Ctenopharyngodon idella). Therefore, the effects of different dietary protein levels on the growth performance, nutritional value, muscle hardness, muscle fiber growth, collagen metabolism and related molecule expression in grass carp were investigated.Nutrient regulation has been proven to be an effective way to improve the flesh quality in fish. As a necessary nutrient for fish growth, protein accounts for the highest proportion in the fish diet and is expensive. Although our team found that the effect of protein on the muscle hardness of grass carp was probably related to an increased collagen content, the mechanism for this effect has not been deeply explored. Moreover, few studies have explored the protein requirements of sub-adult grass crap (n = 25/replicate), and were fed six diets with 15.91%, 19.39%, 22.10%, 25.59%, 28.53% and 31.42% protein for 60 d.A total of 450 healthy grass carp (721.16 ± 1.98 g) were selected and assigned randomly to six experimental groups with three replicates each (Colα1), Colα2, PI3K, Akt, S6K1, La ribonucleoprotein domain family member 6a (LARP6a), TGF-β1, Smad2, Smad4, Smad3, tissue inhibitor of metalloproteinase-2, MyoD, Myf5, MyoG and MyHC relative mRNA levels. The levels of the myostatin-1 and myostatin-2 genes were downregulated, and the protein expression levels of p-Smad2, Smad2, Smad4, p-Akt, Akt, LARP6 and Smad3 were increased.Appropriate levels of dietary protein increased the feed intake, percentage weight gain, specific growth rate, body composition, unsaturated fatty acid content in muscle, partial free amino acid content in muscle, and muscle hardness of grass carp. These protein levels also increased the muscle fiber density, the frequency of new muscle fibers, the contents of collagen and IGF-1, and the enzyme activities of prolyl 4-hydroxylases and lysyloxidase, and decreased the activity of matrix metalloproteinase-2. At the molecular level, the optimal dietary protein increased collagen type I α1  and the muscle hardness (collagen content), respectively.The online version contains supplementary material available at 10.1186/s40104-022-00747-7. Ctenopharyngodon idella) represented the largest world aquaculture finfish production, accounting for 10.5% in 2018 [In 1961–2017, the growth rate of total food fish consumption was nearly twice the population growth rate [ in 2018 . Protein in 2018 . In our MRFs in hybrid catfish (Pelteobagrus vachelli♀ × Leiocassis longirostris♂) and Jian carp (Cyprinus carpio var. Jian), respectively [In grass carp, muscle hardness is positively correlated with the density of muscle fibers . Muscle ectively , 11. TheTGF-β1 gene [Larimichthys crocea), dietary proline and vitamin C increased the activities of P4H and LOX in muscle [TGF-β1 gene in the intestines of grass carp. In human hepatic stellate cells, TGF-β1 inhibited the activity of MMP-2 and upregulated the expression of the TIMP gene by activating Smad3 [In addition to the density of muscle fibers, the increase in muscle hardness is closely related to the content of collagen . Until n-β1 gene . Feeding-β1 gene  which in-β1 gene . A previ-β1 gene . In largn muscle . Therefon muscle , and MMPn muscle . As mentng Smad3 . The aboCtenopharyngodon idella) according to the growth performance and hardness of muscle, which provides significant direction for aquaculture.This study aimed to determine the effect of dietary protein levels on flesh hardness and to explore its regulatory mechanisms by measuring muscle indices related to muscle fiber growth and collagen metabolism, which may provide more theoretical foundation for improving flesh quality by nutritional strategies. In addition, we determined the protein requirements of sub-adult grass carp  were obAt the termination of the feeding trial, fish in each treatment were counted and weighed after anesthesia in a benzocaine bath (50 mg/L), and then the percentage of weight gain (PWG) and specific growth rate (SGR) were calculated. After that, nine of the fish were selected randomly and euthanized by cervical puncture as described by Montenegro et al. . The lefThe moisture, protein, and lipid contents in grass carp muscle were determined by oven drying, the Kjeldahl method, and the Soxhlet exhaustive extraction technique respectively as reported by Horwitz et al. . The fre5′-Inosinic acid  was measured using an ELISA test kit . The contents of hydroxyproline  and lactate (A019-2-1) were measured using test kits . The collagen content was calculated by multiplying the Hyp content by eight according to AOAC . The IGF2 of muscle cross-sectional area according to the method of Johnston et al. [After serial dehydration in gradually increasing concentrations of ethanol, the muscle tissues were equilibrated in xylene and embedded in paraffin. The samples were dissected into 3–5 μm slides, dyed with H&E and observed using a Nikon TS100 light microscope . Morphometric analysis, including myofiber density and frequency of different diameter muscle fibers, was performed using Image-Pro Plus® 4.5 image analysis software. The fiber density was calculated based on the number of fibers per mmn et al. .−ΔΔCT method.Total RNA from fish muscle samples was isolated by an RNAiso Plus kit . The purity and quantity of RNA were identified via agarose gel (1.5%) electrophoresis and NanoDrop 2000 , respectively. Then, the RNA was reverse transcribed to cDNA by a PrimeScript® RT reagent kit . Quantitative real-time PCR was performed by specific primers designed according to the sequences of grass carp , p-Smad2 , Smad4 , total-Akt , and p-Akt  were purchased from Affinity BioReagents . Anti-Smad3  and LARP6  were purchased from ABclonal Technology . The control protein used was β-actin . These antibodies were checked and could successfully cross-react with grass carp proteins of interest.The procedure for muscle protein extraction was performed using lysis buffer . After extraction, the total protein concentrations were determined using the Bio-Rad protein assay kit . The target protein was separated by SDS-PAGE and then transferred to a PVDF membrane for WB analysis. The membrane was blocked with blocking solution (0.5%) for 1.5 h at room temperature and then incubated with primary antibody overnight at 4 °C. Then, the membranes were washed and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody  for 2 h. Finally, the immune complexes were visualized with an ECL kit  under ChemiDoc imaging system . The relative amount of target proteins was analyzed by Image Lab 6.1 software. This experiment was repeated six times.The calculation formula is as follows:P < 0.05. Before statistical analysis, all data were tested for homogeneity of variance and normal distribution by using Levene’s test and Shapiro–Wilk test, respectively (SPSS 21). The linear and quadratic effects of increasing the dietary protein level were conducted by orthogonal polynomial contrasts which was performed by using PROC MIXED (SAS software version 9.4) as described by Lu et al. [Data are presented as the mean ± SD, and were subjected to one-way analysis of variance (ANOVA) and Duncan’s multiple-range test to check for significant differences among the treatment groups at u et al. . The coru et al. .P < 0.05) and then showed no significant difference in higher protein level groups. The FI was significantly increased as dietary protein rose to 22.10% (P < 0.05) and gradually decreased thereafter. The FBW, PWG, SGR and FI showed significant quadratic regression with dietary protein levels (P < 0.05). The FE was the lowest in the dietary protein level 15.91% and the maximum value appeared in the 31.42% group. There was a significant linear regression relationship between FE and dietary protein levels.Growth performance is shown in Table P < 0.06). The maximum muscle crude protein and lipid contents were observed at protein levels of 25.59% and 22.10%, respectively, and were reduced thereafter (P < 0.05). The cooking loss was enhanced with the increase in dietary protein. The pH values in the muscle were the lowest in the 15.91% and 31.42% protein groups, while the lactate content was the highest in these two diets. The moisture and crude lipid contents showed significant quadratic regression with dietary protein levels (P < 0.05). The cooking loss, contents of crude protein and lactate showed significant linear and quadratic regression relationship with dietary protein levels (P < 0.05).The nutrient contents of muscle are shown in Table P < 0.05) with dietary protein increasing, and gradually increased thereafter. The ∑UFA and ∑MUFA were obviously greater at dietary protein level 22.10% compared with that in 15.91% and 31.42% groups (P < 0.05), while the ∑PUFA was opposite. The contents of ∑SFA, ∑UFA and ∑MUFA were observed to be significant linear and quadratic affect by dietary protein levels (P < 0.05). The contents of C14:0, C16:0, C18:0, C14:1, C18:2n6c, C20:2 and C20:3n3 of grass carp muscle showed no obvious differences among all groups (P > 0.05). With the increase of dietary protein, the C15:0 content increased (P < 0.05). The C16:1 content was the highest when the dietary protein was 19.39%. The C17:0 content was increased with the increase in dietary protein from 15.91% to 25.59% (P < 0.05), then plateaued. The C17:1 content increased gradually as dietary protein rose to 25.59% and decreased thereafter (P < 0.05). The maximum of C18:1n9t content appeared in the 31.42% protein group and there were no apparent differences when dietary protein was less than 31.42%. The contents of C20:0, C18:1n9c, C20:1n9, C18:3n3, and C18:3n6 were remarkably increased with the dietary protein increasing from 15.91% to 22.10% (P < 0.05), and plateaued thereafter. The C22:0 content was the highest in the 15.91% dietary protein level (P < 0.05). The contents of C23:0, C20:5n3, C22:6n3 were remarkably higher in the 15.91% protein group compared to 19.39%–31.42% protein groups (P < 0.05). The C22:2 content showed no significant difference when dietary protein levels were 15.91%–28.53%, and the lowest content was observed at the 31.42% dietary protein (P < 0.05). The contents of C15:0, C16:1, C17:0, C17:1, C18:1n9t, C22:2 and C20:5n30 were observed to have a significant linear relationship with dietary protein levels (P < 0.05). The contents of C16:0, C20:0, C18:3n6, C18:3n3, C22:0 were observed to have a significant quadratic effect with dietary protein levels (P < 0.05). The contents of C18:1n9c, C20:3n6, C23:0, and C22:6n3 showed significant linear and quadratic regression relationship with dietary protein levels (P < 0.05).Table P < 0.05). There was a significant quadratic regression relationship between the Arg, Thr, and Asp contents and dietary protein levels (P < 0.05). The maximum