate c-MYC translation also through the delocalization of MALAT1. However, silencing of MALAT1 didn't impact on the m6A modification could mediate also the effects previously observed in breast cancer cells is certainly an intriguing possibility that merits to be verified in future studies. Interestingly, it has been also recently reported that m6A modification of MALAT1 is critical for metastatic ability of cancer cells, reshaping the nuclear speckles [Previously, we have shown that an alternative oncogenic stimulus (e.g. the presence of mutated p53 protein and overexpressed ID4) causes a similar delocalization of MALAT1, in breast cancer cells, with consequent altered splicing . The posspeckles .Although the gold standard treatment for thymic epithelial tumors remains surgery, the use of several innovative agents for TET treatment is under investigation . For thiIn conclusion, our study identified the oncogenic role of METTL3 also in Thymic Ephitelial Tumors, which improves cells proliferation trough the control of c-MYC expression. Moreover, our results highlight METTL3 as a powerful target whose depletion improves the response to different anticancer treatments and open to the possibility of exploiting this protein to improve the treatment of TETs.Human Thymic Carcinoma cell line TC1889, provided by Prof. Ralf J Rieker , was culTC1889 cells were transfected with siRNAs for METTL3 , MALAT1  and contAfter 24 h of METTL3 silencing, TC1889 cells were treated with Cisplatin  , for additional 48 h. The same experiment was performed with ( +)-JQ1  treatment.METTL3 protein expression was analyzed by immunohistochemistry (IHC) in a set of 22 Formalin fixed, paraffin embedded (FFPE) thymic epithelial tumors of different histotypes derived from the Pathology Department of IRCCS Regina Elena National Cancer Institute of Rome, Italy. The thymomas FFPE samples included: 2 type A, 5 type AB, 3 type B1, 6 type B2, and 1 type B3. Moreover, 5 FFPE carcinoma samples were analyzed. Thymomas and thymic carcinoma samples from “Policlinico Umberto I” Hospital of Rome, Italy were used to set up the experimental condition. This study was approved by the Institutional Review Board of “Policlinico Umberto I” Hospital (Rif 3262/26.06.2014 Prot. N° 815/14) and the Regina Elena National Cancer Institute (Rif 383/28.5.2013 Prot. N°5/13 and Rif 447/20.06.2013 Prot. N°7/13). In brief, 5 μm-thick sections from each sample were Haematoxylin and Eosin stained, and consecutive sections were stained with the anti-METTL3 antibody . The METTL3 immunostaining intensity was determined according to Liang et al., 2020 , and the3 cells/well. Medium was changed every four days. 10–14 days after plating, cells were washed with PBS for three times and were stained using crystal violet dye. Cells were counted using ImageJ software.Cells were detached 72 h after METTL3 silencing and seeded into 6-well dishes at a concentration of 1 × 10−ΔΔCt method, using H3 as control. All reactions were performed in duplicate. The following oligo sequences were used: METTL3 For, AAGCAGCTGGACTCTCTGCG; METTL3 Rev, GCACTGGGCTGTCACTACGG; c-MYC For,AGCTGCTTAGACGCTGGATT; c-MYC Rev, AAGTTCTCCTCCTCGTCGC; MALAT1 For, GAGGTCTTTGGTGGGTTGAA; MALAT1 Rev, CCCACCCAGCATTACAGTTC; H3 For, GTGAAGAAACCTCATCGTTACAGGCCTGGT; H3 Rev, CTGCAAAGCACCAATAGCTGCACTCTGGAA.Total RNA was extracted using the TRIzol RNA Isolation System (Invitrogen) according to manufacturer instructions. Reverse transcription to cDNA was performed with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and cDNA was amplified using the SYBR™ Green PCR Master Mix  on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Relative expression levels of targets were determined with the comparative 2Cells were lysed in 2% SDS buffer  and fresh protease inhibitors . Then, cells were sonicated for 10 s and centrifuged for 10 min at 12,000 × rpm. Lysates were quantified using Bradford Assay Reagent . Protein samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated overnight with the following primary antibodies: rabbit monoclonal anti-METTL3 ; rabbit monoclonal anti-H3 ; rabbit monoclonal anti-c-MYC ; mouse monoclonal anti-Puromycin, clone 12D10 ; rabbit monoclonal anti-p70 S6 ; rabbit monoclonal anti-phospho p70 S6 Kinase ; mouse monoclonal anti-S6 ; rabbit monoclonal anti-phospho S6 ; rabbit monoclonal 4EBP1 ; rabbit monoclonal anti-phospho 4EBP1 ; rabbit monoclonal anti-PARP ; mouse monoclonal anti-GAPDH 6C5 ; mouse monoclonal anti-Tubulin B512 . For the detection we used ECL method (Enhanced ChemiLuminescence) (Amersham Biosciences) using ChemiDoc-It Imaging System  instrument.6 cells were incubated 10 min with cycloheximide (Sigma-Aldrich) and then lysed with 400 μL of lysis buffer  supplemented with 10 mM fresh DTT, 100 μg/ml cycloheximide, 1X PIC  and 1X RNase guard (Thermo Scientific). Cells were allowed to swell for 10 min on ice and the lysates were centrifuged for 10 min at 13,000 rpm at 4 °C. The supernatants were collected, loaded on 15–50% sucrose gradient and centrifuged at 38,000 rpm with a SW41 rotor (Beckman) for 1 h 30 min at 4 °C. Fractions were collected with a Bio-logic LP (Biorad). Then, 200 μL of each fraction were pooled together 3 by 3 obtaining four fractions  and total RNA was extracted using Acid Phenol: CHCl3  and RNeasy Mini Kit .After 48 h of transfection with METTL3 and Control siRNA, 20 × 10TC1889 cells were treated with puromycin  at 10ug/ml after 72 h of transfection with siC or siMETTL3. Incorporated puromycin protein level was then analyzed 10, 20, 40 and 60 min after treatment by Western Blot analysis.2) for 5′ at 94 °C. A fraction of fragmented RNA was collected as input control and the rest was incubated with m6A-specific antibody  or with control rabbit IgG (Millipore) antibody for 2 h at 4 °C on rotator. Immunoprecipitated RNA was eluted and resuspended in RNase-free water. RT-qPCR on immunoprecipitated RNA was performed with primers MYC_FW and MYC_REV.Cells were seeded and transfected with METTL3 and Control siRNA. After 72 h, total RNA was extracted and fragmented