Porphyrin derivatives accumulate selectively in cancer cells and are can be used as carriers of drugs. Until now, the substituents that bind to porphyrins (mainly at the meso-position) have been actively investigated, but the effect of the functional porphyrin positions  on tumor accumulation has not been investigated. Therefore, we investigated the correlation between the functional position of substituents and the accumulation of porphyrins in cancer cells using cancer cells. We found that the meso-derivative showed higher accumulation in cancer cells than the β-derivative, and porphyrins with less bulky substituent actively accumulate in cancer cells. When evaluating the intracellular distribution of porphyrin, we found that porphyrin was internalized by endocytosis and direct membrane permeation. As factors involved in these two permeation mechanisms, we evaluated the affinity between porphyrin-protein (endocytosis) and the permeability to the phospholipid bilayer membrane (direct membrane permeation). We found that the binding position of porphyrin affects the factors involved in the transmembrane permeation mechanisms and impacts the accumulation in cancer cells. Regarding biocompatibility, porphyrin is an appealing candidate. It is composed of four pyrrole subunits  and has 18π-electrons that form a plane. Besides, porphyrin is an active center of the four subunits of hemoglobin, which is the main constituent of human blood. Porphyrin can form conjugates with metals 5 which provide electrochemical and oxidation–reduction properties. These conjugates can be used as biosensors to determine the amounts of active oxygen in an organism7. We aim to discover highly versatile and biocompatible porphyrins to reduce the side effects of anticancer agents. Porphyrin can accumulate selectivity in cancer cells8. This accumulation is due to π–π stacking between the π-conjugated structure of porphyrin and the aromatic amino acid residues of blood proteins  and to the activated endocytosis of these proteins facilitated by receptors activated in cancer cells. Altogether, the accumulation of porphyrin in tumors is caused by a combination of the electronic properties of porphyrin and various receptor activities of cancer cells. Based on this, we aim to produce anti-cancer drugs that have low side effects by combining porphyrin with anticancer drugs16.In drug discovery, it is necessary to select a highly biocompatible compound because toxic drugs cannot be clinically used no matter their efficacy19. Porphyrin photosensitivity can generate anti-cancer activity. Porphyrin is excited by light irradiation, and the energy transferred to oxygen when porphyrin returns to the ground state produces reactive oxygen species that have an anti-cancer activity20. Photosensitizers with a porphyrin structure (ex. Photofrin) do not cause damage to normal cells because they are only active when irradiated with light and selectively target tumors thanks to the porphyrin properties. The advantage of tumor accumulation by drug delivery systems (DDS) is the reduction of side effects22. Anti-cancer drug therapy is an effective cancer treatment. However, side effects caused by toxicity in normal cells are problematic, and DDS that transport drug selectively to cancer cells are of particular importance. As described above, porphyrin can deliver drugs selectively to the affected area. Besides, anti-cancer drugs and porphyrin’s photosensitizing effect can act synergistically to enhance anti-cancer effects23. Porphyrins have been used as carriers with a wide variety of substituents. Most studies report the addition of substituents on the meso-position . These studies revealed that the charges of peripheral substituents influence the intracellular distribution, and biomolecule substituents improve the targeting of cancer cells. However, little is known about the influence of these functional positions on porphyrin accumulation in tumors. Here, we focused on β- and meso-positions to elucidate correlations between functional porphyrin positions and accumulation in cancer cells. Understanding this can help define steric effects and lead to the discovery of new molecules with high tumor accumulation.Until now, research on porphyrins as a cancer treatment has focused on their photosensitizing effect27. The affinity between the blood protein and porphyrin at the time of cell membrane permeation must also be considered. Building up from our preceding paper27, we evaluated the following three points.Based on the above, we aim to elucidate the correlation between the tumor accumulation of porphyrin and the functional position of substituent. We previously demonstrated that the functional position of the substituent (β- or meso-position) greatly affects tumor accumulation and thus must be considered when binding drugs to porphyrinsFirst, we monitored porphyrins accumulation in cancer cells at 2, 6, 16, and 24 h and evaluated the correlation between tumor accumulation and substituent position. Second, we examined the intracellular distribution of porphyrin using a confocal laser scanning microscope to evaluate the pathway through which porphyrin penetrates the cell membrane. Third, we assessed the permeability of porphyrin through the phospholipid bilayer using a model cell membrane “liposome”. By adding the above three points to the previous paper, we aimed to elucidate the mechanism of tumor accumulation change caused by functional position from more various angles. This article is based on a study published in the Bioorganic and Medicinal Chemistry Letters, “Correlations between functional porphyrin positions and accumulation in cancer cells.”1), β-P15 (2), meso-P1 (3), and meso-P15 (4). First, compound 6 was converted from Tetraphenylporphyrin (H2TPP) (5) using copper nitrate trihydrate and sulfuric acid28. Compound 6 was reduced with SnCl2·2H2O and hydrochloric acid to yield compound 729. Compound 7 was condensed with the corresponding carboxylic acid chlorides to provide β-P1 (1) and β-P15 (2)30. Compounds were purified by silica gel chromatography (CHCl3). Compound 8 was synthesized by nitration of compound 5 using sodium nitrate31 and treated with SnCl2·2H2O and hydrochloric acid to obtain compound 929. meso-P1 (3) and meso-P15 (4) were synthesized using the same conditions as for β-P1 (1) and β-P15 (2), respectively30. β-P1 (1), β-P15 (2), meso-P1 (3), and meso-P15 (4) were purified by silica gel chromatography (CHCl3) and their identities were confirmed by 1H-NMR, 13C-NMR, and ESI–MS.Scheme 1), β-P15 (2), meso-P1 (3), and meso-P15 (4) showed remarkably different accumulation behavior in human breast cancer cells (MCF-7). Figure As shown in Fig. All compounds reached a maximum accumulation value within six hours, which suggests that the time-dependent performance of porphyrin accumulation in cancer cells is independent of the functional position and substituent.32. Porphyrins may also accumulate in the cytoplasm by diffusing through the phospholipid bilayer membrane due to a concentration gradient17. Accordingly, we investigated how each cell membrane permeation mechanism (endocytosis or concentration gradient) occurs in this experimental system by evaluating the accumulation of porphyrin in lysosomes from fluorescence images.These results indicated that the accumulation of porphyrin in cancer cells depends on the functional position. We hypothesized that such changes in tumor accumulation might be influenced by factors involved in cell membrane penetration. To investigate where porphyrin accumulates in the cell membrane, we observed the subcellular localization of porphyrin using a time-lapse confocal laser scanning microscope. Porphyrin interacts with plasma proteins to form a complex which is then delivered to intracellular lysosomes by endocytosisFigure 8. Serum albumin is the most abundant protein in the blood (approximately 60%) and it selectively forms a complex with porphyrins by hydrophobic interaction or π–π stacking. Hence, the endocytosis of albumin is the primary pathway for tumor accumulation of porphyrins. Accordingly, we expected that the binding affinity of the porphyrin derivatives for albumin to affect their accumulation in cancer cells. To validate this, we determined binding constants between bovine serum albumin (BSA) and the porphyrin derivatives.Meso-derivatives accumulated more than β-derivatives Fig. . Besides33. With this method, binding constants can be calculated using Stern–Volmer plots and Lehrer plots.We used the quenching of the fluorescence of BSA to calculate the binding affinity of the porphyrin derivatives for BSA27. Based on the results of the Lehrer plot 27. These results suggest that π–π stacking between π-conjugated porphyrin derivatives and aromatic amino acid residues of BSA is one of the determining factors of the accumulation into cancer cells.Since we proved in our previous paper, it can be proved that porphyrins form a complex with the protein because Stern–Volmer plots show upward curvature in at higher values of quencher concentration Fig. a27. Baselot Fig. b, the bi35.The results of the fluorescence microscope imagery also revealed that the four porphyrin derivatives localize to the cytoplasm Fig. . TherefoCell membrane permeability was evaluated by quantifying the porphyrin contained in the DOPC liposomes.As shown in Fig. Hence, with these four porphyrins , we found that modifying the position and steric hindrance of the substituent affects their ability to cross the phospholipid bilayer. First, increasing the steric hindrance of substituents decreases their ability to cross the membrane. This behavior is consistent with the results of the accumulation in MCF-7 cells. Second, β-P15 is hardly incorporated into the phospholipid bilayer membrane. This extremely low permeability of β-P15 may be one of the factors of its extremely low accumulation in MCF-7 cells.We found that the meso-derivatives showed higher accumulation in cancer cells than the β-derivatives, and the accumulation depended on the steric hindrance of the substituent Fig. . FurtherNext, we attempted to elucidate the effect of functional position on membrane permeation mechanisms. Thus, the membrane permeation process of porphyrins was evaluated using fluorescence microscopy Fig. . From th36. We suppose that this distorted ring structure decreases the affinity between the porphyrin ring and the aromatic amino acid residues. Next, compounds with a short alkyl substituent showed higher Ka values than compounds with a long alkyl substituent, regardless of position . The steric effects of bulky substituents also inhibit interactions between porphyrins and aromatic amino acid residues.The binding constants between the porphyrin derivatives and BSA were determined by fluorescence quenching. As shown in Table 39. In particularly, regarding the comparison of the membrane permeability of meso-P15 and β-P15, we expected that the porphyrin aggregation in the aqueous solution affects the membrane permeability. It has been reported that association of porphyrins to liposomes was expected to be a two-steps process: firstly dissociation of the aggregates and secondly, binding or permeating of monomers to liposomes40. Therefore, we considered that the aggregation in the aqueous solution is greatly related to the membrane permeability. In addition, the long-alkyl chain substituent of porphyrin increases the hydrophobic interaction between the alkyl chains and increases the self-aggregation42. Furthermore, the β-position derivative has higher aggregation than the meso-position derivative43, and it can be inferred that β-P15, which has a long-alkyl chain and is a β-position derivative, has a structure that promotes aggregation. From the above, it is considered that β-P15 is a most likely to aggregate structure due to the influence of the functional position and the length of the alkyl chain, and it was resulting in a significant decrease in membrane permeability. On the other hand, in the future, the effects of other factors  should be elucidated in more detail.Then, we evaluated the ability of the porphyrin derivatives to diffuse across a phospholipid bilayer using DOPC liposomes as a model of the cell membrane. As shown in Fig. On the whole, the accumulation of porphyrins greatly depends on the functional position and steric