Pro and Met contents were observed in the 25.59% protein group and decreased thereafter. They were observed to have a highly significant quadratic regression relationship with dietary protein levels (P < 0.01). When the level of protein was 31.42%, the contents of Gly and Ala were notably lower than those of the other groups (P < 0.05). The contents of Val, Phe and Leu markedly declined as the dietary protein level increased to 22.10% and then increased gradually (P < 0.05). The Ile content did not change among the 15.91%–25.59% dietary protein levels and significantly increased thereafter. The content of Tyr was lower at 19.39%–22.10% dietary protein levels than that in the other groups, which was observed to have a highly significant quadratic effect with dietary protein levels (P < 0.01). The contents of Lys and Glu were significantly higher at the 25.59%–31.42% dietary protein levels than at the 15.91% group. The contents of Gly, Val, Ile, Leu, and Lys were observed to have a highly significant linear regression relationship with dietary protein levels (P < 0.01). The contents of Ala, Phe, and His were observed to have significant linear and quadratic regression relationships with dietary protein levels (P < 0.05). There was a highly significant quadratic regression effect between TAA, the IMP content and dietary protein levels (P < 0.01).As displayed in Table P < 0.05). These parameters were observed to have significant linear and quadratic regression effects with dietary protein levels (P < 0.05). The frequency of muscle fiber diameter > 50 μm declined with the dietary protein increasing to 22%, and increased thereafter. (P > 0.05).The muscle morphology is shown in Fig. P < 0.05). These parameters were observed to have significant linear and quadratic regression effects with dietary protein levels (P < 0.05).As presented in Table P < 0.05) and then decreased gradually at protein levels higher than 25.59%. The activity of P4H increased gradually when the protein levels of the diet increased from 15.91% to 22.10%, after which it showed no significant differences (P < 0.05). The activity of LOX was gradually enhanced within the range of 15.91%–22.10% protein (P < 0.05) and then showed a downward trend. There was a significant quadratic regression relationship between IGF-1, LOX, P4H and dietary protein levels (P < 0.05). The activity of MMP-2 gradually decreased within the range of 15.91–22.10% and then increased gradually.The content of IGF-1 and the activities of collagen metabolism-related enzymes in grass carp muscle are presented in Table Col1α1, Col1α2, PI3K, Akt, S6K1, LARP6a, TGF-β1, Smad2, Smad4, Smad3 and TIMP-2 genes of muscle are exhibited in Fig. Col1α1, Col1α2, PI3K and Akt were upregulated as dietary protein increased to 22.10% and then decreased at higher protein levels (P < 0.05). The highest mRNA levels of LARP6a, TIMP2 and S6K1 were observed in the 22.10%, 22.10% and 25.59% dietary protein level groups, respectively (P < 0.05), and plateaued at higher protein levels. The mRNA levels of TGF-β1, Smad2 and Smad4 were remarkably increased as dietary protein increased to 25.59%, and decreased between 28.53% and 31.42% protein of diet groups (P < 0.05). The Smad3 gene expression was up-regulated when dietary protein level was increased from 15.91% to 22.10%, and then reduced gradually (P < 0.05). However, MSTN1 and MSTN2 mRNA levels were declined as dietary protein level increase from 15.91% to 25.59% (P < 0.05), and raised thereafter. MyoD, MyoG, MYF5 and MyHC gene expressions were significantly upregulated as dietary protein levels increased and then decreased among 25.59% with 31.42% (P < 0.05). There was a significant quadratic regression relationship between all of these genes and dietary protein levels (P < 0.05). There was also a significant linear regression relationship between Col1α2, S6K1, and TGF-β1 and dietary protein levels.The relative expression of P < 0.05) when the dietary protein levels were up to 25.59%, and all declined thereafter. The Smad3 protein level of muscle was notably elevated as the increase in dietary protein rose to 22.10% and then subsequently reduced (P < 0.05). There was a significant quadratic regression relationship between all of these protein expression levels and dietary protein levels (P < 0.05). There was also a significant linear regression relationship between T-Smad2, Smad4, Akt, LARP6, and Smad3 and dietary protein levels (P < 0.05).As presented in Fig. The present study suggested that 22.10%–25.59% of dietary protein levels increased the growth performance  of grass carp, which was consistent with that in young grass carp . This fiMyHC gene, further supporting that dietary protein indeed promoted muscle fiber growth in grass carp. Muscle fiber growth was positively regulated by MRFs  [MyoD, Myf5 and MyoG in muscle. Correlation analysis suggested that the density of muscle fibers was positively correlated with MyoD , Myf5  and MyoG  mRNA levels, indicating that the proper level of dietary protein might promote muscle fiber growth by regulating MRFs gene expression in grass carp muscle. Unfortunately, there are no more studies about the effect of dietary protein on MRFs, and its specific mechanism needs further research. Furthermore, muscle fiber growth was negatively regulated by MSTN. MSTN1 and MSTN2 are subtypes of MSTN [2C12 myoblasts [MSTN1 and MSTN2 mRNA levels in muscle were reduced by the proper level of dietary protein, indicating that dietary protein might promote the growth of muscle fibers by reducing MSTN mRNA levels. However, the expression levels of MSTN1 and MSTN2 genes were increased at higher protein levels, which was consistent with the result in male Wistar rats (4 weeks old) [MSTN to prevent excessive muscle hypertrophy. These results indicated that proper levels of dietary protein probably promote muscle fiber growth by regulating MRF and MSTN gene expression in grass carp muscle.In fish, muscle hardness is positively correlated with the density of muscle fiber . We founnd MyoG) . The pre of MSTN  that inhyoblasts . Here, wIn addition to the growth of muscle fibers, muscle hardness was correlated with the collagen content, which could be calculated using the Hyp content. The present results were consistent with the previous study which found that the contents of Hyp and collagen were increased in the muscle of grass carp fed optimal dietary protein levels . ActuallCol1α1, Col1α2, TGF-β1, Smad2, and Smad4 and the protein expression of p-Smad2, Smad2 and Smad4, indicating that optimal dietary protein might improve the transcription of collagen by activating the TGF-β1/Smad signaling pathway. The dietary protein regulation of TGF-β1 might be related to methionine. Experiments on grass carp have shown that methionine increased the expression of the TGF-β1 gene in muscle [Col1α1 and Col1α2 are mediated by a member of a superfamily of RNA binding proteins: LARP6, which specifically binds to the 5’ stem-loop in the Col1α1 and Col1α2 mRNAs [LARP6a in grass carp and found that the proper level of dietary protein increased the mRNA and protein expression of LARP6a, indicating that dietary protein changed the synthesis of collagen in part related to its translation. In human aortic smooth muscle cells, the expression of LARP6 and type I collagen is regulated by the endogenous growth factor IGF-1 activating the PI3K/Akt/p70S6k-signaling [IGF-1, PI3K, Akt, S6K1 genes and the protein levels of Akt and p-Akt. Correlation analysis revealed that LARP6a mRNA levels were positively correlated with the IGF-1 content . The regulation of IGF-1 by dietary protein might be related to threonine and arginine. It has been reported that the expression of the IGF-1 gene in the liver of hybrid catfish and blunt snout bream  was upregulated by threonine and arginine, respectively [On the one hand, we focused on collagen synthesis. In grass carp, the main collagen in muscle was type I collagen, which consists of the α1 and α2 peptide chains (Col1α1 and Col1α2) , and itsn muscle . In our α2 mRNAs . We clonignaling . We subsectively , 48. OurBefore collagen maturation, the peptide chains are modified by a series of modifications. P4H catalyzes the production of 4-hydroxyproline, which is crucial for the triple helix of collagen . LOX catCyprinus carpio) muscle [TIMP gene through Smad3 in human hepatic stellate cells [TIMP-2 and Smad3 mRNA levels as well as smad3 protein levels and inhibited the activity of MMP-2. Correlation analysis revealed that MMP-2 activity was negatively related to TIMP-2  and Smad3  mRNA levels, suggesting that proper levels of dietary protein increased the collagen content. This increase is partly by decreasing collagen degradation, probably by TGF-β1/Smad3 and TIMP-2 inhibiting MMP-2 activity. No other research exists on the effects of dietary protein levels on regulating collagen degradation, and the detailed mechanism of this process needs to be studied.On the other hand, the collagen content is correlated with its degradation. MMP-2 degrades the type I collagen in common carp  muscle . In zebr) muscle . TGF-β1 te cells . This rey = –0.0021x2 + 0.1101x–0.4224, P = 0.083, R2 = 0.810) showed that the dietary protein requirement for sub-adult grass carp (721–1381 g) was 26.21%, which was lower than that for young grass carp  [y = –0.0221x2 + 1.0983x–9.5742, P = 0.276, R2 = 0.576), which was slightly lower than the value determined based on growth performance.Considering the cost of protein material and that muscle is the main output of grass carp, it is necessary to determine the protein requirement for sub-adult grass carp according to different indicators. Quadratic regression based on the SGR ( 28.68%) . This diMRFs and MSTN, as well as an increase in collagen content. Furthermore, increased muscle hardness probably be associated with increased collagen synthesis regulated by the TGF-β1/Smad2/4 signaling pathway, IGF-1/PI3K/Akt/p70S6K/LARP6a pathway, and related enzymes (P4H and LOX). In addition, increased muscle hardness is probably associated with decreased collagen degradation regulated by the TGF-β1/Smad3/TIMP-2/MMP-2 signaling pathway. Finally, the dietary protein requirements for sub-adult grass carp were determined to be 26.21% and 24.85%, according to quadratic regression of the SGR and the collagen content, respectively.In conclusion, our study suggested that the optimal levels of dietary protein (22.10%–25.59%) improved the growth performance, nutritional value and hardness of grass carp muscle. We first observed that increased muscle hardness was probably associated with an increase in new muscle fiber regulated by Additional file 1:Table S1. The primer sequences and accession numbers for studied genes.