into ~ 100 nt long fragments in Fragmentation Buffer (100 mM Tris–HCl and 100 mM ZnClFluorescence in situ hybridization to detect MALAT1 lncRNA was performed using a mixture of 48 fluorescent (Quasar® 570) Stellaris™ RNA probes . Cells were fixed with 3.7% formaldehyde in PBS and permeabilized with 70% ethanol overnight. Then, the manufacturer's protocol was followed. MALAT1 distribution was visualized with Confocal microscope.t tests or One-Way Anova test. Differences were considered statistically significant at the p < 0.05 level.The statistical analyses were performed using GraphPad Prism . Statistical analysis to determine significance was performed using Student’s Additional file 1. Supplementary Figure 1. A) After 4 h incubation with 3--2,5 diphenyltetrazolium bromide (MTT), METTL3 depleted samples showed a lower cell viability than control samples, in particular at 72 h and 96 h (left panel) . METTL3 silencing in MTT samples (left panel). B) TC1889 cells after silencing of METTL3 for 72 h were used for Transwell migration assay (24 h of migration). We didn’t observe a decrease in migratory capacity in METTL3 silenced cells compared to control samples. On the right qRT-PCR analysis of METTL3 is shown. (n = 2).Additional file 2. Supplementary Figure 2. A) TC1889 cells were transfected with siC, siMETTL3-6, siMETTL3-7 and siMETTL3 (a pool of four siRNA) and counted with Trypan blue at 96 h (n = 3). B) Representative WB of c-MYC and METTL3 after 24, 48, 72 and 96 h of METTL3-6, METTL3-7 and METTL3 depletion. Quantifications of three independent experiments are shown in the bottom panel. C) Analysis of MALAT1 subcellular localization by RNA FISH in control (siC) and METTL3-silenced  samples at 72 h.Additional file 3. Supplementary Figure 3. A) Puromycin and METTL3 quantification in the samples treated with Puromycin. The samples were normalized on membrane activation lanes (n = 4). B) Representative ribosomal fractionation profile after 72 h of control  and METTL3 silencing  in TC1889 cells. On the right, calculation of area under the polysome and 80S curve in the siC and siMETTL3 samples at 72 h using ImageJ software (n = 2). C) Representative western blot of polysome after 48 h of METTL3 silencing. On the right c-MYC and METTL3 quantification, normalized on GAPDH (n = 3).Additional file 4. Supplementary Figure 4. A) Malat1 expression after 72 h of silencing (n = 3). B) VEGF121 and VEGF165 expression was analyzed by qRT-PCR after 72 h of METTL3 and MALAT1 depletion in TET cells (n = 3). C) Representative Western blot of c-MYC after 24 h of METTL3 depletion (siC+siMETTL3 sample), 72 h of MALAT1 depletion (siC+siMALAT1 sample) and 24 h of METTL3 and 72 h of MALAT1 depletion (siMETTL3+siMALAT1 sample). On the top is represented c-MYC and METTL3 quantification, normalized on Membrane Activation , while on the bottom, c-MYC and MALAT1 expression was analyzed by qRT-PCR (n = 3).Additional file 5. Supplementary Figure 5. A) METTL3 and c-MYC protein levels in TC cells treated with CDDP (top) and JQ1 (bottom) for 48 h. The treatment was performed 24 h after METTL3 silencing (n = 3). B) Representative Western Blot of Cleaved-PARP and PARP and on the right the ratio between Cleaved-PARP and PARP (n = 3).


The chemical and structural properties of the samples were investigated. By the batch uranium adsorption experiments, the magnetic composite GO/Fe3O4/GC exhibits an excellent adsorption performance and fast solid–liquid separation for uranium from aqueous solution. GO/Fe3O4/GC (the maximum adsorption capacity (Qm) was 390.70 mg g−1) exhibited excellent adsorption capacity and higher removal rate (> 99%) for U(VI) than those of glucose-COOH (GC) and magnetic GC (MGC). The effect of the coexisting ions, such as Na+, K+, Mg2+, Ca2+, and Al3+, on the U(VI) removal efficiency of GO/Fe3O4/GC was examined. The equilibrium sorption and sorption rate for the as-prepared adsorbents well fit the Langmuir model and pseudo second-order kinetic model, respectively. The thermodynamic parameters (ΔH0 = 11.57 kJ mol−1 and ΔG0 < 0) for GO/Fe3O4/GC indicate that the sorption process of U(VI) was exothermic and spontaneous. Thus, this research provides a facile strategy for the preparation of the magnetic composite with low cost, high efficiency and fast separation for the U(VI) removal from aqueous solution.In this work, we reported for the first time a novel magnetic composite graphene oxide/Fe These carbonaceous materials generally are fabricated via high temperature reaction5, pyrolysis8, gasfication9, electrospinning technique10, etc. Amongst these techniques, hydrothermal carbonization (HTC) has become a proficient synthesis technique owing to its cheapness, simplification, mild reaction conditions, and lack of any organic solvent and toxic waste. The fabrication of the biomass modification product by HTC process is one of the hot spots in recent years13. As a branch of carbonaceous materials the HTC materials from biomass have emerged since 191314, and exhibited significant potential in various fields, such as adsorption18, catalysis19, fuel cell20, and energy storage/conversion23. Among the potential precursors for the preparation of HTC materials, glucose as a promising candidate has drawn much attention24. Glucose with low-cost and non-toxicity is a natural organic biomass, and reacts with heavy metals and influence their migration behaviour.Nowadays, carbonaceous materials, such as activated carbon25. Adsorption is a popular technique to resolve water contamination problems due to its low energy consumption, easiness, effectiveness, and no secondary pollution27. The glucose-based adsorbents have been proved to be promising for the removal of various pollutants, such as antibiotics28, organic pollutants29, gas pollutants30, dyes31, and heavy metal ions33. However, glucose treated solely by a hydrothermal approach possesses poor pore configuration, undeveloped porosity and low adsorption capacity, which make it being rarely applied in the removal of the pollutants12. Therefore, it is significant to develop