hindrance of the substituent. This interesting phenomenon is caused by the competitive influence of the endocytosis and concentration gradient permeation.We investigated the effects of functional positions  on porphyrins accumulation in cancer cells. We found that the accumulation of the porphyrin derivatives significantly depended on the functional porphyrins position and the substituent’s steric hindrance. We think that such an interesting phenomenon is influenced by the two cell membrane permeation mechanisms of porphyrin, endocytosis, and concentration gradient permeation, and we made great progress in elucidating the mechanism of the effects of functional position substitution by evaluating transmembrane factors. Hence, we found that the functional porphyrin position is crucial when considering the use of porphyrin as a carrier for drug delivery. This study provides new guidelines for the structural design of porphyrin drug products.1H NMR) and carbon nuclear magnetic resonance spectra (13C NMR) were recorded using a JEOL JNM-AL300 spectrometer in the indicated solvent. Chemical shifts (δ) are reported in parts per million relative to the internal standard tetramethylsilane. Elemental analyses were recorded on a Perkin Elmer 2400 II CHNS/O analyzer. High-resolution mass spectra (HRMS) were recorded on a JEOL JMS-T100CS mass spectrometer. The Purification of all tested compounds was performed by HPLC using a Japan Analytical Industry LC-9210 with a JAIGEL 1H column  .Reagents and solvents were purchased from Wako pure Chemical Industries and Tokyo Chemical Industry. Column chromatography was performed using silica gel supplied by Fuji Silysia Chemical. Proton nuclear magnetic resonance spectra (3 (500 mL). The reaction mixture was stirred at r.t. for 16 h, cooled, and water (200 mL) was added to the mixture. The acetic acid was neutralized with aqueous sodium carbonate. The organic phase was washed with water (200 mL) twice. The organic phase was dried with anhydrous sodium sulfate. The solvent was removed under reduced pressure to give β-CuTPNP, which was used for the next reaction without further purification.Glacial acetic acid (16 mL), acetic anhydride (100 mL), and copper nitrate trihydrate  were added to a solution of 5,10,15,20-tetraphenylporphyrin  in CHCl2SO4 (6.8 mL) was added dropwise to a solution of β-CuTPNP in CH2Cl2 (350 mL). The mixture was stirred at r.t. for 30 min, water was added (200 mL) and extracted twice. To neutralize sulfuric acid, a saturated solution of sodium carbonate (200 mL) and water (200 mL) were added to the resulting mixture. The organic phase was extracted with CH2Cl2 and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give β-H2TPNP, which was used for the next reaction without further purification.Concentrated H2TPNP in concentrated hydrochloric acid (50 mL). The mixture was stirred at 65 °C for 5 h under Ar atmosphere, and cooled by adding it to cold water (200 mL). Then, the mixture was adjusted to pH 8 with a saturated solution of sodium carbonate. The organic phase was extracted with chloroform and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The resulting crude solid was purified by chromatography on silica gel (CHCl3 only) to give 2-amino-5,10,15,20-tetraphenylporphyrin .Tin(II) chloride dihydrate  was added to a solution of β-H1H-NMR : δ =  − 2.08 , 4.42 , 7.59–7.84 , 7.88–8.03 , 8.09–8.23 , 8.49–8.63 , 8.67–8.81  ppm.2TPAP  in CH2Cl2 (50 mL). After stirring at r.t. for 15 min, acetyl chloride  was added to the mixture and stirred at r.t. for 3 h. Water (200 mL) was added to the reaction mixture and the organic phase was extracted twice. Saturated sodium hydrogen carbonate (200 mL) and water (200 mL) was added to the resulting mixture. The organic phase was extracted with CHCl3, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The resulting crude solid was purified by chromatography on silica gel (CHCl3 only) to give 2-acetyl-5,10,15,20-tetraphenylporphyrin .Triethylamine (2.4 mL) was added to a solution of β-H1H-NMR : δ =  − 2.83 , 1.84 , 7.62–7.94 , 8.13–8.31 , 8.58–8.66 , 8.77–8.90 , 9.31  ppm. 13C-NMR : 24.0, 116.1, 119.9, 120.5, 121.1, 126.6, 126.7, 126.8, 127.7, 127.8, 128.3, 129.2, 133.3, 134.3, 134.4, 134.5, 140.8, 141.9, 142.1, 142.3, 167.5 ppm. Anal. Calcd for C46H33N5O: C, 82.24; H, 4.95; N, 10.42%. Found C, 81.15; H, 4.89; N, 9.97%. HRMS (ESI) calcd for C46H33N5O 694.2527 [M+], found 694.2583.2TPAP  in CH2Cl2 (30 mL). After stirring at r.t. for 15 min, palmitoyl chloride  was added to the mixture and stirred at r.t. for 3 h. Water (200 mL) was added to the reaction mixture and the organic phase was extracted twice. Saturated sodium hydrogen carbonate (200 mL) and water (200 mL) were added to the resulting mixture. The organic phase was extracted with CHCl3, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The resulting crude solid was purified by chromatography on silica gel (CHCl3 only) to give 2-palmitoyl-5,10,15,20-tetraphenylporphyrin .Triethylamine (0.83 mL) was added to a solution of β-H1H-NMR : δ =  − 2.83 , 0.88 , 1.19–1.34 , 1.48–1.72 , 2.38–2.27 , 7.98–7.95 , 8.15–8.26 , 8.57–8.62 , 8.76–8.56 , 9.36  ppm. 13C-NMR ; 14.1, 22.7, 23.9, 25.5, 29.4, 29.7, 31.9, 37.7, 115.9, 119.9, 120.4, 121.2, 126.6, 126.7, 126.8, 127.7, 127.8, 128.3, 129.2, 133.3, 134.3, 134.4, 134.5, 140.9, 141.9, 142.1, 142.3, 170.5 ppm. Anal. Calcd for: C, 83.01; H, 7.08; N, 8.07%. Found. C, 82.72; H, 6.81; N, 7.83%. HRMS (ESI) calcd for C60H61N5O 890.4773 [M+], found 890.4774.2  were added to a solution of trichloroacetic acid  in acetic acid (30 mL). The resulting solution was stirred at r.t. for 2 h under Ar atmosphere and water (200 mL) was added to the solution. The mixture was extracted with CHCl3. The organic phases were washed with a saturated aqueous NaHCO3 solution followed by water. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give meso-H2TPNP, which was used for the next reaction without further purification.5,10,15,20-Tetraphenylporphyrin  and NaNO2TPNP in concentrated hydrochloric acid (80 mL). After stirring at 65 °C for 2 h under Ar atmosphere, and the mixture was cooled by adding it to cold water (200 mL). Then, the mixture was adjusted to pH 8 with a saturated solution of sodium carbonate. The organic phase was extracted with chloroform and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The resulting crude solid was purified by chromatography on silica gel (CHCl3 only) to give 5-(4-Aminophenyl)-10,15,20-triphenylporphyrin .Tin(II) chloride dihydrate  was added to a solution of meso-H1H-NMR :δ =  − 2.76 , 4.02 , 7.05 , 7.76 , 8.00 , 8.22 , 8.76–8.87 , 8.95  ppm.2TPAP  in CH2Cl2 (50 mL). After stirring at r.t. for 15 min, acetyl chloride  was added to the mixture and stirred at r.t. for 3 h. Water (200 mL) was added to the reaction mixture and the organic phase was extracted twice. Saturated sodium hydrogen carbonate (200 mL) and water (200 mL) were added to the resulting mixture. The organic phase was extracted with CHCl3, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The resulting crude solid was purified by chromatography on silica gel (CHCl3 only) to give 5-(4-Acetophenyl)-10,15,20-triphenylporphyrin .Triethylamine (3.2 mL) was added to a solution of meso-H1H-NMR :δ =  − 2.85 , 2.22 , 7.60–7.80 , 8.02–8.19 , 8.71–8.83  ppm. 13C-NMR : 24.5, 117.9, 119.5, 120.1, 126.6, 127.7, 131.0, 134.5, 134.9, 137.7, 137.8, 142.1, 168.7 ppm. Anal. Calcd for: C, 82.24; H, 4.95; N, 10.42%. Found C, 81.69; H, 4.78; N, 9.88%. HRMS (ESI) calcd for C46H33N5O 694.2573 [M+], found 694.2583.2TPAP  in CH2Cl2 (40 mL). After stirring at r.t. for 15 min, palmitoyl chloride  was added to the mixture and stirred at r.t. for 3 h. Water (200 mL) was added to the reaction mixture and the organic phase was extracted twice. Saturated sodium hydrogen carbonate (200 mL) and water (200 mL) were added to the resulting mixture. The organic phase was extracted with CHCl3, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The resulting crude solid was purified by chromatography on silica gel (CHCl3 only) to give 5--10,15,20-triphenylporphyrin .Triethylamine (1.1 mL) was added to a solution of meso-H1H-NMR : δ =  − 2.75 , 0.87 , 1.69–183 , 2.32 , 1.19–1.45 , 7.57–7.79 , 8.06 , 8.13–8.25 , 8.78–8.90  ppm. 13C-NMR : 14.1, 22.7, 25.7, 29.4, 29.5, 29.7, 31.9, 37.9, 117.9, 119.6, 120.2, 126.7, 127.7, 131.2, 134.5, 135.0, 137.6, 138.0, 142.1, 172.1 ppm. Anal. Calcd for: C, 83.01; H, 7.08; N, 8.07%. Found C, 82.39; H, 7.02; N, 7.88%. HRMS (ESI) calcd for C60H61N5O 890.4752 [M+], found 890.4774.l-Glutamine Stock Solution , 1% (v/v) Antibiotic–Antimycotic Mixed Stock Solution (100x) (Stabilized)  and Dulbecco’s Modified Eagle’s medium  containing 10% (v/v) fetal bovine serum .Human breast cancer (MCF-7) cells, purchased from the Japanese Collection of Research Bioresources , were cultured in 1% (v/v) 100 mM-Sodium Pyruvate Solution (100×) , 1% (v/v) 200 mM-2). Subsequently, porphyrin compound solutions (1.0 mM in DMSO) were added (20 μL/well) and incubated for 2, 6, 16, and 24 h.MCF-7 cells were cultured in a 6-well plate at initial densities of 500,000 cells/well (2 mL/well) for 24 h  was added to the cells for lysis and the cell lysate was centrifuged . The fluorescence intensity of the supernatant of the cell lysate was recorded using a JASCO FP-8300 fluorescence spectrometer.The excitation wavelength was 420 nm. The accumulation concentration of porphyrin in MCF-7 cells was measured by using a calibration curve from the fluorescence intensity.The MCF-7 cells were seeded onto a glass-bottom dish at initial densities of 50,000 cells/well (2 mL/well) and incubated for 24 h. Lysotracker (Thermo Fisher) was then added to reach a final concentration of 50 nM. Cells were incubated for 2 h under growth conditions. Then the loading solution was replaced with fresh medium and the porphyrin compound was added to reach a final concentration of 10 μM and exposed for 24 h. Intracellular localization of porphyrin was evaluated by using a confocal laser scanning microscope FV-10i. Excitation wavelengths of porphyrins and Lysotracker were 440 nm and 645 nm, respectively.BSA was added to Tris–HCl buffer  containing NaCl (50 mM), at the initial concentration of 9.27 μM. The initial volume of the BSA solution was 2.10 mL. The following volumes of porphyrin stock solution (0.80 mM in DMSO) were added to the BSA solution: P1: 0, 0.005, 0.005, 0.005, 0.005, 0.005, 0.01, 0.01, 0.015, 0.02, 0.02, 0.05 mL, P15: 0, 0.005, 0.005, 0.005, 0.005, 0.005, 0.01, 0.01, 0.015, 0.02, 0.02, 0.05, 0.05 mL. The final volume of the porphyrin stock solution was 0.15 and 0.20 mL; final volume of solution in a cell was 2.25 and 2.30 mL. At each step, the fluorescence intensity of the BSA solution was measured (Excitation wavelength: 420 nm).F0 and F are BSA fluorescence intensities in the absence and presence of porphyrin, respectively; Ksv is the Stern–Volmer fluorescence quenching constant; [Por] is the concentration of porphyrin.The fluorescence intensity characteristic of the aromatic amino acid residues of BSA decreases by adding a porphyrin solution to the BSA solution. Stern–Volmer plots were generated using the Stern–Volmer Eq. :1\documeF0 and F are the BSA fluorescence intensity in the absence and presence of quencher (Por), respectively, [Por] is the concentration of quencher, Ka is the binding constant and α is the BSA fraction of the accessible to the quencher fluorophore population.The Stern–Volmer Eq.  was incoLipid films containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)  were prepared by the evaporation of chloroform solutions in glass tubes. The suspension was hydrated with 2 mL of PBS and vortexed for 5 min. The mixture was centrifuged for 5 min at 15,000 rpm, 25 °C, and the supernatant  was discarded. An equivalent volume of buffer solution was added, and the pellet was re-suspended. This procedure was repeated at least twice to prepare multi-lamellar vesicles (MLV).To liposome suspension (2 mL) was added to the porphyrin solution  and incubated for 1 h. Then the MLV suspension was centrifuged for 15 min at 15,000 rpm, and the supernatant was removed and 10 wt% SDS buffer  was added to the sediment to destroy the membrane. The fluorescence intensity of porphyrin in the solution was measured using a JASCO FP-8300 fluorescence spectrometer.


