Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative  strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus  and Bacillus subtilis 168 , compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213  and A0A1Q9FRD3 . The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database. In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive ( Lysozyme is an enzyme also known as muramidase, 1,4-β-N-acetylmuramidase, or N-acetylmuramide glycanhydrolase. It is ubiquitous in animals, plants, and microorganisms , and is Food quality plays a very important role in the world, because substandard food has caused public health problems all over the world. There are many diseases that are spread by unsafe food. They range from diarrhea to various forms of cancer and foodborne diseases. These diseases cause enormous socioeconomic problems for societies ,15,16,17Bacillus is considered the most suitable for the mass production of lysozyme [Bacillus spp., Bacillus subtilis is the most popular species for higher lysozyme production. This is due to its excellent fermentation properties and tremendous ability to secrete proteins (lysozyme) directly into the growth medium, which offers great advantages in downstream processing, and it is nonpathogenic and free of endotoxins [Among all bacteria, the genus lysozyme ,29,30,31dotoxins ,33.Bacillus) were isolated from the soil samples. Following fermentation, various parameters were applied to the Bacillus species to determine the maximum lysozyme production. It was found that Bacillus subtilis BSN314 was able to achieve the highest lysozyme production [Bacillus subtilis BSN314 was purified by salt (ammonium sulfate) precipitation and dialysis methods and characterized by the bactericidal activity of lysozyme. The disintegrating effect of the purified lysozyme was investigated by atomic force microscopy (AFM). The pure form of the lysozyme was isolated, leading to the following three hypotheses for our further studies: (1) Following purification, the enzymatic activity of BSN314 lysozyme should remain higher. (2) The purified lysozyme should exhibit bactericidal action against different pathogenic bacterial strains. (3) Some specific peptides should be present in the purified lysozyme, which are responsible for the enzymatic and bactericidal activities. To evaluate these specific peptides, the extra purified lysozyme was subjected to a liquid chromatography-mass spectrometry (LC-MS) analysis. This study shows the significant importance of the extra-purified lysozyme isolated from Bacillus subtilis BSN314, which could be used in the pharmaceutical and food industries.This study is an extension of our previous study. In the previous study, the lysozyme-producing probiotic bacterial species (oduction . In the 4)2SO4, the amount of purified lysozyme and enzymatic activity were measured  and minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC μg/mL) against both the G-positive and G-negative bacterial strains are shown in Micrococcus luteus (17 mm ZOI), while the lowest activity was measured against both gram-negative species, i.e., Pseudomonas aeruginosa and Salmonella typhimurium (12 mm). The minimum MIC/MBC was found to be 1.25 μg/mL against Micrococcus luteus, while the maximum MIC was measured against Salmonella typhimurium (2.75 μg /mL). To compare the antibacterial activity with the antibiotic (ciprofloxacin), two indices were used: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus  and Bacillus subtilis 168 , as compared to the antibiotic (ciprofloxacin) used in this study.The ultra-purified lysozyme was used for a further antibacterial assay, and atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified lysozyme sample was subjected to antimicrobial activity against five bacteria: To observe the comparative effect of the purified lysozyme on the bacterial strains, the atomic force microscopy (AFM) technique was used. The disintegrating effect of lysozyme on the cells of the bacterial strains is shown in For the evaluation of the different peptides found in the lysozymes, further assessments continued. For this purpose, the resulting protein solution was purified by gel column chromatography. The eluate was collected and its enzyme activity was assessed as 21.93 U. Following the gel column chromatography, the lysozyme was further processed by a native-PAGE analysis, and by this technique, the protein/lysozyme was obtained, whose enzymatic/cell lytic activity was determined to be 40.9 U/mL. This shows that the obtained protein was not denatured at this stage of purification, and the protein (lysozyme) still has cell lytic properties. Following the determination of the cell lytic activity of the purified