novel glucose derivatives with numerous functional group in order to enhance the property.With increasing development of all kinds of industry, severe water pollution caused by reckless discharge into water has critically threatened human health and ecosystem34. However, the excellent dispersion of GO increases difficulty in separation between GO and treated solution, and tend to agglomerate after adsorption which lessens the adsorption capacity of GO35. To overcome these drawbacks of GO, the functionalization of GO with other materials, such as glucose, has become a promising tendency36. The addition of inexpensive glucose onto GO can effectively reduce the production cost of the adsorbent, and obtain abundant active oxygen group simultaneously. Xie et al.37 synthesized glucose-based carbon nanosheets by an integrated GO-confined nanospace directed KOH-activated process for the removal of sulfamethazine. Martín-Jimeno et al.38 prepared HTC xerogels via hydrothermal carbonization of glucose in the presence of GO as morphology directing agent and KOH activation method for CO2 and dye adsorption. However, the preparation methods for these glucose-based composites have some major drawbacks, such as high energy cost, operation complexity, and soli-liquid separation difficulty.Graphene oxide (GO) has been proved a popular candidate as a template for fabricating other functional nanomaterials, owing to its unique layered structure and remarkable physicochemical properties, such as high surface area, hydrophobicity, conductivity, and elasticity. Moreover, GO as an adsorbent can efficiently capture contaminants in water for water remediation3O4, have drawn considerable attention due to their environmentally friendliness and unique magnetic behavior41. The existence of Fe3O4 makes solid substance be rapidly separated from liquid phase only through an external magnetic field to shorten the wastewater treatment period. To our best knowledge, few researches focus on the preparation of the magnetic glucose-based adsorbent with GO as the template for the removal of uranium. Herein, in the present work, a magnetic GO-functionalized HTC adsorbent graphene oxide/Fe3O4/glucose-COOH (GO/Fe3O4/GC) was synthesized using glucose as an initial material via hydrothermal carbonization and magnetization reaction, which aims to develop a novel high-efficiency adsorbent for the removal of U(VI) from nuclear waste influent. The fabrication process and magnetization curve of GO/Fe3O4/GC  are shown in Fig. In order to overcome the separation problem, green and inexpensive magnetic nanoparticles, such as Fe3O4, GC, MGC and GO/Fe3O4/GC were shown in Fig. −1 attributed to the group –COOH was higher than that of glucose, which indicated that GC was successfully obtained after hydrothermal and calcination treatment, and the number of carboxyl, carbonyl and ester groups significantly increased on the surface. These characteristic peaks of GC were similar to those of HTC-COOH reported in the reference32. In the FTIR spectrum of GO/Fe3O4/GC, the characteristic peaks at ~ 567 cm−1 and ~ 352 cm−1 belonged to Fe–O stretching vibration42 appeared, suggesting that the magnetic composite GO/Fe3O4/GC was successfully prepared.43. The structures of MGC and GO/Fe3O4/GC are also amorphous due to the existence of GC. Moreover, the characteristic peaks of the purchased Fe3O4 at 2θ = 29.94°, 35.30°, 42.98°, 53.38°, 56.84°, and 62.46°, corresponded to the (220), (311), (400), (422), (511), and (440) planes of magnetite Fe3O4 with a face-centered cubic structure (JCPDS No. 75-0033)44, were clearly shown in Fig. 3O4/GC and MGC are basically consistent with those of Fe3O4, but the intensity of the peaks has significantly reduced because of the addition of GO and GC, revealing that the decoration process of Fe3O4 did not change the crystal phase of magnetite composite.The Fourier transform infrared (FTIR) spectra of glucose, Fe3O4/GC in the range of temperatures from 30 to 900 °C are shown in Fig. 3O4/GC and MGC inorganic substance Fe3O4 was introduced into their molecular. In general, thermal stability of organic substances is poorer than that of inorganic substances. In the structure of GO/Fe3O4/GC there are more interaction including electrostatic interaction, ionic interaction, and π-π stacking interaction than MGC. Therefore, the thermal stability of GO/Fe3O4/GC is higher than those of glucose, GC and MGC. The composite GO/Fe3O4/GC presented the smallest weight loss (4.28%) when the temperature was up to 900 °C, which showed that GO/Fe3O4/GC had the excellent thermal stability and almost no thermal decomposition took place. While the thermal stability of glucose, GC and MGC was poor, and the weight losses at 900 °C were 76.79%, 49.67% and 16.48%, respectively.To reveal the thermal stability of the samples, the thermogravimetric analysis (TGA) curves of glucose, GC, MGC and GO/Fe3O4, glucose, GC, MGC and GO/Fe3O4/GC was investigated by X-ray photoelectron spectrometer (XPS). The XPS survey spectrum of GO/Fe3O4/GC  ions were formed in the above suspension by adding Fe2+ ions because a part of Fe2+ ions were oxidized to Fe3+ in air by the redox reaction as shown in Eq.  and UO22+ led to the poor adsorption capability for U(VI)14. With the appearance of the positive species (UO2(OH)+, (UO2)3(OH)5+, and (UO2)4(OH)7+) the removal efficiency of U(VI) significantly increased at pH 4.0–7.0 due to the electrostatic interaction between these complex uranium ions with positive charges and the negatively charged adsorbents. When pH is above 7.0, the negatively charged U(VI) species ((UO2)3(OH)7− and UO2(OH)3−) are the dominant U(VI) species in solution which result in the reduction of the U(VI) removal efficiency45. The maximum removal rate for GC, MGC and GO/Fe3O4/GC was 66.30% (pH 6.0), 73.30% (pH 5.0), and 98.70% (pH 5.0), respectively. The sorption efficiency of U(VI) by GO/Fe3O4/GC was much higher than that of GC indicating that the addition of GO in the GC molecular enhanced greatly the adsorption property for U(VI). As a consequence, the optimal pH