Clonorchis sinensis (C. sinensis), is an important food-borne parasitic disease that seriously afflicts more than 35 million people globally, resulting in a socioeconomic burden in endemic regions. C. sinensis adults long-term inhabit the microaerobic and limited-glucose environment of the bile ducts. Energy metabolism plays a key role in facilitating the adaptation of adult flukes to crowded habitat and hostile environment. To understand energy source for adult flukes, we compared the component and content of free amino acids between C. sinensis-infected and uninfected bile. The results showed that the concentrations of free amino acids, including aspartic acid, serine, glycine, alanine, histidine, asparagine, threonine, lysine, hydroxylysine, and urea, were significantly higher in C. sinensis-infected bile than those in uninfected bile. Furthermore, exogenous amino acids could be utilized by adult flukes via the gluconeogenesis pathway regardless of the absence or presence of exogenous glucose, and the rate-limiting enzymes, such as C. sinensis glucose-6-phosphatase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase, exhibited high expression levels by quantitative real-time PCR analysis. Interestingly, no matter whether exogenous glucose was present, inhibition of gluconeogenesis reduced the glucose and glycogen levels as well as the viability and survival time of adult flukes. These results suggest that gluconeogenesis might play a vital role in energy metabolism of C. sinensis and exogenous amino acids probably serve as an important energy source that benefits the continued survival of adult flukes in the host. Our study will be a cornerstone for illuminating the biological characteristics of C. sinensis and the host-parasite interactions.Clonorchiasis, caused by chronic infection with  Clonorchis sinensis, the causative agent of clonorchiasis, long-term inhabits the microaerobic and limited-glucose environment of the bile ducts. Adequate nutrients are essential for adult flukes to resist the adverse condition and survive in the crowed habitat. Studies on energy metabolism of adult flukes are beneficial for further exploring host-parasite interactions and developing novel anti-parasitic drugs. Our results suggest that gluconeogenesis probably plays a vital role in energy metabolism of Clonorchis sinensis and exogenous amino acids might be an essential energy source for adult flukes to successfully survive in the host. Our foundational study opens a new avenue for explaining energy metabolism of Clonorchis sinensis and provides a valuable strategy that the gluconeogenesis pathway will be a potential and novel target for the prevention and treatment of clonorchiasis.Clonorchiasis, closely related to cholangiocarcinoma and hepatocellular carcinoma, has led to a negative socioeconomic impact in global areas especially some Asian endemic regions. Owing to the emergence of drug resistance and hypersensitivity reactions after the massive and repeated use of praziquantel as well as the lack of effective vaccines, searching for new strategies that prevent and treat clonorchiasis has become an urgent matter.  Clonorchis sinensis (C. sinensis) is an important food-borne parasite endemic in Southeast Asian countries, including China, Korea, Japan, and Vietnam. Clonorchiasis, caused by chronic infection with C. sinensis, has seriously afflicted more than 35 million people worldwide, including approximately 15 million people in China . Th. ThCsG-6t method .At 0, 6, 12, and 24 h of feeding, the supernatant of DMEM was collected and centrifuged for 10 min at 12,000 g to remove debris. The processed supernatant samples were stored at -80°C before use. L-amino acid level was quantified with L-Amino Acid Quantitation Colorimetric/Fluorometric Kit  according to the manufacturer’s protocol. Briefly, L-amino acid standard was diluted to a series of gradients in a final volume of 50 μl with L-amino acid buffer in a 96-well microplate. Three μl processed samples were added into the microplate and adjusted to 50 μl with L-amino acid buffer. L-amino acid standard and samples were respectively incubated with 50 μl reaction mix for 30 min at 37°C, protect from light. The fluorescence was measured at Ex/Em = 535/590 nm in a microplate reader. L-amino acid standard curve was obtained based on the standard concentration and the standard reading. The blank reading was subtracted from the sample readings to calculate L-amino acid level of samples by using the standard curve. Every sample was performed in sextuplicate and the experiment was repeated for 3 times.CsFBPase was prepared and purified as described -2-furanyl] phosphonic acid, a specific inhibitor for human FBPase , was synescribed , and solescribed . BrieflyaMax M5) . The reaA total of 720 adult flukes were cultured as described above on 1 × Locke’s as well as high, low, and no glucose DMEM with or without the addition of MB05032 (20 μM). Viability and percent survival of adult flukes with or without MB05032 treatment were measured as described . The picCsFBPase activity in soluble protein of adult flukes with or without MB05032 treatment was measured using the same method as described above. Glucose and glycogen levels in soluble protein of adult flukes were detected with QuantiChrom™ Glucose Assay Kit and EnzyChrom™ Glycogen Assay Kit , respectively. For detection of glucose, glucose standard was diluted to a series of concentration gradients in a final volume of 150 μl with dH2O. Then, 50 μl standards and samples were respectively mixed with 500 μl reagent in appropriately labeled tubes. The tubes were heated in a boiling water bath for 8 min and cooled down in an ice water bath for 4 min. Subsequently, 200 μl processed standards and samples were respectively transferred into a 96-well microplate and read optical density at 620–650 nm. Standard curve was obtained based on the standard concentration and the standard reading. The blank reading was subtracted from the sample readings to calculate glucose level of samples by using the standard curve. For detection of glycogen, glycogen standard was diluted to a series of concentration gradients in a final volume of 200 μl with dH2O. Then, 10 μl standards and samples were respectively transferred into separate wells of a 96-well microplate, and incubated with 90 μl work reagent for 30 min at room temperature, protect from light. The fluorescence was measured at Ex/Em = 530/585 nm. Standard curve was obtained based on the standard concentration and the standard reading. The blank reading was subtracted from the sample readings to calculate glycogen level of samples by using the standard curve. Protein concentration was measured with BCA protein assay kit  and used for the normalization of glucose and glycogen levels as described [At 12 and 24 h of culture, native escribed . Every sP < 0.05 was considered as statistical significance, thereby indicating a probability of error lower than 5%.All data were analyzed using SPSS 13.0 software and the graphs were constructed using GraphPad Prism 5.0 software. Data were represented as mean ± standard deviation (SD) from 3 independent experiments. C. sinensis adults, we compared the component and content of free amino acids between C. sinensis-infected and uninfected bile. It was apparent that total concentration of free amino acids in C. sinensis-infected bile was 0.8 folds higher than that in uninfected bile. In C. sinensis-infected bile, the concentrations of aspartic acid, serine, glycine, alanine, histidine, asparagine , threonine , lysine , hydroxylysine (Hylys), and urea increased to approximately 2.2, 2.5, 1.9, 2.5, 4.4, 2.6, 2.2, 8.9, 4.4, and 2.1 folds, respectively. Additionally, carnosine could be detected in C. sinensis-infected bile, while it could not be detected in uninfected bile (To understand energy source for ted bile .P < 0.01), with median survival time increasing from 10.5 ± 0.5 to 19.1 ± 1.7 d and maximum survival time increasing from 17.8 ± 0.8 to 25.3 ± 1.5 d. Similarly, glycerol feeding significantly extended the survival time of adult flukes , with median survival time increasing from 10.5 ± 0.5 to 17.0 ± 1.5 d and maximum survival time increasing from 17.8 ± 0.8 to 24.2 ± 1.0 d. Gly + Ala feeding respectively extended median and maximum survival times by approximately 81.9% and 42.1%, whereas glycerol feeding respectively extended median and maximum survival times by approximately 61.9% and 36.0%. However, no significant difference was observed between Gly + Ala and glycerol feeding (P = 0.4161)  or 0.5% glycerol (glycerol) . Adult f 0.4161) .CsG-6-Pase, CsFBPase, CsPEPCK, and CsPC in regulating the gluconeogenesis reaction, we analyzed their expression levels by qRT-PCR. Compared with the control, Gly + Ala feeding significantly upregulated the mRNA levels of CsPEPCK and CsPC at 1, 3, 5, and 7 d, and upregulated the mRNA levels of CsG-6-Pase and CsFBPase at 3, 5, and 7 d. However, glycerol feeding downregulated the mRNA levels of CsPEPCK and CsPC at 1, 3, 5, and 7 d, and upregulated the mRNA levels of CsG-6-Pase and CsFBPase only at 3 d . Median and maximum survival times of adult flukes were 20.0 ± 1.0 and 25.8 ± 1.5 d in high glucose DMEM, 27.1 ± 1.5 and 32.2 ± 2.5 d in low glucose DMEM, and 24.9 ± 1.1 and 30.0 ± 2.0 d in no glucose DMEM, respectively, all of which were longer than those in 1 × Locke’s . Accordingly, median and maximum survival times were approximately prolonged by 66.7% and 44.1% in high glucose DMEM, 125.8% and 79.9% in low glucose DMEM, as well as 107.5% and 67.6% in no glucose DMEM, respectively. Moreover, adult flukes in high glucose DMEM survived shorter than those in low glucose DMEM survived (P < 0.05). However, no significant differences were observed between high and no glucose DMEM (P = 0.0974) as well as between low and no glucose DMEM (P = 0.6954)  .CsG-6-Pase, CsFBPase, CsPEPCK, and CsPC in 1 × Locke’s and different DMEM by qRT-PCR to further determine the occurrence of gluconeogenesis inside the body of adult flukes. At 1, 3, 5, and 7 d, mRNA levels of CsG-6-Pase, CsFBPase, CsPEPCK, and CsPC were essentially consistent. At 1 d, mRNA levels of CsG-6-Pase, CsFBPase, CsPEPCK, and CsPC in high, low, and no glucose DMEM were not significantly changed as compared with those in 1 × Locke’s. At 3 d, adult flukes expressed the highest mRNA levels of CsG-6-Pase, CsFBPase, CsPEPCK, and CsPC in 1 × Locke’s. At 5 and 7 d, adult flukes expressed the highest mRNA levels of CsG-6-Pase, CsFBPase, CsPEPCK, and CsPC in no glucose DMEM. However, adult flukes did not express higher mRNA levels of CsG-6-Pase, CsFBPase, CsPEPCK, and CsPC in high and low glucose DMEM than those expressed in 1 × Locke’s , with the relative activity