N-acetylneuraminic acid polymers in the ovaries of Coregonus maraena by immunohistochemistry and found that prevalent clusters of oogonia showed polySia signals on their surfaces. Remarkably, the genome of this salmonid fish contains two st8sia2 genes and one st8sia4 gene, that is, three polysialyltransferases. The expression analysis revealed that for st8sia2-r2, 60 times more mRNA was present than st8sia2-r1 and st8sia4. To compare polysialylation status regarding various polySiaT configurations, we performed a comparable analysis in Sander lucioperca. The genome of this perciform fish contains only one st8sia2 and no st8sia4 gene. Here, too, clusters of oogonia showed polysialylated cell surfaces, and we detected high mRNA values for st8sia2. These results suggest that in teleosts, polySia is involved in the cellular processes of oogonia during oogenesis.In vertebrates, the carbohydrate polymer polysialic acid (polySia) is especially well known for its essential role during neuronal development, regulating the migration and proliferation of neural precursor cells, for instance. Nevertheless, sialic acid polymers seem to be regulatory elements in other physiological systems, such as the reproductive tract. Interestingly, trout fish eggs have polySia, but we know little of its cellular distribution and role during oogenesis. Therefore, we localized α2,8-linked  Sialic acids, a heterogeneous family of acidic monosaccharides, are ubiquitous on the surfaces of eukaryotic cells and are involved in many crucial cellular events . BesidesN-acetylneuraminic acid (Neu5Ac) residues; so far, no polymers with other sialic acids or types of glycosidic bonds  have been found in vivo. In contrast, fish eggs can contain a high variety of different sialic acid polymers. Inoue and colleagues observed that fertilized and unfertilized eggs from Salmonidae contained sialic acid polymers [N-glycolylneuraminic acid (Neu5Gc) homopolymers, as well as hybrid structures of different sialic acid species that could also be O-acetylated [O-glycans of the polysialoglycoprotein (PSGP) [In mammals, sialic acids polymers contain only α2,8-linked polymers ,7,8. Inte.g., α2,-linkagesetylated . The main (PSGP) .st8sia) gene family [st8sia4, two st8sia2 gene loci have been described [st8sia4 took place during evolution, so only one st8sia2 gene now exists [The sialyltransferases, which catalyze the biosynthesis of α2,8-linked polySia, belong to the α2,8-sialyltransferase , a teleost fish with economic relevance in the Baltic area, which belongs to the salmonid family of bony fishes [C. maraena was compared with the polysialylation status of Sander lucioperca (S. lucioperca), since the genome of this percid fish contains one st8sia2 and no st8sia4 gene, whereas the genome of the salmonid fish C. maraena contains two st8sia2 genes and one st8sia4 gene [Whereas the presence of polysialyltransferase genes in various fish lineages  and the y fishes . The hisia4 gene ,16. ThusC. maraena were provided by the Institute of Fisheries of the Mecklenburg-Vorpommern Research Center for Agriculture and Fisheries . Maraena whitefish were raised in a recirculation aquaculture system (RAS), water temperature between 20 and 22 °C, maintained by an automated purification and disinfection system with a 16:8-h day-night cycle. From August to November, water temperature was reduced during 74 days to 4 °C and fish were kept at that temperature for out-of-season reproduction. Day-night light cycles were adapted during that period according to natural conditions. Fish were fed with commercial pellets feed. The fish were caught monthly using a sieve with a net. Specimens were sacrificed following the standards described in the German Regulations for Animal Protection (2006) (TierSChG) and current German Regulations for Animal Protection and Slaughter as of 20 December 2012 (TierSchlV). Ovarian tissues from each fish were used for paraffin histology, and the remaining tissue was snap frozen at −80 °C for future protein extraction and RNA isolation.Female samples from S. lucioperca samples, used for immunohistochemistry, were provided by Fischerei Loch . The ovaries were collected from animals processed for food production. The animals are from cage systems, which are placed in the lake Hohen Sprenz.Ovarian S. lucioperca were provided by the Institute for Fishery of the State Research Center for Agriculture and Fisheries Mecklenburg-Western Pomerania  and maintained in the RAS facilities of the Leibniz institute for farm animal biology (FBN). The temperature of the water was set at 22 °C, with a 12:12-h day-night cycle. Water quality was regularly monitored and maintained by automated purification and disinfection (bio-filter and UV light).For mRNA analysis, C. maraena were extracted with a TriPrep kit following the manufacturer’s instructions . The resulting samples were resolved in 1 × RIPA Buffer, and one aliquot of each sample was treated with endoneuraminidase (endoN) (6.7 µg/mL) for 1 h at 37 °C to degrade α2,8-linked Neu5Ac polymers. In addition, a color prestained protein standard was used . The samples were subjected to 7% SDS/PAGE and subsequently transferred to a PVDF membrane. The membrane was blocked with 0.5% no-fat dry milk in TBS 1× buffer. Immunostaining against polySia was done with monoclonal antibody (mAb) 735 (1 µg/mL) in 5% bovine serum albumin (BSA), which recognizes α2,8-linked Neu5Ac polymers with a degree of polymerization (DP) ≥ 8. Martina Mühlenhoff  provided mAb 735 and endoN [w/v) Coomassie blue in 50% (v/v) methanol, 7% (v/v) acetic acid) to control the protein transfer (loading control), since Coomassie staining is compatible with immunoblotting [TM MP, Feldkirchen, Germany).Proteins from the ovaries of nd endoN ,22. Horsblotting . PictureC. maraena ovaries was purified with the TriPrep kit  according to the manufacturer’s protocol. S. lucioperca ovaries were homogenized using 2.8 mm ceramic beads  at 6000 rpm for 30 s using the Precellys 24 Homogenizer (VWR/Avantor). The whole RNA was extracted using TRIzol Reagent  and subsequently purified with the RNeasy Mini Kit . The quantity of RNA was measured with a NanoDrop Onec photomometer . In addition, the RNA integrity was verified by electrophoresis on 1%-agarose gels. RNA was reverse transcribed to cDNA using the SensiFAST cDNA Synthesis Kit  following the manufacturer’s protocols.The total RNA from st8sia2 r-1, st8sia2 r-2, and st8sia4 for C. maraena and st8sia2 for S. lucioperca. For this purpose, the gene-specific primers were designed using software  to amplify the fragments between 121 and 226 bp. To identify the st8sia2 sequences in maraena whitefish, the orthologous sequences from rainbow trout Oncorhynchus mykiss (O. mykiss) were blasted against the transcriptome of maraena whitefish [Real-time quantitative PCR (RT-qPCR) was used to determine the expression of the polySiaTs: the two duplicated genes hitefish , depositC. maraena:st8sia2 r-1:5´AGCCTCATCAGGAAGAACATCC-3´ (sense)5´-TTCCCTACGATGGCACAGCGT-3´ (antisense)st8sia2 r-2:5´-CGTTCAACAGGAGCCTCTCTAA-3´ (sense)5´-TTCCCTACGATGGCACAGCGC-3´ (antisense)st8sia4:      5´-ATGATAAGGAAGGACGTGCTGC-3´ (sense)     5´-TGTTGAGCGTTCGGCGTCTGT-3´ (antisense)The following primers were used for st8sia2 sequences in pikeperch were obtained from the recently published S. lucioperca genome [st8sia2 in S. lucioperca, the following primers were designed and applied:st8sia2:      5´-GAGGAAGAAACTGCAAATACTGG-3´ (sense)     5´-AGTTGTTTGACGAGAGCTTGACA-3´ (antisense)The a genome . For st8C. maraena and 50 ng/5 µL cDNA for S. lucioperca, 1 µL of sense and antisense primers each, and 6 µL of SensiFast SYBR No-ROX Mix (Bioline). The RT-qPCR program comprised a denaturation phase  followed by 40 cycles of denaturation , annealing , elongation , and fluorescence measurement . PCR products were separated on 3% agarose gels and documented by the iBright FL1000 imaging system . In addition, the specific melting curve of each target gene was evaluated to check for unspecific products. The obtained real-time data were normalized against a factor based on the geometric mean values from three suitable reference genes encoding the eukaryotic elongation factor 1-alpha 1 (gene eef1a1) and two ribosomal protein units (genes rpl9 and rpl32 for C. maraena or rpl32 and rps5 for S. lucioperca) [RT-qPCR was run with the LightCycler 96 System  in a 12-µL reaction volume containing 16.6 ng/5 µL cDNA for ioperca) .v/v/v) for 24 h. In parallel, samples were fixed for 48 h using a Bouin solution with 2% sucrose. Thereafter, the samples were successively dehydrated with a stepwise alcohol gradient and cleared in xylene followed by isopropanol for 27 h in an automatic tissue processor . All the samples were embedded after dehydration in a paraffin station . Paraffin sections (5 µm) were cut with a microtome  and transferred to glass slides. After deparaffinization , the tissues were stained with hematoxylin and eosin (HE). The stained tissue sections were examined with a laser scanning microscope  For histology, tissue samples were cooled on ice and fixed in Bouin  immunoglobulin G (IgG)-free BSA . After three washes, tissue sections were blocked with PBS containing 2% BSA (w/v) for 1 h at 37 °C. To immunolocalize α2,8-linked polySia, mAb 735 (0.4 µg/mL) was used. To confirm the specific binding of mAb 735, we used two control strategies: (1) omitting the first antibody and (2) degrading the α2,8-linked polySia with endoN overnight at 37 °C (1.34 µg/mL). The tissue sections of both controls were on the same slide as the normally stained sample.For immunohistochemistry, the deparaffinized sections were washed with PBS containing 0.2% (w/v) IgG-free BSA at 4 °C. Subsequently, the tissue sections were washed and incubated for 1 h at room temperature (RT) with a secondary antibody . After washing, the sections were revealed using a peroxidase chromogen for immunohistochemistry (IHC) SIGMAFAST 3,3’-Diaminobenzidine (DAB)-tab . The nuclei were stained with hematoxylin for 10 s.The incubation with the primary antibody was prepared overnight in PBS containing 0.2% (2 fragment of goat anti mouse IgG (H + L)  (10 µg/mL in 1% BSA) or with Alexa Fluor 568-conjugated goat anti-mouse IgG (H + L) secondary antibody  (1 µg/mL in 1% BSA). For a parallel staining of DDX4, also known as VASA protein, the tissue sections were blocked with PBS containing 5% BSA and goat serum for 1 h at 37 °C. Thereafter, a rabbit IgG polyclonal antibody against DDX4  (1 µg/mL in 1% BSA) was used (overnight at 4 °C). The antibodies bound against DDX4 were labeled with an Alexa Fluor 647-conjugated goat anti-rabbit IgG (H + L) secondary antibody  (1 µg/mL in 1% BSA) for 1 h at RT. The DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at RT. Finally, a fixation step was performed using 2% paraformaldehyde (PFA) for 20 min at RT. The stained tissue sections were examined with a laser scanning fluorescence microscope .For the visualization of polySia, instead of the HRP-conjugated secondary antibody, the samples were incubated for 1 h at RT with Alexa Fluor 488-conjugated (Fab)st8sia gene loci and their neighbor genes were localized using NCBI BLAST search, and the detection of paralogous genes was assessed by manual chromosome walking. We chose genes physically close to the gene of interest to identify only the syntenic segment of interest.For the synteny (blocks of orthologous genes) and paralogy analyses, the p < 0.05.The calculated values were analyzed with Graph Pad Prism 8.4.3 software  using a Mann–Whitney test. The differences were considered statistically significant at C. maraena, which also belongs to the salmonid lineage, for α2,8-linked Neu5Ac polymers