Hydrophilic extracts from SCG are rich in quinic, ferulic and caffeic acids and showed antioxidant capacity in DPPH, ORAC and FRAP assays. Furthermore, pretreatment of C. viridis brain cells with the polyphenolic extracts from SCG (230 and 460 µg/mL) for 24 h prior to 100 µM H2O2 exposure (1 h) significantly increased antioxidant enzymes activity  and reduced lipid peroxidation (measured by MDA levels). These results suggest that polyphenols found in SCG extracts exert an antioxidative protective effect against oxidative stress in C. viridis brain cells by stimulating the activity of SOD and CAT.Oxidative stress in aquatic organisms might suppress the immune system and propagate infectious diseases. This study aimed to investigate the protective effect of polyphenolic extracts from spent coffee grounds (SCG) against oxidative stress, induced by H Aquaculture is one of the fastest-growing industries and it is becoming one of the main sources of food production worldwide . It has 2−, OH− and H2O2), and the failure of the antioxidant enzymatic defense , to neutralize them [Oxidative stress is a cellular condition caused by an uncontrolled increase in the production of reactive oxygen species , the solid residue of coffee generated during the brewing process, is a rich source of antioxidant polyphenols, which are the main contributors to the antioxidant activity of coffee brews and their by-products . Chlorogic acids ,13. TherCentropomus viridis brain cells was used as a model for studying the protective effects of polyphenolic extracts from SCG against oxidative stress. To determine the protective effect of polyphenolic extracts from SCG on oxidative stress, we successfully established and characterized a marine fish brain cell line, white snook C. viridis. C. viridis is a carnivorous brackish-water-based fish species. This species, which is distributed in the tropical regions of the eastern Pacific Ocean, and possess a high potential for intensive commercial farming [It has been demonstrated that fish species exposed to induced stress present oxidative damage in the brain ,15. The  farming .2O2-induced oxidative stress in C. viridis brain cells.The present study aimed to evaluate the antioxidant effect of polyphenolic extracts from spent coffee grounds against HThe total phenolic content (TPC), of the SCG extract, measured by the Folin–Ciocalteu method, was 892.67 ± 59.12 mg GAE/100 g dry weight. The main compound present in the SCG extracts was quinic acid, which contributed to the 73.59% ± 5.37% of the extract’s TPC, followed by ferulic and caffeic acids .The antioxidant capacity of the polyphenolic extracts from SCG measured using the DPPH, FRAP and ORAC assays is shown in C. viridis brain cells, the MTT cytotoxicity assay was performed. The highest concentrations tested, 2000, 1080 and 640 µg/mL, significantly decreased cell proliferation under 80% (p < 0.05). In contrast, lower concentrations did not affect cell viability when compared with the control (zero), without polyphenolic extracts (2 = 0.9306) [To define the non-toxic concentrations of polyphenolic extracts from SCG on the extracts . The max 0.9306) ,19, and C. viridis brain cells. A significant increase (p < 0.05) by 165.87% ± 7.55% of the MDA level compared with the control group (C) was observed in the C. viridis brain cells when exposed to H2O2 (100 µM) (2O2 (p < 0.05). Furthermore, pretreatment of C. viridis cells with SCG-460 restored the MDA levels at the same level as in the non-exposed control group.Lipid peroxidation is one of the most broadly studied indicators of oxygen toxicity in the biological systems. In this study, the MDA levels were used as a biomarker of lipid peroxidation, reflecting the oxidative stress in the (100 µM) . NeverthC. viridis brain cells to H2O2 (100 µM) significantly (p < 0.05) reduced the activity of SOD to 47.92% ± 3.18% as compared with the control (C) group (p < 0.05) the decrease in SOD activity induced by the H2O2. CAT activity was not significantly affected by the exposure of cells to H2O2 (100 µM)  group a. Pretre(100 µM) b. Nevert2O2-induced oxidative stress in C. viridis brain cells have been demonstrated.In this original manuscript, the antioxidant capacity, in terms of radical scavenging activity, and the non-cytotoxic and protective effects of the polyphenolic extracts from spent coffee grounds against HAquatic organisms might be exposed to oxidative stress due to intensive farming conditions, such as low-quality nutrition of aquafeeds, scarce water quality and drastic changes in temperature, pH or oxygen availability in the growing medium . The oxiPolyphenolic compounds are well known for their antioxidative properties. By-products from coffee processing, such as spent coffee grounds, are rich in hydrophilic phenolic compounds with antioxidant capacity. In this paper, the hydrophilic extract from SCG was obtained and its TPC, phenolic profile and antioxidant capacity were determined. The total phenolic content of the hydrophilic extract from SCG was 892.67 ± 59.12 mg GAE/100 g dry weight. Similarly, Mariotti-Celis et al.  reportedp-coumaric acids to form the chlorogenic acids: caffeoylquinic acids, feruloylquinic acids and p-coumaroylquinic acids, respectively [Quinic acid was the main acid found in the extracts from SCG, followed by ferulic acid and caffeic acid . In coffectively ,28. As pectively , these aectively . In our ectively .The antioxidant capacities of extracts from SCG were measured in terms of radical scavenging activity . Roughlyp-coumaric acid, have demonstrated that in exerting its antioxidant effect, an electron transfer mechanism might occur at the same time as the hydrogen abstraction [Electrochemical studies of hydroxycinnamic acids, such as caffeic acid, ferulic acid and traction . It is itraction . Neverthp-coumaric acid, and therefore, their respective chlorogenic acids, possess -OH groups on their aromatic rings (The antioxidant capacities of phenolic acids and their esters with quinic acid are related to the number of hydroxyl (-OH) groups in the molecule and their position . Caffeicic rings . Furtheric rings  offers tC. viridis. It was observed that extract concentrations below 467.31 µg/mL maintained 80% of the cell viability, manifesting non-cytotoxic effects , the H2O2 can easily degrade to produce the hydroxyl radical and initiate lipid peroxidation [C. viridis brain cells were exposed to 100 µM H2O2, MDA levels significantly increased compared to the non-exposed cells (2O2. Previous studies showed that when PC12 cells were treated with phenolic ethanolic extracts (12.5–50 µg/mL) from Garcinia xanthochymus prior to the exposure to 400 µM H2O2, the MDA levels were significantly lowered [2O2. When SH-SY5Y cells were pretreated with a caffeoylquinic acid derivative (15 and 30 µM) prior to the H2O2 exposure, the MDA levels were significantly reduced in a dose-dependent manner [Hrocedure . In the xidation . Secondaxidation . The MDAxidation . In thised cells . This is lowered . Further lowered  reportedt manner .2+ and Cu1+), quenching or reducing O2−, and breaking the autoxidative chain reaction [It has been mentioned that antioxidants such as phenolic compounds might detain or retard the lipid peroxidation reaction chain by scavenging radical species that might induce peroxidation, chelating metal ions  . It is kg others . Nrf2, tg others . Neverth2 occurs , therefoobserved a compareobserved b, this cpression .2−) to H2O2 and oxygen, while CAT decomposes H2O2 into H2O and oxygen to protect cells from oxidative stress [2O2-exposed C. viridis brain cells pretreated with SCG at 230 µg/mL and 460 µg/mL  reduced lipid peroxidation and increased the activity of the antioxidant enzymes [The enzymes SOD and CAT play a key role in preventing cellular oxidative damage. SOD dismutates the superoxide ion . The SCG were obtained from a well-known commercial coffee shop in Mazatlán, Sinaloa, México. According to the manager of the shop, the variety of coffee was Arabica. The SCG were dried to a constant weight in a forced-air convection drying oven at 35–37 °C for 18–24 h . SubsequOne g of powder from SCG was mixed with four mL of ultrapure water. Then, the mix was stirred at 55–57 °C and 100 rpm for 72 min . The mix2CO3 was added. The mixture was incubated for 2 h in the dark at 25 °C. After incubation, the absorbance at 750 nm was registered using an EPOCH 2NS microplate spectrophotometer . The calculations were completed using a gallic acid standard curve (from 0 to 0.4 mg/mL) and the results were expressed as milligrams of gallic acid equivalents per 100 g of dry sample (mg GAE/100 g). Measurements were made in triplicate (n = 3).Total phenolic content determination by Folin–Ciocalteu assay, was performed according to Swain and Hillis  with a f®® BEH C18 column (1.7 µm × 2.1 mm × 100 mm) maintained at 50 °C, at a flow rate of 0.3 mL/min. The mobile phase consisted of two solvents, 0.1% formic acid (in distilled water) (A) and methanol (B). The gradient system was: 90% A-10% B (0–3 min), 70% A-30% B (3–9 min), 60% A-40% B (9–11 min), 50% A-50% B (11–12 min), 0% A-100% B (12–15 min) and 90% A-10% B (15–17 min) [n = 3).The quantification of phenolic compounds in the extracts from SCG was determined through UPLC using an ACQUITY UPLC H-Class PLUS System  that used a Waters UPLC Pump (QSM) separation module  equipped with an autoinjector flow-through needle and a Waters PDA eλ photodiode array detector. The separation of the phenolic compounds in the extract was achieved using an ACQUITY UPLCn = 3).DPPH radical scavenging activity assay was performed according to Brand-Williams et al. , with sls-triazine, and 20 mM FeCl3·6H2O, at a ratio of 10:1:1, v:v:v). The reaction mixture was incubated at 37 °C for 30 min. The absorbance was read at 593 nm using an EPOCH 2NS microplate spectrophotometer . The calculations were achieved using a Trolox standard curve (from 0 to 500 µM) and the results were expressed as micromoles of Trolox equivalent per 100 g of dry sample (µmol TE/100 g). Measurements were made in triplicate (n = 3).The FRAP assay was carried out following the procedure described by Benzie and Strain  with slin = 3).For the ORAC assay , an aliqMTT Formazan -3,5-diphenylformazan, thiazolyl blue formazan (CAS Number: 57360-69-7) and bovine serum albumin (BSA) were purchased from Sigma Chemical . Dulbecco’s modified Eagle medium (DMEM), L-15 medium (Leibovitz), antibiotic–antimycotic (100X) and fetal bovine serum (FBS) were purchased from GIBCO BRL . Primary fish cell cultures were obtained from the cell and tissue culture laboratory of the Centro de Investigación en Alimentación y Desarrollo, A. C. Mazatlán (Center for Research in Food and Development). All procedures and protocols were performed under the Official Mexican Standard ; the protocols are in accordance with international guidelines for the use of animals in research.C. viridis brain cells were maintained at 27 °C in a humidified 5% CO2 atmosphere with growth medium  changed every other day.Cell cultures of 3) were plated in a 96-well plate and allowed to adhere and spread for 24 h. Polyphenolic extracts from the spent coffee ground were added in varying concentrations (0–2000 µg/mL), and the cells were cultured for 24 h at 27 °C. MTT solution  was added to each well, and the cultures were incubated for an additional 4 h. After incubation, MTT was removed and crystals formed were solubilized with acidified isopropanol. The absorbance at 570 nm