As a result, these k values decreased gradually as the pH of the sample increased from 5.0 to 7.0. The Arrhenius model was applied to describe the heat dependence of the k-values. The activation energy (aE) was calculated for each case of GF2, GF3, and GF4 degradation at pH values of 5.0, 6.0, and 7.0. The result showed that rice bran FOS is very thermostable at neutral pH while more labile at acidic pH.Thermal degradation kinetics of fructooligosaccharides (FOS) in defatted rice bran were studied at temperatures of 90, 100, and 110 °C. FOS extracted from rice bran and dissolved in buffers at pH values of 5.0, 6.0, and 7.0 were prepared for the thermal treatments. The residual FOS (including 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4)) contents were determined using the ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method. The results showed that the thermal degradation kinetics of GF2, GF3, and GF4 followed a first-order kinetic model. Thermal degradation rate constants ( Bifidobacterium spp. and Lactobacillus spp., while reducing or maintaining pathogenic populations  at low levels [‘Fructooligosaccharides’ (FOS) is the common name for fructose oligomers, including three major representatives known as 1-kestose GF2), nystose GF3), and 1F-fructofuranosylnystose GF4). FOS are widely present in a wide variety of foods and feedstuffs. They are naturally occurring sugars with potentially beneficial nutritional effects [, nystose, and 1F-. FOS arew levels ,7,8. Thuw levels ,14,15,16w levels ,18,19. Tw levels ,21,22,23w levels ,25.Oryza sativa) is a global crop that has a long history of safe usage as an indispensable food for humans [Rice . Cellulase preparation was provided by Novozymes  and delivered by Trung Son Technology . The cellulase preparation is in brown color, noted with an activity of 700 EGU/g and the best-storing temperature of 4 °C. Standard FOS set, including GF2, GF3, and GF4, with a purity 99% was purchased from Fujifilm Wako Pure Chemical Corporation . Acetonitril, water, and methanol for UPLC analysis were from Merck .®, Merck, Darmstadt, Germany), being added with 0.1 mL cellulase, and incubated in a water bath shaker at 50 °C for 1 h. The mixture was then blended for 2 min using the IKA T25 disperser (5000 rpm), added with an equal volume of ethanol, stirred, allowed to stand for 15 min, and centrifuged at 5252× g for 12 min . The supernatant was recovered and dried by lyophilization. The dried powder was stored at −20 °C for further use. The yield of fructooligosaccharides extracted from rice bran was about 0.7 g/kg rice bran.The extraction of FOS from defatted rice bran was performed according to the method of Patindol et al.  with min® culture tubes 16 × 100 mm with closed cap . Isothermal treatments were conducted in a temperature-controlled block heater , Tokyo, Japan). After isothemal treatment, the samples were immediately cooled down in ice water and measured for the residual contents of GF2, GF3, and GF4.Rice bran crude extracts (100 mg) and buffers  were added to Falcon 50 mL conical centrifuge tubes  for well mixing. The mixed samples of 5 mL were enclosed in Kimax2 column (150 mm × 2 mm × 5 µm)  [The rice bran-extracted samples thermally treated were analyzed for the residual GF2, GF3, and GF4 contents using an ultra-performance liquid chromatography system equipped with an ESI  coupled to an MS/MS system . The chromatographic separation was performed on a Luna amino—NHCA, USA) . The eluA0 and A are respectively initial- and remaining concentrations at time t (min); and k is the degradation rate constant (min−1).Degradation of GF2, GF3, and GF4 can be described by a first-order kinetic model [k can be determined from a linear regression analysis of ln(A/A0) versus time.Equation (1) is valid under isothermal conditions, whereby the degradation rate constant T is absolute temperature (K); T0 is reference absolute temperature (K); k0 is k at T0 (min−1); aE is activation energy (kJ mol−1), and TR (8.314 J mol−1 K−1) is the universal gas constant.The temperature dependence of the degradation rate constants can be estimated using the Arrhenius equation (Equation (2)):The activation energy can be estimated by linear regression analysis of the natural logarithm of the rate constant versus the inverse of absolute temperature.k value in the present case) and the independent variables  have been formulated [Y is predicted response; β0 is constant; β1, β2, β11, β22, and β12 are unknown parameters of variables for linear, quadratic, and interaction terms, respectively; X1 and X2 are independent variables.Many empirical polynomial models describing the relationship between the predicted response  were constructed using regression analysis.The UPLC-ESI-MS/MS analysis of the standard FOS solutions  was performed with analytical data presented in k values, estimated using linear regression analysis of ln(A/A0) versus t, are reported in The effect of combined temperature and pH on the thermal degradation of rice bran-extracted GF2, GF3, and GF4 dissolved in buffered solutions was studied at 90, 100, and 110 °C and pH values of 5.0, 6.0, and 7.0. As observed, the combined temperature-pH degradation of the rice bran GF2, GF3, and GF4 samples could be adequately described by a first-order model (Equation (1)) in the temperature range of 90–110 °C . Degradaln(k) versus 1/T formed a linear line with a good correlation coefficient , in the temperature range studied, could be estimated using the Arrhenius equation (Equation (2)), with an activation energy range of 61.8–77.7 kJ·mol−1 obtained for GF2, 67.5–92.3 kJ·mol−1 for GF3, and 49.2–72.6 kJ·mol−1 for GF4 , with aX1 as the temperature variable and X2 as the pH variable on the experimental data, the model parameters were estimated using nonlinear regression analysis . Based on the model parameters estimated, however, it was shown that the terms β0 and β22 were redundant, as indicated by the large standard error (~100%). As a consequence, these terms were omitted, and a reduced version of Equation (3) was used ). Model parameters estimated based on Equation (4) are shown in By fitting Equation (3) with k values, respectively) as a function of temperature, pH, experimental k value, and estimated k value (data not shown). In addition, the parity plots of the predicted k values based on Equation (4) versus the experimental k values were established for GF2, GF3, and GF4 ), no tendency was found by graphing residuals (differences between experimental and predicted  and GF4 . The dev version ,35.k of 0.016676, 0.021072, and 0.025567 min−1 corresponding to 8, 10, and 12% loss, respectively, of rice bran GF2, GF3, and GF4 for a total process time of 5 min were simulated and represented in isorate contour plots (By inserting all model parameters of ur plots .Rice bran-extracted GF2, GF3, and GF4 were rather thermally stable compounds at neutral pH while more labile at acidic pH. Among these, GF3 was more heat sensitive compared to GF2 and GF4. A mathematical equation was suggested for a description of the temperature-pH behavior of rice bran-extracted GF2, GF3, and GF4 during the processing of rice bran-based foods. This equation could be useful in designing alternative processing conditions for temperature-pH processing of rice bran-based products. Degradation kinetic studies of rice bran FOS in real food products would be interesting for food processors to evaluate the potential of temperature-pH processing of these products. Similar works can be applied for process stability studies of many other food quality attributes. This type of research is a good approach for the calculation and optimal design of processes for the food processing industry.