with a DP ≥ 8 using mAb 735 [C. maraena. Kitajima and colleagues found that α2,8-linked polyNeu5Ac was present on salmonid fish eggs  and that mAb 735 ,26. To t mAb 735 ,27. The  mAb 735 ,29. In tIt should, however, be noted that it cannot be unambiguously denied that the mAb 735 might additionally visualize other polySia species than α2,8-linked Neu5Ac homopolymers. It has been suggested that anti-polySiaNeu5Ac antibodies  may also bind heteropolymers of Neu5Ac and Neu5Gc residues, even if the binding efficiency is lower ,31. Furtst8sia2 variants and one st8sia4 gene [st8sia2 genes resulted from a fourth duplication of the whole genome in Salmonidae (SGD = R4) after the teleost radiation (TGD = R3) [Following the polySia-positive Western blot results, the expression levels of the polySiaT genes were examined using qPCR. In rainbow trout ovaries, three distinct genes are expressed: two ia4 gene . A recenGD = R3)   and st8sia4 transcripts (about 40 copies/ng RNA) were comparably low (st8sia2-r2 transcripts was more than 60 times higher (about 3110 copies/ng RNA).To determine the relative transcript levels of the polysialyltransferase genes in the ovaries of ably low b. Remarkstx-ov (in C. maraena st8sia2-r1) increase and the transcripts of st8sia4 tend to decrease [C. maraena samples, which were collected during “summer” (from May until August) and “autumn” (from September until December). The mRNA levels for both ST8SiaII enzymes of C. maraena slightly increased, although the changes were not statistically significant  was expressed with copy numbers higher than 1000 , further suggesting that the C. maraena st8sia2-r1 and st8sia2-r2 are co-orthologues of the S. lucioperca st8sia2 and could have the same function in fish ovaries.We conducted synteny and paralogy analyses of the S. lucioperca ovary samples, the Q-PCR analysis of the st8sia2 expression levels revealed that it is more than 10 times higher  than st8sia2-r2, the dominant polysialyltransferase in maraena whitefish ovaries (C. maraena were different compared to S. lucioperca. Thus, a quantitative comparison is hardly possible. Nevertheless, it is obvious that, also in S. lucioperca ovaries, polysialyltransferases are expressed.In  ovaries c. This mS. lucioperca ovaries was also investigated by immunohistochemistry. Consistent with the results from C. maraena, in S. lucioperca, clustered cell populations were polySia positive (S. lucioperca. Thus, despite the absence of ST8SiaIV, polySia is synthesized in these cell populations, likely by the ST8SiaII enzyme. Subsequently, the polysialylation status of positive . OvarianThe heterogeneity of polySia-positive and -negative cells in a cell population is common and depends very often on the state of activation ,3,4,39. st8sia2-r1, st8sia2-r2, and st8sia4, Percidae could only use st8sia2. Interestingly, the st8sia2 gene in Percidae was flanked by genes comparable to st8sia2-r1 and st8sia2-r2 in Salmonidae. In line with this observation, in C. maraena, the mRNA levels of st8sia2-r2 were 60 times higher than the values for st8sia2-r1 and st8sia4. Thus, it seems that polysialylation of oogonia is driven by ST8SiaII variants in both fish families and that polySia is involved in the cellular processes of germ cells during oogenesis in fish. Altogether, these observations further suggest a conserved function for polysialylation found in fish oogonia. Since it is well known that polySia is essentially involved in migration and proliferation processes of neural precursor cells [A special characteristic of dynamic oogenesis in fish is the possibility of oogonia proliferating throughout the fish lifetime . Intriguor cells , a compa


The as-prepared activated carbon presents a large specific surface area of 3025 m2·g−1 resulting from the synergistic effect of KHCO3 and dicyandiamide. As an electrode material, it shows a maximum specific capacitance of 417 F·g−1 at a current density of 1 A·g−1 and very good rate performance. Furthermore, the assembled symmetric supercapacitor presents a large specific capacitance of 314.6 F·g−1 and a high energy density of 15.7 Wh·Kg−1 at 1 A·g−1, maintaining 14.4 Wh·Kg−1 even at 20 A·g−1 with the energy density retention of 91.7%. This research demonstrates that nitrogen-doped hierarchical porous activated carbon derived from paddy has a significant potential for developing a high-performance renewable supercapacitor and provides a new route for economical and large-scale production in supercapacitor application.A facile and environmentally friendly fabrication is proposed to prepare nitrogen-doped hierarchical porous activated carbon via normal-pressure popping, one-pot activation and nitrogen-doping process. The method adopts paddy as carbon precursor, KHCO As a new kind of energy storage device, supercapacitors have been a key technology and research focus in the energy industry. It is generally recognized that the electrode materials are critical for developing high-performance supercapacitors. Among various electrode materials, activated carbon is the most common electrode material used for supercapacitors on account of its large specific surface area, stable chemical properties and low cost, while the low energy density limits its practical applications ,2. There3PO4, KOH or ZnCl2, are also employed to prepare porous carbons with ordered pore structures [Recently, plenty of research work has been devoted to enhancing the performance of activated carbon electrode materials. Activated carbon synthesized with various conducting polymers ,4 and trructures ,10,11,12ructures ,14,15,16Using biomass as raw materials, the popping process has been adopted extensively to prepare three-dimensional carbon material using the Chinese popcorn machine. When the temperature and pressure are high enough, the lid of the container is opened, so the differential pressure between the inside and outside of the rice increases rapidly, and the high-pressure steam in the rice expands rapidly, leading the rice to explode into puffed rice. Lai et al. reported the fabrication of dual-heteroatom-doped carbon nanosheets derived from polished rice and wheat via Chinese popcorn machine for supercapacitor and lithium-ion battery applications . Popcorn3 and dicyandiamide constructs a hierarchical porous structure and also introduces nitrogen atoms to the carbon materials successfully. The obtained material presents a large specific surface area of 3025 m2·g−1, which brings a maximum specific capacitance of 417 F·g−1 (at 1 A·g−1), and exhibits an excellent rate performance too. In addition, the assembled supercapacitor presents a specific capacitance of 314.6 F·g−1, while it has a capacitance retention of 93.3% after 10,000 cycles, which reveals a superb long-term cycle stability, and a remarkable energy density of 15.7 Wh·Kg−1 with 91.7% retention from 1~20 A·g−1. These performances are superior to most previous reports on biomass-derived carbon materials.Inspired by the Chinese popcorn machine, a facile and green fabrication of nitrogen-doped hierarchical porous activated carbon derived from paddy via normal-pressure popping, one-pot activation and nitrogen-doping process, has been demonstrated . Compare3 and dicyandiamide were added to appropriate deionized water according to the mass ratio of 1:3:1, respectively, and then fully stirred and baked at 75 °C for 48 h. Afterwards, the dried mixture was put into a tubular furnace under Ar atmosphere at 800 °C for 2 h with the heating rate of 5 °C/min, and then naturally cooled down to room temperature. Finally, the activated product was washed by using diluted hydrochloric acid to get rid of the residual potassium derivatives and other impurities in the product. Then it was repeatedly filtered with deionized water to neutral. The obtained sample was labeled as PRK3N1. In order to analyze the interaction between KHCO3 and dicyandiamide, pure puffed rice (labeled as PR), puffed rice mixed with KHCO3 (labeled as PRK3), and puffed rice mixed with dicyandiamide (labeled as PRN1) were prepared under the same processes and conditions, respectively. A more detailed recipe of all samples is shown in An appropriate number of paddies were added to a heated pot and stirred continuously for a few minutes. After the paddy exploded, the rice husk was removed with a sieve. Subsequently, puffed rice, KHCOScanning electron microscopy (SEM) images of the samples were recorded on a Hitachi SU8010 SEM system . Transmission electron microscopy (TEM) images were performed on FEI Tecnai G2 F20 S-twin TEM system . Raman spectrums were acquired by using a Renishaw inVia Raman microscope . A Bruker D8 ADVANCE diffractometer  with Cu Kα radiation was used to analyze X-ray diffraction (XRD) patterns. X-ray photoelectron spectroscopy (XPS) analysis was examined by an ESCALAB 250Xi system . The specific surface areas and pore size distribution of the samples were measured by the Brunauere–Emmette–Teller (BET) method on a MicroActive ASAP instrument .−2. An Hg/HgO electrode and a platinum foil were used as the reference electrode and counter electrode in the three-electrode system. In the two-electrode system, two PRK3N1 electrodes were employed to assemble the symmetric supercapacitor with a separator. A Metrohm Autolab PGSTAT302N electrochemical workstation  was applied to carry out galvanostatic charge/discharge (GCD), Cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) tests.The electrochemical properties of the obtained materials were evaluated in two-electrode and three-electrode systems, respectively, using 6 M KOH aqueous solution as the electrolyte. To fabricate the working electrode, the active materials, polyvinylidene fluoride and carbon black were mixed according to the mass ratio 8:1:1 in moderate N-methyl-2-pyrrolidone solvent. After stirring for 6 h, the slurry was coated on a nickel foam and vacuum baked at 80 °C for 24 h. The mass loading of the active material for a single electrode was about 3 mg·cmg) was calculated by the following Equation (1) according to the discharge curve:The specific capacitance  is the specific capacitance in the two-electrode system calculated from the Equation (1), ∆t (s) is the discharge time, and ∆V (V) is the discharge voltage range excluding the IR drop [The power density (P) and energy density (E) for the two-electrode system were calculated by the following Equations (2) and (3): IR drop ,22.3 activation. The structural characteristics and graphitization degrees could be further confirmed by Raman spectroscopy analysis. −1 presents the disordered and defective structures of the carbon lattice, while G band at 1580 cm−1 is related to the in-plane tensile vibration of sp2-hybridized carbon atoms of graphite [D/IG) reveals the degree of graphitization and defect of carbon materials. The ID/IG values of PR, PRK3, PRN1 and PRK3N1 are 2.57, 2.70, 2.59, and 2.71, respectively. The largest ID/IG value of PRK3N1 indicates the highest degree of disorder, which can mainly be ascribed to the heteroatom doping and hierarchical porous structure, which may introduce more electrochemical active sites and enhance the capacitance performance [The XRD patterns of PR, PRK3, PRN1, PRK3N1 are shown in graphite ,25. Geneformance .3. By comparison, PRK3N1 , C-C/C-N bond (285.8 eV), C-O bond (286.5 eV), C=O bond (287.7eV), and O-C=O band (289.7 eV), respectively [s spectrum could be deconvoluted into three peaks centered at 398.4 eV, 399.9 eV and 401.0 eV, related to the pyridinic-N (N-6), pyrrolic-N (N-5) and quaternary-N (N-Q), respectively [s spectrum is well-matched with three peaks, which are attributed to C=O (531.3 eV), C-O-C (532.5 eV) and O-C=O (533.3 eV), respectively [An in-depth study