was determined in each well with a 96-well plate reader. The growth of treated cells was compared with that of untreated cells. Each polyphenolic extract concentration was examined in four replicate wells, and the percentage of viable cells was determined [The thiazolyl blue tetrazolium bromide (MTT) assay was carried out by modifying the original Mosmann . Brain ctermined .6 cells per well in 6-well plates. Cells were pretreated for 24 h with the SCG extract, at 115, 230 and 460 µg/mL. Afterward, 100 µM H2O2 [To examine the effect of the polyphenol extracts from SCG on antioxidant enzymes activity and lipid peroxidation, cells were seeded at a density of 0.5 × 10 µM H2O2 ,57 was aC: control, non-treated cells.2O2 alone.C+: cells treated with 100 µM H2O2.SCG: cells pretreated with the spent coffee ground polyphenolic extracts at 115, 230 and 460 µg/mL, and treated with 100 µM HTreatments are represented as follows:g for 10 min at 4 °C. The cellular pellet was recovered, resuspended in 300 µL of the extraction buffer  and sonicated for 10 min; subsequently, the suspension was again centrifuged at 13,000× g for 10 min at 4 °C and the supernatant was recovered. The supernatant was used immediately for the antioxidant enzyme activity analysis and lipid peroxidation assay.Following incubation, the cells were washed with PBS and detached using a cold solution of PBS/0.5 M EDTA (2%) to achieve a non-enzymatic cellular dissociation. Subsequently, the cell suspension was centrifuged at 13,000× v/v), 50 μL of distilled water and 75 μL of HCl at 37%. This reaction mix was incubated at 45 °C for 40 min. Next, the reaction solution was cooled on ice for 10 min and centrifuged at 3000× g for 15 min at 4 °C. The absorbance was recorded at 586 nm using an EPOCH 2NS microplate spectrophotometer . The calculations were performed by using a 1,1,3,3-tetramethoxypropane (Sigma Aldrich 108383-100ML) standard curve (from 0 to 12 µM) and the results were expressed as micromoles of malondialdehyde per gram of cellular pellet (μmol MDA/g pellet). Measurements were made in triplicate (n = 3).This assay was performed according to Lizárraga-Velázquez et al.  and SoléEnzymatic activity of SOD was determined using the SOD-WST kit (19160 Sigma Aldrich) following the manufacturer’s instructions. Briefly, 20 µL of the enzymatic extract (supernatant) and 200 µL of the WST solution were added to a 96-well plate and mixed by gently pipetting. Later, 20 µL of enzyme working solution was added and mixed again. The plate was incubated at 37 °C for 20 min. Absorbance was read via a 450 nm EPOCH 2NS microplate spectrophotometer  and the results were expressed as units of SOD activity per milligram of protein (U/mg protein). One unit of SOD activity was defined as the amount of enzyme that inhibits formazan formation by 50%.2O2 dissolved in 100 mM phosphate buffer (pH 6.5). Subsequently, 10 µL of the supernatant was added and reaction kinetics were immediately carried out for two minutes with readings every 10 s. Readings were done at 240 nm in an EPOCH 2NS microplate spectrophotometer . The results were expressed as units of CAT activity per milligram of protein (U/mg protein). One unit of catalase activity was defined as the amount of enzyme needed to catalyze 1 mmol of H2O2 per minute at 25 °C.Catalase activity was determined according to Aebi  with slin = 3).In order to express the enzymatic activity as U/mg protein, the concentration of protein in the supernatant was determined according to Bradford . Briefly(p < 0.05).Data were tested for normality using the Anderson–Darling test. The rest of the data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test using the software Minitab version 17.1 . The results are expressed as means ± standard deviations (SD). Different letters show significant differences


It was observed that the structural, morphological, and magnetic properties of obtained specimens are influenced by the amount of introduced La3+ ions. Surprisingly, only the compound with a composition of BiFe0.85Mn0.15O3 was not monophasic, and the presence of neighboring phases was determined from X-ray diffraction analysis and Mössbauer measurements. Structural transitions from orthorhombic to cubic and back to orthorhombic were also observed depending on the La3+ amount. Antiferromagnetic behaviour was observed for all of the samples, with the highest magnetisation values for Bi0.5La0.5Fe0.85Mn0.15O3. Additionally, structural attributes and morphological features were evaluated by Raman spectroscopy and scanning electron microscopy (SEM), respectively.In this work, lanthanum and manganese co-substitution effects on different properties of bismuth ferrite solid solutions Bi Therefore, few mechanisms  explaining ferroelectricity are known [Since the work of Spaldin in 2000, where the existence of only a few magnetic ferroelectrics was questioned , multifere known .3) can be considered one of the most investigated perovskite-type multiferroic materials. This compound demonstrates magnetoelectric properties in both thin-film [3 structures, which display different morphologies, are also explored thoroughly since these nanostructures showed an increase in magnetisation values [3) [3 is difficult to prepare, LaFeO3 can be synthesised by different types of techniques, including sol-gel combustion [Bismuth ferrite  . While pmbustion , solid-smbustion , hydrothmbustion , co-precmbustion .3. One of the examples can be La3+ intercalation to the BiFeO3 system, leading to an increase in magnetisation, conductivity, and improved ferroelectric properties [3+ ions with Mn3+ ions can lead to structural transition, a better magnetoelectric coupling effect, and enhancement of magnetisation [2+ ions, which could improve the electrical properties [1−xLaxFe1−yMnyO3/Ti3C2 hybrids have high catalytic activity towards Congo Red degradation [1-xLaxFe1-yMnyO3 focus on intercalation of small amounts of La3+ up to 30% [Substitution of A-site, B-site, or both cations can lead to improvement in physical properties or formation of phase-pure BiFeOoperties ,18,19. Stisation ,21,22. Coperties . Furtherradation . While mp to 30% , we aime1-xLaxFe0.85Mn0.15O3 (with different x steps) solid solutions by an environmentally friendly, cost-effective, and simple sol-gel technique using only ethylene glycol as a complexing agent. Various characterisation techniques were used to evaluate structural, morphological, and magnetic properties regarding chemical composition.In this work, we prepared a series of Bi1-xLaxFe0.85Mn0.15O3 solid solutions, bismuth (III) nitrate pentahydrate (Bi(NO3)3∙5H2O, Roth , 98%), lanthanum (III) nitrate hexahydrate (La(NO3)3∙6H2O, Alfa Aesar , 99.9%), iron (III) nitrate nonahydrate (Fe(NO3)3∙9H2O, Alfa Aesar, 99.9%), and manganese (II) nitrate tetrahydrate (Mn(NO3)2∙4H2O, Alfa Aesar, 99.9%) were used as starting materials. Firstly, required amounts of lanthanum, iron, and manganese nitrates were dissolved in 20 mL of distilled water. Before addition of bismuth nitrate, the pH of solution was adjusted to 1 by the addition of nitric acid . After the dissolution of bismuth nitrate, the complexing agent, ethylene glycol , ≥99.5%), was added to the mixture. The molar ratio between total metal ions and ethylene glycol was 1:2. The obtained solution was homogenised under constant stirring at 90 °C for 1 h. Next, the temperature was increased to 120 °C for solvent evaporation and the formation of a gel. The resulting gel was dried in the oven at 120 °C for 6 h, ground in agate mortar, and annealed at 650 °C with a heating rate of 1 °C/min for 1.5 h in air.For the preparation of Biλ–X-ray wavelength, β–full width at half maximum in radian, θ–Bragg diffraction angle. The β was measured for corundum standard in order to evaluate the instrumental broadening. Alpha FT-IR spectrometer  was used for FT-IR analysis of compounds. All spectra were recorded at ambient temperature in the range of 4000–400 cm−1. The morphology of samples was examined using a scanning electron microscope (SEM) . Raman spectra were acquired using LabRam HR800  Raman spectrometer equipped with thermoelectrically cooled (−90 °C) CCD camera (DU920P-BR-DD), 600 lines/mm grating and microscope. Spectra were excited with a 532 nm beam from the CW diode-pumped solid-state (DPSS) laser . The laser power at the sample was restricted to 0.2 mW to avoid laser-induced sample heating and photodegradation. The 50×/0.50 NA long working distance (LWD) objective was employed during the measurements. The overall integration time was 600 s. The position of the Raman bands on the wavenumber axis was calibrated by the Si Raman band at 520.7 cm−1. Parameters of the bands were determined by fitting the experimental spectra with Gaussian-Lorentzian shape components using GRAMS/A1 8.0  software. Magnetometer consisting of the lock-in amplifier SR510 , the Gauss/teslameter FH-54 (Magnet Physics) and the laboratory magnet supplied by the power source SM 330-AR-22  was applied to record magnetisation dependences on an applied magnetic field. Mössbauer spectra were measured using 57Co(Rh) source and Mössbauer spectrometer . Closed-cycle He cryostat  was applied for low-temperature measurements. The doublets/singlets, sextets, and hyperfine field distributions were used to fit Mössbauer spectra applying WinNormos Site and Dist software. Isomer shift is given relative to α-Fe.Thermal decomposition of precursor gel was investigated by thermogravimetric and differential scanning calorimetric (TG/DTG-DSC) analysis using PerkinElmer STA 6000 Simultaneous Thermal Analyzer. About 5–10 mg of dried sample was heated from 30 °C to 900 °C at 10 °C/min heating rate in dry flowing air (20 mL/min). X-ray diffraction (XRD) analysis of obtained products was performed with a Rigaku Miniflex II diffractometer using a primary beam Cu Kα radiation (λ = 1.541838 Å). The 2θ angle of the diffractometer was set in the range from 20° to 70° while moving 10°/min. The obtained diffraction data were refined by the Rietveld method using the Fullprof suite. Crystallite size was calculated by applying Scherrer’s equation: 0.1La0.9Fe0.85Mn0.15O3 composition compound are presented in Thermogravimetric analysis was employed to evaluate thermal decomposition behaviour and determine optimum annealing temperature, which is required for the formation of solid solutions. TG/DTG/DSC curves of precursor gel for Bi1-xLaxFe0.85Mn0.15O3 solid solutions. The obtained results are demonstrated in 0.85Mn0.15O3. It is known that Fe3+ substitution by the small amount of Mn3+ can lead to stabilisation of the perovskite phase while avoiding the formation of neighbouring phases [3 is considered to be metastable with respect to two Bi-rich Bi25FeO39 sillenite and Fe-rich Bi2Fe4O9 mullite phases [0.85Mn0.15O3 sample. By intercalating La3+ into the perovskite structure, few trends can be observed in the XRD profiles. The intensity of the peaks labelled (110), (022), and (310) decreases, while the intensity of the (220) peak increases for La-rich solid solutions. Since the ionic radius of the La3+ ion (ionic radius 1.16 Å in VIII-fold coordination) is slightly smaller than the Bi3+ ion (ionic radius 1.17 Å in VIII-fold coordination) [X-ray diffraction analysis was carried out to determine phase purity along with structural changes for the Big phases . On the e phases . Those t22), and 0 decreas3 compound, which has a trigonal unit cell with rhombohedral or hexagonal axes. Fitting was performed with