for GC, MGC and GO/Fe3O4/GC was selected as 6.0, 5.0 and 5.0 in the next U(VI) adsorption tests, respectively.The effect of the solution pH on the adsorption efficiency of the as-prepared adsorbents toward U(VI) is exhibited in Fig. +, K+, Ca2+, Mg2+, and Al3+) on U(VI) sorption by GO/Fe3O4/GC at 25 °C and pH 5.0 was shown in Fig. +, K+, Mg2+ and Ca2+ had no significant competition effects on the sorption of U(VI). In contrast, the presence of Al3+ had a suppressive effect on U(VI) sorption. The results showed that the binding ability of cations to U(VI) followed the priority sequence: + 3 valence cations  < + 2 valence cations  < + 1 valence cations  which indicated that the better electrostatic interaction between high valence cations and the adsorbent GO/Fe3O4/GC result in the decrease of the adsorption efficiency for U(VI).The influence of some important co-existing cations  46:5\docmentclass2pt{minimQm) of U(VI) on GC and GO/Fe3O4/GC was determined to be 396.85 mg g−1 and 390.70 mg g−1, respectively, higher than those of the previously reported glucose-based materials  GO/Fe3O4/GC can be rapidly separated from the liquid phase through external magnetic fields due to the presence of magnetic Fe3O4.According to Langmuir isotherm fit result, the maximum sorption capacity  obtained in this study suggested that the adsorption process was mainly physical adsorption, which was in accordance with the reference47.The Dubinin–Radushkevich (D–R) model is adopted to better explain the U(VI) adsorption behaviour  onto the adsorbents. According to the D-R isotherm parameters, the obtained −1 by GC, MGC and GO/Fe3O4/GC. From Fig. 3O4/GC toward U(VI) indicated a fast adsorption process, and the remove of U(VI) could reach above 98% within 30 min. But the remove of U(VI) by GC could reach 97% after 24 h. Moreover, as presented in Fig. 22+ ions onto GO/Fe3O4/GC was well fitted by the pseudo-second-order model. Adsorption kinetic parameters of the pseudo first-order and pseudo second-order model for GC, MGC and GO/Fe3O4/GC were given in Table 3O4/GC50.The adsorption kinetic mechanism is controlled by a mass transfer process involving equilibsrium time as well as physical and chemical adsorption characteristics. Figure Kd versus 1/T onto GC and GO/Fe3O4/GC were shown in Fig. H0), entropy (ΔS0) and standard free energy (ΔG0) from 303 to 333 K in the adsorption processes were calculated according to Eqs.   Kd versu3O4/GC was examined in this study. After the adsorption experiments, the obtained U-loaded GO/Fe3O4/GC was rinsed and washed with the regenerant (3 M HNO3) and the deionized water (DW) thoroughly until U(VI) ions were not detected in the rinse solution. Then, the dried and regenerated GO/Fe3O4/GC was reused for the further adsorption experiments  = 10 mg L−1, T = 25 ℃, adsorbent dosage = 0.15 g L−1, and contact time = 24 h). The results proved that GO/Fe3O4/GC was used repeatedly for the U(VI) adsorption, and the U(VI) removal rate reached 85.45% after five cycles.Regeneration is an important aspect in the process of wastewater treatment in view of the cost saving. The reuse of GO/Fe2(NO3)2·6H2O) was purchased from Xi’an Dingtian Chemical Reagent Co. (China). The stock solutions of uranium (5–150 mg L−1) were prepared by dissolving UO2(NO3)2·6H2O in DW and acidified with a small amount of concentrated HNO3. Glucose was obtained from Chengdu Keshi Reagent Co. (China). All reagents were of analytical grade and used without further purification. DW was used throughout the experiments.Uranyl nitrate  was synthesized using glucose via a hydrothermal method. Briefly, 6 g of glucose were dissolved in 60 mL DW and placed in a Teflonlined autoclave at 180 ℃ for 24 h. After the autoclave was cooled to room temperature, the solid product HTC was filtered and washed with DW until the filtrate was colorless, and finally dried at 60 ℃ under a vacuum. Finally, carboxyl-rich glucose-COOH (GC) was obtained by heating HTC in a muffle for 5 h at 300 ℃.3·H2O solution was added to the GC solution until the solution pH becomes 11. 1.25 g of FeSO4·7H2O was added slowly to the mixture under stirring. After stirring for 3 h, the black product (MGC) was collected by magnetic separation, and completely washed with DW and ethanol. Finally, MGC was dried at 50 ℃ in vacuum.Firstly, 0.5 g of GC was dissolved in 100 mL DW. Then, 30% NH51. In a typical synthesis of GO/Fe3O4/GC, the mixture of 0.25 g of GO and 0.25 g of GC was dispersed in 100 mL DW under ultrasonic radiation for 3 h. Then, 30% NH3·H2O solution was added to the GO/GC solution until the solution pH becomes 11. 1.25 g of FeSO4·7H2O was added slowly to the mixture with continuous stirring. After stirring for 3 h, the magnetic black product (GO/Fe3O4/GC) was collected by magnetic separation, and washed with DW and ethanol. Finally, the product was dried at 50 ℃ in vacuum.Firstly, GO was prepared from natural graphite by the modified Hummers method3O4 and GO/Fe3O4/GC were conducted at 27℃ under a varying magnetic field . XPS  was used to analyze the chemical composition of the samples. Thermal stability of the products was studied by a TGA system  from 30 to 900 ℃ at a heating rate of 10 K min−1 under an argon flow.The FTIR spectra of the as-prepared adsorbents were obtained using a FTIR spectrometer . The crystal phases of the samples were characterized by the XRD pattern . The magnetic measurements of FeR, %) and adsorption capacity  were calculated according to Eqs. (c0 (mg L−1) is the initial U(VI) concentration; ct (mg L−1) is the U(VI) concentration at time t; V is the volume of the solution (L); W is the dosage of the adsorbent (g).The influence of pH, co-existing cations, contact time, initial U(VI) concentration, and temperature on the U(VI) removal was investigated. The U(VI) solution pH was adjusted to the desired value using HCl and NaOH. The as-prepared adsorbent was added to 20 mL solution and shaken in a shaker . After filtration, the U(VI) concentrations in solutions were determined by an MUA micro-quantity uranium analyzer . The removal rate ( to Eqs.  