was applied to analyze the atomic compositions and surface chemical properties of all samples, the fully scanned XPS spectra are shown in C-C bond 4.6 eV, Cectively . The higectively ,30. It iectively ,31. Furtectively . Besides3 activation, PRK3 possesses typical type I adsorption–desorption isotherms with a steep increase at very low relative pressure, indicating the existence of abundant micropores [2·g−1/0.018 cm3·g−1, 6 m2·g−1/0.003 cm3·g−1, respectively), while PRK3 synthesized by KHCO3 activation has a higher surface area (1436 m2 g−1) and larger pore volume (0.614 cm3·g−1), which is attributed to the intense activation properties of KHCO3. As previously reported, after KHCO3 is completely decomposed at about 200 °C, the formation of CO2 and H2O (Equation (4)) positively contributes to creating the macropores [2·g−1) and largest pore volume (2.381 cm3·g−1), suggesting that the activation ability is obviously strengthened as the simultaneous presence of KHCO3 and dicyandiamide at high temperature, although the reaction mechanism is still unclear and needs to be further verified.The nitrogen adsorption–desorption measurements were performed to estimate the porous structure of all samples. According to cropores ,32. The cropores ,34. Morecropores . When thcropores . HoweverThe pore size distribution (PSD) of all samples measured by the NLDFT model is shown in 3 and dicyandiamide has a significant effect on forming a hierarchical porous architecture and doping nitrogen atoms, which is propitious to improving the electrochemical performance of the as-prepared materials.From the above discussion, the conclusion can be drawn that the synergistic effect of KHCO−1 are displayed in The CV curves of PR, PRK3, PRN1 and PRK3N1 at the scan rate of 50 mV·s−1 are illustrated in −1 at 1 A·g−1, with the capacitance retention of 79% even at 20 A·g−1 (330 F·g−1). The high specific capacitance and outstanding rate capability of PRK3N1 may be attributed to the hierarchical porous structure and heteroatomic doping resulting from the coordinative impact of KHCO3 and dicyandiamide, which would further induce extra pseudo-capacitance, shorten the ion diffusion pathway and create more active sites in favor of rapid charge transfer [The GCD curves of all samples measured at a current density of 1 A·gtransfer .s represents the serial resistance related to the electrolyte, Rct represents the charge transfer resistance, Zw represents the Warburg impedance arising from diffusion limitation, while C1 and C2 represent the pseudo-capacitance and electrical double-layer capacitance, respectively. The simulation results of PRK3 and PRK3N1 are shown in s values are 1.08 Ω and 0.50 Ω for PRK3 and PRK3N1, respectively. Meanwhile, the charge transfer resistance Rct of PRK3N1(2.12 Ω) is smaller than that of PRK3 (2.5 Ω), which implies the good conductivity and rapid charge transfer of PRK3N1 is due to efficient transport channels brought by abundant mesopores and macropores [The Nyquist plots of all samples tested in the range of 10 mHz to 100 kHz with 5 mV voltage amplitude are shown in cropores .−1, and the GCD curves from 1 to 20 A·g−1, are shown in −1 at the current density of 1 A·g−1, and 286.2 F·g−1 at 20 A·g−1  and possesses an outstanding rate performance. Furthermore, the assembled symmetric supercapacitor presents a high specific capacitance of 314.6 F·g−1 and a superb capacitance retention of 93.3% after 10,000 cycles, as well as remarkable energy density with 91.7% retention from 1 to 20 A·g−1. The prominent capacitor behavior illustrates that nitrogen-doped hierarchical porous activated carbon derived from paddy has a brilliant foreground in high-performance renewable energy storage devices.In summary, nitrogen-doped hierarchical porous activated carbon derived from paddy was synthesized via normal-pressure popping, one-pot activation and nitrogen-doped process. Due to the synergistic effect of KHCO


PPOs are commonly present as an isoenzyme family. In walnut (Juglans regia), two different genes (jrPPO1 and jrPPO2) encoding PPOs have been identified. In this study, jrPPO2 was, for the first time, heterologously expressed in E. coli and characterized as a tyrosinase (TYR) by substrate scope assays and kinetic investigations, as it accepted tyramine and L-tyrosine as substrates. Moreover, the substrate acceptance and kinetic parameters  towards 16 substrates naturally present in walnut were assessed for jrPPO2 (TYR) and its isoenzyme jrPPO1 (TYR). The two isoenzymes prefer different substrates, as jrPPO1 shows a higher activity towards monophenols, whereas jrPPO2 is more active towards o-diphenols. Molecular docking studies performed herein revealed that the amino acid residue in the position of the 1st activity controller  is responsible for the different enzymatic activities. Additionally, interchanging the 1st activity controller residue of the two enzymes in two mutants (jrPPO1-Asn240Gly and jrPPO2-Gly240Asn) proved that the amino acid residue located in this position allows plants to selectively target or dismiss substrates naturally present in walnut.Polyphenol oxidases (PPOs) are ubiquitously distributed among plants, bacteria, fungi and animals. They catalyze the hydroxylation of monophenols (monophenolase activity) and the oxidation of  PPOs consist of tyrosinases (TYRs), catechol oxidases (COs) and aurone synthase (AUS). TYRs catalyze the ortho-hydroxylation of monophenols to o-diphenols  as well as their subsequent oxidation to o-quinones , whereas COs are solely able to perform diphenolase activity 5 and a reduced economic value6.Polyphenol oxidases (PPOs) are metalloenzymes with a type-III copper center widely distributed among archaea, bacteria, fungi, animals and plantsjrPPO1) has been studied extensively12. In vivo, jrPPO1 is expressed as a latent 66.8 kDa pre-pro-enzyme composed of the catalytically active domain (~ 39 kDa) which is flanked by an N-terminal chloroplast transit peptide (~ 12 kDa) and a C-terminal domain (~ 16 kDa) that is shielding the entrance to the catalytic pocket and requires removal for the enzymatic activity to occur8. As shown for apple tyrosinase (MdPPO1), this can be achieved by a self-cleavage reaction13. Alternatively, PPOs can be activated in vitro by fatty acids14, acidic pH15, proteases17 and detergents such as sodium dodecyl sulfate (SDS)18. The crystal structure of jrPPO1 unveiled the architecture of the di-copper center in which each of the two copper atoms (CuA and CuB) is coordinated by three conserved histidine residues9. Comparison of the active centers of jrPPO1 and other plant PPOs shows a high level of conservation, however, two amino acid residues, named 1st (HisB1 + 1 = His239 + 1) and 2nd (HisB2 + 1 = His243 + 1) activity controller residue , which is, for the first time, investigated within this study.In 2016 Martínez-García et al.jrPPO1 (C0LU17) and jrPPO2 (A0A2I4DDQ0) share an amino acid sequence identity of 73% , whereas jrPPO2 displays a glycine (Gly240). Asn in the position of the 1st activity controller (HisB1 + 1) has previously been associated with increased monophenolase activity as it stabilizes a water molecule which is activated by a conserved glutamic acid and reacts as a base for the deprotonation of incoming substrates. The deprotonation of the substrate is imperative for the hydroxylation of monophenols20. Thus, an asparagine in the position of the 1st activity controller is also present in the sequences of MdPPO221  and VvPPOg22 . In contrast, the deprotonation of a substrate can also occur by a hitherto unknown mechanism as glycine in the position of the 1st activity controller is compatible with monophenolase activity as well. This has already been proven by the sequences of MdPPO321 , ToPPO123, ToPPO224  and VvPPOcs-3 25, which all accept the standard substrates L-tyrosine and/or tyramine.73% Fig.  and espe73% Fig. . The mosJuglans regia) is known for being rich in phenolic compounds valuable to the cosmetic and pharmaceutical industries26, however, PPO side reactions in walnut limit the availability of these compounds28. The rich abundance of phenolic compounds in walnut has been investigated thoroughly within the last years33, leading to the identification of numerous small phenolic compounds  as the cause for the different reactivities in these two enzymes, which was further substantiated by kinetic measurements and docking studies using two mutants targeting the 1st activity controller residues of the two isozymes .Herein, we report the cloning of the gene encoding latent 42 as well as 20 mM sodium ascorbate to suppress PPO side reactions since in situ produced quinones oxidize DNA.Genomic DNA (gDNA) was isolated from walnut leaves using a cetyltrimethylammonium bromide (CTAB) assisted cell lyses method, which produced a total yield of 250 ng gDNA (~ 40.000 base pairs)/g frozen plant material. The co-extraction of phenolic compounds substantially decreases downstream applicability (PCR) of DNA extracts. Thus, 2% (w/v) PVP (polyvinylpyrrolidone) was added to the DNA extraction bufferjrPPO2 gene19 encompasses an N-terminal chloroplast transit peptide, an active domain and a C-terminal domain. Using Q5 High-Fidelity DNA polymerase and specific primers  vs. California19). Minor structural variability has already been reported for jrPPO1, however, enzymatic activity, pH optimum and SDS dependent activation of the latent enzyme has been shown to remain unaffected by these variations10.The predicted rs Table  a ~ 1,70jrPPO2  cells and used for protein expression. The enzyme was expressed as a fusion protein with an N-terminal GST-tag, which on the one hand facilitates its purification and on the other hand increases the solubility and, thus, reduces the formation of inclusion bodies43.Based on the sequencing results, a second primer pair was designed binding to the starting region of the active domain and the end of the C-terminal domain Table , which pjrPPO110 and other plant PPOs44, expression at low temperatures (~ 20 °C) in combination with prolonged expression times and the usage of a nutrient-rich medium (2xYT) results in an increased overall yield. jrPPO2 produced the highest yield with 70 mg/l purified, latent enzyme, followed by jrPPO2-Gly240Asn (63 mg/l), jrPPO1 (41 mg/l) and jrPPO1-Asn240Gly (34 mg/l). All enzymes were expressed at a purity level of at least 95% 49. Thus, the pH dependence was assessed in increments of 0.5 pH units ranging from pH 3.0 to pH 8.0 . The maximum activity was observed at pH 6.0 10.ine Fig.  as a sub50 as activity can be measured only in the presence of an additional activator. SDS has been proven suitable in activating plant PPOs51 and was previously shown to overcome their latency. Thus, the activation of PPOs is achieved with SDS molarities ranging from 0.35 mM51 to 4.0 mM21. We tested the concentration-dependent activation of jrPPO1 with SDS molarities ranging from 0.5 to 5.0 mM. The highest activity was observed at 2.5 mM SDS  and Km (mM) values were determined for tyramine, l-tyrosine, dopamine and l-DOPA  compared to the monophenols  of jrPPO2 was considerably higher for the less polar substrate tyramine (kcat/Km = 18.7 s−1 mM−1) compared to the carboxylic substrate L-tyrosine (kcat/Km = 0.69 s−1 mM−1), which held also true for the diphenolic substrates : pyrogallol, protocatechuic acid, gallic acid, ethyl gallate, 4-hydroxyphenylacetic acid, coumaric acid, caffeic acid, quercetin and taxifolin , in contrast to their non-methoxylated homologs (vanillic acid / 4-hydroxybenzoic acid and ferulic acid/coumaric acid)  but rejected by jrPPO1 (after 24 hours). Similarly, gallic acid  Fig. . ConsequPO2 Fig. . Severalcid Fig.  was accecid Fig.  was oxidurs