various structural models , previously completed by Kumar and Kar [1-xLaxFe1-xMnxO3 (x varying from 0 to 0.3) solid solutions. They observed that the structure of all co-doped samples consists of a mixture of two different phases: orthorhombic with Pbnm space group and rhombohedral with R3c space group. In our case, the BiFe0.85Mn0.15O3 sample has an orthorhombic unit cell with a Pbnm space group. B-site substitution with Mn3+ [3+ [3+ has an identical structure to the BiFe0.85Mn0.15O3 sample, with a decrease in cell volume and slight variations in lattice parameters. Further increase in La3+ up to 75% resulted in a structural change to a cubic unit cell with Pmm space group. Moreover, the appearance of new diffractions peaks labelled (111), (211), (221), and (131) for a sample containing 90% of La3+ confirms another structural transition from cubic to the orthorhombic unit cell with the Pbnm space group. Interestingly, in comparison with the solid solution with 90% of La3+ ions, the LaFe0.85Mn0.15O3 compound has an increase in unit cell parameters and cell volume. Analogous results were observed for Bi1-xLaxFeO3 solid solutions, where an increase in La3+ concentration resulted in an increase in cell parameters [Rietveld refinement was carried out for all synthesised solid solution specimens. The splitting of the most intense (200) peak was not observed for any of our samples, which means that the perovskite structure deviates from the crystal symmetry of the BiFeO and Kar  for Bi1-ith Mn3+  or Ga3+ Mn3+ [3+  ions canrameters . This wa3+ leads to a small increase in crystallite size. After the structural change from orthorhombic to cubic unit cell, the crystallite size increased by 25% in comparison with the previous compound. The compound with 90% of La3+ showed a sudden decrease in crystallite size, which can be associated with the structural change. Furthermore, LaFe0.85Mn0.15O3 showed a similar crystallite size to the Bi0.25La0.75Fe0.85Mn0.15O3 solid solution.The most intense diffraction peak (200) was chosen for the calculation of crystallite size. The obtained results are presented in 3 and BiFe0.85Mn0.15O3 samples. BiFe0.85Mn0.15O3 was chosen because this compound is known for possessing different structures with a variety of space groups. Pure BiFeO3 exhibits a characteristic pattern of bands related to a rhombohedrically distorted perovskite structure; the main bands are located at 78 cm−1 , 141 cm−1 (A1-1), 173 cm−1 (A1-2), 221 cm−1 (A1-3), 270 cm−1 (E-3), 348 cm−1 (E-5), 366 cm−1 (E-6), 469 cm−1 (A1-5), and 527 cm−1 (E-8) [−1 might be associated with the E-9 mode or related with second-order vibrational transition [1 symmetry bands located at 141, 173, and 221 cm−1 and one E symmetry mode located at 78 cm−1 are the most intense features in the spectrum of pure BiFeO3 compound.The short-range structure of studied compounds was probed by Raman spectroscopy by using a 532 nm excitation wavelength. The technique is very sensitive to structural distortions and could provide additional structural information on unit cells as well as space-group type. −1 (E-8) ,36,37,38ansition ,40. The 3+ ions results in drastic changes in the Raman spectrum  to orthorhombic (Pbnm space group) [3) (Pnma space group for oxygen octahedra) [Doping with Mnspectrum . A stronbrations ,42. Thusng bands ,42. These group) . A similtahedra) . These o1-xLaxFe0.85Mn0.15O3 solid solutions, and results are demonstrated in −1 and 700–200 cm−1, respectively [−1 interval, which indicates the absence of residual organic species or carbonates in solid solutions. Our previous study [1-xMnxO3 solid solutions revealed that the addition of Mn3+ could result in the most intense peak shift to higher wavenumbers. Samples with orthorhombic structure have two absorption bands: a strong and sharp band located at 533–552 cm−1 region as well as a weak and broad one at 436–474 cm−1. Only one peak at around 550 cm−1 can be observed for compounds with cubic structures. These bands can be ascribed to the stretching mode of the Fe–O bond in the FeO6 octahedra. With the increase of La3+ concentration, weaker absorption band shifts to higher wavenumbers, while a strong intensity peak only shifts to higher wavenumbers until the La3+ amount reaches 50%. Larger substitution results in a monotonic shift to lower wavenumbers. Furthermore, no stretching or bending vibrations belonging to Bi2O3 or La2O3 can be visible for all our samples, which indicate no presence of these impurity phases.FT-IR spectroscopy analysis was carried out to further investigate structural changes in Biectively . In our us study  on BiFe13+ and Mn3+ co-doping on the morphology of mixed-metal ferrite powders, the SEM analysis was executed for solid solutions of four different compositions, and the results are demonstrated in 0.9La0.1Fe0.85Mn0.15O3 sample  to account for line broadening. At low temperature  and LaFBi2Fe4O9 .1-xLaxFe0.85Mn0.15O3 (where 0 ≤ x ≤ 1) solid solution specimens. Nearly all compounds were phase-pure, with the exception of BiFe0.85Mn0.15O3, where the presence of Bi2Fe4O9 and Bi25FeO40 impurity phases was determined from X-ray diffraction analysis data and Mössbauer measurements. Depending on La3+ concentration, few structural transitions from orthorhombic to cubic and back to orthorhombic were observed in the series. Raman and FT-IR spectroscopies also provided additional structural information in good agreement with XRD analysis results. SEM analysis demonstrated that intercalation of Mn3+ ions limited the growth of the particles, and the largest particles were observed for Bi0.9La0.1Fe0.85Mn0.15O3. All samples demonstrated antiferromagnetic behaviour, with the highest magnetisation values observed for the Bi0.5La0.5Fe0.85Mn0.15O3 sample.An aqueous sol-gel method was successfully applied for the preparation of Bi


It is active against several types of formin from different species. Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and found that SMIFH2 inhibits activity of myosin ATPase and the ability to translocate actin filaments in the gliding actin in vitro motility assay. Inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 compared with that needed to inhibit retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, including bovine myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficiently than it inhibits formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions.The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization This article has an associated First Person interview with Yukako Nishimura, joint first author of the paper. Summary: The inhibitor SMIFH2 inhibits a variety of myosins almost as efficiently as formins, its designated targets. In vitro, formin activities include nucleation and elongation of actin filaments , S. pombe (Cdc12 and Fus1), S. cerevisae (Bn1), and M. musculus (mDia2) are also inhibited, with IC50 values ranging from 5–15 µM SMIFH2, suggesting that the inhibitor is generally applicable to all formins , albeit at relatively high concentration of SMIFH2 . In the absence of actin, myosins have very low basal ATPase rates that are activated 10–1000-fold by the addition of actin (in vitro. We first investigated the effect SMIFH2 might have on actin-activated magnesium-dependent ATPase (MgATPase) activity of human non-muscle myosin 2A (heavy chain encoded by MYH9), whose enzymatic activity depends on phosphorylation of its regulatory light chain subunit through myosin light chain kinase (MLCK). We found that SMIFH2 does, indeed, inhibit activity of this myosin in a dose-dependent manner, with an IC50 of ∼50 µM (Table S2). SMIFH2 also inhibited the actin-activated ATPase activity of rabbit skeletal muscle myosin 2  with an IC50 of ∼40 µM , demonstrating that the drug targets myosin rather than indirectly inhibiting it via binding to actin.Two methods are primarily used to assess actomyosin function of actin . In somef ∼50 µM A. Thereff ∼40 µM B. At a cin vitro motility assay. Here, skeletal muscle myosin 2 HMM is bound to a nitrocellulose-coated coverslip and its ability to translocate Rhodamine–phalloidin-labeled actin filaments observed (−1 (mean±s.e.m.). This activity was completely abolished at a SMIFH2 concentration of 150 µM and could not be reversed, even by extensive washout using motility buffer without SMIFH2. The lack of reversibility could be due to covalent modification of myosin by SMIFH2. Interestingly, under these conditions, immobile actin filaments were tethered to the surface but fewer filaments were present. This was in contrast to conditions of without or reduced concentrations of SMIFH2. At intermediate concentrations of SMIFH2 (50 and 100 µM) average velocity was reduced , Drosophila myosin 7a (heavy chain encoded by crinkled) and bovine myosin 10 (heavy chain encoded by MYO10) (summarized in Table S3). SMIFH2 inhibited the ATPase activity of each of these myosins with various degrees of potency, i.e. Drosophila myosin 7a with an IC50 of ∼30 µM  very rapidly reduces the traction forces exerted by fibroblasts on their substrates. Further, by using cells confined to micropatterned circular adhesive islands, we demonstrated that SMIFH2 inhibits the retrograde flow of myosin filaments in the course of centripetal movement of contracting transverse arcs. By themselves, these findings were not alarming since the dynamics of the actin polymerization /depolymerization and interaction of myosins with actin are thought to be tightly linked . HoweverIn vitro, gliding of actin filaments due to myosin 2A was also suppressed by SMIFH2. Thus, direct inhibition of myosin 2A by SMIFH2 could be involved in the effects of SMIFH2 observed in cells. In our experiments, the concentrations of SMIFH2 sufficient to stop the flow of non-muscle myosin 2A filaments in living and even permeabilized cells were, however, still below the concentrations needed to substantially inhibit non-muscle myosin 2A function in vitro. Of note, the effect of high-dose (150 µM) SMIFH2 on gliding of actin filaments via skeletal non-muscle myosin 2 was irreversible, whereas effects of lower doses of SMIFH2 were shown to be reversible in vivo . REF52 cells were transiently transfected with the mEos3.2-Actin expression vector  by using jetPRIME transfection reagent  in accordance with the manufacturer's protocols. HFF cells were transfected with GFP-tagged myosin regulatory light chain (GFP-MRLC) expression vector  cell line  and human vector  . The computation algorithm as published by Traction force microscopy with embedded beads was performed as described previously . Briefly4 cells/ml on the hydrophobic uncoated μ-dish 35 mm  with fibronectin micro-patterns and incubated 3-8 h prior to the experiment. For stress fiber imaging, REF52 cells were transfected with mEos3.2-Actin expression plasmid and seeded onto a 35 mm glass bottom dish  24 h prior to the assay.For HFF cells, circular adhesive islands of fibronectin were fabricated by stencil patterning as described previously . GFP-MRL2, 0.1 mM EDTA, 1 mM EGTA, 1 mM 2-mercaptoethanol, 250 nM phalloidin  and 2 μg/ml protease inhibitor cocktail ] supplemented with 0.1% Triton X-100 and 4% PEG MW35000 for 10 min at room temperature, followed by three washes with extraction buffer A. Cytoskeleton contractility assay was carried out at 37°C using buffer A supplemented with 2 mM ATP with or without the appropriate drugs, i.e. SMIFH2  or para-aminoblebbistatin . All drug remained in the buffer during the entire period of