and 9),,R, %) an3O4/GC were facilely prepared using the inexpensive and environmentally benign glucose as a raw material for U(VI) capture via the simple hydrothermal carbonization and magnetization reaction. The optimum adsorption conditions for U(VI) with the initial concentration of 10 mg L−1 was at a pH of 5.0, a dosage of 0.15 g L−1, and contact time of within 30 min when using GO/Fe3O4/GC as the adsorbent. The existence of co-existing ions in solutions such as Na+, K+, Mg2+, Ca2+ and Al3+ had different influence on the removal of uranium by GO/Fe3O4/GC. Adsorption data of U(VI) by GO/Fe3O4/GC were in good agreement with Langmuir isotherm model and pseudo-second-order kinetic model. The composite GO/Fe3O4/GC exhibited excellent U(VI) sorption capacities (390.70 mg g−1), and a faster adsorption rate than those of GC, MGC and the previously reported glucose-based materials. The superior U(VI) uptake and fast solid–liquid separation after adsorption were mainly attributed to the abundant presence of GO and magnetic Fe3O4 particles in the molecule of GO/Fe3O4/GC. The facile production and high stability of GO/Fe3O4/GC reinforce its potential in the industrial purification of various pollutants, which paves the way for a new route to develop a novel glucose-based composite as a low-cost and highly efficient adsorbent to remove U(VI) from uranium-containing waste influents. The as-prepared GO/Fe3O4/GC has good regenerability which is very important in the practical application.In summary, three adsorbents GC, MGC and GO/Fe


Brevibacillus choshinensis (B. choshinensis).Phospholipase D (PLD) has significant advantages in the food and medicine industries due to its unique transphosphatidylation. However, the high heterologous expression of PLD is limited by its cytotoxicity. The present study sought to develop an efficient and extracellular expression system of PLD in the non-pathogenic 2+ stress, with the highest activity of 10 U/mL. The inductively coupled plasma–mass spectrometry (ICP-MS) results elucidated that the over-expression of PLD by P2 promoter without Mg2+ stress induced the ionic homeostasis perturbation caused by the highly enhanced Ca2+ influx, leading to cell injury or death. Under Mg2+ stress, Ca2+ influx was significantly inhibited, and the strengths of P2 promoter and HWP gene expression were weakened. The study results revealed that the mechanism of Mg2+ induced cell growth protection and PLD expression might be related to the lowered strength of PLD expression by P2 promoter repression to meet with the secretion efficiency of B. choshinensis, and the redistribution of intracellular ions accompanied by decreased Ca2+ influx.The extracellular PLD was effectively expressed by the strong promoter (P2) under Mg2+ stress. By ICP-MS and qPCR analysis combined with other results, the mechanism of the efficient extracellular PLD expression under Mg2+ stress was demonstrated. The relatively low-speed PLD expression during cell growth alleviated cell growth inhibition and profoundly improved PLD production. These results provided a potential approach for the large-scale production of extracellular PLD and novel insights into PLD function.The PLD production was highly improved under MgThe online version contains supplementary material available at 10.1186/s12934-022-01770-z. Phospholipase D PLD, EC 3.1.4.4) could synthesize novel medicinal bioactive phospholipids , 3, phos.1.4.4 coEscherichia coli (E. coli)  and [Fe2+/3+] decreased, while [K+], [Mg2+], [Ca2+], [Mn2+], and [Zn2+] increased, resulting in similar cell growth. Compared with the EP group, higher [Mg2+] and [Mn2+] with an increasing rate of 67.86% and 61.62%, and lower [K+] and [Ca2+] with a decreasing rate of 23.91% and 54.17% were observed in the EPMg group. At 36 h, most of the ions maintained a steady-state, while [Ca2+] and [Zn2+] showed an increase of 101.93% and 140.32% , [Ca2+] reduced sharply, indicating that [Ca2+] might regulate cell growth. Comparing the changes of ions in three groups at 36 h, [K+] and [Mg2+] were found to have the lowest concentration in the EP group and the highest concentration in the EPMg group. On the contrary, [Ca2+] had the highest value in the EP group and the lowest value in the EPMg group. The combined results of Fig. 2+ in the PLD-imposed cells could keep the cells with low [Ca2+], high [K+], and [Mg2+] contents, which is beneficial for cell growth.The changes of ion contents in the cells were not consistent at different growth periods under Mg2+] of the EP group with the lowest growth state was significantly higher than the other two groups at 24 h and 36 h. However, the EPMg group with the best growth state at 36 h was significantly lower than the EP and NP groups, indicating that PLD significantly increased the intracellular [Ca2+] content, which was harmful to the cells. In contrast, the addition of Mg2+ could significantly inhibit the increase of Ca2+ induced by PLD expression to alleviate the toxic effect of PLD on cells, resulting in the continuous increase of cell growth and PLD expression. Meanwhile, the growth of B. choshinensis was inhibited under Ca2+ stress  were investigated from 12 to 60 h to further analyze the effect of ion changes. Compared with the NP group, [Na+] showed a similar decreasing trend at the first 24 h, while [K+] had a similar increase in the EP and EPMg groups  alleviated cell growth inhibition and profoundly increased PLD production by redistributing the ions inside and outside the cell. The effect of salt stress on PLD expression was consistent with our study results. However, PLD expression in B. choshinensis showed a different phenomenon without any continuous influx of Na+. Therefore, it was speculated that the effect of Mg2+-induced protection on cell growth and PLD expression might be related to the P2 promoter expression.Xiong et al. expressed PLD in B. choshinensis, whose expression could also be regulated by the P2 promoter  could cause damage to the cells, even cell death. Besides, [Mg2+] needs further attention as the significant decrease