Fig. .jrPPO1 and jrPPO2, kcat and Km values were determined for substrates that showed activity towards jrPPO1 and/or jrPPO2. Molar extinction coefficients were reported previously52 or were determined herein  and higher efficiency values (kcat/Km ratio) towards jrPPO2 than towards jrPPO1. The only exception was protocatechuic acid  with jrPPO1. However, since the Km value for protocatechuic acid , it showed a substantially higher catalytic efficiency towards jrPPO2 (kcat/Km = 109 s−1 mM−1), compared to jrPPO1 (kcat/Km = 3.95 s−1 mM−1)  showed a higher turnover rate towards jrPPO1, compared to jrPPO2. Monophenolase/diphenolase activity ratios (kcat monophenol/kcat diphenol) of corresponding mono- and diphenols were higher for jrPPO1 than for jrPPO2. The activity ratio of tyramine/dopamine for jrPPO1 was 0.27, compared to 0.05 for jrPPO2 (Table kcat/Km) monophenol/(kcat/Km) diphenol).cid Fig. , which wcid Fig.  increasejrPPO1 favors monophenolic substrates, whereas jrPPO2 targets diphenolic substrates. This trend also correlates with the flavonoid substrates and was particularly pronounced for the diphenolic substrate taxifolin , compared to jrPPO1  of jrPPO1 towards monophenols, compared to jrPPO2. To clarify the molecular cause for the increased diphenolase activity of jrPPO2, compared to jrPPO1, docking studies were employed.Thus, our data show that lin Fig. , which wjrPPO2 was built using the SWISS-MODEL server54  as a template. Molecular docking was performed for jrPPO1 as well as jrPPO2. Binding poses were calculated for all kinetically investigated substrates, which included the standard substrates tyramine, l-tyrosine, dopamine and l-DOPA and the natural substrates pyrogallol, protocatechuic acid, gallic acid, ethyl gallate, 4-hydroxyphenylacetic acid, coumaric acid, caffeic acid, quercetin and taxifolin. The results offered highly valuable information detailing the molecular basis for the different reactivities of jrPPO1 and jrPPO2.For the docking studies, a homology model of r54 Fig.  and the jrPPO2 exhibited a high level of structural homology, compared to the crystal structure of jrPPO1 (RMSD = 0.487 Å). However, the amino acid in the position of the 1st activity controller residue represents a notable difference between the architectures of the active centers of jrPPO1 and jrPPO2 . Thus, orienting substrates into the active center of jrPPO2 with the phenolic ring facing the di-copper center appears to be entropically more favorable, compared to jrPPO1, due to the spatially less demanding 1st activity controller residue (Gly240). This explains the significantly higher turnover rates and efficiency values of diphenolic substrates for jrPPO2, compared to jrPPO1 . Semiquantitative in-gel activity tests demonstrated that the mutant VvPPOcs-3-Gly241Asn (1st activity controller: Asn241) showed increased activity rates towards the monophenolic substrates tyramine and p-tyrosol 25, which is in accordance with our results. Moreover, it has been demonstrated, based on the crystal structure of the bacterial tyrosinase from Bacillus megaterium (BmTYR)55, which also features an Asn in the position of the 1st activity controller (HisB1 + 1 = Asn205), that Asn205 forms a polar bond with the first CuB coordinating histidine (HisB1 = His204) and, thereby, stabilizes His20455. In BmTYR, the Nδ1 atom of the imidazole ring of His204 (HisB1) is located at a distance of 2.7 Å from the amide group of Asn204 (1st activity controller residue). Similarly, the Nδ1 atom of the imidazole ring of His239 (HisB1) in jrPPO1 is located at a distance of 2.9 Å from the amide group of Asn240 (1st activity controller residue)9. Thus, in jrPPO1 Asn240 probably shows a stabilizing effect on His239. The combination of these effects explains the higher activity rates of monophenolic substrates with jrPPO1, compared to jrPPO2 , whereas the activity towards the monophenol tyramine was reduced   and an increased activity towards tyramine . Accordingly, jrPPO1-Asn240Gly (tyramine/dopamine activity ratio = 0.03) had a stronger preference of diphenols over monophenols than jrPPO2-Gly240Asn  in an upright orientation.Docking studies were performed for ine Fig.  and all ine Fig. , S10–S13on Figs.  and S13.jrPPO1 (targeting monophenols) and jrPPO2 (targeting diphenols). The substrate scope is dependent on the amino acid present in the position of the 1st activity controller. Moreover, an asparagine in the position of the 1st activity controller increases monophenolase activity, whereas diphenolase activity is reduced (compared to the presence of glycine in the same position). Substituting asparagine with a spatially less demanding amino acid (such as glycine) increased the kcat value of dopamine considerably. Our results lead to the conclusion that jrPPO1 and jrPPO2 in vivo target different substrates and, thus, most probably fulfill different physiological tasks. The common appearance of plant PPOs as a family of isoenzymes suggests that they are involved in several cellular pathways, covering a diverse spectrum of functionalities. We hope that our work will inspire the deciphering of the different tasks assigned to PPOs, thereby illuminating their elusive reactivities.This data show that the amino acid residue in the position of the 1st activity controller is responsible for the different substrate preferences of jrPPO2 was, for the first time, heterologously expressed, purified and characterized. Activity tests using standard substrates  clarified that jrPPO2 is a TYR, as it accepted l-tyrosine and tyramine. Moreover, substrate scope assays using 16 natural substrates showed a more expansive substrate scope for jrPPO2 as it accepted 4-hydroxybenzoic acid and gallic acid, compared to jrPPO1, which was inactive with 4-hydroxybenzoic acid and showed only marginal activity towards gallic acid. Kinetic parameters were determined for jrPPO2 and its isoenzymes jrPPO1, which pointed towards differences in substrate preference. jrPPO2 showed a higher catalytic efficiency for diphenols whereas jrPPO1 was more active on monophenols. Docking studies revealed that the amino acid in the position of the 1st activity controller can increase the activity towards monophenolic substrates, as it has previously been proposed, by stabilizing a conserved water molecule or reduce enzymatic activity towards diphenolic substrates by sterically impeding substrate orientation. The two mutants jrPPO1-Asn240Gly and jrPPO2-Gly240Asn proved the key role of the 1st activity controller as jrPPO1-Asn240Gly showed an enzymatic profile similar to jrPPO2, whereas jrPPO2-Gly240Asn resembled jrPPO1.In this study, Our results demonstrate that, in vivo, different PPOs within the same plant target different substrates, which is achieved by the variability of one crucial amino acid residue (1st activity controller). This novel understanding of the functionality of PPO isoenzymes in plants will hopefully allow controlling their reactivity and, thereby, enhance the nutritional and economic value of plant products.42. The mixture was incubated in a 70 °C water bath for one hour followed by centrifugation at 20.000 rpm for 10 min. The supernatant was extracted with 1 volume of phenol:chloroform:isoamyl alcohol  and subsequently the aqueous layer was washed two times with 1 volume 100% chloroform. The aqueous layer was precipitated by adding 1 volume of EtOH (96%) and incubation at 0 °C for 2 hours. The pellet resulting from centrifugation at 20.000 rpm for 10 min at 4 °C was washed two times with EtOH (70%) at 0 °C, dried and resuspended in 100 µl TE buffer . The quality and quantity of the DNA extract were checked by 0.6% agarose gel electrophoresis , pH 8.0)sis Fig. .jrPPO2 published previously19  was used for the amplification and a ~ 1,700 base pair amplicon was produced (for detailed PCR setup see supplementary information). The PCR product was cloned into the pENTRY-IBA51 vector and sequenced to reveal the full-length sequence of jrPPO2. Thereafter, the gene encoding jrPPO2  was amplified with the second pair of primers  and transformed into E. coli BL21 (DE3) cells.The first pair of primers binding outside of the open reading frame of the active domain and the C-terminal domain was designed  from the sequence of 19 Table . Q5 HighjrPPO1 and jrPPO2, cloned into the pENTRY-IBA51 donor vector, respectively, served as templates for the mutagenesis experiments. Q5 High-Fidelity DNA polymerase was used to introduce the mutations into the sequence by back to back annealing primers (Table T4 Polynucleotide Kinase (NEB) and T4 DNA Ligase (NEB) were used to create cyclic plasmids (pENTRY-IBA51). The open reading frames were then sub-cloned into the pGEX-6P-1 expression vector using the Esp3I restriction enzyme and expressed as described previously10 (see supplementary information).The genes encoding rs Table  with thejrPPO1, jrPPO2, jrPPO1-Asn240Gly and jrPPO2-Gly240Asn were measured on an LTQ Orbitrap Velos mass spectrometer  equipped with a nanospray ion source using an ion transfer capillary temperature of 300 °C and an electrospray voltage of 2.1 kV. 5 µl of the sample was loaded on a trap column of an UltiMate 3000 nano HPLC-system (Dionex) using 0.1% trifluoroacetic acid. The separation was carried out at a flow rate of 300 nl/min on a C4 analytical column 50 cm × 75 µm Accucore C4, 150 Å, 2.6 µm (Thermo Fisher Scientific) using mobile phase A  and mobile phase B . Full MS scans were acquired in positive ion mode ranging from 400 to 2000 m/z at a resolution of 7,500 (FWHM at 400 m/z).Mass spectra of jrPPO2 using the diphenolic substrate dopamine. The highest activities were measured at pH 6.0 (50 mM sodium phosphate buffer) and 2.5 mM SDS. Kinetic measurements of jrPPO2 and jrPPO2-Gly240Asn were performed under these conditions by measuring the increase of the colored reaction products photometrically  in 96 well plates at 25 °C using the latent enzyme and SDS as an activator. pH and SDS optima were determined for lly Fig. . For jrPjrPPO1 and/or jrPPO2, 100 µg of the purified, latent enzyme were mixed with 1 mM substrate in 50 mM sodium phosphate buffer and 2 mM (jrPPO1 and jrPPO1-Asn240Gly) or 2.5 mM (jrPPO2 and jrPPO2-Gly240Asn) SDS in 200 µl solution at 25 °C. Due to their limited solubility, the assays for the flavonoid substrates  or 2.5 mM (jrPPO2 and jrPPO2-Gly240Asn) SDS and 10% DMSO in 200 µl at 25 °C. Substrates that exhibited no visually detectable change in color within 24 hours were flagged as inactive. A control assay was performed for each substrate containing 1 mM substrate in 50 mM sodium phosphate buffer and 2.5 mM SDS in 200 µl at 25 °C.To determine which substrates showed activity with kcat and Km value), the maximum reaction rate was measured at 7–8 different substrate concentrations in a total volume of 200 µl containing 50 mM sodium phosphate buffer at pH 6.0, 2 mM (jrPPO1 and jrPPO1-Asn240Gly) or 2.5 mM (jrPPO2 and jrPPO2-Gly240Asn) SDS and variable amounts of enzyme  was prepared for molecular docking by adding missing side chains using Coot57. A homology model of jrPPO2 was built using the SWISS-MODEL server54. The exhaustiveness was set to 100 and 20 poses were calculated for each target and substrate . Binding poses were searched in a grid box enclosing the two copper ions of the active site, the 1st and 2nd activity controller and Phe260 , the 2nd activity controller residue  and Phe260, were defined as flexible residues. Poses that significantly deviated from the binding pose of l-tyrosine were flagged as ’unreasonable’ poses. All visualizations were created using PyMOL 2.358.Molecular docking was performed using AutoDock Vinae Tables  and S5. 60 Figs.  and S2. Supplementary information