observation.The protocol of the cell permeabilization and cytoskeleton contractility assay has been described previously . Brieflyz-stacks, step-size 0.35 μm were acquired. For REF52 cells, time-lapse images were acquired for 30 min at 5 min intervals at the basal plane of cells.Super-resolution SIM imaging was performed by using a W1-spinning-disc confocal unit coupled with the live super-resolution (SR) module  analysis was used to measure average instantaneous speed of filaments of non-muscle myosin 2 visualized by expression of GFP-MRLC. PIV analysis was performed using P-value) were calculated using two-tailed unpaired Student's t-test. Bar graphs and scatter plots show mean±s.d. for the respective groups of data.Plotting and statistical analysis were done by using GraphPad Prism 7 (GraphPad Software). Significant differences -like fragment of human non-muscle myosin 2A was prepared by expression in Sf9 insect cells as described . Cells wmyosin 5 , Drosophyosin 7a  and a foyosin 7a  were alse myosin . Rabbit e myosin .2, 0.1 mM EGTA, 1 mM ATP, 10 mM MOPS pH 7.0 at 25°C. The buffer also contained 40 units/ml l-lactic dehydrogenase, 200 units/ml pyruvate kinase, 200 μm NADH, and 1 mm phosphoenolpyruvate. Absorbance was monitored at 340 nm. Non-muscle myosin 2A was first phosphorylated by incubation in 0.3 M KCl, 4 mM MgCl2, 0.2 mM CaCl2, 0.1 mM EGTA, 0.1 mM ATP, 1 μM calmodulin, 2 nM MLCK for 10 min at room temperature.Actin-activated ATPase assays were carried out using an NADH-linked assay in a Cary 50 spectrophotometer as previously described . SMIFH2 in vitro motility assay was conducted at 30°C in 50 mM KCl, 4 mM MgCl2, 0.1 mM EGTA, 30 mM MOPS pH 7.2, 0.5% methylcellulose, 1 mM ATP, using an oxygen-savaging system consisting of 2.5 μg/ml glucose oxidase, 45 μg/ml catalase, 2.5 mg/ml glucose and 50 mm DTT  with a Zorbax 300SB-C18  and introduced into the mass spectrometer as described . Positiv


Crystal structure, optical properties, and color of the La3LiMnx1−TixO7 (0 ≤ x ≤ 0.03) samples were characterized. Strong optical absorption was observed at a wavelength between 400 and 550 nm, and a shoulder absorption peak also appeared around 690 nm in all samples; orange colors were also exhibited. Among the samples synthesized, the most brilliant orange color was obtained at La3LiMn0.97Ti0.03O7. The redness (a*) and yellowness (b*) values of this pigment were higher than those of the commercially available orange pigments. Therefore, the orange color of this pigment is brighter than those of the commercial products. Since the La3LiMn0.97Ti0.03O pigment is composed of non-toxic elements, it could be a new environmentally friendly inorganic orange pigment.La However, the use of the cadmium and molybdate orange pigments has been forbidden or restricted because they contain toxic elements which have harmful effects on the human body and the environment. Although the bayferrox orange pigment is environmentally friendly, the vividness of this pigment is not sufficient. Therefore, development of environmentally friendly inorganic orange pigments is required, and several studies have been reported  octahedra surrounded by six O2− ions. 3LiMnx1−TixO7 (0 ≤ x ≤ 0.05) samples. Cell volume increased as the Ti4+ content increased in the range of x ≤ 0.03. This result indicates that some Mn4+  . The Li+0.053 nm ) ions in.0605 nm ) ones. T3LiMnx1−TixO7 (x = 0 and 0.03) samples are shown in 4+.The SEM images of the La3LiMnx1−TixO7 (0 ≤ x ≤ 0.03) samples, which were obtained in a single-phase form, were analyzed by XRF, and the results are summarized in The element ratios of La, Mn, and Ti for the La3LiMnx1−TixO7 (0 ≤ x ≤ 0.03) samples, which were obtained in a single-phase form, are depicted in 2p-Mn3d charge transfer transition, while those around 500 and 690 nm in the visible region were attributed to the d-d transition of Mn4+. The former was spin-allowed 4A2g–4T2g transition and the latter was spin-forbidden 4A2g22122Eg and 4A2g–2T1g transitions, respectively  octahedra, which was caused by the Ti4+ substitution, because Ti4+ (0.0605 nm 1/2. The hue angle (h°) ranges from 0 to 360° and is estimated according to the following formula: h° = tan−1(b*/a*).The optical reflectance spectra were measured with a UV-Vis-NIR spectrometer , using a standard white plate as a reference. The color properties were evaluated in terms of the Commission Internationale de l’Éclairage (CIE) 3LiMnx1−TixO7 (0 ≤ x ≤ 0.05) samples were synthesized using a solid-state reaction technique as environmentally friendly inorganic orange pigments. The La3LiMnx1−TixO7  samples were obtained in a single-phase form, but an impurity phase was observed for x = 0.05. In the visible light region, the optical absorption band at a wavelength below 550 nm and the shoulder absorption peak around 690 nm were attributed to the spin-allowed and spin-forbidden d–d transitions of Mn4+, respectively. These absorption intensities were increased by the Ti4+ doping, because the [MnO6] octahedra were more distorted. Accordingly, the sample color became more vivid orange. Among the samples synthesized in this study, the La3LiMn0.97Ti0.03O7 pigment exhibited the most brilliant orange color. In addition, the orange color of the present pigment was brighter than those of the commercially available orange pigments, because the a* and b* values of this pigment were higher than those of the commercial ones. Since the orange color of the La3LiMn0.97Ti0.03O7 pigment has chemical stability, it has a potential to be a novel environmentally friendly inorganic orange pigment.La


The purpose of the current study was to investigate the biological function of cell division cycle-associated protein 7 (CDCA7) on ovarian cancer (OC) progression and analyze the molecular mechanism of CDCA7 on OC cellular processes and angiogenesis. CDCA7 expression in OC tissues and adjacent normal tissues was obtained from Gene Expression Profiling Interactive Analysis (GEPIA) and in various cancer cell lines was obtained from Cancer Cell Line Encyclopedia (CCLE). Moreover, CDCA7 expression in adjacent normal tissues and tumor tissues of OC patients as well as in normal ovarian epithelial cells (NOEC) and ovarian cancer cells  was further confirmed via Western blot assay and Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, Immunohistochemistry (IHC) was also applied for determination of CDCA7 expression in tissues of OC patients. Then, SKOV3 cells were introduced with shRNA-CDCA7 for functional experiments. GeneMANIA database analysis and coimmunoprecipitation (Co-IP) assay verified the interaction between CDCA7 and enhancer of zeste homolog 2 (EZH2) to probe the potential mechanism. CDCA7 expression was elevated in tumor tissues of OC patients and OC cell lines. CDCA7 silencing restrained the proliferative, migrative and invasive capacities and arrested cell cycle of OC cells. In addition, CDCA7 knockdown induced a weaker in vitro angiogenesis of HUVECs. Mechanistically, CDCA7 interacted with EZH2. Downregulation of CDCA7 arrested angiogenesis by suppressing EZH2 expression. To sum up, the current study revealed the impact and potential mechanism of CDCA7 on OC cellular processes, developing a promising molecular target for OC therapies.  Cancer is the most serious disease that endangers human health and life at present, among which ovarian cancer (OC) is one of the three most common malignancies of female reproductive system . OC, accTumor is a class of cellular cyclical disease. A variety of cytokines participate in the regulation of cell cycle . The celThe imbalance of epigenetic pathways often occurs in cancers. Understanding underlying mechanism of epigenetic factors helps to develop effective cancer therapies . PolycomHere, this current work was carried out to identify the biological role of CDCA7 in OC. The impact of CDCA7 on OC cellular processes was analyzed through a series of functional experiments. What’s more, potential molecular mechanism of CDCA7 on angiogenesis in OC was investigated in vitro.http://gepia.cancer-pku.cn/index.html). Cancer Cell Line Encyclopedia  database was applied to interrogate CDCA7 expression in OC cell lines.Expression levels of CDCA7 in OC tissues and normal tissues were obtained at Gene Expression Profiling Interactive Analysis , ovarian cancer cells , and human umbilical vein endothelial cells (HUVECs) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences . Cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum , penicillin (100 U/mL)/streptomycin (100 μg/mL)  in a humidified chamber at 37°C with 5% CO5/well) were seeded into 24-well plates. About 4 µg vectors were mixed with 20 µl Lipofectamine 2000 in Opti-MEM  at room temperature for 15 min. Then, the mixture was added into the plate and transfections were performed at room temperature for 6 h. Subsequently, the medium was replaced with fresh medium and transfected cells were then cultured for 48 h before the subsequent experiments.The lentivirus  carrying EZH2 gene (OV-EZH2), shRNA-CDCA7, or negative control (OV-NC or shRNA-NC) were transfected with Lipofectamine 2000 transfection reagent  following the manufacturer’s instructions. Briefly, cells . The secondary antibody was used to incubate the sections for 20 min at room temperature. Diaminobenzidine  was applied for staining development and sections were counterstained with hematoxylin for 10 min. The staining results were observed under a light microscope .Briefly, tumor tissues were fixed and cut into 4-μm-thick sections. Then, sections were deparaffinized in xylene and dehydrated in a graded ethanol series. Next, sections were immersed in sodium citrate and heated for antigen retrieval. After incubation with 3% H4 cells/well. Cells were cultured for 24, 48, and 72 h and then 10 μL CCK-8 reagent  were added into each well. Next, cells were continuously cultured for 2 h. Finally, OD450  values were detected by a microplate reader .SKOV3 cells were collected and seeded into 96-well plates at a density of 10In brief, SKOV3 cells were digested with trypsin and washed with PBS. Then, cells were fixed with 70% ice-cold ethanol at 4°C for 4 h. Afterward, cells were stained with propidium iodide  at room temperature in the dark for 30 min and assessed by a FACS flow cytometer .The migrative ability of OC cells was evaluated by wound healing assay. Briefly, SKOV3 cells were spread onto 6-well plates. When reaching 90% confluence, SKOV3 cells were divided with a 200 μl of pipette tip into a cell-free area. Next, serum-free medium was added for culture. After 24 h incubation, the migration distance was imaged and evaluated under an inverted microscope .5 cells was appended to the upper chamber, and 500 μL complete medium was added to the lower chamber. Post 24-h incubation, transwell chambers were washed with PBS and fixed by 4% paraformaldehyde for 10 min. The invaded cells were stained with 0.1% crystal violet  and observed under an inverted microscope .Transwell chambers precoated with Matrigel  were placed into 24-well plates. Serum-free culture solution containing 1 × 105/ml. Then, 100 µl of HUVECs was seeded on the Matrigel and cultured for 6 h. Tube formation was observed and photographed using a light microscope . The number of tube-like structures was determined using Image J software .96-well plates were pre-coated with 100 µl Matrigel. HUVECs were resuspended with serum-free medium at a density of 2 × 10−ΔΔCt method.TRIzol Reagent  was used to extract total RNA from OC