Wolbachia is a Gram-negative endosymbiont associated with several species of arthropods and filarioid nematodes, including Dirofilaria immitis. This endosymbiont may elicit a Th1 response, which is a component of the immunity against Leishmania infantum.Wolbachia of D. immitis and L. infantum in naturally infected dogs and cytokine circulation, dogs without clinical signs (n = 187) were selected. Dogs were tested for microfilariae (mfs) by Knott, for female antigens of D. immitis by SNAP, and for anti-L. infantum antibodies by IFAT and assigned to four groups. Dogs of group 1 (G1) and 2 (G2) were positive for D. immitis and positive or negative to L. infantum, respectively. Dogs of group 3 (G3) and 4 (G4) were negative to D. immitis and positive or negative to L. infantum, respectively. Wolbachia and L. infantum DNA was quantified by real-time PCR (qPCR) in dog blood samples. A subset of dogs (n = 65) was examined to assess pro- and anti-inflammatory cytokine production using an ELISA test.To investigate the interactions between D. immitis with circulating mfs, 85% were positive to Wolbachia, with the highest amount of DNA detected in G1 and the lowest in dogs with low mfs load in G1 and G2. Among dogs positive to L. infantum, 66% from G1 showed low antibody titer, while 48.9% from G3 had the highest antibody titer. Of 37 dogs positive to Wolbachia from G1, 26 (70.3%) had low antibody titers to L. infantum (1:160). Among cytokines, TNFα showed the highest mean concentration in G1 (246.5 pg/ml), IFNγ being the one most represented (64.3%). IL-10 (1809.5 pg/ml) and IL-6 (123.5 pg/ml) showed the highest mean concentration in dogs from G1. A lower percentage of dogs producing IL-4 was observed in all groups examined, with the highest mean concentration (2794 pg/ml) recorded in G2.Of 93 dogs positive to D. immitis and Wolbachia with the lower antibody titers of L. infantum in co-infected dogs, suggesting the hypothesis that the endosymbiont may affect the development of the patent leishmaniosis. However, due to the limitations associated with the heterogeneity of naturally infected dogs in field conditions, results should be validated by investigation on experimental models.Results show the association of  Wolbachia and filarioid nematodes  has important implications in the biological processes of reproduction, development, molting and embryogenesis of filarioids [Dirofilaria immitis, the causative agent of the canine heartworm disease (HWD), harbors Wolbachia endosymbiont in all its developmental stages, from adult to microfilariae (mfs) [Wolbachia is released into the bloodstream of hosts after death of D. immitis [Wolbachia associated to D. immitis infection may increase the severity of the clinical signs of HWD by triggering inflammatory response [Onchocerca volvulus [D. immitis may stimulate Th2 anti-inflammatory response, while Wolbachia triggers the Th1 pro-inflammatory response [Wolbachia surface protein (WSP) and other molecules exert on antigen presenting cells and cytokine production [Asaia-WSP engineered bacterium was shown to elicit the Th1 cellular response against Leishmania infantum, with a protozoal killing effect [The endosymbiotic relationship described between larioids –4. Dirofae (mfs) . In addi immitis , also be immitis , 8. Furtresponse , 9–11. Avolvulus , D. immiresponse , 13, 14.oduction , 15–18. g effect .L. infantum and D. immitis in many geographical areas around the world [D. immitis infection increased in areas of southern Europe, where canine leishmaniosis (CanL) was historically endemic, as a consequence of pets traveling with their owners (i.e. from endemic to previously non-endemic regions) and changes in arthropod vector ecology and distribution [D. immitis and L. infantum led to potential immune interactions [D. immitis may be affected by the presence of Wolbachia endosymbiont [Wolbachia in dogs naturally infected by D. immitis and/or L. infantum and to assess the relationship between pathogen infection and cytokine circulation.Given the sympatric occurrence of he world –24, the he world . For exaribution –30. Thisractions , 28. Indractions  depends ractions –35, whicractions . In thissymbiont , 27, 32.n = 84) was enrolled from a previous study aiming to control D. immitis and L. infantum infection [A total of 187 dogs presenting no apparent clinical signs were selected to reduce other factors that could interfere with the analyses. All dogs, living in two municipal shelters in southern Italy  were subjected to physical examination to establish their health status. Among them, a cohort of dogs (nfection .g). Individual blood samples were screened by modified Knott test for the detection of mfs of D. immitis, as previously described [D. immitis by SNAP® 4Dx® Plus Test , according to the manufacturer's instructions.Blood samples were collected from either the cephalic or jugular veins and placed in a K3 EDTA tube (2 ml) and in a clot activator tube (5 ml) to obtain serum after centrifugation  using as antigen the promastigotes of L. infantum zymodeme MON-1, as previously described [To assess the exposure of dogs to escribed . SamplesD. immitis and to anti-Leishmania antibodies. Specifically, dogs positive for mfs and positive and/or negative to IFAT were grouped in G1 (n = 47) and G2 (n = 46), respectively, while dogs negative for mfs and for female antigens of D. immitis and positive or negative to IFAT were included in G3 (n = 47) and G4 (n = 47), respectively , selected based on their negativity and/or positivity to IFAT with a titer ≥ 1:160 and for mfs, were examined by analyzing the production of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) and interferon-gamma (IFNγ) and of anti-inflammatory cytokines such as interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10) using an ELISA kit,   Table . The posL. infantum kDNA minicircle by real time-PCR (qPCR) using the primers, probes and cycle protocol described elsewhere [L. infantum isolate cultured zymodeme MON-1 was used as positive control, whereas gDNA extracted from blood sample from a healthy dog was used as negative controls.Genomic DNA (gDNA) was extracted from each blood sample using the GenUPgDNA commercial kit  according to the manufacturer’s instructions. All samples were tested for lsewhere . gDNA frWolbachia of D. immitis by qPCR using primers  and probe wDimm.ftsZ.523p (6FAM-CGTATTGCAGAGCTCGGATTA-TAMRA) targeting the ftsZ gene as previously described [Samples were tested for escribed  with minD. immitis and that from blood samples of pathogen-free dogs were used as positive and negative controls. The positivity for Wolbachia was established based on the threshold cycle (Ct) value up to 38.5.Briefly, all qPCR reactions were carried out in a final volume of 20 μl, consisting of 10 μl IQ Supermix , 6 μl diethyl pyrocarbonate (DEPC)-treated pyrogen-free DNase/RNase-free water , 2.5 μl of template DNA (except no-template control), primers and probe at 50 μM and 20 μM concentration, respectively. The run protocol consisted of a hot-start at 95 °C for 3 min and 40 cycles of denaturation (95 °C for 5 s) and annealing-extension (60 °C for 30 s). The qPCR was performed in a CFX96 Real-Time System , and the increase in the fluorescent signal was registered during the extension step of the reaction and analyzed using CFX Manager Software, version 3.1 . All the samples were tested in duplicate, and DNA of adult of χ2 test was used to compare percentages of positivity among categories of the same independent variables. Collinearity among independent variables was assessed using Pearsonʼs correlation coefficient. A P-value < 0.05 was considered as statistically significant. Statistical analyses were performed using StatLib for Windows  Quantitative Parasitology 3.0 [Associations between infections and variables were assessed through univariate analysis. Exact binomial test established the confidence intervals (CI) with 95% confidence level. The Chi-square logy 3.0  and GrapWolbachia DNA  was detected in a dog from G1 (mfs load ≥ 600) and the lowest  in dogs with low mfs load (i.e. 1 ≤ mfs ≤ 200) in both G1 and G2  was recorded in dogs with a WolbachiaCt values ranging from 25 to 30  and vs. Ct > 35  having a high mfs load and between low vs. high mfs score   was observed between dogs with WolbachiaCt value of 25–30 and 30–34 vs. > 35, having high mfs load  tested positive for NA Table , with 37Leishmania antibodies, 31 were from G1  with low antibody titers (up to 1:160), followed by 16 dogs  with higher titers (from 1:320 to 1:2560). In G3, 23 dogs  had a high antibody titer (from 1:320 to 1:2560) (Table L. infantum (4.3%) with a minimum and maximum Ct value  detected for dogs from G1  had low antibody titers  against L. infantum  and those with antibody titers ranging from 1:320 to 1:2560   was recorded analyzing the percentages of IFNγ-positive sera between dogs from G1 (64.3%) and G4 (14.2%), with the same percentage of positive dogs in G2 and G3 (50%) (Table IL-10 (55.4%) and IFNγ (44.6%) were the most prevalent cytokines produced throughout dogs examined Table , Fig. 1.χ2 = 5.1, P = 0.023), IFNγ vs. IL-4 , IL-10 vs. IL-4  and IL-4 vs. IL-6 . Similarly, in G4 a statistically significant difference was observed between IFNγ and TNFα vs. IL-10 and between IL-10 vs. IL-4 . No statistically significant difference was observed in cytokine production within groups G2 and G3 , IL-4  and IL-6   is coherent with the hypothesis that this endosymbiont might control the Leishmania infection. Overall, the prevalence of Wolbachia observed in co-infected dogs is in line with data described in a previous study [Wolbachia DNA in relationship to the mfs load  exclusively observed in co-infected dogs (63.2%) differed from a previous study from Spain, where Wolbachia was more frequently detected in microfilaraemic dogs not infected with L. infantum [Wolbachia [Data herein presented suggest that echanism . Indeed,us study , where tinfantum . In addiolbachia . Furtherolbachia .Wolbachia in controlling the development of CanL may be highlighted by the high prevalence of co-infected dogs (70.3%) showing a low anti-Leishmania antibody titer (up to 1:160), likely due to the low parasitic load detected by qPCR in the blood [L. infantum.The role of he blood , while aWolbachia amount recorded in co-infected dogs may trigger an elevated TNFα production , which was not recorded in dogs infected with only L. infantum. The strong effect of Wolbachia in producing this pro-inflammatory cytokine has also been demonstrated in an in vitro experiment [L. infantum. However, the absence of TNFα observed in dogs positive for L. infantum agrees with previous studies where the cytokine was detected only in a few dogs with active leishmaniosis [Furthermore, the absence of clinical signs in dogs may be also determined by the stimulation of a Th1 immune response triggered by the endosymbiont bacterium