N-sulfated glucosamines at the NRE. Uronic NRE  have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in  Heparin is a complex heterogeneous linear animal polysaccharide used as a drug for its anticoagulant properties. Heparin is also the starting material for the synthesis of the widely used antithrombotic drug, Low Molecular Weight Heparins (LMWH). Heparins can be extracted from various tissues  and different animal species . However, porcine mucosa is currently the only approved starting material for LMWH manufacturing ,2. The cChemically speaking, heparin is a highly sulfated heterogeneous linear polysaccharide made of disaccharide repeating units comprising an uronic acid (either iduronic or glucuronic) and a glucosamine. Building block analysis, realized either by nuclear magnetic resonance (NMR) spectroscopy ,6 or by For purified samples where quantitative polymerase chain reaction (qPCR)  cannot b18 bonds of the stationary phase. Interestingly, the Carbopack AS11 SAX columns , typically insufficiently retentive to resolve all disaccharide building blocks, was within effective retention range after reductive amination by sulfanilic acid. Ion pair chromatography ) and 120 µL of 100 mM sodium acetate buffer (pH 7.0) containing 2 mM Ca(OAc)2 and 0.1 mg/mL BSA.Digestion of heparin (20 µL of a 20 mg/mL solution in water) was performed at room temperature for 48 h in a total volume of 160 µL containing 20 µL of a mixture of the three heparinases  containing 4–6 mg of sulfanilic acid and 6–10 mg of picoline borane. The reaction was complete after 8 h at 37 °C. The remaining reagents were removed on Sephadex G10 (column 30 × 2.6 cm) circulated with H2PO4 at pH 2.8, and mobile phase B was an aqueous solution of 2.5 mM NaH2PO4 with 1 M NaClO4 adjusted to pH 3.0. A linear gradient  was applied for elution at a flow rate of 0.22 mL/min. Diode array detection was used. Double UV detection was performed at 265 nm and 232 nm. An NRE building block-specific signal was obtained by the reconstruction of 265 nm − 2.21 × 232 nm. In the first part of this study, our conventional AS11 method ,41 was u2PO4 at pH 3.2. All other parameters were unchanged. In the second part of the study, full resolution of building blocks was obtained with optimized pH for the mobile phase and connection of two AS11 columns (25 × 0.21 cm) in series. Mobile phase A was 2.5 mM NaHHeparin digests were injected on ion-pair LC/MS chromatography using Acquity UPLC BEH C18 column, 2.1 × 150 mm, 1.7 μm (Waters). Mobile phase A was water, and mobile phase B water/acetonitrile (30:70). The ion pairing reagent, HPTA (7.5 mM) and a buffering agent, HFIP (50 mM) were added to both A and B. A linear gradient  was applied for elution at a flow rate of 0.22 mL/min. Column temperature was set at 30 °C and diode array detection used. Double UV detection was performed at 265 nm and 232 nm. An NRE building block-specific signal was obtained by the reconstruction of 265 nm − 2.6 × 232 nm.E mode [Electrospray ionization (ESI) mass spectra were obtained using a Waters Xevo Q-Tof mass spectrometer. The electrospray interface was set in negative ion mode with a capillary voltage of 2000 V and a sampling cone voltage of 20 V. The source and the desolvation temperatures were 120 °C and 300 °C, respectively. Nitrogen was used as desolvation (750 L/min) and cone gas (25 L/min). The mass range was 50–2500 Da (scan rate = 0.8 s). Acquisition was performed in MSE mode  with low2O, 50–80 mg of the fraction was added to 50 mg of sulfanilic acid mixed with 50 mg of picoline borane. After 8 h at 37 °C, the reagents were eliminated using Sephadex G10. Semi-preparative purification was performed on AS11 columns (25 × 2.1 cm) at room temperature and a flow rate of 20 mL/min. An aqueous NaClO4 (0–0.6 M) mobile phase at pH 2.5 was used for elution. Fractions were neutralized and controlled on analytical AS11. The selection of fractions was based on the NRE selective UV signal.Semuloparin was injected (2 g per injection) on a column (200 × 5 cm) packed with Bio Gel P30. The tetrasaccharide fraction was collected and desalted on a column packed with Sephadex G10 (100 × 7 cm). The reductive amination of the tetrasaccharide batch was a slightly modified version of the procedure used for digests. In 1 mL HIn a second step, the two main NRE disaccharides were also isolated to check their retention behavior in both ion-pair and AS11 methods. They were isolated from disaccharide fractions of BLH and PMH heparins digested by heparinase 1, purified by conventional methods, as already described . The semd-glucuronidase. Briefly, 100 µL of the oligosaccharide solution was diluted 1/3 in 50 mM sodium acetate pH 4.5, added with 100 units of enzyme and incubated overnight at 30 °C.Sulfanilic tagged heparin digests and semuloparin tetrasaccharides were treated with β-2HPO4 pH 7 before being treated for one day at room temperature with Δ4-5 glycuronidase (20 milliunits).The sulfanilic tagged heparin digests (100 µL) were diluted 1/2 in 5 mM NaDerivatization by reductive amination sulfanilic acid tagging was applied to heparinase digests of heparin. Two chromatographic methods were proposed for the separation of the tagged building blocks: SAX chromatography on AS11 columns with UV detection and ion-pair liquid chromatography on a C18 column with MS detection. In addition to known heparin classical di and tetrasaccharides, new building blocks issued from the NRE are described. The latter include sulfated glucosamines, uronic acids, and trisaccharides. NRE glucuronic acids were specifically detected in PMH. A marker of porcine heparin, GlcA-GlcNS,3S,6S was isolated and heparin building blocks assayed using SAX chromatography on AS11 columns. The stability of this stationary phase enabled good control of chromatographic separation, which is superior compared to the classical method of separating Δ4-5 unsaturated building blocks on a silica based SAX column . This me


Toxoplasma gondii in macrophage and human foreskin fibroblast (HFF) cell line infection models. However, the mechanism of action of RSV has not yet been determined. Thus, with the aim of identifying a possible mechanism of the anti-T. gondii activity of this compound, we analyzed the effects of RSV on histones H3 and H4 lysine 16 acetylation (H4K16). We also analyzed RSV-induced DNA damage to intracellular tachyzoites by using the DNA damage marker phosphorylated histone H2A.X (γH2AX).Resveratrol (RSV) is a multitarget drug that has demonstrated activity against T. gondii tachyzoite growth at concentrations below the toxic threshold for host cells. The IC50 value after 24 h of treatment was 53 μM. RSV induced a reduction in H4K16 acetylation (H4K16ac), a marker associated with transcription, DNA replication and homologous recombination repair. A similar deacetylation effect was observed on histone H3. RSV also increased T. gondii H2A.X phosphorylation at the SQE motif (termed γH2A.X), which is a DNA damage-associated posttranslational modification. Our findings suggest a possible link between RSV and DNA damage or repair processes that is possibly associated with DNA replication stress.RSV inhibited intracellular  Toxoplasma gondii is an important pathogen affecting animal and human health, particularly during pregnancy and in immunocompromised patients [patients . In humapatients . The curpatients . In recepatients –7.Resveratrol  is a natural polyphenolic phytoalexin produced in plants. RSV can alter the activities of the histone deacetylases (HDACs) I and II, DNA methyltransferases (DNMTs) and is an activator of Sir2, a sirtuin belonging to HDACIII . Sir2 moT. gondii infection in mice would confer protection of the host [T. gondii tachyzoites.RSV administration during acute the host , regulatthe host . Chen etthe host  observedthe host  identifiT. gondii replication and histone posttranslational modification (PTM) and its effects on H3 and H4K16 acetylation levels. In addition, we investigated whether γH2A.X levels differ between control and treated intracellular tachyzoites. Collectively, our results show that RSV inhibited T. gondii replication and induced H4K16 and H3 deacetylation. Furthermore, γH2A.X levels were highly increased in tachyzoites treated with RSV relative to control tachyzoites, suggesting an association between RSV treatment and DSB damage.In this work, we evaluated the effects of RSV on Red Fluorescent Protein) strains were cultured under standard conditions in vitro in monolayers of immortal human foreskin fibroblasts . The RH RFP was kindly provided by Dr. Silvia N. Moreno . Cell monolayers were infected with tachyzoites and incubated with high-glucose Dulbecco’s modified Eagle medium  supplemented with 10% (10X) or 1% (1X) fetal bovine serum  and penicillin -streptomycin (10 mg/ml) solution  at 37 °C in a 5% CO2 atmosphere. Resveratrol  solution was prepared in DMSO  vehicle and stocked at 10 mg/ml.Tachyzoites of RH wild-type and RH RFP , anti-H3ac , anti-H4  and anti-H4K16ac  were used as primary antibodies, and Alexa Fluor goat anti-mouse 488  and anti-rabbit 595  were used as secondary antibodies. Alkaline phosphatase–conjugated antibodies were used in Western Blot assays .T. gondii γH2A.X was obtained from Eurogentec (Belgium) based on the peptide NH2-C + GKHGV-S(PO3H2)-QEF–COOH, designed from the T. gondii H2A.X amino acid sequence. Rabbit anti-rH2A.X was obtained from the Animal Facility at Facultad de Ciencias Exactas y Naturales  on the basis of the recombinant rH2A.X [A serum sample of rabbit anti-t rH2A.X . Murine t rH2A.X .4 cells/well) in 96-well plates and incubated for 24 h. After this period, confluence was assessed by microscopy, and the medium was replaced with fresh medium containing RSV at one of several concentrations. For the experiments in which the cells were incubated with RSV for 24 h, the concentrations ranged between 0 and 200 µM. Cell viability was determined based on the reduction of 3--2,5-diphenyltetrazole bromide . The absorbance at 540 nm was measured from the bottom of the plate using a BioTek Synergy plate reader . The graphs display the viability of the cells relative to the viability of control cells treated with 0.5% DMSO in culture media (100% viability).To evaluate the cytotoxic effect of the drugs on host cells, fibroblasts (hTERT) were seeded at 40% confluence  following previously described methods [Replication was performed and assessed using RFP-expressing RH tachyzoites as previously described . For int methods . Data weIntracellular tachyzoites were incubated for 24 h with 100 μM RSV, washed with 1X PBS and fixed with methanol for 10 min at −20 °C. Then, the cells were permeabilized with 0.2% v/v Triton X-100 in PBS for 10 min and blocked with 3% w/v BSA in PBS for 30 min at room temperature (r.t.). Then, primary antibodies diluted in 0.5% w/v BSA in PBS were added, and the cells were incubated for 60 min at r.t. The cells were then washed three times with PBS and then incubated with the corresponding Alexa Fluor-conjugated secondary antibodies for 60 min at r.t. Samples were mounted on coverslips with mounting medium  containing 10 µg/µl DAPI  to stain the nuclei. Primary antibodies were diluted 1:200, whereas secondary antibodies were diluted 1:2000. Samples were analyzed by epifluorescence microscope  equipped with a 63X objective and a Zeiss Axiocam 506 mono microscope camera. Images and fluorescence intensity analysis were obtained with the Fiji package of ImageJ .7 parasites/lane). Nonspecific binding sites were blocked with 5% nonfat dry milk in TBS containing 0.1% v/v Tween-20 (TBS-T) for 6 h, and the membranes were then incubated overnight with primary antibodies: mouse anti-H4, rabbit