tissues and cells. Then, 5 μg of total RNA was used to prepare complementary deoxyribose nucleic acid (cDNA) by PrimeScript™ RT Reagent Kit . Real-time quantitative PCR was performed using ABI 7500 quantitative PCR instrument . The PCR thermocycling conditions were as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The following primer sequences were used for qPCR: CDCA7: forward, 5ʹ-CCAGGCTCCGACTCACAATCAAG-3ʹ and reverse, 5ʹ-GTACTTATCCTCTTCCTCCTCCTCCTC-3ʹ; EZH2: forward, 5ʹ-AATCAGAGTACATGCGACTGAGA-3ʹ and reverse, 5ʹ-GCTGTATCCTTCGCTGTTTCC-3ʹ; GAPDH: forward, 5ʹ-TGACTTCAACAGCGACACCCA-3ʹ and reverse, 5ʹ-CACCCTGTTGCTGTAGCCAAA-3ʹ. GAPDH served as the internal reference. Relative gene expression of CDCA7 and EZH2 was analyzed by 2Total protein from OC tissues and cells was extracted using protein extraction lysate . Then, protein concentration was evaluated by BCA Protein Assay Kit . Protein samples were separated by 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes . Next, PVDF membranes were blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with primary antibody against CDCA7 , EZH2 , CyclinE1 , CyclinE2 , MMP2 , MMP9 , VEGFA , VEGFR1 , VEGFR2  and GAPDH . The next day, membranes were washed with TBST and incubated with the corresponding secondary antibody  for 2 h at room temperature. Enhanced chemiluminescence reagents  were applied to visualize the protein bars. Finally, protein bands were detected by a Bio-Rad imaging system .http://genemania.org/) was an analytical tool to analyze the interconnection of gene clusters. We constructed gene interaction networks for CDCA7 using GeneMANIA.GeneMANIA . The supernatant was incubated overnight at 4°C with anti-CDCA7, anti-EZH2 antibody or IgG as negative control. Then, 20 μL protein A/G-beads was coincubated with the supernatant at 4°C for 1 h. The beads were extracted and washed three times with lysis buffer. The immunocomplexes were used for western blotting assay.All data analyses were processed with SPSS 21.0 statistical software  and measurement data from three independent experiments were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for comparisons among multiple groups. The p value < 0.05 indicate a statistically significant difference.Quantification analysis of CDCA7 levels in tumor tissues and adjacent normal tissues of OC patients was done in GEPIA database. A relatively higher level of CDCA7 was observed in tumor tissues of OC patients ). FurtheCDCA7 expression in various cancer cell lines were assessed in CCLE database. Importantly, a relatively higher level of CDCA7 was seen in OC cell lines ). MoreovTo investigate the biological functions of CDCA7 on OC progression, shRNA-CDCA7-1 or shRNA-CDCA7-2 were introduced into SKOV3 cells. About 48-h posttransfection, CDCA7 protein ) and mRNIt was observed that CDCA7 silencing strongly restrained the migration of SKOV3 cells. In addition, the invasive abilities of SKOV3 cells showed the similar tendency to be suppressed by CDCA7 knockdown . MoreoveIt is well known that tumor growth and metastasis need glorious angiogenesis for nutrition provision. Obvious reduced VEGFA, VEGFR1, and VEGFR2 expression indicated that CDCA7 silencing arrested angiogenesis ). MoreovGeneMANIA database analysis presented that CDCA7 interacted with EZH2 ). Here, It was seen that suppressing effects of CDCA7 silencing on VEGFA, VEGFR1, and VEGFR2 expression were abolished upon EZH2 elevation ). What’sIn recent years, OC treatment mainly focuses on targeted therapy and surgery. Accompanied by in-depth research on the pathogenesis of OC, great progress has been made in OC therapies . HoweverMultiple genes and proteins are involved in the process of cell cycle regulation, and CDCA7 is one of them. More and more studies have found that abnormal expression of CDCA7 plays an important role in the occurrence and development of tumors ,7. In adTumor metastasis is the main cause leading to poor prognosis of cancer patients. The influence of CDCA7 on tumor metastasis has gradually attracted the attention of researchers . A growiAs an important component of polycomb group proteins (PcGs), EZH2 has been confirmed to be overexpressed in a variety of cancer types with lethal outcomes, including OC cases ,14. The To conclude, the current study validated that CDCA7 was highly expressed in OC tissues and cell lines. Furthermore, functional experiments evidenced that knockdown of CDCA7 exhibited inhibitory effects on OC cell growth, migration, invasion, and angiogenesis in vitro. Mechanically, downregulation of CDCA7 arrested angiogenesis by suppressing EZH2 expression. These findings prompted that CDCA7 may be a promising molecular target for OC therapies.


The expression of eIF5A2 was examined by immunohistochemistry in PTC tissues and its adjacent tissue (n = 39) from 207 PTC patients. Functional analysis of eIF5A2 was performed in PTC cell lines in vitro and in vivo. The results showed that eIF5A2 was overexpressed in PTC tissues compared with the adjacent tissues. Enhanced eIF5A2 expression was significantly correlated with extrathyroidal extension (p = 0.012), lymph node metastasis (p = 0.002), TNM stage (p = 0.006), T classification (p = 0.047) and BRAF V600E mutation (p = 0.036). EIF5A2 inhibition prevented PTC cell growth, invasiveness and migration and induced cell apoptosis in vitro. Furthermore, eIF5A2 depletion inhibited tumor growth and metastasis in vivo. The data indicated that eIF5A2 could be employed as a novel prognostic marker and effective therapeutic target for PTC.Eukaryotic translation initiation factor 5A2 (eIF5A2) plays an important role in tumor progression and prognosis evaluation. However, the potential role of eIF5A2 in human papillary thyroid cancer (PTC) is unknown. In this study, we aim to investigate the association between eIF5A2 expression and PTC clinical outcomes and underlying its Biological function in PTC cells   Thyroid cancer is a common endocrine malignancy, of which papillary thyroid carcinoma (PTC) accounts for 85–90%. However, no reliable and specific markers are used for the diagnosis of PTC . UltrasoFor more than twenty years, identifying suitable molecular markers to guide surgery or to wait for patients with FNA biopsy (FNAB) with uncertain thyroid nodules has been the key to thyroid nodules research. Many potential genetic diagnostic markers, such as mutations in RAS, PIK3CA, PTEN, P53, TERT and BRAF genes and marker combinations have been studied in thyroid or lympy nodes and FNAB specimens . HoweverOnly two proteins in eukaryotic cells have the unique amino acid tyrosine, namely eIF5A1 and eIF5A2, which are covalently linked to the lysine 50 residues of these proteins through post-translational modifications on hypnosis, guided by two highly conserved and essential enzymes-deoxyhysupsine synthase (DHPS) and deoxyhysupsine hydroxylase (DOHH) . Basic rin vitro and in vivo. We also determine whether eIF5A2 expression relates with clinical catchratics in patients with PTC. Finally, we determine its role in regulating the malignant cell phenotype in PTC cell lines.Here we investigate the expression of eIF5A2 in patients with PTC and the function in PTC cells 2 at 37°C.The PTC cell line BCPAP was purchased from the European Collection of Cell Cultures . It was maintained in 10% fetal bovine serum and 5% COPrimary papillary thyroid cancer (PTC) from 207 consecutive patients who underwent surgical therapy and adjacent tissue in 39 patients between 2018 and 2019 at the affiliated hospital of Qingdao University. Written informed consent was obtained from all patients. The Institutional Review Board of the affiliated hospital of Qingdao University approved the study. Detailed history was recorded and examination was done. No patients received neoadjuvant chemotherapy or radiotherapy. The age of the patients ranged from 17 to 76 years (mean age was 48.7 years). All these specimens were pathologically confirmed as PTC.Formalin-fixed paraffin-embedded tissue sections from excised specimens were processed according to standard procedures. Immunohistochemical staining and positive expression judgment for eIF5A2 was performed as described previously ,19.A total of 20 µg protein was separated by 10% SDS-PAGE and transferred to a 0.2 µm polyvinylidene fluoride membrane using the Bio-Rad SemiDry instrument (Bio-Rad), and incubated with anti-eIF5A2 and anti-GAPDH  at 4°C overnight. After washing with 0.1% TBST for 15 minutes and performing 3 times, the membrane is coupled to the secondary stage of HRP Dilute the antibody at a ratio of 1:10,000 for 1 h at room temperature. Then, it was washed again with 0.1% TBST 3 times, 15 minutes each time. The enhanced chemiluminescence detection  was used for semi-quantitative analysis.The PLVX-IRES-ZsGreen vector containing eIF5A2-short hairpin RNA (eIF5A2-shRNA) was designed and amplified using the standard method. The recombinant viruses were infected into BCPAP cells and Puromycin (0.4 μg/mL) was used to select stable transfected colonies.siRNA oligonucleotides were purchased from GenePharma . siRNA sequences against eIF5A2 were as follows: 5ʹ CAUUCAAGAUGGUUACCUUtt 3ʹ and 5ʹAAGGUAACCAUCUUGAAUGca 3ʹ. The siRNA oligonucleotides were transfected into BCPAP cells using lipofectamine™ 2000  as the manufacture’s instructure.Cell invasion/migration was performed using BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to a previously described method . ExperimCell viability was determined by the 3--2,5-diphenyltetrazolium bromide (MTT) assay, using an assay kit as the manufacturer’s instructure. After 96 h incubation, cell viability was evaluated by adding 50 μl of 0.15% MTT  to each well.Cells were plated at 500 cells/well into 6-well plates  and cultured for 10 days, then stained with crystal violet using the standard method. The colony formation rate was calculated as the (number of clones)/(number of seeded cells)×100.Cell apoptosis was performed using an Annexin V-PE/7-AAD Apoptosis Detection Kit  according to the manufacturer’s instructions.eIF5A2-shRNA cells (7 x 106/0.2ul PBS) or Lv-NV cells (7 x 106/0.2ul PBS) were injected sc into the flank of mice (n = 6). Tumor sizes were measured and calculated weekly for 5 weeks using a caliper. Tumors were retrieved and placed in 10% formalin for histology and TUNEL assay.Lv-5 BCPAP-Lv-eIF5A2-shRNA cells via the tail vein. The mice were killed under anesthesia after 4 weeks. The lungs were fixed in 4% polyoxymethylene.4-6-week-old SCID mice  were injected with 2 × 10eIF5A2 and anti-Ki-67 antibody for immunohistochemistry staining as the manufacture’s protocol.Paraffin-embedding retrieved tumors were washed and dehydrated. 4 μm thick sections were mounted on poly-lysine slides, and stained with H&E for the light microscopy analysis. 