reducing to less than 85% at 12 h and less than 60% at 24 h  (P < 0.05 or P < 0.01)  . In addi < 0.01) .At 5 d, untreated adult flukes were active with continuous muscle contraction and expansion as well as body wriggle and migration. In contrast to this, adult flukes with MB05032 treatment showed reduced activities with body stiffness, shrink, and curliness regardless of what kind of culture solutions they were maintained  Videos.P < 0.0001; low glucose DMEM versus low glucose DMEM + MB05032, P < 0.0001; no glucose DMEM versus no glucose DMEM + MB05032, P < 0.0001; 1 × Locke’s versus 1 × Locke’s + MB05032, P < 0.01). Under high glucose DMEM condition, MB05032 treatment remarkably decreased median survival time from 20.0 ± 0.6 to 7.5 ± 0.2 d and maximum survival time from 26.2 ± 2.0 to 14.1 ± 1.7 d. Under low glucose DMEM condition, MB05032 treatment similarly decreased median survival time from 25.9 ± 1.0 to 7.0 ± 0.1 d and maximum survival time from 33.9 ± 3.0 to 12.0 ± 0.7 d. Under no glucose DMEM condition, MB05032 treatment also decreased median survival time from 23.0 ± 0.5 to 5.9 ± 0.1 d and maximum survival time from 29.3 ± 1.5 to 10.7 ± 0.3 d. Likewise, under 1 × Locke’s condition, remarkable decreases of median survival time from 13.0 ± 0.3 to 3.1 ± 0.1 d and maximum survival time from 17.5 ± 1.5 to 7.0 ± 0.1 d were observed with MB05032 treatment. Hence, in these solutions, MB05032 treatment showed a 62.5%-76.2% decrease in median survival time and a 46.2%-64.6% decrease in maximum survival time  An uninfected bile sample. (C) The standard sample. Concentration of free amino acids in bile samples was calculated by using the standard curves generated by the standard sample.(A) A (PDF)Click here for additional data file.S2 FigCsFBPase by 0–50 mM AMP or 0–500 μM MB05032. (B) Percent inhibition of native CsFBPase in soluble protein of adult flukes by 0–50 mM AMP or 0–500 μM MB05032. (C) Inhibition of rCsFBPase and rCsFbA-2 by 10 μM MB05032. The 50% inhibiting concentration (IC50) of AMP (positive control) and MB05032 against rCsFBPase or native CsFBPase were calculated using four-parameter logistics nonlinear regression, respectively. The activities of rCsFBPase or rCsFbA-2 with or without the addition of MB05032 were compared by student’s t test. **P < 0.01 was represented as statistical significance; n.s., not significant.(A) Percent inhibition of r(PDF)Click here for additional data file.S1 Video(AVI)Click here for additional data file.S2 Video(AVI)Click here for additional data file.S3 Video(AVI)Click here for additional data file.S4 Video(AVI)Click here for additional data file.S5 Video(AVI)Click here for additional data file.S6 Video(AVI)Click here for additional data file.S7 Video(AVI)Click here for additional data file.S8 Video(AVI)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableP < 0.05 and P < 0.01 were represented as statistical significance.Survival time was evaluated by the log-rank test. (PDF)Click here for additional data file.S3 TableP < 0.05 and P < 0.01 were represented as statistical significance.Survival time was evaluated by the log-rank test. (PDF)Click here for additional data file.S4 TableP < 0.01 and P < 0.0001 were represented as statistical significance.Survival time was evaluated by the log-rank test. (PDF)Click here for additional data file.


The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X  was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a ‘single pot’ phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site . The library contained ~1.73 × 10The online version of this article (10.1007/s10719-020-09925-z) contains supplementary material, which is available to authorized users. H) and light chain (VL) of an immunoglobulin, joined together by a linker. In the Nissim library, the VH chain is variable, whereas the VL is fixed [H dp-38 germline gene segment [H chain, especially the complementarity determining region 3 (CDR3) [H chain [Heparan sulfate (HS) is a long heterogeneously sulfated glycosaminoglycan, predominantly present in the extracellular matrix and at the cell surface. During its biosynthesis, HS is modified by sulfation and epimerization reactions, resulting in a high structural and functional diversity , 2. Specis fixed , 16. Abo segment , 17–21. 3 (CDR3) . The CDR3 (CDR3) , 23, 24.[H chain , 17–21. [H chain , 25, 26.[H chain .H germline segment (dp-38) and a CDR3 consisting of an XBBXBX heparin binding consensus site. As a template we used a previously obtained scFv antibody (HS4C3) which harbors both a dp-38 germline gene segment and a CDR3 sequence containing an XBBXBX heparin binding consensus site (GRRLKD) [To expand the number of antibodies against HS, we here engineered and evaluated an antibody phage display library targeted for HS with a single V(GRRLKD) , 13.H3 dp-38 germline with an XBBXBX CDR3 amino acid sequence. Using the well-characterized HS4C3 scFv antibody from the Nissim library as template, the library was constructed in a series of 5 PCR procedures (PCR 1-PCR 5)  of the HS4C3 gene was amplified. In PCR 2, the VL (including the c-Myc tag), the linker sequence and the framework 4 of the VH of the HS4C3 gene were amplified. The PCR 3 procedure was performed to introduce a new CDR3 into the VH gene using an 87 nucleotide degenerate reverse primer. This primer contained the CDR3 encoded as 5’-SNN NYK SNN NYK NYK SNN -3′, where S encodes for G or C; Y for C or T; K for G or T; and N for A, C, G, or T, to obtain the XBBXBX sequence (Table H genes and the VL gene in PCR4 using overlapping sequences (FR4 of the VH). The product of PCR 4 was evaluated on 1% agarose gel and isolated using the QiaEX II agarose gel extraction kit . The purified product was amplified in PCR 5. The final product was purified after 1% agarose gel electrophoresis using phenol/chloroform extraction. DNA was precipitated using ethanol, the pellet was washed with 70% ethanol and dissolved in 50 μL water. The final product was digested using NcoI and NotI restriction enzymes  and ligated overnight at 16 °C into a NcoI and NotI digested pHEN1 vector  using the Sureclone Ligation Kit . The ligation product was purified using phenol/chloroform extraction. The ligation product was transformed via electroporation into E. coli TG1  electroporation competent cells, efficiency ≥1.0*1010 cfu/μg . To calculate the titer of transformants, dilutions of the original electroporated bacteria were plated onto 2xTY agar plates containing 0.1% (w/v) ampicillin and 1% (w/v) glucose and incubated overnight at 37 °C.The phage display library was constructed to contain only scFv antibodies harboring the Vures PCR -PCR 5  ampicillin and 1% (w/v) glucose and incubated overnight at 37 °C. Subsequently, 240 clones were randomly picked from the plate and cultured overnight in LB medium containing 1% (w/v) glucose and 0.1% (w/v) ampicillin. Plasmid DNA was collected using the QIAprep spin Miniprep Kit  and the Vw/v) (NH4)2SO4 [w/v) ampicillin.Phage display biopanning procedures was performed as described previously . Tubes wNH4)2SO4 . Phages w/v) (NH4)2SO4 containing 10 μg/mL HS from bovine kidney (Seikagaku), HS from porcine intestinal mucosa, heparin from porcine intestinal mucosa, dermatan sulfate from pig skin, chondroitin sulfate A (CSA) from bovine trachea, chondroitin sulfate C (CSC) from shark cartilage, thymus DNA, and hyaluronic acid from rooster comb . After washing, plates were blocked with 3% (w/v) bovine serum albumin (BSA) in 0.01 M phosphate buffered saline pH 7.2 (PBS) with 1% (v/v) Tween 20 for 60 min (200 μL/well). After induction of picked clones in 200 μL 2xTY with 0.1% (w/v) glucose and 0.1% (w/v) ampicillin in a 96-wells polystyrene plate, plates were centrifuged and 50 μL of the supernatant was mixed with PBS with 0.1% (v/v) Tween 20 (PBST) containing 2% (w/v) BSA in each well and incubated for 2 h. Bound antibodies were detected using a mouse monoclonal antibody against the c-Myc tag (9E10), followed by a goat-anti-mouse IgG conjugated with alkaline phosphatase  in 1% (w/v) BSA/PBST. Enzyme activity was detected by addition of 100 μL of 1 mg/mL p-4-nitrophenylphosphate in 1 mM diethanolamine, with 0.5 mM MgCl2, pH 9.8. Absorbance was measured at 405 nm. Positive clones were inoculated in 15 mL LB-medium (0.1% (w/v) ampicillin and 1% (w/v) glucose) and cultured overnight at 37 °C. Plasmid DNA was collected using the QIAprep spin Miniprep Kit  and the CDR3 was sequenced using the PelB, forlinkseq and VL3 primers  glucose). Subsequently, 10 mL of the overnight culture was added to 1 L induction medium (2xTY medium containing 0.1% (w/v) glucose and 0.1% (w/v) ampicillin). At an OD600 of 0.6, the antibody expression was induced by addition of isopropyl-β-D-thiogalactopyranoside  in a final concentration of 1 mM, and incubated for three h at 30 °C. The cultures were centrifugated for 15 min at 3561 g to separate bacteria from culture medium. Bacterial pellet was resuspended and incubated for 15 min on ice in 10 mL 0.2 mM sodium borate buffer (pH 8.0) containing 0.16 M NaCl to accommodate osmotic shock. This buffer was enriched with protease inhibitors , supplemented with 1% (w/v) NaN3. After centrifugation for 15 min at 10,000 g at 4 °C, supernatant was passed over a PBS wetted 0.45 μm filter and subsequently dialyzed against PBS, using a Visking 12-14 kDa MWCO dialysis hose .For further characterization of the antibodies, scFv were obtained from the periplasmic fraction as described . BacteriELISA. Epitope specificity of the scFv antibodies was further analyzed using an ELISA setting. The wells of a 96-wells flat bottom polystyrene microplate  were coated by incubating with 100 μL of 90% (w/v) (NH4)2SO4 containing 10 μg/mL heparin, N-desulfated/N-acetylated heparin, 2-O desulfated heparin or 6-O desulfated heparin  Tween 20 in PBS. Periplasmic fractions containing the scFv antibodies were diluted 1:1 in 1% (w/v) BSA/PBST and incubated for 1.5 h. Bound scFv antibodies were detected as described above, using mouse anti-c-Myc antibody 9E10 and alkaline phosphatase conjugated goat anti-mouse antibodies, and visualized using alkaline phosphatase substrate disodium p-4-nitrophenylphosphate.n, Italy , 31). CoImmunohistochemistry. Rat kidney specimens  were snap-frozen in liquid isopentane and stored at −80 °C. Cryo sections (5 μm) were rehydrated and blocked in 1% (w/v) BSA/PBST for 60 min and incubated with selected scFv antibodies in 1% (w/v) BSA/PBST for 90 min, with antibody HS4C3 as a positive control. ScFvs were detected by a 45 min incubation with mouse monoclonal anti c-Myc antibody 9E10 (1:10) in 1% (w/v) BSA/PBST, followed by a 45 min incubation with goat anti-mouse IgG, Alexa 448 conjugated (Molecular Probes Inc.) (1:300) in 1% (w/v) BSA/PBST. After