by production of high levels of pro-inflammatory cytokines , 27, 44.periment , regardlmaniosis , 46.Wolbachia amount detected in co-infected dogs, and consequently its effect on controlling the L. infantum infection, may also be supported by the high prevalence of dogs (64.3%) producing IFNγ, a similar mean cytokine concentration value being observed between these dogs (88.2 pg/ml) and those positive for L. infantum (97.23 pg/ml) or D. immitis (105.4 pg/ml). Accordingly, IFN-γ was associated with the absence or low antibody titer against L. infantum [L. infantum or stimulated with L. infantum antigen, an increase in IFNγ, IL-10 and IL-4 mRNA expression levels were recorded [D. immitis with circulating mfs associated to the presence of Wolbachia [D. immitis (507.8 pg/ml) or L. infantum (677.4 pg/ml). However, though IL-10 is considered a predictive parameter of the evolution of CanL and active visceral leishmaniosis in humans [L. infantum infected (254.9 pg/ml) or uninfected dogs (111.7 pg/ml). However, other studies also described these contrasting results, IL-4 being detected readily or not in L. infantum infected or asymptomatic dogs [Wolbachia amount recorded in the co-infected dogs may have affected the IL-6 production (123.5 pg/ml) in these dogs. Indeed, the role of this endosymbiont in stimulation of IL-6 production was also supported by an in vitro experiment [. However, though IL-6 is generally regarded as a Th2 cytokine with disease progression, other studies described its protective role in some forms of leishmaniosis [The infantum . Besidesrecorded , 48, 49. immitis 5.4 pg/mlolbachia . All then humans –53, othen humans –56. Furtn humans , where tn humans . Indeed,nfected 2.9 pg/ml tic dogs , 58. Theperiment . Howevermaniosis , thus inmaniosis , 48, 60.Overall, interpreting the cytokine expression profile in CanL is still problematic, and the differences observed regarding the role of cytokines may be due to the different methods used for the analyses  , 54, 56.Wolbachia in clinical leishmaniosis, pointing at a possible role of this endosymbiont in the modulation of the Th1 immune response. However, future studies based on the simultaneous combination of different approaches of analysis (i.e. expression of the mRNA vs. quantification of cytokine production) is mandatory.Though the present work has some limitations, such as the low number of dogs included and their heterogeneity, and the lack of a follow-up study, the results presented suggest the involvement of


A), suggestive of plasmacytic differentiation. Immunohistochemical stains showed that the large lymphoma cells were positive for CD20 (B), CD138 , IgM (D), BCL2, BCL6, and MUM1, and negative for CD3, CD5, and IgG. By in‐situ hybridization study, the neoplastic cells demonstrated immunoglobulin lambda (E) light chain restriction and negative for kappa (F). Ki‐67 proliferation index was 40–50% (G). Lambda and IgM stains also highlighted the Russell bodies. Flow cytometry immunophenotype analysis detected an abnormal lambda restricted population of large B‐cells which were positive for CD19, CD20dim, CD22, CD38, CD79b, and negative for CD5, CD10, CD23, CD30, CD43, and CD200 (H). FISH showed no “MYC” rearrangements. The morphology and immunophenotype were diagnostic of diffuse large B cell lymphoma  with marked plasmacytic differentiation and Mott cells. The patient was switched to blinatumomab and reached CR 5 months later, but relapsed after one year. He further received six cycles of R‐CHOP plus venetoclax and reached CR until now.The patient is a 75‐year‐old man with a history of chronic lymphocytic leukemia (CLL), status post three courses of chemotherapy, and reached complete remission (CR). He presented with a right elbow mass. PET‐CT showed FDG‐avid nodular lesion in the soft tissue, most notably in the right upper elbow and left gluteal region, and also in the left lower lobe of the lung. The largest nodule in the right elbow measured 5.5 × 4.5 cm, which was biopsied. The biopsy showed diffuse infiltration of large neoplastic cells with round nuclei, fine chromatin, and prominent nucleoli. Admixed are many plasmacytoid lymphocytes and plasma cells, some showing abundant cytoplasmic Russell bodies (Mott cells) , not bright CD38, and surface light chain restriction are features that support a diagnosis of DLBCL with marked plasmacytic differentiation, while lack of B‐cell markers and surface light chain expression and bright CD38 and diffuse CD138 favor a plasma cell neoplasm.Shaoying Li designed and performed the research and wrote the paper, Jie Xu, C. Cameron Yin and Sergej Konoplev wrote the paper.


The concentrations of 100 µM of caffeine, 20/50 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression under TGF-β1 stimulation (p ≤ 0.024). LUM and ALDH3A1 expression remained low under TGF-β1 stimulation, independently of AFM supplementation. Immunocytochemistry showed that 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone reduce the conversion rate of aCSK to SMA+ MYO-SF. In conclusion, in aCSK, 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation, with no influence on cell counts. However, the AFMs were unable to protect aCSK from characteristic marker loss.We evaluated the small molecules (AFM) caffeine, curcumin and pirfenidone to find non-toxic concentrations reducing the transformation of activated human corneal stromal keratocytes (aCSK) to scar-inducing myofibroblasts (MYO-SF). CSK were isolated from 16 human corneas unsuitable for transplantation and expanded for three passages in control medium (0.5% FBS). Then, aCSK were exposed to concentrations of caffeine of 0–500 μM, curcumin of 0–200 μM, pirfenidone of 0–2.2 nM and the profibrotic cytokine TGF-β1 (10 ng/mL) for 48 h. Alterations in viability and gene expression were evaluated by cell viability staining (FDA/PI), real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. We found that all AFMs reduced cell counts at high concentrations. The highest concentrations with no toxic effect were 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone. The addition of TGF-β1 to the control medium effectively transformed aCSK into myofibroblasts (MYO-SF), indicated by a 10-fold increase in α-smooth muscle actin (SMA) expression, a 39% decrease in lumican (LUM) expression and a 98% decrease in ALDH3A1 expression ( Corneal stromal scarring remains a major cause of blindness worldwide, with limited treatment options ,2. DamagHuman CSK are quiescent in vivo and typically express stromal crystalline proteins, including aldehyde dehydrogenases , a-enolase, lactic dehydrogenase and transketolase, contributing to corneal transparency ,5. They Though undoubtedly useful for the goal of shrinking the injury site, wound contraction in the cornea disturbs the shape and curvature of this critical optic and its ability to precisely focus light onto the retina. Loss of transparency, largely in the form of haze during corneal wound healing, results from several factors: (1) infiltration of the cornea by inflammatory cells, (2) SMA-positive MYO-SF, which are less transparent than quiescent CSK, likely due to their decreased crystallin synthesis , and (3)Expanding human CSK is challenging and this has been a limiting factor in their application in cellular research, corneal tissue engineering and cell therapy ,12. MediClinically, attempts to control corneal scarring have mostly involved the use of steroids or mitomycin C (MMC) . While eConsequently, there is an urgent need for antifibrotic agents that effectively impede CSK from MYO-SF conversion and corneal scarring, while exerting as little toxicity as possible, in vivo and in vitro.In recent years, efforts were undertaken to investigate whether small antifibrotic molecules (AFM), capable of manipulating intracellular signaling downstream of TGF-β receptor activation, might represent a better alternative to steroids, MMC and topical application of anti-TGF-β antibodies to the eye ,19,20. ICaffeine inhibited TGF-β activation in lung epithelial cells and interrupted lung fibroblast responses to TGF-β in concentrations between 50 µM and 100 µM .Curcumin at concentrations of 5–20 μmol/L significantly inhibited UVB-induced secretion of IL-6 and IL-8 by human limbus epithelial cells in a dose-dependent manner, while curcumin alone did not affect the secretion of IL-6 and IL-8 .Jiang et al. showed that pirfenidone inhibited human umbilical vein endothelial cell (HUVEC) viability at concentrations of ≥300 μg/mL, but eye drops containing 1000 μg/mL of pirfenidone administrated four times per day for 14 days reduced corneal edema, promoted corneal wound healing and inhibited neovascularization formation in a rat alkali burn model .However, the effects of caffeine, curcumin and pirfenidone on primary human aCSK have not yet been investigated. Hence, we investigated their antifibrotic potential in concentrations of caffeine of 0–500 μM, curcumin of 0–200 μM and pirfenidone of 0–2.2 nM (0–400 µg/mL).To evaluate the potential toxic effects of the AFMs, aCSK were incubated in different media  for 48 hp = 0.027).Following 48 h of incubation, caffeine showed significantly reduced aCSK counts in comparison to the control in the highest tested concentration of 500 µM  and 200 µM of curcumin .In curcumin, a significant cell reduction effect was seen at concentrations of 50 µM and above, in comparison to the control  significantly reduced the aCSK cell count  pirfenidone.p < 0.001, p < 0.001) and a 98% decrease in ALDH3A1 expression (p < 0.001) in aCSK after 48 h of incubation.TGF-β1 addition to the control medium led to a 10-fold increase in SMA expression .aCSK incubated in caffeine 100 µM + TGF-β1 showed a significantly lower SMA expression compared to the control + TGF-β1 group .The incubation of aCSK in pirfenidone 1.1 or 2.2 nM medium led to no significant difference in SMA expression compared to control aCSK. SMA expression levels were significantly lower in pirfenidone 1.1 nM + TGF-β1 compared to control + TGF-β1  of pirfenidone. TGF-β1 addition to the control medium induced the conversion of aCSK to SMAStromal cellular responses such as CSK activation and apoptosis after surgical trauma have been studied in the corneas of mice , rats 55, rabbitHence, investigating the effect of caffeine, curcumin and pirfenidone on primary human aCSK is crucial in the search for a means of preventing haze and MYO-SF differentiation, while stimulating the regeneration of the corneal stroma and epithelium, and preserving or restoring normal ocular optics.Caffeine  is one of the most consumed food additives worldwide and has wide-ranging pharmacological activities including effects on the central nervous, cardiovascular and respiratory systems . It can In our study, we showed, for the first time, that 100 µM of caffeine significantly reduced SMA expression and MYO-SF conversion in primary human