2 μm thick sections were incubated with the anti-Cell apoptosis was detected by Terminal Deoxynucleotidyltransferase (TdT)-Mediated dUTP Nick End Labeling (TUNEL) Staining  using an in situ cell death detection kit, all according to the supplier’s instructions.Values were reported as means ± SD and compared by Student’s t-test or chi-squared test. Statistical analysis was performed using SPSS.22.0 . P < 0.05 was considered as statistical significance. Each assay was repeated three independent times in triplicates.P = 0.018).We first analyzed the expression of eIF5A2 in 207 cases of PTC and 39 cases of adjacent tissues using immunohistochemical staining. As shown in p = 0.012), lymph node metastasis (p = 0.002), TNM stage (p = 0.006), T classification (p = 0.047) and BRAF V600E mutation (p = 0.036). No significant differences were found between the two groups in terms of other clinicopathological variables, including patient age, gender, distant metastasis, Follicular variant and multifocality.The knockdown of eIF5A2 expression in BCPAP cells was confirmed by Western blot analysis. High basal eIF5A2 expression was shown in BCPAP cells, eIF5A2 siRNA completely inhibited eIF5A2 expression in BCPAP cells, and no affect was shown in control NC siRNA transfected BCPAP cells ). MTT asThe affect of targeting eIF5A2 by eIF5A2 siRNA on the migration and the invasive potential of BCPAP cells was evaluated by Transwell assays. As shown in in vivo.The stably transfected BCPAP/eIF5A2 shRNA cells or BCPAP/NC shRNA cells were inoculated into nude mice subcutaneously for 5 weeks. All of the mice developed xenograft tumors at the subcutaneous injection sites. The BCPAP/eIF5A2 shRNA groups had less tumor mass compared to the BCPAP/NC shRNA groups ). FurtheThe expression of eIF5A2 is up-regulated in a variety of malignant tumor tissues, and the up-regulation of eIF5A2 is related to the poor prognosis of the patients with malignant tumors ,15. In tin vitro and in vivo [in vivo and in vitro. Taking BRAF V600E mutant BCPAP cells as an example, the study found that targeting eIF5A2 inhibits the proliferation of BCPAP cells in vitro and promotes BCPAP cell apoptosis. At the same time, the invasion and metastasis ability of BCPAP cells is significantly inhibited with eIF5A2 knockout. Further studies demonstrated that targeting eIF5A2 has a significant inhibitory effect on the growth and metastasis of BCPAP cells in vivo. The above research data indicated that eIF5A2 is related to the progress of PTC, and eIF5A2 may be the gene target for PTC therapy.Previous study has shown that targeting eIF5A2 suppressed cell proliferation in pancreatic cancer cells  in vivo . Enhance in vivo . We nextAlthough eIF5A2 was upregulated in PTC tissues, the mechanism of up-regulation of eIF5A2 expression in PTC is still unclear. The BRAF V600E mutation occurred about 45–50% in PTC, 25–30% in ATC, and none in FTC and benign thyroid neoplasm . BRAF V6PEAK1 is a non-receptor tyrosine kinase that regulates the activity of Src kinase and plays an important role in the occurrence and development of malignant tumors . In pancin vivo and in vitro. eIF5A2 provided a novel prognostic marker and therapeutic strategy for PTC patients.Our results demonstrated that eIF5A2 was overexpressed in PTC tissues and eIF5A2 overexpression was associated with metastasis and poor prognosis in patients with PTC. We found eIF5A inhibition reduced the PTC cell proliferation and invasive ability


Igf2bp1 is an oncofetal RNA binding protein whose expression in numerous types of cancers is associated with upregulation of key pro-oncogenic RNAs, poor prognosis, and reduced survival. Importantly, Igf2bp1 synergizes with mutations in Kras to enhance signalling and oncogenic activity, suggesting that molecules inhibiting Igf2bp1 could have therapeutic potential. Here, we isolate a small molecule that interacts with a hydrophobic surface at the boundary of Igf2bp1 KH3 and KH4 domains, and inhibits binding to Kras RNA. In cells, the compound reduces the level of Kras and other Igf2bp1 mRNA targets, lowers Kras protein, and inhibits downstream signalling, wound healing, and growth in soft agar, all in the absence of any toxicity. This work presents an avenue for improving the prognosis of Igf2bp1-expressing tumours in lung, and potentially other, cancer(s). To idenKras fragments for the 3ʹUTR RNA probes were synthesized by PCR amplifications of a pGEM-T_Easy plasmid containing part of the Kras cDNA, using the following primer sets:in vitro transcription reactions, using the MEGA Script T7 High Yield Transcription kit (Invitrogen). For FP experiments, the 5ʹEndTag Nucleic Acid Labelling System (Vector Laboratories) was used to label the RNA with fluorescein at its 5′end.BclRNAoligo/5ʹ6FAM/CCCGUUGCUUUUCCUCU GGGAAGGAUGGCGCACGCUGGG, was synthesized and HPLC purified by IDT.The amplified DNA fragments contain overlapping sequences of approximately 200bp from the Kras-coding region and 5ʹ end of the 3ʹUTR with a T7 promoter (in bold). These fragments were then used as templates in 2, 0.05% IGEPAL) at room temperature for 15 min. 7 μL of this reaction mix was aliquoted with GNF WDII (USA) into wells of 1536 well plates, with a final concentration of 20 μM compound or DMSO. The plates were spun down and read in a BMG Pherastar FS (Germany) plate reader, in FP mode with a 485/520/520 polarization filter, calibrated to 35 mP for free fluorescein with similar total intensity as experimental samples. The G9a FP assay was based on using an S-adenosylhomocysteine (SAH)-FITC probe and antibody to SAH to produce a high FP signal when the antibody binds to SAH-FITC. Unlabelled SAH produced by incubation of G9a enzyme with S-adenosylmethionine (SAM) and peptide substrate competes for the antibody and results in a low FP signal. One hundred per cent activity means that G9a is fully active and therefore a low FP signal is observed (SAH is produced)For the small-molecule screen, compounds were obtained from the collection maintained at the Grand Israel National Center for Personalized Medicine, consisting of molecules purchased from commercial vendors. Labelled RNA (10 nM) was incubated with recombinant Igf2bp1 protein (400 nM) in FAM-RNA buffer /(mPMax − mPMin))) ×100, where the assay minimum (mPMin) is fluorescence Kras RNA alone and the assay maximum (mAMax) is fluorescence Kras with Igf2bp1 protein. Any small molecule that causes a change of more than three standard deviations from the mean is determined appropriate for further investigation.nd generation (NanoTemper). Labelled proteins were spun at 15,000 g for 5 min to eliminate aggregates. All samples were 2X labelled protein mix of Igf2bp1 (200 nM) in PBST and were combined (1:1) with serial dilutions of compound or Kras6 RNA in binding buffer  in final volume of 20 μL, room temperature for 15 min. Samples were then loaded into standard capillaries (NanoTemper) and read in the Monolith NT.115 scanner (NanoTemper) using the red filter with reading conditions of 95% LED power and 40% MST power, 5 sec fluorescence before, 30 sec MST on, 5 sec fluorescence after and 25 sec delay between samples. Results were analysed using the MO Control software (NanoTemper) for curve fitting to KD.Recombinant full-length Igf2bp1 protein was labelled with RED-tris-NTA dye using a Monolith NT His-Tag Labelling Kit (NanoTemper) while RRM12, KH12 and KH34 di-domain peptides were labelled using the Protein Labelling Kit RED NHS 2D calculations.Recombinant Igf2bp1 protein was serially diluted (1:1) starting at an initial concentration of 10,000 nM in 0.75 μg heparin, in a total volume of 13 μL. Fluorescently labelled Kras 6 RNA (750 nM) diluted in reaction buffer , 0.5 mM DTT) was heated to 65°C for 5 minutes, kept on ice for 1 minute and RNasin (200 U/mL)  was then added. 2 μL RNA was added to each well protein sample, and after a 30 min incubation at room temperature in the dark, loading dye (3 μL)  was added to each mixture. For compound inhibition experiments, compounds or DMSO were incubated for 4 hours at 37 °C with the protein prior to adding the RNA. Samples were electrophoresed on native TBE polyacrylamide gels  that had been pre-run for 30 min. Gels were run at 120 V in 0.5X TBE, at 4°C in the dark. They were then carefully washed and scanned using a Typhoon FLA9500 imager using Alexa 488 laser and LPB1/2 filter. Igf2bp1-RNA complex formation was quantified using the ImageJ software. Prism6 software was used for curve fitting and KH1299 cells were maintained in RPMI medium, and ES2, RKO, LKR-M-Fl, LKR-M-GFP, and HEK293 cells were maintained in DMEM . Both media contained 10% FCS  and 10 μg/mL ciprofloxacin (Bayer).Cells were seeded in 96 IncuCyte® ImageLock plates (20X10^3 cells per well) for 24 hours, to a near confluency of 95%, before the addition of increasing concentrations of compound. After a further 24 hours, wells were scratched using the IncuCyte® 96-well WoundMaker Tool, and the cells cultured for an additional 48 hours using the IncuCyte® S3 Live-Cell Analysis System (Essen BioScience). The plate was imaged at increments of 120 minutes for a period of 48 hours, and then analysed for relative wound healing.ES2, H1299 and HEK293 cells were seeded in a 96 well plate (5X10^3 cells per well) for 24 hours before adding the compound at increasing concentrations. After an additional 24 hours, the plate was incubated in an IncuCyte® S3 Live-Cell Analysis System (Essen BioScience). The wells were filmed for 72 hours, and then analysed for proliferation rate Using IncuCyte® S3 Live-Cell Analysis System (Essen BioScience)Vectors expressing different fragments of split luciferase reporters with flexible linker were procured from Addgene  . The vecIGF2BP1 Forward Primer- CCGGAATTCGCCACCATGA ACAAGCTTTACATCGGCAACIGF2BP1 Reverse Primer without STOP Codon- CCGCTCGAGCTTCCTCCGTGCCTGGGCCTGGTTIGF2BP1 Reverse Primer with STOP Codon- CCGCTCGAGTCACTTCCTCCGTGCCTGGGCCTGThe RKO cells were seeded in DMEM with 10% calf serum 24 hrs prior to the transfection. Transfection was done using Lipofectamine 2000 with reporter plasmids and Renilla-luciferase expressing vector . After 24 hrs, cells were lysed using passive lysis buffer . The lysates were cleared by centrifugation and its 20 μL volume was mixed with 100 μL of luciferase substrate solution for luminescence recording which was followed by addition of 100 μL of Stop-glo solution  with an additional luminescence reading. Both readings were detected by using GloMax 20/20 Luminometer (Promega), and the final value for each sample was the ratio of their two respective readings. To test the effects of 7773, transfected cells with reporter plasmids were washed with PBS after 24 hrs of transfection and incubated with medium containing 7773.Western blots were performed as described . For fluThe following primary antibodies were used: mouse anti-total ERK (p44/42 MAPK (Erk1/2) L34 F12 Cell Signalling 4696s), mouse anti-Kras  rabbit anti-dpERK, , rabbit anti-alpha/beta tubulin , goat anti-rabbit IgG-HRP (Jackson), goat anti-mouse IgG-HRP (Jackson). Secondary antibodies for fluorescent westerns: donkey anti-mouse 800 (Rockland 610–732-124), goat anti-rabbit 680 (Molecular Probes A21076).Primers were calibrated for several Igf2bp1 RNA targets:Cells were grown for 12 hours in 12 well plates prior to incubation with the compound at different concentrations for an additional 12 or 24 hours. Total RNA was then extracted using EZ-RNA total RNA isolation kit  and cDNA prepared from the RNA using the First Strand cDNA Synthesis kit (Quanta bio). Real time PCR was performed with Fast SYBR Green Master Mix (Thermo Fisher Scientific), and cDNA expression analysed with the Bio-Rad CFX Manager 3.1.Cells were grown for 12 hours in 6 well plates prior to incubation with the compound or DMSO for an additional 48 hours, 2000 cells were suspended in 0.3% agar-complete medium and then seeded in triplicates onto a 0.6% agar-complete medium base in a 6 well plate. After one week, the colonies were fed with 0.3% agar-complete medium, and after 2 additional weeks, imaged and quantified.E. coli (NEB) and successful cloning was confirmed by sequencing (Source Bioscience). Igf2bp1 di-domain KH12 (V194-N369) cDNA sequence was previously cloned in pETM30 (EMBL) [The open reading frame of Igf2bps were cloned into the pET-21(d) plasmid (Novagen) between the Nco1 and Xho1 sites, as previously described . Human ITo induce expression of recombinant Igf2bps-His tag proteins, a single transformed BL21(DE3) colony was grown in LB medium supplemented with ampicillin (100 g/ml) at 37°C for 7 hours, with agitation. This starter was then transferred to 500 ml fresh LB and incubated with agitation for 1 hour. Protein expression was induced by the addition of IPTG to a final concentration of 0.5 mM for an additional 3 hours. Cells were harvested by centrifugation at 4700 rpm for 20 min at 4°C. The pellet was suspended in HNTA buffer , 1% TritonX-100) and Protease Inhibitor Complex (1:100) (APExBIO). Samples were lysed by sonication and spun at 13,000 RPM in cold for 20 min. The collected supernatant was incubated for 2 hours on a high speed rotator at 4°C with 10 mM imidazole and 0.5 mL Nickel beads (Adar Biotech) which had been prewashed 3X in NTA buffer . The nickel beads were then loaded on a Poly-Prep Chromatography Column (BioRad) and the recombinant protein was eluted through sequential washes of imidazole (1 ml), at the following concentrations: 20 mM, 40 mM, 50 mM, and 250 mM. Samples from all steps of the protein production process were collected and analysed by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining. The fractions which contained the protein were dialysed at 4°C overnight using SnakeSkin Dialysis Tubing, 1000 MWCO (Thermo Scientific) in TGKED buffer . Glycerol (20%v/v) was added to the protein following the dialysis.E. coli cells (Invitrogen). 