each incubation, sections were washed in PBST (3 × 5 min). Sections were fixated in 100% ethanol, air-dried and embedded in 10% (w/v) Mowiol in 0.1 M Tris-HCl, pH 8.5, 25% (v/v) glycerol and 2.5% (w/v) NaN3. Sections were examined using a Leica DM 6000 fluorescence microscope and images were obtained using the Leica Microsystems software.8 unique clones, encoding for 1.73 × 106 unique full-length proteins. To obtain an impression of the actual diversity of the library, 222 randomly picked clones were sequenced, yielding 194 full sequences of which 189 (97.4%) were unique (Table 8 clones encoding for 1.68*106 (97% of 1.73 × 106) unique full-length proteins, which is 97% of the theoretical diversity. Screening of CDR3 sequences demonstrated that 23.3% (44/194) of the randomly selected clones contained a XBBXBX amino acid sequence, similar to what was theoretically estimated (24.4%) (Table p < 0.01)  at the ‘B’ site, the other possibilities being Q, S or N. The latter amino acids are also frequently involved in the binding of heparin/HS , 32. TheAfter biopanning against immobilized HS, 25 unique antibodies were selected against bovine kidney (10 min elution), and 30 after an additional 20 min elution . MP4A11’10.b.k. was able to detect both 6-O desulfated and 2-O desulfated heparin, but did not bind to the N-acetylated form of heparin, indicating N- sulfation to be essential for binding.Reactivity of the anti-HS antibodies was further studied using modified heparin preparations  heparin, and stained Bowman’s capsule, but not the glomerulus. Antibody MP4F11 reacted with all desulfated heparin preparations and stained the glomerulus as well as Bowman’s capsule. This suggests that e.g. antibody MP3B2’20.b.k. recognizes a highly sulfated HS domain, whereas antibody MPF11 recognizes a low sulfated region. Clearly, more in depth studies are needed to specify the exact nature of the HS structure(s) recognized by the antibodies. New developments in HS microarrays and cell-based HS libraries offer potential to do such analyses [From the library, 67 new scFv antibodies against HS were selected, indicating the versatility of the library. The biosynthesis of HS allows for a large number of motifs and patterns within the molecule. The initial HS backbone consists of repeating disaccharide units composed of a glucuronic acid and an N-acetyl glucosamine. The acetyl group of the N-acetyl glucosamine can be exchanged for a sulfate group, initiating further modifications including epimerization of a glucuronic acid into an iduronic acid, the addition of a sulfate group to the C2 hydroxyl group of the uronic acid, and the addition of sulfates to the C6 and C3 hydroxyl groups of the glucosamine. Since these modifications occur in clustered regions, HS consists of low and highly sulfated domains. As a result, HS has a large structural variability and many different sulfation patterns are possible , 2, 41. analyses –44.H and/or VL may result in the generation of anti-HS scFvs recognizing specific sulfation patterns. These tools may be instrumental in elucidating of the function of HS sulfation patterns in (patho)physiological processes.In this study we aimed to obtain new anti-HS scFv directed towards different HS-epitopes by engineering the CDR3. Since this approach was successful, applying specific modifications to the anti-HS scFvs may prove to be effective to obtain antibodies against specific sulfation patterns. These modifications may not be limited towards the CDR3, since regions located outside the linear heparin binding site have been demonstrated to influence HS binding –47. KnowIn conclusion, we successfully constructed a new anti-HS scFv library, engineering the CDR3 to contain the heparin-binding consensus site XBBXBX. The library may be a rich source of new antibodies directed against different HS-epitope, and may allow the design of novel antibodies recognizing specific sulfation patterns.ESM 1(PDF 238 kb)ESM 2(DOCX 12.2 kb)


Gallic acid (GA), a plant-derived ubiquitous secondary polyphenol metabolite, can be a useful dietary supplement. This in vitro study’s primary purpose was to assess the anti-aging properties of GA using rat embryonic fibroblast (REF) cells, antidiabetic effects via pancreatic islet cells, and finally, elucidating the molecular mechanisms of this natural compound. REF and islet cells were isolated from fetuses and pancreas of rats, respectively. Then, several senescence-associated molecular and biochemical parameters, along with antidiabetic markers, were investigated. GA caused a significant decrease in the β-galactosidase activity and reduced inflammatory cytokines and oxidative stress markers in REF cells. GA reduced the G0/G1 phase in senescent REF cells that led cells to G2/M. Besides, GA improved the function of the β cells. Flow cytometry and spectrophotometric analysis showed that it reduces apoptosis via inhibiting caspase-9 activity. Taken together, based on the present findings, this polyphenol metabolite at low doses regulates different pathways of senescence and diabetes through its antioxidative stress potential and modulation of mitochondrial complexes activities. The generation of such free radicals induces oxidative stress that leads to DNA, lipid, and protein damages [Reactive oxygen species (ROS) are oxygen-containing chemically reactive species, such as superoxide, peroxide, and hydroxyl radicals. The dismutation of superoxide generates hydrogen peroxide , or 3,4,5-trihydroxy benzoic acid, is a plant-derived polyphenolic compound. It has demonstrated antidiabetic function in an animal model . GA is a2O2-induced aging using the rat’s embryonic fibroblast (REF) cells. The level of oxidative stress biomarkers was measured to determine the protective effect of GA in the isolated pancreatic islet cells. Furthermore, the optimum dose of GA is determined, i.e., required for the survival and function of pancreatic islet cells in an ex vivo model. StatsDirect 3.3.4 was used to calculate the statistics.The purpose of this in vitro study is to evaluate the attenuating property of GA in Hp < 0.001 -2,5-diphenyltetrazolium bromide (MTT) assay showed that the concentration of GA resulted in a 150% viability of cells compared to the control group . All con < 0.001 A. In conectively B. EC50 v2O2 represented a significant increase in β-galactosidase concentration compared to the control group (p < 0.001). In contrast, GA and H2O2 + GA treatment of REF cells showed a significant reduction in the β-galactosidase concentration compared to the H2O2 group  β-galactosidase concentration: REF cells treated with H2O2 alone and in combinations. The status of REF cells in three phases of the cell cycle, including G0/G1, S, and G2/M, was interpreted (2O2 group (p < 0.01). Interestingly, in comparison to the H2O2 group, a significant enhancement in G2/M arrest was observed in GA  and H2O2 + GA  groups.(b) Cell cycle analysis: The cell cycle distribution of REF cells was determined after treatment of GA and Herpreted . The pop2O2, the mitochondrial complexes I, II, and IV activities were reduced (p < 0.001). On the other hand, treating REF cells with GA showed a significant enhancement in mitochondrial complexes activity compared to the H2O2 group (p < 0.001). Furthermore, exposure to H2O2 + GA significantly enhanced the level of mitochondrial complexes I, II, and IV .(c) Mitochondrial activity: As shown in p < 0.001). GA treatment showed a considerable increment in both Ferric reducing antioxidant power (FRAP) and thiol levels. A significant rise in FRAP levels in GA vs. control groups, i.e., 106.83 vs. 56.60 mM (p < 0.001) was observed. Likewise, a considerable increase in thiol levels in GA vs. control groups, i.e., 5 vs. 3 µM (p < 0.001) was observed  levels was observed after GA treatment compared to the control group (observed . There wectively .2O2 (p < 0.001). While in comparison to H2O2, there was a significant decrease in the level of TNFα of GA (p < 0.001) and H2O2 + GA (p < 0.001) groups. Similarly, a higher amount of interleukin (IL)-1β and IL-6 were observed when REF cells were exposed to H2O2 (p < 0.001)—while in comparison to H2O2, a considerable decrease in the level of IL-1β of GA (p < 0.01) and H2O2 + GA (p < 0.01) groups was observed. In the case of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), there was a significant increase in the H2O2 (p < 0.001) compared to control, while in comparison to H2O2, a considerable decrease in the level of NFκB of GA (p < 0.001) and H2O2 + GA (p < 0.01) groups was observed (A higher tumor necrosis factor α (TNFα) was observed when REF cells were exposed to Hobserved .p < 0.001). In contrast, a significant decrease in insulin secretion was observed in the stimulated phase when the cells were treated with GA. The decrement in the control vs. GA group was from 22.10 ± 0.36 to 18.10 ± 0.80 mU/mg protein/h (p < 0.01)  and stimulated (16.7 mM) phases. A significant increase in insulin secretion was observed in the basal phase when the cells were treated with GA. The increment in control vs. GA group was from 2.2 ± 0.11 to 7.3 ± 0.11 mU/mg protein/h ( < 0.01) .p < 0.01) group was observed. Similarly, a significant decrease in the number of necrotic cells in the GA vs. control (p < 0.01) group was observed ((a) Acridine orange (AO) and ethidium bromide (EB) double staining: The absorption of AO and EB shows the number of viable cells and necrotic cells, respectively. A significant decrease in the number of viable cells in the controls (49.50%) vs. GA (64.82%) . In comparison, the number of necrotic cells was significantly decreased in GA (3.73%) vs. the control group (20.5%) (p < 0.01) ((b) Flow cytometry assay: The flow cytometry assay has shown a significant increase in the percentage of live cells in the GA (78.2%) vs. the control (71.1%) group ( < 0.01) B. Figurep < 0.05) and 46.4% (p < 0.001), respectively. A more noticeable decline in the activity was observed with the caspase-9 ((c) Caspase-3 and 9 activities: In comparison to control groups, a decrease in the caspase-3 and 9 activities was observed in the GA groups. The activity of caspase-3 and 9 was reduced to 85% . Such a decrease in the β-galactosidase activity supports the protective anti-aging potential of GA. A few studies have shown a beneficial role of GA in limiting the activity of redox-sensitive transcription factors.Cellular senescence is a major cause of aging. It is characterized by numerous biochemical changes, such as the increased activity of β-galactosidase . It is wnt cells . Similar2O2-mediated cellular senescence on the P53 transcription factor, P21, P16, Rb protein [2O2-mediated cell cycle arrest G1 phase of REF cells. Furthermore, in comparison to H2O2, GA