aCSK. However, caffeine was unable to protect from CSK marker loss.Caffeine is not known to undergo a significant first-pass metabolism and generally reaches its peak plasma concentrations within 30–120 min of its administration. Studies have shown that serum caffeine levels from moderate coffee consumption usually range between 20 and 50 µmol/L and do not tend to exceed levels of 70 µmol/L . In geneConsequently, the topical, rather than systemic, application of 100 µM of caffeine eye drops in a corneal scarring model could be a coherent next investigative step.Fibrosis within the cornea tends to be dynamic, with ongoing MYO-SF generation and apoptosis. Once mature MYO-SF develop, they persist until the requisite source of TGF-β1 and/or TGF-β2 to maintain viability is sufficiently reduced by repair. Once the levels of these TGF-β isotypes drop in the stroma, then IL-1 produced by surrounding cells ), unopposed by TGF-β1 or TGF-β2, triggers increased apoptosis of the MYO-SF . In our + MYO-SF conversion show different responses to fibrosis inhibition has been shown before.Interestingly, the antifibrotic caffeine did not protect aCSK from the loss of their characteristic markers  during stimulation by 10 ng/mL of TGF-β1. The fact that CSK marker expression and SMASeidelmann et al. demonstrated that the expression of the MYO-SF marker SMA decreased with incremental human platelet lysate (hPL) substitution in CSK and SF, implying an antifibrotic effect, which they attributed to the basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) found in hPL. Nevertheless, CSK markers decreased with higher hPL substitution .Similarly, Jester et al. incubated primary rabbit CSK with 10 ng/mL of bFGF and found a fibroblast-like phenotype but negative SMA immunocytochemistry after 7 days of culture .Curcuma longa L., belongs to the polyphenols. It was isolated over 140 years ago by Vogel and was synthesized in 1913 by Lampe -pyridone) is a non-peptide pharmacologic anti-fibrotic, anti-inflammatory and anti-oxidant compound that was approved by the Food and Drug Administration (FDA) for patients with idiopathic pulmonary fibrosis (IPF) ,54. SeveIn the eye, pirfenidone has been shown to inhibit the activity of human Tenon’s fibroblasts in vitro , and hasPirfenidone nanoparticles also improved corneal wound healing and prevented corneal fibrosis in Sprague Dawley rats in vitro and in vivo . In addiIn our study, we showed, for the first time, that 1.1 nM (200 µg/mL) of pirfenidone significantly reduced SMA expression and MYO-SF conversion in primary human aCSK. However, pirfenidone was unable to protect from CSK marker loss under TGF-β stimulation.Pirfenidone is commercialized as an oral immediate release formulation with an administration of 801 mg/day during the first week, followed by an increase to 1602 mg/day during the second week and a subsequent dose increase to reach 2403 mg/day after 15 days of treatment. Pirfenidone use, however, is limited by the occurrence of adverse events . GastroiIn a pharmacokinetic study on 28 patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis, pirfenidone showed a rapid absorption rate, reaching the maximal concentration at around 0.86 ± 0.37 h and a peak concentration of 19.81 µg/mL .Jiang et al. previously demonstrated that pirfenidone inhibited human umbilical vein endothelial cell (HUVEC) viability at concentrations of ≥300 μg/mL, but eye drops containing 1000 μg/mL of pirfenidone administrated four times per day for 14 days reduced corneal edema, promoted corneal wound healing and inhibited neovascularization formation in an alkali burn rat model . ConsequInterestingly, aCSK incubated in media containing 1.1 or 2.2 nM 200 or 400 µg/mL) of pirfenidone showed a significant increase in lumican expression, which could indicate a protective effect against CSK marker loss during 0.5% FBS stimulation [00 µg/mL + MYO-SF is an integral part of organ fibrosis, and is frequently used to screen for the antifibrotic effects of different components in the cornea and other organs in and ex vivo [As a limitation, in this in vitro cell culture pilot study, we investigated the antifibrotic effects of the AFMs through alterations in CSK marker and SMA expression. Nevertheless, corneal fibrosis is a complex dynamic process influenced by various genes and proteins ,73, amon ex vivo ,23. FurtTo summarize, we found that 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation, with no influence on cell counts. However, they were unable to protect from CSK marker loss. Further studies are necessary to investigate the ideal concentrations and application modes of caffeine, curcumin and pirfenidone for the inhibition of corneal scarring, as well as their potential for CSK ex vivo expansion for tissue-engineering and research purposes.Human CSK were isolated from 16 corneas (8 donors) unsuitable for transplantation  supplied by the Cornea Bank Aachen. CSK were isolated and cultivated as previously described ,74. BrieThe CSK were cultured in aCSK basal medium containing 0.5% FBS until passage 3. After 24 h, the medium was exchanged for new medium containing the according substitutes . The bas2 on collagen-I-coated 24-well plates  and incubated in different media . After 48 h of culture in different media  and incubated in different media (−ΔΔCT) method for 6 different donors [The cells were seeded at 9000 cells/1.8 cmnt media  for 48 hnt media : glycerat donors . Relativp value of ≤0.05 was considered statistically significant.All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed with SPSS version 22.0 . The Mann–Whitney U test or Wilcoxon rank-sum test were used to compare cell numbers and gene ratios. A


BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO Cell lines are one of the most valuable tools in experimental biology for research development. In addition, cellular models are beneficial for disease research, especially cancer, testing candidate drugs, personalized therapeutic strategies, and signaling pathways in analysis at gene and protein levels  In additThe application of cellular models in mentioned research is often related to genetic modifications that allow precise control of specific genes. In most cases, genetic manipulations allow silencing or overexpression of the gene, but latter-day gene-editing tools, i.e., CRISPR/Cas9 and TALEN, are used to induce gene-specific insertion or deletion . The impElectroporation can be used for inserting any molecules, e.g., drugs, chemicals, or foreign genetic material (DNA or RNA), in cells and is characterized as the application of brief electric pulses to permeabilize the cell plasma membrane. EP is widely used, and much interest has been given to increasing transfection efficacy by varying pulse parameters and electroporation medium properties. Transfection efficiency and viable cell rates are the main parameters to consider. It is well known that increased transfection efficiency results drop in cell viability . Thus, dBCR/ABL negative myeloproliferative neoplasms or chronic myeloid leukemia. Several significant studies were carried out with the UT-7 cell line and its subtypes to evaluate their response to chemotherapy drugs, anthracyclines, and antimetabolites. Moreover, next to the cell viability analysis, signaling pathways, e.g., MAPK/ERK, PI3K/AKT, KIT, JAK/STAT, and NGF/TRKA, that contribute to blood cancers, are being investigated at gene and protein levels [UT-7 cell line was established in a patient with acute myeloid leukemia . UT-7 cen levels ,25,26,27However, UT-7 cells are known for their low efficiency of transfection . Since nLately, nucleofection, a new electroporation-based method, was developed. It is indicated that nucleofection enables DNA to directly enter the nucleus for the high-efficiency transfection and high viability of a wide variety of cells. According to the manufacturer, nucleofection of the UT-7 cell line results in high levels of transfection and cell viability  . However4. Finally, we evaluated the influence of varying plasmid DNA concentrations. In addition, the design of our methodology allowed us to assess the distribution of cells among four different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead, which led us to a better understanding of the relation of GET efficacy and cell death of UT-7 cells.Hence, in this work, we describe protocol optimization for the electroporation of the UT-7 cell line. In addition, this study will describe the methodologies we have developed for optimizing EP settings to balance the highest possible transfection efficiency with robust cell viability and growth post-gene electrotransfer. UT-7 cells were electroporated using square-wave pulses, following the systemic screening of pulse strength and duration for best GET efficacy. Furthermore, the impact of DNase inhibitors on GET efficacy was tested by electroporating UT-7 cells in the presence of ZnSO2. UT-7 cells were passaged 1:10 twice a week and always 24 h before the experiment. For some of the experiments, the medium was complemented with 80 µM and 100 µM ZnSO4 .The UT-7 human cell line was purchased from the German Collection of Microorganisms and Cell Cultures . Cell line authentication was performed by DSMZ. The UT-7 cell line was maintained in alpha-MEM  supplemented with 20% fetal bovine serum , 100 μg/mL streptomycin, 100 units/mL penicillin , and 2 mM L-glutamine . Recombinant human GM-CSF  was added to UT-7 cells that require the bioactive protein for survival and proliferation at a final concentration of 5 ng/mL. The cells were cultured in a water-jacketed incubator at 37 °C with 5% COEscherichia coli, DH5-α strain, selected with ampicillin , and purified with the Plasmid Maxi-Prep Kit . The plasmid was produced at a concentration of 1 μg/μL. Before electroporation, plasmid vectors were loaded at a final concentration of 20 μg/mL (unless otherwise explicitly stated).The pEGFP (3400 bp)  encoding a green fluorescent protein was used as an indicator for the assessment of transfection efficiency. The plasmid was extracted from competent 6 cells/mL. Next, a volume of 50 µL (9 × 104 cells) was transferred between stainless steel plate electrodes separated with a 2 mm gap. A BTX T820 electroporator (Harvard Apparatus)  or laboratory-made electroporator was used for pulsing, changing the voltage, and pulse duration. A square wave 1 HV  pulse, 1200–1800 V/cm pulse strength, and 100–750 µs pulse duration were delivered. After pulsing, UT-7 cells were transferred into 24 well plates  and left for 20 min for recovery. After the recovery period, cells were seeded into two 24-well and 96-well plates  for transfection, propidium iodide staining, and MTS assays, respectively. 