15N-labelled N-6xHis-RRM12 and -KH34 and N-6xHis/GST-KH12 fusion proteins were obtained by growing the cells in M9 minimal media supplemented with 15NH4Cl as the sole nitrogen source. Cells were cultured at 37°C and expression was induced overnight at 18°C by adding 0.5 mM Isopropyl β-d-1-thiogalactopyranoside. After cell lysis and sonication in 10 mM TRIS-Base pH 8.0, 10 mM imidazole, 1 M NaCl, 5% glycerol, 2 mM 2-Mercaptoethanol, a tablet of cOmplete™ protease inhibitor cocktail (Roche) per 50 mL of buffer, 0.01% Triton-X, 200 μg/mL lysozyme (Sigma), KH12 and KH34 were initially purified by immobilized metal affinity chromatography (IMAC) using a HisTrap™ FF Nickel Sepharose Column . RRM12 expressing cells were lysed and sonicated in the same lysis buffer as above added with 8 M urea. Initial purification was performed with a nitrilotriacetic acid agarose matrix (ThermoFisher Scientific) using the same buffers used in IMAC added with 8 M urea. Refolding was obtained by step-wise dialysis at 4°C. After this first step of purification, the tags were removed from the three proteins by overnight cleavage with 5 µM TEV protease at 4°C in 50 mM TRIS-Base pH 7.5, 150 mM NaCl, 2 mM 2-Mercaptoethanol. The proteins were then loaded onto a cation-exchange Hi-Load SP-Sepharose 26/10 column  and eluted by applying a 0–100% gradient of 10 mM TRIS-Base, 1 M NaCl, 2 mM 2-Mercaptoethanol (pH 6.25 for RRM12 and pH 7.3 for KH12 and KH34). The final step of purification was performed using a Hi-Load 16/600 Superdex 75 pg , equilibrated with 10 mM Na2HPO4 pH 7.4, 50 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine) (TCEP). The purity of the purified protein peak was assessed using SDS-PAGE [The three Igf2bp1 di-domains RRM12, KH12 and KH34 were expressed in BL21(DE3) SDS-PAGE , whilst SDS-PAGE . The >95A La protein plasmid was graciously provided by Tilman Helse and purification was performed as described .1H frequency. 1H-15N-Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance (15N-HSQC) experiments [15N-RRM12, 15N-KH12 and 15N-KH34, obtaining protein-to-7773 molar ratios of 1:1, 2, 4, 8, 14, in 10 mM Na2HPO4 pH 7.4, 50 mM NaCl, 1 mM (TCEP), 10% D2O, 0.02% NaN3. Some non-specific signal loss is observed during the titration of RRM12 but not of the other di-domains, most likely because of a small amount of aggregation. For 15N-KH34, an additional equivalent titration was performed in the same buffer but higher salt concentration (150 mM NaCl). All NMR spectra were processed using NMRpipe [NMR experiments were recorded at 25°C on a Bruker Avance spectrometer operating at 800 MHz  NMRpipe  and anal NMRpipe  and TopS1H and δ15N are the chemical shift differences of the 1H and 15N dimensions, respectively. The published 15N-HSQC resonance assignments [15N-HSQC spectra.where δignments  were obtPrimary sequence alignments of KH12 and KH34 were carried out with T-COFFEE multiple sequence alignment server  and aligD values are reported with standard deviation. FP experiments for the HTS  were performed once ; 504 molecules showed a standard deviation greater than 3. Of these, 48 compounds passed a further quality control screen (see text) and then were tested in a dose-response assay performed in triplicate in parallel to a counterscreen of an unrelated protein/RNA pair using the same FP reaction. Seven molecules demonstrated specific, dose-dependent inhibition of Igf2bp1-Kras 6 RNA binding. One molecule, 7773, was resynthesized twice and inhibited Igf2bp1 binding of Kras 6 RNA with an IC50 of ~30 μM. EMSA experiments were repeated 3 times, and representative gels are shown. Three biological repeats were used for each point in the wound healing and proliferation assays, and each experiment was performed at least twice. For each RT-PCR experiment, 3 biological repeats were performed, with 3 technical repeats for each sample. Western blots of phosphoERK and Kras proteins were performed twice, with 2 biological repeats for each point. Growth in soft agar was performed twice, with 3 biological repeats for each experimental group.All MST experiments were repeated at least three times, with every point performed in triplicate. KIn accordance with the UK Medical Research Council policy, the authors will make available data, software and materials related to this study. Plasmids for the expression of IMP1 RRM12, KH12, and KH34 are available from the authors upon request.G12V is known to be a driver mutation for lung adenocarcinomas [Krasrcinomas , and we rcinomas . Previourcinomas ,18. We trcinomas . As seenrcinomas . Igf2bp1rcinomas . As expeD) between Igf2bp1 and Kras 6 mRNA is ~20 nM  is a quantitative method for assessing interactions between molecules . Differes ~20 nM . These rs ~20 nM , even thRNA Recognition Motif) 1 and 2, KH (hnRNP-K homology domain) 1 and 2, and KH3 and 4. Two of these di-domains, KH12 and KH34, were shown to bind to a range of RNA cognate sequences in vitro and are required for the functional interaction with different targets in cells [D of ~200 nM. This affinity is only ten-fold weaker than that of the entire protein, and, as no binding to the other isolated di-domains is observed, these data suggest that the key interactions between Igf2bp1 and Kras 6 RNA are mediated by KH34. The higher affinity of the full-length protein indicates that additional, weaker interactions with the other domains are also possible. As for many other Igf2bp1 targets in highly proliferating cells, the exact binding site of KH34 is difficult to define, with multiple putative imperfect sites for the individual KH domains present in the Kras6 sequence.Igf2bps contain 6 putative RNA binding domains, arranged in di-domains from the N- to C-terminus: RRM  binding to its target (Bcl2 RNA)   or on a cl2 RNA) . A very cl2 RNA) , showed f ~90 μM . These mand (E).Figure 3.D of 17 μM. When the less effective 393 compound was used, an approximately seven times lower affinity was observed (KD of ~120 μM). The KD values calculated from the MST data are consistent with the IC50’s obtained from the dose response FP curves and confirm a direct interaction between the inhibitors and Igf2bp1. Compound 7773 showed no binding in the MST assay to the control RNA binding protein, La  and identified the amide-protons’ resonances that shift upon addition of 7773  revealed that the inhibitor-binding region is predominantly hydrophobic . To valied above , which iTo validate that 7773 disrupts Igf2bp1 binding to its RNA target, we made use of both EMSA and MST assays. Igf2bp1 protein was pre-incubated with either 7773 (dissolved in DMSO) or DMSO and then tested for its ability to bind Kras 6 RNA, as assayed by gel shift. As Igf2bp1 concentrations are increased, slower-migrating RNA-protein complexes appear on the gel in a protein concentration-dependent manner, even in the presence of DMSO compare . In the in vitro.To further verify the activity of 7773, we tested its ability to inhibit Igf2bp1 binding to Kras 6 RNA in a dose-dependent manner using the MST assay. As seen in D was significantly higher (52 μM) than that observed with Igf2bp1 (17 μM). The gel shift assay also confirmed that 7773 weakly inhibited Igf2bp3 binding of Kras 6 RNA compared to the DMSO control , expressing high levels of Igf2bp1  Fig. 10. There wAnchorage-independent growth is a hallmark of lung carcinomas and other neoplastic cells . H1299 cERT2; Igf2bp1loxP/loxP and Rosa26-CreERT2; Igf2bp1loxP/loxP mice) are healthy (data not shown). For these reasons, we undertook a screen to identify small-molecule inhibitors of Igf2bp1. Evidence presented here indicates that a hit identified in the screen is a very promising lead molecule. In vitro, 7773 binds Igf2bp1 and inhibits its ability to bind Kras 6 RNA. When incubated with cells, 7773 targets Igf2bp1 and causes a reduction in Kras mRNA and other RNA targets, reduces Kras protein and downstream signalling, and represses cell migration and growth in soft agar. We anticipate that further refinement of this compound will lead to a new class of drugs that can be used in a clinical setting for treating lung adenocarcinomas as well as other neoplasias. We have previously described a role for Igf2bp1 in lung adenocarcinoma progression as well as in cell migration and metastasis [Therapies directed at inhibiting Igf2bp1 function constitute a potentially powerful approach for fighting cancer, given the correlation between elevated Igf2bp1 expression and poor clinical outcomes ,44, the tastasis . In additastasis ,47–51. WStructural analysis of the binding of 7773 to Igf2bp1 has begun to shed some light on how this molecule functions. NMR analysis indicates that 7773 interferes with the binding of Igf2bp1 to its Kras RNA target by binding to a planar, elongated surface in the KH34 di-domain interface. Interestingly, binding is mainly hydrophobic but is not mediated by the two canonical base-recognition grooves, which are also hydrophobic. As shown previously, KH34 binds bipartite RNA sequences, with each domain contributing to the binding and the RNA spacer between the target sequences spanning the two canonical RNA-binding grooves. It seems possible that 7773 prevents RNA binding by occupying the path the RNA must trace to connect the two grooves. Alternatively, 7773 binding may result in local conformational changes in the residues framing the hydrophobic grooves. The results of the gel shift and MST experiments  demonstrKras is the most commonly mutated oncogene in cancer and is thought to drive more than 30% of all tumours . It has in vitro or in vivo, was not significantly affected by the expression of either of the constructs. Wound healing, however, was increased by FL-Igf2bp1, and decreased by DN-Igf2bp [in vivo as well as in vitro. Selective targeting was further validated by the ability of 7773 to inhibit the split-luciferase reporter constructs fused to Igf2bp1, but not to control luciferase. Consistent with these findings is the fact that 7773 satisfies Lipinski’s Rule of 5 for drug solubility and cell uptake, having a molecular weight under 500 daltons and a cLogP of 2.5 (<5) [These data are consistent with those reported previously using a mouse lung adenocarcinoma cell line, LKR-M, in which either a human full length (FL-)Igf2bp1 or a dominant negative (DN-)Igf2bp construct was overexpressed. When these cells were xenografted subcutaneously into syngeneic mice, FL-Igf2bp1 overexpression significantly enhanced, and DN-Igf2bp overexpression significantly repressed, the number and burden of LKR-M derived lesions in the lungs. Proliferation of these cells, either N-Igf2bp  due to iN-Igf2bp . 7773 ap2.5 (<5) .Several other Igf2bp1 target RNAs  are reduced in lung and ovarian carcinoma cells incubated with the compound . These rin vitro (termed BTYNB) has been identified using fluorescence polarization [A small molecule that inhibited Igf2bp1-cMyc RNA interaction rization . AlthougBy virtue of their ability to bind a wide range of pro-oncogenic mRNAs, Igf2bp proteins represent an attractive target for directed therapies. Indeed, these proteins have been described as part of an epigenetic switch that occurs during oncogenesis . Igf2bp