anti-H4K16ac, mouse anti-H3, rabbit anti-H3ac, rabbit anti-γH2A.X, rabbit anti-H2A.X and mouse anti-SAG1. Band intensities were quantified from scanned images using ImageJ software, and the value obtained from each band was normalized to the loading control, SAG1. Then, each histone PTM was normalized to its respective histone (H2A.X or H4).Immunoblotting was performed as previously described with minor modifications . Intracet test (*p ≤ 0.05 and ****p ≤ 0.0001) with GraphPad Prism 6.1 software .Data were expressed as mean ± SD from three to four experiments. The variations in the data were analyzed using unpaired Student´s T. gondii lytic cycle, transgenic RH RFP tachyzoites were grown intracellularly for 24 h in the presence of RSV. RSV showed a dose-dependent effect on intracellular T. gondii replication  after 72 h of incubation in comparison with pyrimethamine. The authors also observed that HFF cells were 100% viable at an RSV concentration of 2 μg/ml (8.5 μM). The differences in IC50 values among studies may be due to differences in the duration of drug exposure.To test our study model, uninfected or infected hTERT host cells were incubated for 24 h with different doses of RSV Fig. a, b. Neaion Fig. a, yieldin et al. , who foui et al.  identifiTrypanosoma cruzi [T. gondii transcription and/or replication through H3 and H4K16 deacetylation. However, further studies are required to confirm this possibility.Since HDAC enzymes are one of the multiple targets of RSV, we analyzed the acetylation levels of H3 and H4K16. Tachyzoites treated with RSV presented significantly weaker H3ac and H4K16ac labeling  than tachyzoites treated with DMSO Figs. a. We decma cruzi . Furtherma cruzi . In addima cruzi . Our resT. gondii phosphorylated peptide in the SQE C-terminal motif of T. gondii H2A.X was prepared. To confirm its specificity, a Western blot assay against recombinant nonphosphorylated T. gondii H2A.X was performed. Rabbit anti-TgγH2A.X antibody did not recognize T. gondii H2A.X  was determined by Western blot. A specific rabbit anti-A.X Fig. a but reaate Fig. b. After ate Fig. c.Fig. 3ET. gondii replication , genomic localization when present in the nucleus, etc., have not yet been reported. This lack of knowledge impairs our ability to infer whether the observed effects of RSV are associated with these sirtuins. Our results and those of others show that RSV exhibits anti-T. gondii activity in vitro. Studies in mice should be conducted to further evaluate RSV’s potential as a therapeutic candidate against toxoplasmosis. Very importantly, at low doses, RSV has beneficial effects on a wide variety of human diseases; however, at high doses and under long-term application, it can have adverse effects [T. gondii therapy. Here, we observed that in vitro, the IC50 of RSV was lower than the threshold of hTERT toxicity. Perhaps the dose and treatment duration could be managed by combining RSV with other drugs  to achieve a synergistic effect.Since RSV is a multitarget drug, it is difficult to establish which targets in activity . Thus, i effects . This asAdditional file 1: Quantification of H4K16ac mark with or without RSV treatment. The panels show ten nuclei of intracellular tachyzoites under RSV or DMSO control treatment labeled by indirect immunofluorescence with α-H4K16ac (1:200). Nuclei were also stained with DAPI. Antibody fluorescence and DAPI signals were quantified and plotted in different graphs , anti-TgH2A.X (1:5000) or anti-TgγH2A.X (1:100). Lysates were obtained from purified intracellular tachyzoites previously treated with RSV 50 μM (R), Sirtinol 50 μM (S) or DMSO 0.5% v/v (C) for 24 h. Three independent experiments with similar results are shown.


The objective of our study was to retrospectively analyze the safety of 2 cycles of systemic HD-MTX administered as an ambulatory regimen. Between January 2013 and December 2016, 103 patients were carefully selected on 6 criteria, including age < 60, albumin > 34, performance status 0 or 1, normal renal and hepatic functions, good understanding of practical medical guidance, and no loss of weight. Strict procedures of HD-MTX infusion were observed including alkalinization, urine pH monitoring, and leucovorin rescue. Renal and hepatic functions were monitored at days 2 and 7. MTX clearance was not monitored. Toxicities and grades of toxicity were collected according to the NCI-CTCAE (version 4.0). Among the 103 selected patients, 92 (89%) patients successfully completed the planned 2 cycles of HD-MTX on an outpatient basis. Eleven patients completed only 1 cycle, 3 because of lymphoma progression and 8 because of toxicity including 3 grade II hepatotoxicity, 2 grade I/II renal toxicity, 1 grade III neutropenia, 1 active herpetic infection, and 1 grade III ileus reflex. Reported adverse events (AE) included 92 (84%) grade I/II and 18 (16%) grade III/IV. Grade III hepatotoxicity, mostly cytolysis, was the most frequent AE observed with 8 (8%) events. Grade III/IV hematologic toxicities concerned 9 patients with 8 grade III/IV neutropenia and 1 thrombocytopenia. Renal toxicity was rare, mild, and transient, observed with 4 (4%) grade I/II events. Ambulatory administration of HD-MTX at 3 g/m2 without MTX clearance monitoring is safe with strict medical guidance. It requires careful selection of patients before administration, and a renal and hepatic monitoring after the administration.High-dose methotrexate (HD-MTX) at 3 g/m CNS relapse is a serious event in patient with aggressive non-Hodgkin lymphoma (NHL) and associated with poor outcomes. CNS relapses in DLBCL occurs in 1 to 31% depending on the series and risk factors studied . In peri> 3 g/m2 seems to be the best alternative in this context [The incidence of CNS relapses in both brain parenchyma and meninges is usually observed during the first 2 years of follow-up in DLBCL , 6. The  context . This st context . The val context  in 2016 2 [2) is used for a variety of pediatric and adult cancers including osteosarcoma, acute lymphoblastic leukemia, and primary or secondary CNS lymphoma.An exhaustive review of available data about CNS prophylaxis highlights the efficiency of HD-MTX as CNS prophylaxis at a dose superior or equal to 3 g/m2 , 13. HD-2). When patients experience delayed MTX elimination, the prolonged exposure to toxic MTX concentrations can lead to significant morbidity. All of these toxicities may lead to non-reversible adverse events and mortality [MTX is an antimetabolite-targeting folate metabolism and penetrates through cell membranes, particularly at doses where it crosses the blood-brain barrier. MTX is mainly bound (50 to 80%) to albumin in the plasma circulation and its essentially renal clearance explains the possible occurrence of severe toxicity after high-dose administration  or T cell (n = 5) non-Hodgkin’s lymphoma (NHL) between January 2013 and December 2016 at Saint-Louis Hospital, Paris, France. All these patients were treated as first-line treatment with CHOP or ACBVP, in association with anti-CD20 for B cell lymphoma and were eligible for HD-MTX to benefit of CNS relapses prophylaxis [We performed a retrospective analysis of HD-MTX administration in the outpatient clinic among patients with aggressive B cell  on day 1, and 60 mg/m2 prednisone on days 1–5. In the (R)-ACVBP group (95 patients), the 4 cycles consisted of an induction part, each cycle containing 375 mg/m2 rituximab if B cell NHL, 75 mg/m2 doxorubicin, and 1200 mg/m2 cyclophosphamide on day 1; 2 mg/m2 vindesine and 10 mg bleomycin on days 1 and 5; and 60 mg/m2 prednisone on days 1–5. CNS prophylaxis was included in the sequential consolidation part, starting 4 weeks after completion of the fourth cycle of R-ACVBP, consisting of 2 cycles of MTX (3 g/m2), with four subsequent cycles of rituximab (375 mg/m2) combined with etoposide (300 mg/m2) and ifosfamide (1500 mg/m2).In the (R)-CHOP group (8 patients), HD-MTX was administered 21 days after the 4th (5 patients), or 6th cycle (3 patients) of R-CHOP − 375 mg/mAll procedures performed were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Our strategy for ambulatory HD-MTX administration was to monitor renal and hepatic functions only and not MTX clearance, as this MTX clearance is not available in the outpatient setting. We then carefully selected the patients based on 6 criteria. These 6 mandatory criteria were (1) patient younger than 60 years; (2) performance status of 0 or 1 at the HD-MTX time infusion; (3) normal renal ≥ 60 ml/min and hepatic functions in the 7 days prior to HD-MTX; (4) albumin level strictly greater than 34 g/l; (5) an absence of significant weight loss (less than10% compared to baseline); and (6) a good understanding of practical medical guidance such as oral hydratation at 2 l of alkaline water per day for 3 days, discontinuation of all drugs with potential for interaction with MTX , and guiThe HD-MTX infusion was managed with 3 well-defined periods: the period before infusion, the period during infusion, and the period after the infusion of the MTX. On the day before the HD-MTX infusion, patients were asked to initiate at home the urine alkalinization by drinking 2 l of alkaline water per day, as Vichy St Yorre, as well as during the 24 h following infusion. Cotrimoxazole was discontinued 2 days before HD-MTX until the end of calcium folinate administration. On the day of infusion, a 14% sodium bicarbonate solution was administered to obtain a urine pH > 7.5 within 1 h after administration. If necessary (pH > 7.5 not obtained after 1 h), we pursued alkalinization by increasing the infusion rate. Ondansetron was administered immediately before the infusion. Then, the HD-MTX was infused over a period of 2 h and followed by 1 l of 14% sodium bicarbonate over 1.5 h. After the HD-MTX infusion, the patient started the first dose of oral calcium folinate at the dose of 50 mg at H24 and pursued this treatment every 6 h during 3 days (until day 4) for a total of 12 administrations. An alkaline hyperhydratation with 2 l of alkaline water per day was also maintained during 24 h after administration. Biological analysis of creatinine clearance, ALT, AST was performed 48 h after the administration of HD-MTX and additional tests including a blood count; complete hepatic biology including ALT, AST, gamma GT, and phosphatase alkaline; and creatinine clearance was programmed once a week until the next cycle of chemotherapy. In case of increase in renal function greater than 50%, hepatotoxicity grade ≥ II, or clinical grade ≥ 2 reported toxicity , the patient was contacted by the unit for inpatient hospitalization. In this procedure, MTX plasma levels were not monitored as renal and hepatic functions were precisely controlled after HD-MTX infusion  presented comorbidities. These included 7 arterial hypertension, 3 prior history of solid tumors , 3 type 2 diabetes (1 insulino-requiring—2 non insulino-requiring), 2 psychiatric disorders (1 anorexia and 1 depression), 2 pulmonary diseases (1 asthma and 1 sleep apnea syndrome), 3 non-active chronic viral infections , and 11 other diseases . Three patients presented 2 comorbidities. All the patients had normal hepatic and renal function at time of HD-MTX infusion.n = 80, 78% patients) did not receive any concomitant treatment. Two patients received an antidiabetic treatment, 5 an antihypertensive drug, 1 an antiviral treatment, 3 a psychiatric treatment, and 7 others . Five female patients received oral contraception.A majority of patients , a disseminated stage , and elevated LDH levels . The age-adjusted IPI (aaIPI) was 2–3 for 54 patients (52%). The CNS-IPI score was retrospectively calculated. A low risk (score 0–1) was found in 40 (39%) patients, an