lysozyme, the next step was to assess the composition of the lysozyme using various techniques, e.g., SDS-PAGE and LC-MS. Following the gel column chromatography, the lysozyme was also analyzed by the SDS-PAGE technique. The SDS-PAGE analysis revealed three protein bands, indicating that the BSN314 lysozyme was purified to a monomeric protein . The resThe bands of BSN314 lysozyme obtained by the SDS-PAGE analysis were cut-off, precipitated with acetic acid, enzymatically digested, desalted, and then analyzed by LC-MS. The chromatogram is shown in MVNIIQDFIP VGANNRPGYAMTPLYITVHNTANTAVGADA AAHASYLKNP DTATSWHFTVDDTEIYQHLPLNENGWHAGDGNGSGNRASIGIEICENADGDFAQAANAQLIKTLMSEHNISLANVVPHKYWSGKECPRKLLDTWDSFKAGIGGGGSQTYVVKQGDTLTSIARAFGVTVAELQEWNNIEDPNLIQVGQVLIVSAPSYAEEPELYPLPDGIIQLTTPYTSGEHVFQVQRALAALYFYPDKGAVNNGIDGIYGPKTADAVARFQSVNGLTADGIYGPTTKAKIAAQLS. MNIKKCKQLLMSLVVLTLAVTCLAPMSKAKAASDPIDINASAAIMIEASSGKILYSKNADKRLPIASMTKMMTEYLLLEAIDQGKVKWDQTYTPDDYVYEISQDNSLSNVPLRKDGKYTVKELYQATAIYSANAAAIAISEIVAGSETKFVEKMNAKAKELGLTDYKFVNATGLENKDLHGHQPEGTSVSEESEVSAKDMAILADHLITDYPEILETSSIAKTKFREGTDDEMDMPNWNFMLKGLVSEYKKATVDGLKTGSTDSAGSCFTGTAELNGMRVITVVLNAKGNLHTGRFDETKKMFDYAFDNFSMKEIYAEGDQVKGHKTISVDKGKEKEVGVVTNKAFSLPVKNGEEKNYKAKVTLNKDTLTAPVKKGTKVGKLTAEYKGDEKDYGFLNSNLAGVDLVTKENVEKANWFVLTMRSIGGFFAGIWGSIVDTVTGWF.B. subtilis is of great interest to the secretome because it has a powerful machinery to produce proteins that are secreted from the cell and are free of toxins [B. subtilis without denaturation. In this study, a multi-step methodology was used to purify the lysozyme product (protein) biosynthesized from the previously isolated strain Bacillus subtilis BSN314 [4)2SO4 was added to the aqueous protein solution at a low concentration, the solubility of the proteins in the solution gradually increased due to the ion concentration of the ammonium sulfate, which is called the salting-in effect. As the addition of (NH4)2SO4 increases, the solubility of the proteins begins to decrease, resulting in protein precipitation. Now, more protein is precipitated from the solution while less protein is soluble. This is called the salting-out effect [Currently, f toxins ,36,37. Rs BSN314 . This mus BSN314 ,38. A his BSN314 . Howevers BSN314 . Therefot effect ,41. The t effect . Bacillus subtilis BSN314 was purified in three sequential steps: sulfate precipitation, dialysis, and ultrafiltration. To determine the bactericidal potential of the purified lysozyme, the purified protein/lysozyme was used for bactericidal activity against some pathogenic bacteria . The results of ZOI and MIC/MBC showed that lysozyme had a higher antibacterial activity against gram-positive , rather than gram-negative bacteria . In the previous literature, the same kind of results were recorded for the bactericidal activity of lysozyme [Lysozyme produced by lysozyme ,44,45,46lysozyme ,12,13. Alysozyme . Atomic lysozyme . The baclysozyme ,50,51.The resulting purified protein (lysozyme) was further processed by gel column chromatography and then further processed to isolate the un-denatured protein by native-PAGE analysis. The purified/isolated lysozyme was found to still have cell lytic/enzymatic activity. To separate the expected peptides responsible for the biological activities , the SDS-PAGE technique was used (to separate the peptides with masses between 5 and 250 kDa). The sequences of the primary protein standards were monitored by SDS-PAGE . FollowiBacillus subtilis BSN314 lysozyme might be responsible for the antibacterial and enzymatic activities. They prevent the growth of bacteria. The results of antibacterial activity, the AFM analysis, and cell lytic activity indicated that BSN314 lysozyme has a strong potential against bacteria and can disintegrate the bacterial cell. The same type of observation was made in previous studies [The PSM data showed that among the 309 matched peptide spectra, only two components with molecular masses 31.2 kDa and 48.6 kDa, were considered particularly relevant. These two components were; A0A1Q9G213  and A0A1Q9FRD3 . They may be responsible for antibacterial and enzymatic activities ,55,56. T studies ,58,59. P studies ,63,64,65glacial acetic acid were purchase from Beijing Chemical Plant. While yeast extract PL0021  and soy peptone  were purchased from Beijing Aoboxing Biotechnology Co. Ltd. . Analytical grade sodium chloride (≥99.5%), sodium dihydrogen phosphate dodecahydrate (≥99.0%), disodium phosphate (≥99.0%), sodium hydroxide (≥98.0%), ammonium sulphate (≥99.0%), ethanol, 5× protein loading buffer, and Agar  was provided from CHEMBASE Company ; 50 × TAE, Tris-HCl, glycine, SDS, Coomassie brilliant blue, IPTG, kanamycin, agarose gel electrophoresis, and surfactant Tween 20 and Tween 80 were