The material constant (B) of the nanocrystalline nickel manganite fibers applied by drop-casting on test interdigitated electrodes on alumina substrate, dried at room temperature, was determined as 4379 K in the 20–50 °C temperature range and a temperature sensitivity of −4.95%/K at room temperature (25 °C). The change of impedance with relative humidity was monitored at 25 and 50 °C for a relative humidity (RH) change of 40 to 90% in the 42 Hzπ1 MHz frequency range. At 100 Hz and 25 °C, the sensitivity of 327.36 ± 80.12 kΩ/%RH was determined, showing that nickel manganite obtained by electrospinning has potential as a multifunctional material for combined humidity and temperature sensing.Nickel manganite nanocrystalline fibers were obtained by electrospinning and subsequent calcination at 400 °C. As-spun fibers were characterized by TG/DTA, Scanning Electron Microscopy and FT-IR spectroscopy analysis. X-ray diffraction and FT-IR spectroscopy analysis confirmed the formation of nickel manganite with a cubic spinel structure, while N Monitoring humidity and moisture levels is essential in many industrial processes, such as the food, medical and semiconductor industries, as high-quality products often need to be produced in controlled conditions . Humidit3+ and Mn4+) [2O4 as a humidity sensing material [2O4 as a result of the synthesis method have a noticeable influence on electrical properties [2O4 nanoparticles, such as sol–gel [2O4 nanofibers were obtained by Bhagwan et al. [Nickel manganite is a well-known NTC thermistor oxide material, low-cost, non-toxic and abundant , with a nd Mn4+) ,20. It hnd Mn4+) ,21,22,23nd Mn4+) ,25 in phnd Mn4+) , or as and Mn4+) . Gawli ematerial . The str sol–gel ,29 and h sol–gel  resultinn et al.  using el2O in the response phase of sensor use and does not hinder the desorption of H2O molecules in the recovery phase [Mesoporous metal oxides are widely applied as gas sensing materials, including humidity sensing . A largery phase . Accordiry phase , an incrry phase . Optimizry phase .2O4 nanocrystalline fibers with a high specific surface area and mesoporous structure with potential as a multifunctional material, investigated here for humidity and temperature sensing.In this work, we used electrospinning to obtain NiMn3)2⋅6H2O, purum p.a., purity ≥ 97%), manganese acetate tetrahydrate ((CH3COO)2Mn⋅4H2O, ACS reagent, purity ≥ 99%), N,N-Dimethylformamide , absolute ethanol (ACS reagent), polyvinylpyrrolidone  and deionized water were purchased from Merck  and used as received.Nickel nitrate hexahydrate  on a TESCAN Electron Microscope VEGA TS 5130MM (Czech Republic).Simultaneous TG/DTA analysis of as-spun fibers was performed on a SETSYS Evolution 24,000 Setaram Instrumentation device  up to 1000 °C with a heating rate of 5°/min in air. FT-IR spectroscopy of the as-spun fibers was performed on a FT-IR Nicolet 6700 ATR device  with the resolution of 4 cm2O4 nanofibers was analyzed using X-ray diffraction  in the 10–90° range. Functional groups were determined by FT-IR spectroscopy (on the same instrument and conditions as above). The fiber morphology was observed by field emission scanning electron microscopy (FESEM) on a TESCAN MIRA3 XM (Czech Republic). The nitrogen adsorption–desorption isotherm was collected on a Sorptomatic 1990 Thermo Finigen  at 77 K. Samples for measurement were prepared by degassing in a vacuum at 110 °C for 36 h.The crystal structure of NiMn2O4 nanocrystalline fiber powder was ground for a short time with an agate mortar and pestle, mixed with a small amount of deionized water to prepare a paste that was drop cast onto alumina substrate with small test interdigitated PdAg electrodes , previously prepared by screen printing PdAg paste onto alumina substrate combined with firing in a conveyor furnace at 850 °C for 15 min, as shown in detail in [NiMnetail in . The thi® software as 238 nm with values ranging from 130 to 430 nm.As-spun fibers were smooth and randomly oriented with a uniform surface . The mea−1 range are noted, belonging to residual water in the fibers [−1 [−1 of the carbonyl group in PVP [2 group in PVP were noted at ≈2959 cm−1 and 1420 cm−1, respectively [−1 [−1 [2 rocking vibrations of PVP can be noted at ≈845 cm−1 [−1 and ≈1462 cm−1 can be assigned to asymmetric and symmetric vibrations of C−O originating from the acetate ion in the manganese acetate precursor [−1 are masked by the C=O stretching vibration and C−H bending vibrations of PVP, respectively [−1 can be attributed to the bending mode of the nitrate group [−1 can be due to both the vibrational mode of nitrate groups in the nickel nitrate precursor, and Mn−O vibration in the manganese acetate precursor [The FT-IR spectrum of as-spun fiber a shows be fibers ,37,38. Tp in PVP . C−H strectively . Tertiarvely [−1 , while a845 cm−1 . The banrecursor . Vibratirecursor .2O) [The measured TG/DTA in 2O)  correspo2O) ,37.2O4 nanocrystalline fibers. The characteristic reflections on the measured X-ray diffraction pattern  higher than 0.8, reveals the presence of high diameter mesopores, as well. The pore size distribution curve is given as an inset in 3/g), determined by the BJH method, represents 52% of the total pore volume determined by the Gurevich method for p/p0 = 0.99. Additionally, the most significant contribution to the total specific surface area of 25.3 m2/g, calculated using the Brunauer–Emmett–Teller (BET) equation, originates from the mesopores’ specific surface area of 21.5 m2/g . In contrast, the volume of micropores determined by the t-plot method is small; 0.005 cm3/g. Overall, similar to Bhagwan et al. [2O4 nanocrystalline fibers.The measured nitrogen adsorption–desorption isotherm b is of tn et al. , we obta2O4 [R∞ is the reference resistance of the thermistor, representing resistance at an infinite temperature when 1/T = 0, T is the temperature expressed in K, and B is the material constant  of the thermistor.As noted before for NiMn2O4 ,19,29 as2O4 , as showIn our case, the change in resistance was monitored in a temperature and humidity climatic chamber, maintaining a constant relative humidity (RH) of 40%  and changing the temperature, as the change in RH had an influence on overall resistance values.2O4 powder obtained by sol–gel combustion [3-NiMn2O4 nanocomposite thick films [The material constant (B) of the thermistor  was estimated from the linear fit  [2O4 nanocrystalline fibers in temperature sensing, especially as these values were obtained for drop-cast powder with no further temperature treatment. NiMn2O4 nanocrystalline fibers could be applied in temperature sensors in the form of ink on flexible substrates that can tolerate only low-temperature treatment of the sensing layer [The temperature sensitivity at room temperature (25 °C) was determined as α = −4.95%/K = 1/ (−4%/K) ,49 and cng layer .At both operating temperatures (25 and 50 °C), the measured impedance decreased with the increase in relative humidity (RH), and also with the change in frequency, as shown in 2O4 with a rise in RH leads to an increase in ionic conductivity in accordance with the humidity sensing mechanism [One dielectric relaxation process was noted for complex impedance. This has been noted before for metal oxide semiconductors, including nickel manganite ,28,55,56echanism ,60. This2 electrospun fibers [2SnO4/SnO2 composite [2O4 nanocrystalline fibers for application in humidity sensing.The sensitivity, defined as the ratio between the change of sensor impedance and RH (ΔZ/ΔRH) at 100 Hz, was determined as 327.36 ± 80.12 kΩ/%RH at 25 °C. It decreased with an increase in frequency, and was 259.33 kΩ/%RH, 93.34 kΩ/%RH, 40.71 kΩ/RH at 200, 500 and 1000 Hz, respectively. At the working temperature of 50 °C, the sensitivity at 100 Hz was determined as 69.88 kΩ/%RH. A similar decrease in sensitivity with frequency was noted by Zhang et al. . The obtn fibers  and nanoomposite , confirm2O4 synthesized by refluxing nickel oleate and manganese oleate [3 [2TiO5 [n) are shown in The measured complex impedance spectra  at both working temperatures, the dependence of complex impedance on frequency represented an ideal semicircle . As the relative humidity increased the n value decreased, though it remained relatively close to 1, reaching 0.96038 and 0.95068 for RH 90% at the working temperatures of 25 and 50 °C, respectively. This shows that the behavior of NiMn2O4 nanocrystalline fibers was close to perfect Debye, with the measured complex impedance semicircle showing only a slight depression that increased with an increase in RH from 60 to 90%. Similar values of the critical exponent (between 0.97 and 1) were noted before for NiMn2O4 thin films, but for the change in temperature in the 180–500 K range [The capacitance was calculated taking into account the case of parallel resistance and CPE element, as described in detail in . The detmbustion , or Ni0.intering . In the  K range .relf = 1/R⋅C and it shifted towards higher values with an increase in RH, and also with working temperature  is used to describe the conductivity [3O+ to a water molecule that is close by and ionized, forming H3O+ again. This results in proton hopping between water molecules, increased conductivity, reduced resistance, shrinking the complex impedance semicircles and a shift in the dielectric relaxation frequency towards higher values.At low RH, water molecules are chemisorbed by the mesoporous nanocrystalline NiMnuctivity . A proto2/g) mesoporous nanocrystalline nickel manganite fibers with a cubic spinel structure were obtained using the electrospinning method, followed by calcination at 400 °C with a slow heating rate of 1°/min. Drop-casting a paste made from the obtained powder and water on test interdigitated alumina electrodes enabled determination of the B constant as 4379 K in the 20–50 °C temperature range, and a temperature sensitivity of −4.95%/K at 25 °C. The sensitivity of 327.36 kΩ/%RH in the relative humidity range of 40 to 90% confirmed the potential of electrospun nanocrystalline NiMn2O4 fibers as a multifunctional material for combined temperature and humidity sensing.High surface (25.3 m