intermediate risk (score 2–3) in 53 (51%), and a high risk (score ≥ 4) in 10 (10%) patients.Histological subtypes were DLBCL or transformed follicular lymphoma for 79 patients (77%), PMBL for 19 patients (18%), T cell lymphoma (NOS or ALK anaplastic lymphoma) for 5 patients (5%). At presentation most of the patients had a good performance status , and 8 (8%) a CHOP associated with anti-CD20 for 6 patients. Based on Cheson criteria 2014 [Regarding the chemotherapy induction regimen, 95 (92%) patients received an ACVBP associated with anti-CD20 for 92 patients (89%)  were grade I/II and 18 (16%) grade III/IV. Thirty-three (32%) patients presented no toxicity.n = 78 toxicities, 71%), including 67 toxicities grade I/II, and 11 grade III/IV, and 32 after cycle 2 including 25 grade I/II and 7 grade III/IV ; this toxicity was mainly an increase in blood liver enzymes. Among them, 14 toxicities were grade I, 14 grade II, and 8 grade III. There were no grade IV hepatic toxicities. Hepatic toxicity occurred mainly after the 1st cycle . Four patients presented hepatic toxicity after both 2 cycles, including 1 that presented grade III hepatic toxicities after both. All hepatic toxicities were reversible in less than 15 days.n = 4) occurred in 11 patients (11%) with 8 grade I toxicities, 3 grade II toxicities, and no grade III/IV toxicities, mainly after the first cycle (9/11).Skin and mucosal toxicities including mucositis and conjunctivitis  patients presented a digestive toxicity with a total of 18 events. Digestive toxicity concerned 16 grade I, 1 grade II, and 1 grade III toxicities (reflex ileus). This occurred either after cycle 1 , or after cycle 2 . Three patients presented digestive toxicities after cycle 1 and 2.Twenty-three patients (22%) presented a hematological toxicity with a total of 26 events, either after cycle 1 (14 events) or after cycle 2 (12 events). Seventeen toxicities were grade I/II  and 9 were grade III/IV (8 neutropenia with only 1 grade IV and 1 patient with grade III anemia). Three patients presented a cytopenia after both cycles 1 and 2 and 1 patient presented a bicytopenia after cycle 1.Transient grade I paresthesia occurred in 9 patients (9%) mainly after cycle 1  patients. There was no grade III/IV neurological toxicities reported.Renal toxicity occurred in 4 patients (3.9%) and was grade I in 2 patients and grade II in 2 patients and after the first cycle for 3 of the 4 patients. There was no grade III/IV renal toxicity.The other toxicities included arthralgia grade I (1 patient), edema grade I (1 patient), and dyspnea grade II (1 patient).Overall, only 2 patients (2%) were hospitalized after systemic HD-MTX. The reasons were a grade III reflex ileus  for one patient, and one because of hepatic cytolysis .Among the 103 pts. of our study, 78 pts. remained in CR after 2 years of follow-up (76%). Five pts. in CR at the end of treatment were lost, with no follow-up. Twenty patients presented a relapse with 5 CNS relapses (4.9%)  and 15 others relapses (15%). Three CNS relapses occur less than 6 months after the end of treatment, 1 at 1 year of follow-up, and 1 after 3 years.All patients except for 11 (89%) received the 2 cycles of systemic HD-MTX as an ambulatory administration. Among these 11 patients, 7 received only 1 cycle of HD-MTX because of toxicities and 4 because of lymphoma progression (3 patients) and viral infection (1 patient). The toxicities that induced an arrest of treatment was a grade I/II renal toxicity in 2 patients, a cytolysis in 2 patients , a grade III neutropenia in 1 patient, and a grade III digestive toxicity in 1 patient.2 in these series [n = 103), supports a safe outpatient administration of HD-MTX in a highly selected population of patients, without MTX clearance monitoring, but with a very strict and careful monitoring of the renal and hepatic functions. Kidney function is a good indicator to monitor proper elimination of methotrexate [Results of our study highlighted the safety of outpatient HD-MTX administration associated with renal and hepatic monitoring only, in a highly selected population of patients aged less than 60. Overall, 89% of the patients completed the 2 cycles of HD-MTX, and 80% of the patients presented no toxicity or grade I/II toxicities. These results can be compared to other studies with patients with primary CNS lymphoma , or CNS e series . Only one series  reportedotrexate  and shouA key aspect of our work was to identify the selected population of patients eligible for an outpatient administration of H-MTX with no MTX clearance monitoring. This type of surveillance based on renal and hepatic monitoring is a novelty and requires a careful selection of the patient population. Young age less than 60, an albumin level ≥ 35 g/L, a good performance status (0–1), and a stable weight (< loss of 10% compared to baseline) are key parameters to avoid MTX clearance abnormalities , 32. As The number of any grade toxicity has been observed to be more frequent after cycle 1 than after cycle 2 of HD-MTX. This can be explained by the cumulative toxicity of prior cycles of immunochemotherapy during the induction period. A majority of patients received 4 cycles of R-ACBVP or 4 to 6 cycles of R-CHOP before the HD-MTX administration. Toxicity may be cumulative during this first cycle, especially for hematological and mucositis toxicities , 33. To DLBCL is a very aggressive lymphoma and most patients had a poor quality of life during the treatment, often related to repeated hospitalizations. Thanks to outpatient administration of HD-MTX, patients spent more time at home with a lower impact on their social functioning. Younger patients with DLBCL presented worse quality of life scores than more elderly patients, mainly because of social functioning alterations , 37. ShoIn conclusion, we demonstrated that HD-MTX outpatient administration based on renal and hepatic monitoring only was feasible and safe in a selected population of patients. The selection is based on very practical and simple criteria. The organization necessary for this treatment can be easily adapted to different hospital settings.


APOE ε4 is the major genetic risk factor for Alzheimer’s disease (AD). A precise role for apolipoprotein E (apoE) in the pathogenesis of the disease remains unclear in part due to its expression in multiple cell types of the brain. APOE is highly expressed in astrocytes and microglia, however its expression can also be induced in neurons under various conditions. The neuron-like cell line SK-N-SH is a useful model in the study of the cellular and molecular effects of apoE as it can be differentiated with retinoic acid to express and secrete high levels of apoE and it also shows the same apoE fragmentation patterns observed in the human brain. We previously found that apoE is cleaved into a 25-kDa fragment by high temperature-requirement serine protease A1 (HtrA1) in SK-N-SH cells. To further understand the endogenous functions of apoE, we used CRISPR/Cas9 to generate SK-N-SH cell lines with APOE expression knocked-down (KD). APOE KD cells showed lower APOE and HTRA1 expression than parental SK-N-SH cells but no overt differences in neuritogenesis or cell proliferation compared with the CRISPR/Cas9 control cells. This research shows that the loss of apoE and HtrA1 has a negligible effect on neuritogenesis and cell survival in SK-N-SH neuron-like cells. APOE ε4 allele is the major genetic risk factor for the disease [Alzheimer’s disease (AD) is the most common neurodegenerative disorder and the presence of the  disease . Apolipo disease ,3.in vitro mediated by the high temperature-requirement serine protease A1 (HtrA1) enzyme is allele selective, with increased fragmentation of apoE3 compared with apoE4 [ApoE is fragmented in the human brain in an isoform-specific manner  generatith apoE4 .HTRA1 knockdown in SK-N-SH cells reduced apoE 25, whilst incubation of recombinant apoE 25 in SH-SY5Y cells promoted neuritogenesis [The SK-N-SH neuroblastoma cell line has been previously used to test neuroprotective compounds against some AD-relevant features, such as amyloid β ,8, oxidaogenesis .APOE ε3/3 background of the SK-N-SH cells limits the model when trying to understand the apoE4 isoform and its fragmentation process. The aim of this research was to knock-down APOE expression using CRISPR/Cas9 genome editing to provide a cellular model for investigating the role of apoE and its effect on HtrA1 in SK-N-SH cells.The 2. The medium was changed every 2–3 days and cells were passaged when 80% confluence was reached. Cells were incubated with Trypsin (0.05% Trypsin-Ethylenediaminetetraacetic acid (EDTA)) (Life Technologies) for 3–5 min at 37°C and stopped using DMEM/F12 with 10% (v/v) FBS, after which cells were centrifuged at 800×g for 5 min and split 1:10.Human SK-N-SH neuroblastoma cells were obtained from American Type Culture Collection . Cell line identity was confirmed by short tandem repeat (STR) profiling at the Garvan Molecular Institute , with results matching ATCC HTB-11. Analysis for potential mycoplasma contamination of the parental SK-N-SH and the clones tested negative. SK-N-SH cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (v/v) (1:1)  supplemented with 10% (v/v) Foetal Bovine Serum (FBS)  and 100 IU/ml Penicillin, and 100 µg/ml Streptomycin  at 37°C and 5% COtrans retinoic acid (ATRA). Cells were plated at the density specified for each experiment using DMEM/F12 with 5% (v/v) FBS unless otherwise specified. Cells were grown for 24 h before ATRA treatment started, and full media changes were performed every 3 days until day 6 or 9 as specified in each experiment. Live cell imaging of the cultures, cell confluence and neurite outgrowth measurements over time were performed using the Incucyte Zoom Imaging System .SK-N-SH cells were differentiated into a neuronal phenotype with the addition of 10 µM all-APOE gene as shown in in silico on- and off-target score screening of gRNA1 was performed using the online platform Benchling [The CRISPR lentivirus plasmid with guide RNA (gRNA) sequence 5′-CTCGCCGCGGTACTGCACCAGG-3′ was designed by Sigma–Aldrich to target exon 4 of the enchling ,25.The plasmid, as shown in SK-N-SH cells were plated at 16,000 cells/well on a 96-well plate before treatment with puromycin (Sigma–Aldrich). The following puromycin concentrations were added in triplicate 24 h after seeding: 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0 µg/ml as control. Cell death was tracked using the Incucyte Zoom System every 2 h for a period of 6 days. After 5 days, all cells from the 2.5 µg/ml or higher concentration were dead, and only <5% of the 1.25 µg/ml had survived. Therefore, the concentration of 2 µg/ml was chosen to perform the antibiotic selection of cells after testing that all the cells died in a 5-day period.