treatment confirmed cell cycle arrest in the G2/M phase (p < 0.01). In line with the obtained results, Ou et al. showed that GA causes cell cycle arrest at the G2/M phase in the human bladder transitional carcinoma cell line through checkpoint kinase 2 (Chk2)-mediated phosphorylation of Cdc25C [Controlling cellular senescence in the G1 phase of the cell cycle is the main target for regulating the aging process ,28. Stud protein . Further protein . Similarf Cdc25C .Generally, superoxides are deactivated by superoxide dismutase and glutathione peroxidase. The deficiency of mitochondrial complex I resulted in increased superoxide production followed by subsequent induction of superoxide dismutase . A studyp < 0.001) and inflammatory cytokines, including TNFα (p < 0.001), IL-1β (p < 0.01), and IL-6 (p < 0.001), compared to the H2O2 group. The present results are similar to the previous ones suggesting the protective role of GA against inflammation via suppressing mast and cancer cells [Inflammation acts as a defensive response by cells after exposure to a toxic agent. The cell inflammatory response involves the secretion of various inflammatory cytokines ,35. Traner cells ,39.2O2 [2O2 group (p < 0.001). Also, GA significantly improved antioxidant capacity by enhancing FRAP and TTM levels in both REF and pancreatic islets cells after GA treatment.It was demonstrated that phenolic compounds significantly decline oxidative stress generated by neutrophil-mediated ROS . Antioxi2O2 . Like otCaspase-3 and 9 are the group of enzymes regulating inflammation and apoptosis. As a cancer-selective agent, GA in apoptosis of normal and cancer cells can act dually . In thisStudies on the effects of GA in diabetes show that this compound inhibits diet-induced hypertriglyceridemia and hyperglycemia and protects pancreatic β-cells. GA induces a nuclear transcription factor, which causes differentiation and insulin sensitivity in adipocytes, called peroxisome proliferator-activated receptor-γ (PPAR-γ). GA also enhances the cellular glucose uptake by stimulating the phosphatidylinositol 3-kinase (PI3K)/p-Akt signaling pathway and translocating GLUT1, GLUT2, and GLUT4 as insulin-stimulated glucose transporters . FurtherThis study has demonstrated the protective role of GA on the REF and rat pancreatic islets. Therefore, we found that GA at all studied concentrations attenuates oxidative stress and is safe. Moreover, GA reduces apoptosis, suppresses caspases activity, and improves insulin secretion, thus demonstrating antioxidant, antidiabetic, and antiapoptotic potential. Hence, GA is the right candidate for anti-aging in skincare products or a dietary supplement, warranting future human studies.® fluorescein isothiocyanate (FITC) kit  were used in this study.All chemicals were purchased from Sigma-Aldrich  unless otherwise mentioned. Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), interleukins (IL-6 and IL-1β), ELISA kits , Senescence β-galactosidase staining kit , rat-specific β-galactosidase kit , rat-specific enzyme-linked immunosorbent insulin ELISA kit , and ApoFlowExEthical approval for the animal study: This study is performed according to the NIMAD Ethics Committee Approval (IR.NIMAD.REC.1397.028). All ethical protocols related to the use of animals in research were carefully noted.Isolation of rats’ embryonic fibroblast (REF) cells: Rats were kept for induction of pregnancy. The isolation and culture of primary REF cells were done as per the standard protocol explained previously . Pregnan2.2H2O, 2.38 g/L HEPES, 0.27 g/L KCl, 0.05 g/L MgCl2, 8 g/L NaCl, 0.42 g/L NaH2PO4, and 0.5 g/L glucose.1H2O. The pancreas was cut into small pieces over the ice, washed twice, and centrifuged at 3000× g for 60 s. They were exposed to collagenase enzyme solution (0.0025 g/5 mL Krebs buffer) at 37 °C to digest the attached tissues for about 10 min. Krebs buffer containing 0.5% bovine serum albumin (BSA) was then added to stop the digestion, and the pancreatic pieces were again centrifuged twice at 3000× g for 60 s. The pancreatic islet cells of approximately 100 to 150 µm were isolated using a sampler under a stereomicroscope. Standard RPMI-1640 culture medium comprising 5% FBS, 0.5% penicillin-streptomycin, and 8.3 mmol/L glucose was added to the isolated cells. The pancreatic islets at a density of 10 islets/200 µL culture medium were kept for 24 h at 37 °C under 5% CO2 for further use [Isolation of pancreatic islet cells: Pancreas was removed and washed using the Krebs buffer to remove the attached lymph nodes, blood vessels, and fat. Krebs buffer solution at pH 7 consists of 0.22 g/L CaClther use . For nor4 REF cells/well in each group) and 10, 100, and 1000 μM (in case of 10 islet cells in each group). Cultured cells were treated with 20 μL MTT reagent, i.e., 0.5 mg/mL and 50 μL culture medium. After incubation at 37 °C and 5% CO2 humidified atmosphere for 4 h, the formazan dye was made soluble by adding 150 μL dimethyl sulfoxide (DMSO) to each sample. Finally, the absorbance was determined at 570 nm using a microplate reader. The probit regression model was employed to estimate the EC50 of GA [MTT assay: The viability of REF and pancreatic islet cells were determined after different concentrations of GA treatment, i.e., 0, 1, 10, 100, and 1000 μM  1 × 104 REF cells in DMEM-HG (Control), (b) REF cells exposed to 600 μM H2O2 for 2 h (H2O2), (c) REF cells in EC50 of GA (GA), and (d) REF cells with EC50 of GA + 600 μM H2O2 (GA + H2O2). A concentration of 600 µM H2O2 was observed as a potent inducer of cellular senescence [50 of GA on pancreatic islet cells. All cells were incubated at 37 °C and 5% CO2 humidified atmosphere.Study design: After determining the ECnescence . Simulta5 cells/well for samples  and different concentrations of βGAL  as standards were incubated at 37 °C for 2 h. The supernatant was removed, and 100 µL of biotin antibody was added to each well and set at 37 °C for another hour. After washing the cells by filling each well with 200 µL of wash buffer for a total of three washes, 100 µL of Avidin horseradish peroxidase (HRP) was added to each well, and plates were incubated at 37 °C for another hour. After aspiration, 90 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) was added to the plates and set again for 30 min. The 50 µL of stop solution was then added, and the optical density was determined at 450 nm. Along with the quantitative assay, β-galactosidase was also investigated qualitatively. REF cells were washed, and then they were exposed to the fixative solution containing 2% glutaraldehyde and 20% formaldehyde in 10× PBS for about 15 min at room temperature. Next, cells were washed with PBS and then stained with b-galactosidase stain. The bluish-green color of senescent cells was observed under the light microscope  at 200× magnification [Senescence-associated β-galactosidase (SABG) concentration: The β-galactosidase concentration in REF cells was determined using a rat-specific SA-βGAL kit. The well plates containing the REF cells at the density of 1 × 10fication .5 cells/well. The cells were exposed to 600 µM H2O2 and EC50 of GA alone or in combination for 24 h. After the incubation period, the REF cells were appropriately harvested, washed twice with PBS, and fixed in ice-cold 70% ethanol. After centrifuging at 10,000× g for 5 min, pellets were washed with ice-cold PBS and were redistributed in 200 µL (from 50 µg/mL stock solution) propidium iodide (PI) containing 20 µg/mL RNAse-A and incubated at 37 °C for 30 min. The cells were then washed with PBS, and cell cycle distribution at G0/G1, S, and G2/M phases was determined by a flow cytometer . The data were processed using FlowJo analysis software [Cell cycle analysis by propidium iodide staining: REF cells were seeded in 6 well plates at the density of 5 × 10software .4 cells/well for each group was used for assessing mitochondrial complexes. (a) Mitochondrial complex I (NADH dehydrogenase): Activity of mitochondrial complex I was determined by measuring NADH’s consumption, which is determined by the translocation of electrons initially to complex I and subsequently to an electron acceptor, such as synthetic ubiquinone. Electrons through NADH passed to mitochondrial complex I and were then accepted by synthetic ubiquinone. In this method, 100 to 200 μg/mL of total mitochondrial protein were added to a reaction mixture containing potassium phosphate buffer , 25% BSA, MgCl2 (5 mM), decylubiquinone (2.8 mM), NADH (5.7 mM), antimycin-A (3.7 mM), and KCN (2 mM). The absorbance of NADH was performed spectrophotometrically at 340 nm. Soon after, the rotenone was added to the reaction mixture, and the rotenone-insensitive activity of NADH-cytochrome b oxidoreductase was evaluated. Finally, the rotenone-insensitive activity was subtracted from the total activity, and the overall net rate was determined. The obtained results of enzyme activity were expressed as nanomoles of NADH per minute per milligram of mitochondrial protein (nmol/min/mg protein) at 340 nm [2, and succinate. Then, antimycin A, rotenone, KCN, and DCPIP were added, and baseline was recorded for 3 min. The enzyme-dependent reduction of DCPIP was then measured for 3 to 5 min at 600 nm. The activity of mitochondrial complex II was calculated using a DCPIP standard curve. (c) Mitochondrial complex IV (cytochrome C oxidase): The cytochrome C was reduced by adding 60 µL of sodium hydrosulfite (10 mg/mL). Then, the mitochondrial protein and Lubrol-PX in potassium phosphate buffer were added to further reduce the cytochrome C. The decrease in optical absorbance at 550 nm was recorded for 3 to 6 min. The results were presented as the natural logarithm of the absorbance and reported as the first-order rate constant, i.e., (k) min/mg of mitochondrial protein [Activity assay of mitochondrial complexes: Density of 1 × 10t 340 nm ,48. (b)  protein .4 REF cells or 10 islet cells for the pancreatic section, was homogenized with 100 µL phosphate buffer and was then centrifuged at 2375× g for 15 min. Next, the supernatant of the extractions was used for the following oxidative stress markers.Each group of cells, containing 1 × 10g, the 600 µL supernatant was removed, and cell deposition was combined with 150 µL TBA (1% w/v). The mixture was incubated in a boiling water bath for 15 min, followed by 400 µL n-butanol. Finally, the absorbance was recorded at 532 nm [Lipid peroxidation measurement: The thiobarbituric acid (TBA) was used to determine lipid peroxidation in REF cells. Following homogenization, the REF cells were mixed with 800 µL trichloroacetic acid. After 30 min centrifuging at 3000× ROS assay: This involves converting a fluorescent dye, i.e., 2,7-dichlorofluorescein diacetate (DCFDA), to a non-fluorescent compound. Cellular esterase deacetylates, DCFDA, and ROS then oxidized the deacetylated compound into 2,7-dichlorofluorescein (DCF). Finally, the changes in the fluorescence intensity were detected by a microplate reader with excitation and emission spectra at 488 and 525 nm, respectively .3+TPTZ -s-triazine) complex to the ferrous form was determined at 593 nm [Ferric reducing antioxidant power (FRAP) assay: This is based on assessing antioxidant potential. The reduction of Fet 593 nm .g for 10 min. The supernatant’s absorbance was measured at 412 nm, and results were presented in µmol/mg protein [Assay of total thiol molecules (TTM): This involves the addition of 0.6 mL Tris-EDTA buffer  to 0.2 mL supernatant of the cell extraction, and 40 μL 5-5′-dithiobis-2-nitrobenzoic acid (10 mM in pure methanol). The final volume of 4 mL was made using pure methanol. This mixture was incubated at room temperature for 15 min and then centrifuged at 3000× 4 cells/well for each group) was performed using rat-specific inflammatory cytokine ELISA kits. In brief, the specific antibody was coated to the wells. Then, standards and all samples were added to the wells. After washing the wells, the enzyme streptavidin-HRP, which binds to the biotinylated antibody, was added. After the washing step and incubation time, the TMB substrate solution was added. Finally, the stop solution was added to change the color from blue to yellow. The absorbance of yellow color was detected at 450 nm.Measurement of Inflammatory Cytokines  and Transcription Factor (NFκB): Quantitative measurement of inflammatory cytokines and NFκB levels in the REF cells  Acridine orange (AO) and ethidium bromide (EB) double staining: 10 pancreatic islets for each group were used to perform AO/EB staining. The antiapoptotic effect of GA was determined by identifying morphological apoptotic and necrotic cells through the use of DNA-binding AO and EB dyes. Viable and nonviable cells both emit green fluorescence after absorbing an intercalated AO in the DNA. However, only nonviable cells emit red fluorescence after absorbing an intercalated EB in the DNA. After treating pancreatic islet cells with GA for 24 h, the cells were washed and stained with a mixture of 100 μg/mL of AO and EB and kept at room temperature for 5 min. Finally, the fluorescence of stained cells was observed through fluorescence microscopy  at a magnification of 40×, and the staining intensity was measured by ImageJ analysis software.® FITC kit. Cells with an approximated density of 3 × 105 cells/100 μL were incubated with 5 μL each of annexin V-FITC and PI at room temperature for 15 min. Then, the samples were analyzed with a flow cytometer .(b) Flow cytometry assay: This was used to determine the apoptotic vs. necrotic cells. The isolated pancreatic islet cells were treated with trypsin and then with BSA to stop the digestion. The suspended cells were washed twice with PBS, and dual staining was done using the ApoFlowEx(c) Measurement of caspase-3 and 9 activity: The activity of caspase-3 and 9 was determined via a colorimetric assay, as reported previously . After aFor normalizing data obtained from the above tests, each group of islets or REF cells was homogenized in 1 mL of each test’s related buffer. Then, 10 µL Bradford reagent was added to 100 µL of diluted samples, and after 5 min, the absorbance was read at 595 nm by the spectrophotometer. BSA was used as the standard.p < 0.05.All experiments were repeated three times, with six groups for each condition. Results were recorded as Mean ± standard error (SE). One-way analysis of variance (ANOVA) and Tukey’s tests were used to compare treatment and control groups. The differences were considered significant if This study has concluded GA with a potential of being an (a) anti-aging agent, through the reduced β-galactosidase activity and cell cycle arrest in G2/M phase, (b) antioxidant agent, via enhancing the level of mitochondrial complexes I, II, and IV, (c) anti-inflammatory agent, by decreasing levels of NFκB, TNFα, IL-1β, and IL-6, (d) antidiabetic agent, through increasing insulin secretion in pancreatic islet cells, and (e) antiapoptotic agent, by decreasing caspase-3 and 9 activities. The facts mentioned above suggest GA’s therapeutic benefits for the formulations of anti-aging skincare products or dietary supplementations for metabolic disorders, e.g., diabetes, through parallel molecular mechanisms.


Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production.Application of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C.A total of 3532 Overall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing. Plant biomass biorefineries use renewable and relatively inexpensive raw materials as feedstocks for processing into value-added biofuels and chemicals, with potential benefits for industry and the environment. Biorefinery processes can help alleviate problems associated with industrial chemicals and fossil fuels, such as increasing energy costs, environmental pollution and climate change , 2. A maBioethanol production by cassava starch biorefineries has attracted considerable interest because of the competitive advantages of cassava, such as abundant availability, low cost, and non-competition with direct food/feed supplies . Conventhttps://www.cazy.org/). α-Amylase and glucoamylase are commonly used in combination for starch processing. α-amylase breaks α-1,4-glycosidic bonds into amylopectin, or amylose straight chains, to release straight-chain and branched oligosaccharides of various lengths. Glucoamylase can cleave both α-1,4- or α-1,6-glucosidic bonds at the non-reducing ends of starch chains, or dextrins, to release glucose [Amylase for starch processing accounts for the majority of the industrial enzyme market and consists of four related enzymes, α-amylase (EC 3.2.1.1), glucoamylase (EC 3.2.1.3), α-glucosidase (EC 3.2.1.20), and 1,4-a-glucanbranching enzyme (EC 2.4.1.18)  that allow them to bind to the surface of raw starch granules , therebyClassical physical or chemical mutagenesis is an efficient strategy for breeding of fungal strains, in particular, for improvement of enzyme yields. The main mutagens used are ethyl methanesulfonate (EMS), nitroglycerin, UV-radiation, and “atmospheric and room temperature plasma” (ARTP) –12. The P. oxalicum. The starting strain used was the engineered strain OXPoxGA15A, derived from the parental strain ∆PoxKu70 via over-expressing a raw-starch-degrading glucoamylase gene PoxGA15A [In this study, we employed multiple rounds of ARTP/EMS-combined mutagenesis to improve RSDE production in PoxGA15A , 15, 16.PoxGA15A in OXPoxGA15A [PoxGA15A and the parental strain ∆PoxKu70, the clear zones around the OXPoxGA15A colonies being slightly bigger  and ball-milled Avicel, at a series of different ratios, as the top layer, and modified minimal medium (MMM), without carbon source as the bottom layer. RNCF flour was ground directly from freshly harvested cassava tubers after drying, without any other pretreatments, such as cellulose removal. RNCF is very similar to the cassava flour used in the starch industry and different from edible raw starch available from farmer’s markets. As expected, Avicel stimulated the expression of cellulase and xylanase genes, as well as induction of the pPoxEgCel5B promoter, which controls over-expression of the RSDG gene, PoxGA15A was determined at 1, 2, 4, 8, and 12 h after treatment. Treatment with 2% EMS for 8 h resulted in 93.6% lethality , whereas treatment for 12 h resulted in 100% lethality  three rounds of EMS, 2) one round of ARTP, and (3) one round of ARTP combined with EMS  was also determined, after 350, 400, 450, 500, and 550 s of treatment at a radio-frequency power input of 130 W and a flow rate of 10 L/min. The lethality increased with treatment time and treatment for 500 s resulted in 95.1% lethality  inoculum with 108 spores/mL , respectively. This was also far higher RSDE production than by P. oxalicum strain GXU20 (20 U/mL), using the same substrate [The secretion of plant biomass-degrading enzymes  by filamentous fungi is closely related to the characteristics of the lignocellulosic substrates in media, such as physical properties and chemical composition, as well as culture-related factors, such as the culture set-up and substrate loading , 17. ThePoxGA15A. The optimum pH and temperature, with RNCF as substrate, were 4.5 and 65 °C, respectively, which were similar to those of crude enzymes from OXPoxGA15A  87.6% and 98.1% after 72 h, respectively Fig. PoxGA15A and A2-13 enzymes, respectively , but the PoxAmy13A band was not significantly different between A2-13 and OXPoxGA15A , glucoamylase gene, POX02412, as well as α-amylase gene, PoxAmy13A (POX09352), and its key regulatory gene, PoxAmyR (POX03890). After 12 h of culture, transcription of PoxAmyR was up-regulated by 62.0%. The other three were down-regulated after 12 h of culture, by 40.8–91.9% (p < 0.05), but between 24 and 48 h, they were up-regulated by 33.9–559.5%  and cultured for 2‒6 days. The A2-13 colonies on wheat bran plus Avicel, Avicel, PDA, and glucose plates were smaller than those of OXPoxGA15A to varying degrees, appeared dense, thick with mycelia, and darker in color. A2-13 colonies on the other carbon sources were darker in color, but not different in size . Of the 35 SNVs in CDSs, 25 non-synonymous SNVs were detected, including one stop, non-synonymous mutation in gene POX03359 that encodes a hypothetical protein  in P. oxalicum strain 114–2 and 64.4% with Set1 in Saccharomyces cerevisiae. Set1 performs the methylation of histone H3 lysine 4 (H3K4) [S. cerevisiae, Set1 contributes to assembly of the elongation complex associated with RNA polymerase (Pol) II, by binding nascent RNA [P. oxalicum, by modulating H3K4me1 and H3K4me2 signals, as well as regulating colony diameter and spore number. POX01661 encoded the subunit Tho2 of the THO complex that is required for RNA transcript elongation, mRNA biogenesis, and export in S. cerevisiae [POX09438 was annotated as formin Bni1p, which is involved in the polarized localization in S. cerevisiae, controlled by the Rho GTPase Cdc42, via the effector Gic2p [POX05164. To our surprise, a β-glucosidase gene, POX03641 was found with a unique SNV and another gene, POX02958-encoded calcium/calmodulin-dependent protein kinase type I, and two ribosomal protein-encoding genes, POX01113 and POX01133.Comparative genomic analyses detected 230 single-nucleotide variations (SNVs) and 131 insertion/deletions (InDels) in the genome of A2-13, compared with that of OX4 (H3K4) . In S. crevisiae , 33. POXor Gic2p . Cdc42 iPOX02086, encoding a zinc finger protein of CCHC-type, two sugar/inositol transporter