of [Ca2+] was accompanied by the significant increase of [Mg2+] in the EPMg group, which was conducive to cell recovery and growth. The increase of [Ca2+] by PLD expression in B. choshinensis was effectively alleviated under Mg2+ stress, which redistributed the intracellular ions, especially decreased [Ca2+] influx, and increased the growth status of B. choshinensis.Studies have elucidated the mechanism underlying the toxicity of PLD overexpression to cells and protection of the PLD-imposed cells by Mgof cells , 38 and of cells , resultiof cells , 41. Simlum (ER) . These sBacillus brevis 47 [Stv. Cinnamoneus PLD in B. subtilis resulted in the production of inclusion bodies due to excessive promoter strength [Streptomyces racemochromogenes PLD in B. subtilis WB600, the extracellular PLD activity controlled by the highest-strength P43 promoter was lower than the relatively weak PHpall promoter [B. choshinensis, it was found that Mg2+ reduced the transcription level of the pullulanase gene, leading to the extracellular secretion of pullulanase in the correct form after folding [2+ on PLD in this study was consistent with its effects on pullulanase expression, indicating similar mechanism. In EP group, the P2 promoter exhibited a high transcriptional level of the PLD gene, leading to a higher protein synthesis and hasty cytotoxicity. As for the EPMg group, Mg2+ specifically inhibited the P2 promoter activity to maintain an appropriate transcriptional level during the growth stage, which ensured that PLD was properly secreted into the culture medium, thereby the cytotoxicity induced by high PLD expression was alleviated.The secretion promoted by the signal peptide in the vector pNCMO2-PLD, derived from the MWP of revis 47 , led therevis 47 . Gong restrength . Huang epromoter . Moreove folding . The eff2+ stress, more PLD accumulated in cells  was used to express phospholipase D. Plasmid pNY326 and pNCMO2 (Takara Bio Inc.) were used as the expression vector. The strain E. coli JM109 (Takara Bio Inc.) was used as the cloning host. Other chemicals, unless otherwise noted, were purchased from Sinopharm Chemical Reagent Co. Ltd. .E. coli JM109 was cultured on the Luria broth (LB) medium containing 10.0 g/L tryptone, 5.0 g/L yeast extract, and 10.0 g/L NaCl, with the introduction of 50 mg/L Ampicillin (Amp) to each culture. In the shake-flask culture, the primary inoculum of B. choshinensis was grown for 24 h in the 250 mL baffled flasks containing 50 mL of TM medium at 30 ℃ and 120 rpm in a shaker until OD 600 = 2 ~ 3. Subsequently, the seed culture (0.5 mL) was diluted to 50 mL of medium A in a 250 mL shake flask at 30 ℃ and 120 rpm. The TM medium contained 10.0 g/L glucose, 10.0 g/L tryptone, 5.0 g/L beef extract, 2.0 g/L yeast extract, 10.0 mg/L FeSO4·7H2O, 10.0 mg/L MnSO4·4H2O, and 1.0 mg/L ZnSO4 7H2O. Medium A contained: 30 g/L glucose, 30.0 g/L beef extract, 25.0 g/L yeast extract, and 100 mM MgSO4 (It was the optimum concentration after optimization). The contents were modified according to the medium reported by Chun Zou et al. [u et al. .Streptomyces antibioticus  with removed signal peptide was synthesized by Sangon Biotech (pUC57-PLD anti). The PLD gene was amplified by PCR using the primers 5ʹ-TGCTCTAGAGCGGACACACCGCCCACC-3ʹ, containing an Xba I site, 5ʹ-CCGGAATTCTCAGCCCGCCTGGCGAGCCGGGC-3ʹ, containing an EcoR I site, and the plasmid pUC57-PLD anti as the template. The product was ligated into the vector pNY326 (P5 promoter) via T4 ligase (Takara Bio Inc.), then the plasmid pNY326-PLD was transformed into B. choshinensis. As for another expression vector, the PLD gene fragment amplified by PCR was ligated into the vector pNCMO2 (P2 promoter) via T4 ligase. The hybridized fragment was transformed into E. coli JM109. After being confirmed by DNA sequencing, the plasmid obtained from the transformant was transformed into B. choshinensis through New Tris-PEG method . Finally, these two recombinant strains (B.choshinensis/pNCMO2-PLD and B.choshinensis/pNY326-PLD) were used for phospholipase D expression.The PLD gene from g for 10 min to determine the dry cell weight (DCW). The pellet was resuspended in 0.9% (w/v) NaCl and re-pelleted by centrifugation. Later, it was frozen by liquid nitrogen for 1 min, which was immediately freeze-dried to constant weight by a vacuum freeze drier. Finally, the biomass standard curve was plotted according to OD600 and DCW values.Cell growth was monitored by measuring the optical density of the culture broth at 600 nm using a microplate reader. Accurately, 4 mL of the culture broth was pelleted by centrifugation at 13,000×The PLD activity was determined using phosphatidylcholine as a substrate according to the previously described method . One uniThe celles harvested at 48 h were separated into two parts by centrifugation: the medium supernatant and the cell pellets. After pretreated, the extracellular and intracellular protein samples were added with 4 × SDS-PAGE loading buffer with DTT  and boiled for 10 min, respectively. The proteins were separated in 10% SDS-PAGE. ColorMixed Protein Marker 5-245KD  was used to determine the apparent molecular weight of separated proteins. Proteins were visualized with Coomassie Brilliant Blue.B. choshinensis was cultured in 500 mL shake flasks. 