24-well plates were supplemented with a culture medium up to 450 µL and 96-well plates up to 150 µL. Afterwards, plates were placed in an incubator for 48 h. The electroporation regimen was carried out at room temperature conditions.UT-7 cell line was cultured in T75 flasks  and pelleted by gentle centrifugation at 300 RCF for 5 min. Cells were counted using a Neubauer hemocytometer  under an optical microscope. An appropriate cell number was suspended in a laboratory-made electroporation medium at a concentration of 1.8 × 102 , 5.59 mM Na2HPO4 , 3.00 mM NaH2PO4 , and 242.20 mM sucrose . EP medium was filtered out through 0.22 µm syringe filters before usage.The characteristics of the laboratory-made EP medium were 0.1 S/m conductivity, 270 mOsmol osmotic pressure, and 7.2 pH. The medium contained 1.73 mM MgClUT-7 cell line was observed for viability and fluorescence 48 h post-transfection. The analysis was performed using BD Accuri C6  flow cytometer. 22 μm core size and 66 μL/min flow rate were used for all measurements.The cell culture was pelleted by centrifugation at 300 RCF for 3 min in a centrifuge to remove the growth medium. Then, UT-7 cells were resuspended in 200 μL 1xPBS  buffer. For pEGFP transfection efficiency assessment, cells were excited using a 488 nm laser, and data was collected by counting 10,000 events/samples using a 533/30 nm FL1-A emission filter for green color.Propidium iodide (PI) staining was used to determine dead cells after electroporation. First, the cell samples were centrifugated at 300 RCF for 3 min to remove the growth medium. Afterwards, cells were diluted in 90 µL 1xPBS  containing 10 µL PI (400 µM) solution . 3 min after dilution, UT-7 cells were measured by flow cytometry. Cell permeability to propidium iodide was analyzed using a 585/40 nm FL2-A emission filter for red color. Cells negative for PI were considered living cells.+ and PI+ cell populations were acquired for compensation. The gating strategy is depicted in the Results section.Color compensation was used by subtracting 6% of the pGFP channel from the PI channel to avoid color spillover of transfected cells into the population of dead cells. Separate controls of only pGFPGET stands for transfection efficacy and N for cell number.The transfection efficacy was calculated as the ratio of transfected/viable cells vs. the number of viable cells in the control sample (Equation (1)):2. The absorbance was measured at 490 nm by a microplate reader .MTS tetrazolium  assay was performed for metabolism assessment of the UT-7 cell line. After electroporation, UT-7 cells were seeded into a 96-well plate at a density of 9000 cells/well for up to 48 h following previously described conditions. The relative viability of the UT-7 cell line was analyzed by measuring the amount of MTS converted by the cells. Briefly, 40 µL MTS (5mg/mL in 1xPBS) was added to each well and incubated for another 3 h at 37 °C, 5% COp < 0.05 value was considered statistically significant. Experiments were performed in duplicates and repeated at least two times independently.IBM SPSS Statistics 22.0  software was used to perform statistical tests. The data were analyzed using an independent sample t-test and one-way analysis of variance (ANOVA), followed by post hoc Tukey multiple comparison tests when appropriate . The dat+/PI−), untransfected/viable (pEGFP−/PI−), transfected/dead (pEGFP+/PI+), and untransfected/dead (pEGFP−/PI+) cell subpopulations.We established a gating strategy for a proper assessment of transfected cells co-stained with PI for distinguishing among four different cell subpopulations in the sample . Using s+ (transfected) from untransfected cells (GFP−) (− (viable) cells from PI+ (dead)  vs. forward scattering-height (FSC-H) A. Then Gs (GFP−) B and PI−+ (dead) C cells w+ (dead) F in the + (dead) E.The effect of the EP buffer on transfection efficiency was verified by performing GET in three different electroporation buffers, laboratory-made EP medium, BTXpress Cytoporation Medium T , and Opti-MEM . A laboratory-made EP medium resulted in the same number of viable and transfected cells compared to different manufacturers’ EP buffers (results not shown). Therefore, the following experiments were performed in a laboratory-made EP medium.Different electroporation conditions were tested, including variations in electric field strength  and pulse duration . In each experiment, a single high voltage pulse was applied, and 20 μg/mL of the plasmid was used.We exploited three different assays to determine the viability of UT-7 cells to discriminate between live and dead cells since it is impossible to wash out dead suspension cells from the samples without losing viable ones. For this reason, we used Propidium iodide (PI) to stain dead cells with a leaky plasma membrane. Then, the overall cell number was obtained by counting all cells in the sample using flow cytometry, and PI-positive (dead) cells were excluded from the population. Furthermore, different gene electrotransfer parameters were tested, and the overall cell number, including PI results, was compared to the MTS assay .p < 0.007).Results demonstrated that the cell death rate increased by increasing the voltage and duration of the electric pulse. Moreover, we determined that the overall cell number compared to the number of viable (PI negative) cells was significantly higher throughout the whole range of parameter sets from 1.6 kV/cm 100 µs. Only the 1.2 kV/cm 250 µs parameter set showed no difference . NotablyThese results confirmed that the best solution for further experiments was to follow the PI staining of dead cells since PI staining and metabolic activity demonstrated similar results. However, the PI method was considered superior to the MTS method in our case due to the ability to use the PI methodology for simultaneous cell viability and transfection efficiency measurements.Next, we evaluated pEGFP plasmid electrotransfection efficiency in UT-7 cells under the same EP parameter sets  as in FiOur data indicate that increasing pulse intensity and duration was the significant cause of the growing number of transfected/dead (pEGFP+/Dead) cells A. Such rAn opposite pattern was noticed in the untransfected/viable subpopulation, where the number of cells diminished notably with increasing pulse intensity and duration. The same pattern was observed in the overall cell number decrease A. For inIn contrast, the increase in mean fluorescence intensity in the transfected/viable cell subpopulation was more pronounced than the transfected/viable cell number. These results suggest that increasing pulse strength and duration helps more plasmid molecules enter electroporated cells.4 on GET efficiency under conditions from previous experiments that demonstrated the highest efficiency. Subsequently, UT-7 cells were electroporated with 1.4 kV/cm 250 µs, 1.6 kV/cm 250 µs, 1.8 kV/cm 250 µs, 1.2 kV/cm 500 µs, 1.4 kV/cm 500 µs, and 1.2 kV/cm 750 µs pulses in the presence of 80 µM and 100 µM ZnSO4 , 1.2 kV/cm 250 µs 100 µg/mL pEGFP , and 1.2 kV/cm 250 µs 200 µg/mL pEGFP . Where 1.4 kV/cm 250 µs 20 µg/mL pEGFP vs. 1.4 kV/cm 250 µs 100 µg/mL pEGFP gave 6.81 ± 0.58% vs. 14.94 ± 1.68%, p < 0.001 and 1.4 kV/cm 250 µs 20 µg/mL pEGFP vs. 1.4 kV/cm 250 µs 200 µg/mL pEGFP .Here, we determined that the concentration of pEGFP plasmid is a key factor in increasing GET efficiency in UT-7 cells. The increase of plasmid concentration helped to elevate the amount of transfected/viable (pEGFP+/Viable) cells per sample: 1.2 kV/cm 250 µs 20 µg/mL pEGFP vs. 1.2 kV/cm 250 µs 50 µg/mL pEGFP  which were obtained when UT-7 cells were electroporated using 1.6 kV/cm 500 µs and 1.2 kV/cm 500 µs pulses in the presence of 20 µg/mL pEGFP plasmid, respectively. To our knowledge, only several studies have demonstrated gene electrotransfer using the UT-7 cell line ,29,30. U4 as a nuclease inhibitor on the electroporation process. Nucleases are responsible for plasmid degradation before entering the nucleus and subsequently lower cell proliferation rates [2+. However, we did not use other substances, i.e., EDTA or EGTA, as DNase inhibitors because they cause elevated cell death indices and lower transfection efficiency [2+ ions on electropermeabilization and the plasmid DNA–plasma membrane interaction, the ZnSO4 was instantly added into the growing medium after the electric pulses were delivered. However, we did not observe any effect of ZnSO4 on transfection efficiency and UT-7 cell viability. As we know, there is no report on UT-7 cell line transfection improvement with DNase inhibitors. Only Delgado-Canedo et al. (2006) reported a comprehensive analysis of electrotransfection conditions improvement in the suspension K562 cell line. In the study, better efficiency of electroporation was achieved by using DNase inhibitor ZnSO4 at a concentration of 80 µM, adding the reagent immediately after the pulses [Having defined the best conditions, we further tested the effect of ZnSOon rates ,37. Hereficiency . Thus, Ee pulses .p < 0.001). Several studies have reported that an increased plasmid concentration (from 50 to 300 µg/mL) enhances transfection efficiency; however, at the same time, cell viability was reduced significantly [Our data suggest that another critical factor for effective gene electrotransfer is the concentration of plasmid DNA added to the cell suspension before pulsing. We evaluated the impact of various amounts of pEGFP, i.e., 20, 50, 100, and 200 µg/mL, on gene electrotransfer efficiency. It was noticed that increased concentration of the plasmid DNA contributed to a drastically elevated amount of transfected/viable UT-7 cells (pEGFP+/Viable) per given sample. Using a 1.4 kV/cm 250 µs pulse with 200 µg/mL pEGFP resulted in the best combination of the cell transfection and viability ). Such a formula will allow the reader to predict in one step how many transfected/viable cells one may expect after the GET procedure following our protocol.It can be concluded that an increase in electric pulse duration, but not the intensity, elevates gene electrotransfection efficiency into suspension UT-7 cells. However, an increase in pulse intensity and duration substantially diminishes cell viability without a notable increase in transfected/viable cell number. On the other hand, an increase in plasmid concentration considerably enhances GET efficiency with a slight impact on cell viability.An increase in HV pulse duration results in more efficient gene electrotransfer into suspension UT-7 cells compared to pulse intensity;Additional tools or conditions should be taken into consideration for achieving higher pDNA electrotransfer into suspension UT-7 cells since increasing HV pulse intensity and duration results in higher cell killing rather than transfected/viable cell number;For instance, an increase in pDNA concentration results in a more prominent increase in transfected/viable cell subpopulation compared to pulse duration and intensity;However, DNAase inhibitor ZnSO4 had no impact on pDNA electrotransfer into suspension UT-7 cells nor affected cell viability.