encoding genes POX05515 and POX08241 and an α-glucosidase gene POX07319.In addition to the SNVs in CDSs, of 193 SNVs located in intergenic regions, 38 and 10 were localized in 1500 bp upstream- and 300 bp downstream regions of the coding genes, respectively  that encoded endo-β-1,4-xylanase Xyn11A, alkaline phosphatase, ABC transporter, and a hypothetical protein, respectively. InDels in POX00063  and POX09551  resulted in a frameshift mutation in A2-13, whereas deletion of nucleotides in POX02669  and POX07393 (G and TACTCCCC deleted at positions 877 and 878) resulted in removal of a few amino acids at the C-terminus  production in P. oxalicum strains were cultured on potato-dextrose agar (PDA) plates for spore production. The starting strain, OXPoxGA15A, was from the China Center for Type Culture Collection, Wuhan, China  were cultured in liquid MMM (g/L: (NH4)2SO4 4.0, KH2PO4 4.0, CaCl2 0.6, MgSO4·7H2O 0.60, FeSO4·7H2O 0.005, MnSO4 0.0016, ZnCl2 0.0017, CoCl2 0.002, and 1 mL/L of Tween 80) [g for 15 min and the supernatant used for measurement of enzymatic activity and hydrolysis of natural raw starch flour.All ween 80) , supplemP. oxalicum strains (1.0 × 108 spores) were pre-cultured in MMM containing glucose as the sole carbon source for 24 h, then transferred into MMM containing 2% w/v wheat bran, plus 3% w/v Avicel in a shaker, at 180 rpm and 28 °C, for 12–48 h. The mycelia were harvested by filtering for RNA extraction every 12 h.For RT-qPCR analyses, P. oxalicum strains OXPoxGA15A and A2-13 were inoculated into Completed Medium containing (in g/L) yeast extract 1.0, glucose 10.0, casein hydrolysate 1.0 g, NaNO3 6.0, KCl 0.52, MgSO4·7H2O 0.52, KH2PO4 1.52, FeSO4·7H2O 0.005, MnSO4 0.0016, ZnCl2 0.0017, and CoCl2 0.002, pH 6.5, followed by shaking at 180 rpm and 28 °C, for 48 h.For genome re-sequencing, A two-layer agar gel was prepared, containing raw natural cassava flour  and ball-milled Avicel , at a series of different ratios as the top layer, and MMM, without a carbon source as the bottom layer. RNCF was ground directly from freshly harvested cassava tubers after drying, without any other pretreatments, such as cellulose removal.P. oxalicum strains using chemical methods, as reported previously [Total DNA and RNA were extracted from hyphae of eviously .8 spores/mL. EMS  was added to the suspension and incubated in a shaker at 180 rpm and 28 °C, for 1–12 h, to determine the optimum treatment time. An equal volume of Na2S2O3 solution was used to stop reaction. The treated spores were separated by centrifugation at 11,300×g for 10 min at 4 °C and re-suspended at different concentrations (between 102 and 108/mL) with sterile water.For EMS treatment, fresh spores were re-suspended in phosphate buffered saline (PBS) at 106 spores/mL. Spore suspension (10 µL) was spread on carrier plates and treated for 0 to 550 s at a flow rate of ten liters per minute and radio-frequency power input of 130 W, in a Type M ARTP Mutagenesis Bio-breeding Machine , to determine the optimum treatment time. The treated spores were washed from the carrier plates using sterile water and adjected to concentrations of 102 to 108 spores/mL.For ARTP treatment, fresh spores were re-suspended in 5.0% glycerol at 10ARTP/EMS-combined treatment was performed thereafter, as described above, with the optimum treatment times of 8 h by EMS and 500 s by ARTP.Treated spore suspension (~ 100 μL) was spread on plates containing MMM supplemented with RNCF (1% w/v) plus Avicel (0.5% w/v). After incubation for 8 days at 28 °C, the diameter ratio between each colony and its clear zone was measured and calculated. Spores without EMS and/or ARTP treatment were used as controls.P. oxalicum cultures were diluted with citrate buffer . Diluted enzyme solution (50 μL) was added to RNCF , incubated at 65 °C for 30 min; then the reaction mixtures were transferred to boiling water for 10 min. Enzymes inactivated by boiling were used as controls. Amounts of released reducing sugars and p-nitrophenol were determined using the 3,5-dinitrosalicylic acid method [The activity of RSDEs was determined as described previously , using Rd method  and specFor further enhancement of RSDE production by the mutant A2-13, culture parameters, including the initial medium pH, incubation temperature, carbon source composition, and nitrogen source, were optimized, respectively, using MMM containing wheat bran (4% w/v) plus Avicel (1% w/v). The effect of the initial medium pH on RSDE production by A2-13 was determined at pH 2.5, 3.5, 4.5, 5.5, 6.5, and 7.5, at 28 °C. Subsequently, incubation temperatures of 20, 24, 28, 32, and 36 °C were evaluated at the previously determined optimum pH, to determine the optimum incubation temperature.Furthermore, at the optimal pH and temperature, MMM containing wheat bran plus other carbon sources  that replaced Avicel at a ratio of 4:1 (w/w) was assessed for their effects on RSDE production. Subsequently, various ratios of wheat bran to the identified carbon source  were evaluated.4NO3, Peptone, (NH4)2SO4, and NaNO3, and the optimum concentration of the best nitrogen source was determined.Similarly, several nitrogen sources were evaluated, including soybean cake powder, Urea, NH2, 103, 104, 105, 106, and 107) on RSDE production were determined. All optimization experiments were carried out in a shaking incubator at 180 rpm. P. oxalicum was cultured for 6 days and the crude enzymes were collected for RSDE activity measurement.Finally, the effects of different-sized inoculations of spores (10RSDE activity against raw cassava flour (1% w/v) was measured in 0.1 M citrate–phosphate buffer at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0, at 65 °C for 30 min, as described above. RSDE activity at each pH was expressed as relative to that at the optimum pH (designated as 100%). The optimum reaction temperature was then determined at the optimum pH, by measuring the activity at various temperatures . RSDE activity at each temperature was expressed as relative to that at the optimal temperature.2HPO4 buffer (pH 3.0–7.0), Tris–HCl buffer (pH 7.0–9.0), and glycine–NaOH buffer (pH 9.0–11.0); then the residual RSDE activity was determined at the optimum pH and temperature for activity. Activity of untreated RSDE was defined as 100% and used as the positive control. Similarly, to ascertain temperature stability, crude RSDE was added to buffer at the optimum pH for stability, and the mixture was incubated at various temperatures from 30 to 65 °C, for 1 h. The residual RSDE activity was calculated as described above.To evaluate pH stability, crude RSDE was incubated at 4 °C for 24 h in buffers at various pHs, i.e., 0.1 M citrate-NaP. oxalicum A2-13 and OXPoxGA15A, hydrolysis reactions with 10% (w/v) solid substrate and an enzyme loading of 50 U/g substrate were conducted in citrate–phosphate buffer (pH 4.5) at 40 °C for 72 h. The reducing sugars produced were determined using the DNS method [RNCF and natural raw corn flour  were used as substrates for enzymatic hydrolysis. For raw starch hydrolysis by RSDEs from S method .P. oxalicum strains with commercial α-amylase  at a ratio of 1:1 was also tested, as described above. RSDEs were added at several concentrations .A combination of RSDEs from the P. oxalicum strains A2-13 and OXPoxGA15A were extracted and subsequently used for construction of the read libraries with a length of 400 bp. Libraries were sequenced on a BGISEQ-500 platform at the Beijing Genomics Institute . After removing low-quality reads and adaptors, the generated clean reads were mapped onto the genome of P. oxalicum strain HP7-1 [Genomic DNAs of in HP7-1  using Boin HP7-1  to detecPOX09428 was used as reference. The expression level of each tested gene was calculated relative to that of POX09428, and subsequently normalized against that for the parental strain ΔPoxKu70. Each experiment was repeated at least in triplicate.RT-qPCR was carried out as described previously . The tesP. oxalicum strains OXPoxGA15A and A2-13 cultured in MMM, containing wheat bran plus Avicel, for 6 days, were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The relative quantity of each protein was measured by GS-900TM calibrated densitometer  with a software ImageLab.Proteins secreted by P. oxalicum colonies on agar plates. In addition, harvested mycelia were transferred to microscope slides, and photographed using an Olympus DP480 microscope . Photomicrographs were analyzed using cellSens Dimension digital imaging software (Olympus).A Canon EOS 6D digital camera  was used to photograph t test.Statistical analysis was performed using Microsoft Excel  by Student’s Re-sequenced data are available from the Sequence Read Archive database under accession number SRA493765.Additional file 1: Fig. S1. ARTP/EMS-mediated mutagenesis and screening for RSDE hyperproducers. The P. oxalicum isolates were grown on the two-layer agar gel plates for 8 days. *: the isolates were screened and used for the next round of mutagenesis, by comparative analysis of their RSDE activities in MMM, containing wheat bran plus Avicel as the carbon source, for 6 days. RSDE activity was determined using RNCF as the substrate. ARTP: atmospheric and room-temperature plasma; EMS: ethyl methyl sulfonate; RNCF: natural raw cassava flour; MMM: modified minimal medium.Additional file 2: Fig. S2. Curve showing lethality against P. oxalicum strains OXPoxGA15A and E3-16, treated by ethyl methyl sulfonate (A) and atmospheric and room-temperature plasma (B). P. oxalicum spores were spread on PDA plates and incubated at 28 °C for 4 days. Each data point represents mean ± SD. Each experiment contained three biological replicates.Additional file 3: Fig. S3. Effects of culture conditions and inoculated spore number on RSDE production of P. oxalicum strain A2-13. (A) Initial pH of medium; (B) incubation temperature; (C) Carbon source; (D) Proportions of wheat bran and Avicel; (E) Nitrogen source; (F) NH4NO3 concentration; (G) inoculated spore number. P. oxalicum strains were cultured at 28 °C and 180 rpm for 6 days. Each data point represents mean ± SD. Each experiment contained three biological replicates.Additional file 4: Fig. S4. Colonic (A) and mycelial (B) analysis of the P. oxalicum strains A2-13 and OXPoxGA15A on plates containing various carbon sources. RNCA: natural raw cassava flour plus Avicel, RNCF: natural raw cassava flour, PDA: potato dextrose agar. Scale bar = 100 μm.Additional file 5: Table S1. List of 230 single-nucleotide variants of the mutant A2-13 compared with the starting strain OXPoxGA15A. Additional file 6: Table S2. Mutation sites in coding sequences (CDS) in the mutant A2-13 compared with that in OXPoxGA15A.Additional file 7: Table S3. Primers used in this study.