20 mL of the culture broth was pelleted by centrifugation at 6000×g for 20 min at different time intervals. The pellet was resuspended in 1 × PBS, and re-pelleted by centrifugation for three times, then dried to a constant weight. The dried cells with 8 mL of nitric acid were completely digested by microwave digestion apparatus CEM MARS6 . Later, the liquid was completely evaporated and the leftover was re-dissolved in 2% nitric acid. The liquid sample was filtered, and then investigated under an inductively coupled plasma mass spectrometry (ICP-MS) .EasyScript®. All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  , following the manufacturer’s instructions.Total RNA was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit , following the manufacturer’s instructions. The extracted RNA was used as the template for complementary DNA (cDNA) synthesis. The first-strand cDNA was synthesized using TransStart® Top Green qPCR SuperMix (+ Dye I/ + Dye II), 0.4 μL of forward primer, 0.4 μL of reverse primer, 1 μL of cDNA, and 8.2 μL of nuclease-free water. The cycling program for qPCR was as follows: 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s, 60 °C for 30 s, then dissociation. The data were analyzed by using the 2−ΔΔCT model [The expression levels of the two genes encoding PLD and HWP were evaluated. The 16S rRNA gene was used as an internal control to normalize the results. The sequences of the primers used for quantitative real-time PCR (qPCR) are listed in Table CT model .Table 1PAdditional file 1: Table S1. Ingredients comparison of three mediums. Fig. S1. Effects of three mediums on B. choshinensis /pNCMO2-PLD cell growth (A) and PLD enzyme activity (B). All the medium contained 60 mM MgSO4. Fig. S2. Comparison of cell growth and phospholipase D production using A. different beef extract concentrations; B. different yeast extract concentrations; Biomass (open bar), PLD activity (filled bar). Fig. S3. Identification of reconstructed plasmid pNY326-PLD. Lane M: DNA marker; lanes pNY326-PLD: Sample of recombinant plasmid after Xba I and EcoR I double enzyme digestion. Fig. S4. Identification of reconstructed plasmid pNCMO2-PLD. Lane Marker: DNA marker; lanes 1-6: Sample of recombinant plasmid after Xba I and EcoR I double. Fig. S5. Transformation results of B. choshinensis under different conditions. Transformation of pNY326-PLD in TM with neomycin (A); transformation of pNCMO2-PLD in TM with neomycin (B); transformation of pNCMO2-PLD in TM with neomycin and Mg2+.enzyme digestion. Fig. S6. Identification of B. choshinensis/pNCMO2-PLD. Lane Marker: DNA marker; lanes 1-3: Sample of recombinant plasmid from the recombinant strains after Xba I and EcoR I double enzyme digestion.


Aspergillus carbonarius PC-4 strain was used to optimize the β-fructofuranosidase production in a cost-effective process and the sucrose hydrolysis was evaluated to produce inverted sugars.Microbial β-fructofuranosidases are widely employed in food industry to produce inverted sugar or fructooligosaccharides. In this study, a newly isolated Optimization of nutritional components of culture medium was carried out using simplex lattice mixture design for 72 and 120 h at 28 °C. One-factor-at-a-time methodology was used to optimize the physicochemical parameters. Crude enzyme was used for sucrose hydrolysis at different concentrations.m/V) and yeast extract  after 72 h with an enzyme activity of 9.4 U/mL, obtaining R2=91.85%, R2adjusted=85.06%, highest F-value (13.52) and low p-value (0.003). One-factor-at-a-time used for optimizing the physicochemical conditions showed optimum temperature (20 °C), pH (5.5), agitation (180 rpm) and time course (72 h) with a 3-fold increase of enzyme production. The invertase-induced sucrose hydrolysis showed the maximum yield (3.45 mmol of reducing sugars) using 10% of initial sucrose concentration. Higher sucrose concentrations caused the inhibition of invertase activity, possibly due to the saturation of substrate or formation of sucrose aggregates, making it difficult for the enzyme to access sucrose molecules within the created clusters. Therefore, a cost-effective method was developed for the invertase production using agroindustrial waste and the produced enzyme can be used efficiently for inverted sugar production at high sucrose concentration.The optimized conditions of enzyme production were achieved from cultivations containing pineapple crown waste (1.3%, Aspergillus carbonarius PC-4 strain. This enzyme exhibited a good potential for inverted sugar production at high initial sucrose concentration, which is interesting for industrial applications.This study presents an efficient utilization of pineapple crown waste to produce invertase by a newly isolated  These enzymes naturally occur in plant, bacteria, yeasts and filamentous fungi  and incubated at 30 °C for five days. Isolated colonies were inoculated in the center of Petri dishes to ensure the purity of fungal strains. Samples of this strain with a portion of the solid culture medium (5 mm×10 mm) were transferred to sterile glass flasks (6 mL) and filled with 4 mL sterile distilled water, which were labelled and hermetically closed with rubber stoppers and aluminium seals 2(SO4)2·6H2O 10, CuSO4·5H2O 2.5, MnSO4·H2O 0.05, H3BO3 0.05 and Na2MoO4·2H2O 0.05. Salt solution (solution B) was prepared containing (g/L): Na3C6H5O7·5H2O 150, KH2PO4 250, NH4NO3 100, MgSO4·7H2O 10 and CaCl2·2H2O 5. To 1 L of solution B, 5 mL of biotin (0.1 mg/mL), 5 mL of solution A and 0.2 mL of chloroform were added. All solutions were stored at 4 °C. Medium preparation consisted of 50-fold dilution of solution B. Inoculum was obtained from PDA slant agar cultivations by preparing conidial suspensions with distilled water and adjusted to 106 spore/mL. A volume of 1 mL was used to inoculate 20 mL Vogel’s salt medium at pH=6 in Erlenmeyer flasks (125 mL), which was supplemented with pineapple crown as carbon source, besides ammonium chloride and yeast extract as nitrogen sources. Pineapple crown waste was obtained from producers from Miracema do Tocantins, Tocantins, Brazil. It was initially dried and ground to a powder (30 mesh). Then, this powder was washed extensively to remove free soluble sugars. All cultivation media were sterilized at 121 °C for 15 min . Shake flasks were incubated at 28 °C and 180 rpm for 72-120 h. After cultivation, biomass was separated by vacuum filtration system  and determined gravimetrically, which was expressed in gram per liter of culture medium.Vogel’s medium was used for enzyme production , pH=6.0. The second set of experiments was conducted to evaluate the influence of different initial pH values on invertase production. The initial medium pH was adjusted by the addition of 1 mol/L NaOH or HCl with values ranging from 3.0 to 8.0. These cultivations were also performed for 72 h at 180 