Emerging evidence identifies enhancer RNAs (eRNAs) as a class of regulatory ncRNAs that can contribute to the transcription of target genes. In this study, we used an integrated data analysis method to identify the important role of eRNAs in ovarian cancer (OC).Gene expression profiles and clinical information from The Cancer Genome Atlas (TCGA) database were used for this study. Based on expression analysis using GEPIA2 gene and Kaplan–Meier survival was performed to ensure the significance of the selected enhancer RNA ADCY10P1 in OC. Next, we explored the correlation and clinical significance between ADCY10P1 and target gene NFYA. Furthermore, we evaluated the effects of overexpression of ADCY10P1 on the proliferation, migration, invasion and epithelial-mesenchymal transformation (EMT) of OC cell lines. We also investigated the biological function enrichment score of ADCY10P1 and verified it with OC cell lines. Finally, external validation was conducted, and the prognostic value of the ADCY10P1 in different tumors was demonstrated.We selected the eRNA ADCY10P1 associated with OC prognosis, with NFYA as its predicted target gene. Low ADCY10P1 expression was found to be associated with poor overall survival, high histological grade, and advanced stage of OC. Additionally, overexpression of ADCY10P1 inhibited the proliferation, migration, invasion and EMT phenotype of OC cell lines. Furthermore, ADCY10P1 was observed to inhibit glycolysis and fatty acid metabolism, thereby affecting OC progression. Meanwhile, OC tissue samples were externally validated. In addition, the pan-cancer analysis revealed that ADCY10P1 had prognostic value in other cancers.This study showed that ADCY10P1 plays a key role in OC progression and may facilitate prognosis prediction. Ovarian cancer (OC) is the third most common type of cancer in women and is the leading cause of mortality in gynecological malignancies. In 2020, in the United States alone, approximately 21,750 new cases of OC were diagnosed, and 13,940 women died from the disease [Emerging evidence demonstrates that human RNA transcripts are mainly non-coding RNAs (ncRNAs) consisting of small interfering RNAs (siRNAs), microRNAs, and long non-coding RNAs (lncRNAs) . Among tRecent studies have identified enhancer RNAs (eRNAs) as a class of regulatory ncRNAs that can increase the transcription of target genes . There hTherefore, we aimed to explore the potential clinical utility of eRNAs with respect to their functions, mechanisms, association with OC prognosis, and potential therapeutic targets. This study found that the eRNA ADCY10P1 is strongly associated with overall survival (OS) and clinical features among patients with OC. Additionally, overexpression of ADCY10P1 inhibited the proliferation, migration, invasion and EMT of OC cell lines. Furthermore, when expressed at a high level, ADCY10P1 was found to act as a protective lncRNA that can inhibit glycolysis and fatty acid metabolism, crucial for the aggressive proliferation of cancer cells.P = 0.035). Furthermore, ADCY10P1 expression was correlated with the predicted target gene NFYA expression . In addition, to further establish whether ADCY10P1 expression is involved in OC progression, our results showed it was negatively correlated with Federation of Gynecologists and Obstetrics (FIGO) stage  and normal ovary cell line (IOSE80) using qRT-PCR. The expression levels of ADCY10P1 were significantly lower in the OC cell lines compared with the IOSE80 cell line, while NFYA expression levels were higher in SKOV3 and OVCAR3 cell lines than in the IOSE80 cell line  and OC samples . In addition, we found that ADCY10P1 was more highly expressed in normal tissues than in tumour tissues, such as in cervical and endocervical cancers (CESC), kidney chromophobe (KICH), and lung adenocarcinoma  and a normal ovary cell line (IOSE80) were purchased from the Cell Bank of Chinese Academy of Sciences . All cell lines were authenticated using the short tandem repeat (STR) profiling test. Then, IOSE80, A2780, OVCAR3 cells were cultured in RPMI-1640 Medium  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 μg/ml streptomycin . SKOV3 cell was maintained in McCoy’s 5A Medium  supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.https://cancergenome.nih.gov/). The clinical data of patients with OC, including survival time, age, survival state, survival grade, and gene expression, were obtained from TCGA. Using these data, we explored the association between putative eRNAs and OS in patients with OC using the “survival” and “survminer” packages of the R software (v4.0.0: http://www.r-project.org) (P < 0.05). Co-expression analysis was performed between the putative eRNAs and their predicted targets. Furthermore, we identified the expression levels of the eRNAs and their predicted targets in OC and other types of cancers based on TCGA and Genotype-Tissue Expression (GTEx) data from the gene expression profiling interactive analysis (GEPIA) 2 database (http://gepia2.cancer-pku.cn), [The gene expression profiles for OC and 33 other types of cancers were downloaded from the TCGA database (pku.cn), . Subsequhttp://software.broadinstitute.org/gsea/index.jsp). The result was obtained using the “pheatmap”package in the R software.Gene set variation analysis (GSVA) was performed using the default parameters of the GSVA package in R to explore the relationship between the selected eRNA and the biological function enrichment score . Additio−∆ct or 2−∆∆ct method.Total RNA was extracted from tissue samples or cell lines using TRIzol™ reagent  to synthesise cDNAs using the PrimeScript RT reagent kit  according to the manufacturer’s instructions. Quantitative real-time PCR analysis was performed using the SYBR Green PCR kit . The primer sequences are shown as follows: ADCY10P1 , NFYA , ACLY , SCD1 , HK2 , LDHA  and GADPH . The relative expression of ADCY10P1 and NFYA were calculated by 2The pLV3-CMV-ADCY10P1 was used to construct the plasmid expressing ADCY10P1in mammalian cells. Then, the plasmid was transfected in A2780 and SKOV3 cells using Lipofectamine 3000  to overexpress ADCY10P1. Meanwhile, an empty plasmid was used as a negative control.3 cells/ well) or SKOV3 cells (3× 103 cells/well) were seeded into a 96-well plate with 100 µL of complete medium. After grown at 37 °C in a humidified incubator containing 5% CO2 for 0, 24, 48, 72, 96 and 120 h, 10 μL CCK-8 solution  was added, and the cells were incubated for 3 h in a dark cell incubator at 37 °C with 5% CO2. The absorbance was measured at 450 nm using a microplate reader. We used an EdU assay kit  to measured OC cell proliferation, following the manufacturer’s protocol. Finally, fluorescence microscopy  was used to visualized cell fluorescence.Cell growth was assessed using the Cell Counting Kit-8 (CCK8) and Ethynyl-2-deoxyuridine (EdU). A2780 cells  was measured using Image J software. For OC cell invasion assays, 200 μL cell suspension without FBS was seeded in transwell upper chambers coated with Matrigel , and medium with 20% FBS was added into the lower chambers. After incubation for 48 h, the invasive cells were fixed with methanol, stained with 0.1% crystal violet and imaged.Cells were lysed with radioimmunoprecipitation (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF)  and PhosSTOP™ . Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with TBST containing 5% skim milk and incubated overnight at 4 °C with the MMP9 (NO. 13667), HK2 (NO. 2867) and LDHA (NO. 3582) antibodies from Cell Signaling Technology , and N-cadherin (NO. AF4039), vimentin (NO. AF7013) and SCD1(NO. DF13253) antibodies from Affinity Biosciences , and E-cadherin (NO. 20874–1-AP) and ACLY (NO. 15421–2-AP) antibodies from Proteintech . Thereafter, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h, and the signals were visualized with an enhanced chemiluminescence ECL kit .t-test, Student’s t-test, or One-way ANOVA as appropriate. Additionally, the correlation strength was evaluated using Pearson’s correlation coefficient (r). Statistical significance was set at P < 0.05, and two-tailed P values were assumed.Statistical analyses were carried out using SPSS 22.0 . Differences between groups were assessed using Chi-square test, paired sample