−2 to 10−6 dilution were performed as recommended by the manufacturer together with a no-lentivirus control well. Full media change with base media was performed 24 h after to remove the lentiviral particles from the culture. Antibiotic selection using 2 µg/ml puromycin started 48 h after removal of the lentiviral particles until all the cells in the control well were dead. Transduced cells were kept in culture without antibiotic to allow their expansion; cells were passaged into a six-well plate, allowing them to grow from individual cells into groups of ten colonies and then they were manually picked for monoclonal selection. Clones were cultured in 96-well plates and expanded into 24- and 12-well plates when they reached 80% confluency until there were enough cells to extract gDNA, harvest protein and make stocks.SK-N-SH neuroblastoma cells were plated at 100,000 cells/well in a six-well plate. Base media (DMEM/F12 media with 10% (v/v) FBS, 100 IU/ml Penicillin and 100 µg/ml Streptomycin) was supplemented with 8 µg/ml of polybrene to enhance the transduction efficiency. Serial dilutions of the CRISPR lentivirus over a range of 10APOE F (5′-CTACAAATCGGAACTGGAGGAAC-3′) and APOE R (5′- ACCTGCTCCTTCACCTCGTC-3′) and water up to 25 µl reaction. PCR was amplified in the MyCycler™ Thermal Cycler System  using the following program: initial denaturation at 95°C for 1 min, followed by 35 cycles of 15 s at 95°C for denaturation, 15 s at 66°C and 10 s at 72°C for extension and final extension at 72°C for 5 min.Genomic DNA was extracted using the ISOLATE II Genomic DNA Kit (Bioline). DNA concentration and purity were determined using the Nanodrop 2000C instrument. PCR was performed by mixing 100–200 ng DNA with 12.5 µl MyTaq™ HS DNA Polymerase, 10 µM of primers g for 1 min. Flow through was discarded, 600 µl wash buffer were added and centrifuged at 12000×g for 1 min. Flow through was discarded and columns were centrifuged at maximum speed for 2 min to remove any buffer. After transferring the column into a 1.5 ml microcentrifuge tube, 20 µl of elution buffer was added and samples were centrifuged at 12000×g for 1 min to collect the purified PCR product.Electrophoresis was performed using 4 µl of each sample mixed with GelRed Nucleic Acid Gel Stain and run on 1.5% agarose gel at 100 V for 30 min run along with Hyperladder 100 bp and visualised on Gel Logic 2200 Pro Imaging System. After confirming the specific DNA amplification of 489 bp, PCR products were purified using a modified protocol of Pure Link Quick Gel Extraction kit (Invitrogen): 16 µl PCR product were mixed with 20 µl water and 120 µl buffer L3, transferred into the columns and centrifuged at 12000×Sequencing reactions were performed by mixing 2 µl of 5× Sequencing buffer, 1 µl ABI BigDye Terminator v3.1 Ready Reaction Mix , 1 µl Primer F or R (2 µM), 4 µl water and 2 µl of purified PCR product. Samples were run on the MyCycler™ Thermal Cycler System following the program: 96°C for 30 s followed by 35 cycles of 96°C for 10 s, 66°C for 5 s and 60°C for 4 min.g for 20 min at 22°C. The supernatant was removed, the pellet was rinsed with 180 µl ice-cold 70% ethanol and the samples were centrifuged for 20 min at 4°C. The supernatant was removed and the samples were dried in the dark before being processed.Ethanol precipitation was performed by mixing 50 µl 100% ethanol, 10 µl water, 2 µl 125 mM EDTA pH 8.0, 2 µl 3 M sodium acetate pH 5.2 and 10 µl sequencing reaction. The samples were incubated at 22°C in the dark for 20 min after mixing before they were centrifuged at 16000×Samples were run in the 3500×L Genetic Analyzer (Applied Biosystems). SeqA6 Software was used to convert raw files into .ab1 files and electropherograms were analysed using BioEdit software.260/230 and A260/280 ratios. DNAse I treatment was performed using Turbo DNA-free kit (Invitrogen) following the manufacturer’s instructions. The mRNA was purified by ethanol precipitation: mRNA was diluted up to 200 µl RNAse-free water and mixed with 20 µl of 3 M Na acetate and 220 µl isopropanol. After vortexing, samples were stored at −80°C for 1 h and then centrifuged at 16000×g for 20 min at 4°C. The supernatant was discarded, and the pellet was rinsed with 70% ethanol before being centrifuged again at maximum speed for 2 min. The supernatant was removed, and the pellet was dried before it was resuspended in RNAse-free water to yield a final concentration between 100 and 500 ng/µl. Purity and concentration of the isolated mRNA were measured using the Nanodrop 2000C.Cell samples for mRNA extraction were washed twice with 1× Phosphate Buffered Saline (PBS) before they were harvested in TriSURE (Bioline). Samples were kept at −80°C until mRNA was extracted, following the manufacturer’s instructions. The mRNA concentration was measured using the NanoDrop 2000C and purity was assessed based on the A18 and incubating samples for 30 min at 45°C followed by 30 min at 85°C in MyCycler Thermal Cycler. The samples were stored at −20°C until use.Up to 5 µg of mRNA were reverse transcribed to generate complementary DNA (cDNA) using Tetro cDNA Synthesis kit (Bioline). The protocol was followed as per manufacturer’s instructions using Oligo (dT)Ct values were in the range between cycles 20 and 32, for APOE and HTRA1, and between cycles 15 and 22 for the housekeepers, GAPDH and HPRT1.Each cDNA sample was run in duplicates or triplicates using between 25 and 50 ng cDNA per reaction. A ‘no reverse transcriptase’ RNA control and ‘no template’ control were also run as negative controls. The qRT-PCR machine LightCycler 480  or RotorGene 3000  were used following the program: denaturation at 95°C for 2 min, followed by 35 or 40 cycles of denaturation at 95°C for 10 s, annealing at specific primer Tm for 10 s and extension at 72°C for 10 s. The melting step started at 95°C for 5 s followed by ramp from 65 to 97°C at 0.1°C/s before cooling. Primer sequences are shown in CT (ΔΔCt) method using the efficiency obtained for each set of primers. GraphPad Prism 7 was used to generate graphs and perform statistical analysis.Raw data from LightCycler 480 or RotorGene 3000 instruments were extracted and analysed using LinRegPCR  to obtaig for 5 min to remove cell debris and dead cells, transferred into a 1.5 ml microcentrifuge tube and stored at −80°C until use.Conditioned medium from cell culture was collected to analyse extracellular proteins released into the media. The conditioned medium was collected directly from the plate, centrifuged at 10000×g for 5 min. The supernatant was transferred into a 1.5 ml microcentrifuge tube avoiding the small debris pellet at the bottom of the tube. The cell lysate was stored at −80°C until use.Intracellular protein was harvested in RIPA buffer  mixed with 1× protease inhibitor cocktail  and 0.1 M phosphatase inhibitor sodium orthovanadate. Once conditioned medium was collected, cells were rinsed with 1× PBS and incubated with 100 µl RIPA buffer per well of a 24-well plate for 20 min on ice. Cell lysate was vortexed for 10 s and centrifuged at 10000×Total protein quantification was performed using the DC Protein Assay (Bio-Rad), following the manufacturer’s instructions. Absorbance was measured at 750 nm using a Spectramax Plus Plate Reader . The sample concentration was calculated based on a standard curve generated using Bovine Serum Albumin (BSA) (Sigma–Aldrich) diluted in RIPA buffer at concentrations of 0, 200, 400, 600, 800 and 1200 µg/ml.Samples were mixed with 5× Laemmli Sample Buffer (Bio-Rad) and proteins were denatured for 5 min at 95°C. Samples were loaded into SDS/PAGE gels along with the Precision Plus Protein Dual Color (Bio-Rad) molecular weight marker and separated by electrophoresis at 120 V for 80 min. Protein samples were transferred to 0.45-µm nitrocellulose membranes at 100 V for 35 min using ice-cold 1× Western blot transfer buffer. Successful transfer and equal sample loading were determined by staining the membranes with Ponceau S Solution Bioreagent (Sigma–Aldrich) for 2 min and the excess stain was washed off with distilled water. The membranes were scanned on Epson Perfection 3170 Photo , then washed off for 5 min with 1× PBS-0.1% (v/v) Tween 20 (PBS-T) followed by incubation for 5 min with boiling 1× PBS and another 5 min with 1× PBS-T. Blocking for 1 h with 5% (w/v) milk in 1× PBS was performed before incubation with primary antibodies made up in 5% (w/v) milk in 1× PBS and 0.001% (v/v) sodium azide. Antibody details are shown in LI-COR Image Studio was used for quantification of Western blot bands, Excel was used for data analysis, and GraphPad Prism 7 was used to generate graphs and perform statistical analysis.APOE knock-down SK-N-SH cell lines to compare with the parental APOE ɛ3/3 SK-N-SH cells.The SK-N-SH neuroblastoma cell line is a useful model to study apoE fragmentation (as reviewed in ). To furin silico on- and off-target screening of the gRNA sequence was performed. The prediction of the on- and off-target specificity scores range from 0 to 100, where higher values indicate a more specific sequence [TMEM175 gene had a score of 1.43 and 4 mismatches in the sequence and a non-coding region in chromosome 1 obtained a score of 1.41 and 3 mismatches, compared with the APOE gene having a score of 100 and no mismatches in the sequence. This indicated a good specificity of the selected gRNA to proceed.To predict the gRNA specificity, an sequence ,25. The The pmax-GFP vector was used as a control for positive lipofectamine transfection, giving a 21% transfection efficiency after 24 h, and puromycin selection was performed until 100% of the cells in the control condition were dead (data not shown).APOE sequence  were further characterised to confirm an APOE knock-down (KD) after the DSB. After neuronal differentiation with 10 µM ATRA, the parental SK-N-SH cells up-regulate apoE expression [APOE KD were analysed via qPCR after ATRA treatment (or DMSO as a vehicle control) for 6 days  and in the conditioned medium (extracellular apoE) in the selected clones . The parondition B.The SK-N-SH cells and the selected clones 1–4 showed a similar morphology before differentiation . After 6HTRA1 levels were analysed in parental SK-N-SH cells and the selected CRISPR clones after 6 days of ATRA treatment or DMSO control  were differentiated along with 4 CRISPR control clones  that had undergone the CRISPR process and the monoclonal selection but had no alteration in the APOE sequence (APOE KD clones) and SH-SY5Y cells showed only neuroblastic morphology, whereas SK-N-SH and CRISPR control clone 1 (representative of CRISPR control clones) showed a combination of neuroblastic and substrate-adherent cells  and 4 CRISPR control clones was performed as shown in APOE KD clones showed a significant reduction in intracellular apoE when compared with the parental SK-N-SH cells; however, as expected, the CRISPR control clones did not show a significant reduction  has been published as a cell line to study the fragmentation of apoE [APOE in this cell line thus provides a cell model to understand the role of apoE. CRISPR/Cas9 technology has revolutionised genome-editing over the last decade. Compared with previous genome-editing techniques such as zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR is considered to be a simpler and more flexible method with a high editing efficiency [TMEM175 obtaining 1.43 and had 4 mismatches in the sequence, that compared with APOE gene having a score of 100 and no mismatches indicates high specificity to bind the APOE gene over other sequences of the genome [APOE expression. The four selected clones showed a reduction in APOE expression after ATRA differentiation, and the KD was confirmed by a reduction in protein level  [Previous studies have modified the expression of apoE in mouse models  ). Some oc system , learninc system  or cholic system . Expressneration . One of neration . Moreoveneration . In thisneration . Based o. (2018) . Thus, wAPOE ɛ4 allele is the major genetic risk factor of AD [APOE knock-down clones generated here showed a reduction in HTRA1 expression concomitant with a reduction in APOE expression (APOE knock-down clones generated in this study can be used for such studies.The presence of the or of AD ,38 and aor of AD . Howeverpression  and 5. UAPOE KD expression that can be used as models to study apoE function, including fragmentation by exogenous addition of apoE isoforms and the interaction between apoE and HtrA1.In conclusion, we used CRISPR/Cas9 technology to generate different clones of SK-N-SH cell lines with