rpm and at an optimum temperature of 20 °C. The third experimental set was accomplished aiming at the analysis of different agitation rates: 130, 150, 180 and 210 rpm. These experiments were conducted at 20 °C and initial pH=5.5 for 120 h. Samples from all experimental sets were taken at intervals of 12 h. Cell-free broth was used for enzyme and protein assays.m/V) sucrose diluted in 0.1 mol/L citrate-phosphate buffer, pH=5.0. Enzyme assays were performed at 50 °C for 5 min. Samples of 0.2 mL from the enzyme reaction solution were transferred to reaction tubes with 0.2 mL of the reagent 3,5-dinitrosalisylic acid and heated at 100 °C for 5 min in a water bath , according to Miller  in 0.1 mol/L citrate-phosphate buffer (pH=5.0) were submitted to enzyme hydrolysis. A volume of 10 mL sucrose solution was maintained for 5 min at 50 °C with subsequent addition of 0.1 mL crude enzyme to the reaction tubes, and then sucrose hydrolysis was performed for 180 min. Samples of 0.4 mL were taken at different intervals and subjected to enzyme inactivation by boiling at 100 °C for 5 min, then 0.2 mL were added to 3,5-dinitrosalicylic acid to determine the amount of reducing sugars produced after hydrolysis reaction. Results were expressed in µmol of reducing sugar. All experiments were carried out in triplicate.Sucrose solutions of 1, 5, 10, 15, 20 and 30% (1), yeast extract (X2) and pineapple crown (X3). The experimental set considered three levels of concentration for each input variable . The experimental design totalized 12 experiments, which were performed in triplicates. The data factors were chosen after a series of preliminary assays (data not shown). A simplex lattice mixture design was performed for culture medium optimization for invertase production. The model was fitted to evaluate the influence of three factors on invertase production: ammonium chloride , q is the number of ingredients for the design experiment, XFor the analysis of physicochemical conditions for invertase production, the data were subjected to analysis of variance (one-way ANOVA) by BioEstat 5.0 (Aspergillus carbonarius PC-4 showed an invertase activity of 6.7 U/mL with a production yield of 587 U/g of substrate and a productivity of 1.6 U/h (Preliminary experiments with Yp/s) and enzyme productivity (rp) for varying compositions of substrates in each experimental run. The invertase activity ranged from 1.0 to 9.7 U/mL during cultivation for 72 h and from 2.0 to 10.0 U/mL during cultivation for 120 h, which showed the effect of component variables on fermentative parameters for enzyme production. Analyses based on t-test and p-value (<0.05) for invertase production as the response showed that yeast extract and pineapple crown were significant ingredients of the mixture for cultivation for 72 and 120 h, whereas ammonium chloride was significant only after 120 h of cultivation. The effect of evaluated factors on enzyme activity is given in the Pareto charts  and a low p-value (0.003), which is an interesting result since a shorter period of cultivation with similar enzyme activity values resulted in better enzyme production yields. The obtained second order models express the empirical relationship between the invertase production and the ingredients of the mixture  during 72 and 120 h of cultivation, respectively:ANOVA results for quadratic model showed an Rf 80.43% . From th1 is ammonium chloride, X2 is yeast extract, and X3 is pineapple crown concentrations.where y is invertase activity (U/mL), XA. carbonarius PC-4 to produce invertase under submerged culture condition was observed. The invertase production by Aspergillus strains isolated from agroindustrial residues and by-products has been extensively studied in the last decades. These substrates are rich in nutrients such as carbon and nitrogen sources for the microbial growth and they are also inexpensive, which is economically interesting  U/mL). The validation experiments corroborate the model and show an invertase production of similar value ((9.4±0.4) U/mL) suggested by the second order model (data not shown).The results predicted by the second order model developed for invertase production by A. carbonarius PC-4. Additional culture conditions were previously established and maintained  . The increase of incubation temperature to 20 °C resulted in the highest invertase production ((15.5±0.8) U/mL), with yield and productivity values of 783.2 U/g and 2.92 U/h, respectively . The invertase production reached the maximum at pH=5.5 ((18.1±0.4) U/mL) , with a yield of 1140.9 U/g and a productivity of 4.3 U/h , with maximum invertase production occurring at pH=5.0 . The invertase production and additional fermentation parameters increased as the rate of agitation increased up to 180 rpm for 72 h (invertase production of (18.7±0.1) U/mL, yield of 1184.2 U/g and productivity of 4.4 U/h) . Dinarvand et al. , 50 °C and pH=5.0. The reactions were initiated with the addition of a total amount of 0.15 U in the reaction system. At low amount of sucrose , the hydrolysis reactions by invertase from A. carbonarius PC-4 reached the maximum production of reducing sugars of 498 µmol after 90 min of reaction. By Increasing the sucrose up to 10%, the maximum reducing sugar production was observed after 150 min of reaction (3451.7 µmol)  . Furtheret al.  and parametric experimental sets leading to 3-fold increase of invertase production from low-cost substrates (pineapple crown and yeast extract). The β-fructofuranosidase produced by A. carbonarius PC-4 showed a promising potential for the synthesis of inverted sugars from different initial sucrose contents, which is particularly interesting to the food industry, especially for the production of sweeteners for confectionery and beverage industries. Therefore, the results obtained in this study revealed A. carbonarius PC-4 as a new and adapted microbial strain suitable for the synthesis of invertase from an agrowaste of pineapple industry.Pineapple crown waste for production of β-fructofuranosidase was efficiently used with a newly isolated