purchased from Biotopped Technology Co. Ltd . Analytical pure 5 × non-denatured protein loading buffer, 30% acrylamide, and 10% ammonium persulfate were purchased from Fujian Ming Lin Technology Co. Ltd . While tetramethylethylene diamine (TEMED) ≥ 99%, 1.5 M Tris-Hcl (pH 8.8), 1.0M Tris-Hcl (6.8), non-denatured protein running buffer, a BCA protein assay kit, were provided by Biyuntian Co..Protein marker was purchased from Thermo Fisher Scientific . SDS-PAGE gel preparation kit, DNA Marker DL 2000, and DL 5000 were purchased from Beijing Zhuangmeng International Biogenomics Division Technology Co. Ltd. . Nucleic acid dyes T4 was obtained from TAKARA Company  and DNA ligase, BamHI restriction enzyme, XhoI restriction enzyme, and DNA agarose gel recovery kit from OMEGA company .TIANamp Bacteria DNA Kit (DP302) was purchased from Tiangen Biotech. (Beijing) Co. Ltd., while analytical grade agarose was obtained from Biowest , and SDS-PAGE kit from Beijing Zhuangmeng International Biogenomics Division Technology Co. Ltd. Glucose anhydrous (≥99.0%), glycerin, and hydrogen peroxide (≥35.0%) were purchased from Sinopharm Chemical Reagent Co. Ltd. , while Ex Red and DL2000 plus were purchased from Beijing Zhuangmeng Bio. PCR pre-mix (Prime STA R HS) was purchased from Takara Bio Company .An analytical balance (model No. BSA 224S), manufactured by Sartorius Scientific Instruments  Co., Ltd.; an ultra clean work table, type SW-CJ-1FD, made by Sujing Group Suzhou Antai Air Technology Co., Ltd.; a high-pressure steam autoclave (model No. HVA-100), made by Hirayama ; a pH meter (model No. FE20) equipped with a Lab Pure Pro-ISM probe, made by Mettler instruments Shanghai Co., Ltd.; a visible spectrophotometer (model No. 722), made by Shanghai Jinghua Technology Instrument Co., Ltd.; a mini spin centrifuge (model Eppendorf AG 2231), made by Germany; a nucleic acid electrophoresis apparatus (model No. DYY-6C), produced by the Beijing Liuyi Instrument Factory; a gel imaging system (model No. UVP Bio Spectrum 510), supplied by Ultra-Violet Products Ltd. Cambridge, UK; a bench shaking incubator (model No. IFORS AG CH-4103), made in Bottmingen Switzerland; a thermal cycler type PCR (model No. S1000) instrument, made by Bio-Rad , and a microwave digestion system (model No. 910980) were used for this study. Furthermore, a benchtop refrigerated centrifuge (model no CT15RE), made by HITACHI , a biochemical incubator (model Bluepard), manufactured by Shanghai Yiheng Scientific Instrument Co., Ltd.; a magnetic stirrer (model RCTBS025 IKA) and dialysis bag, manufactured by Beijing Boya Red Star Biotechnology Co. Ltd.; a protein purifier (model No 25), made by AKTA Pure company; an Infinite microplate reader (model No M200PRO) from Switzerland TEC company; a decoloring shake, made by Beijing Liuyi Instrument Factory; a protein electrophoresis system, by BIO-RAD ; an ice maker (model No FM40), produced by the snow ice making company, and an ultrasonic disruptor, manufactured by Galanz , were also used in this study.2HPO4, and Tween-20 0.1%. For the solid medium: 1.8% (w/v) agar powder was added. While the LB liquid medium was prepared by adding 0.5% (w/v) yeast extract, 1% (w/v) peptone, and 1% (w/v) sodium chloride. All of the media were sterilized at 121 °C for 20 min. For the kanamycin solution, 50 mg/mL mother liquor was prepared, after that it was diluted with sterile distilled water until a 50 µL/mL concentration was obtained. The modified medium (MM) ingredients were; 0.5% yeast extract, 2.5% soy peptone, 1.5% glucose, 0.5% KA 5 × Tris-glycine buffer solution was made by mixing 15.1 g Tris, 94 g glycine, and 5 g SDS; it was then stirred with 1 L of ionized water and then stored at room temperature. A phosphate buffer (PB) was prepared by mixing of 1.217 g of sodium dihydrogen phosphate dihydrate, 4.370 g of disodium hydrogen phosphate dodecahydrate, and 1 L of deionized water, to obtain 20 mM phosphoric acid with pH 7.0 buffer. While the phosphate buffered saline (PBS) was formulated by adding 0.609 g sodium dihydrogen phosphate dihydrate, 2.185 g disodium hydrogen phosphate dodecahydrate, 29.220 g sodium chloride, and 500 mL deionized water, to obtain 20 mM phosphoric acid with pH 7.0. The Coomassie brilliant blue staining solution was prepared by adding 0.1 g Coomassie brilliant blue R-250, 25 mL of isopropanol, and 10 mL of glacial acetic acid. Then, it was stirred well, to make a constant volume of 100 mL, while the Coomassie brilliant blue staining decolorizing solution was made with 100 mL acetic acid and 50 mL ethanol. Then, 850 mL deionized water was added and the solution was then stored at room temperature. Then, 24.2 g Tris, 5.7 mL glacial acetic acid, and 10 mL 0.5 mol EDTA, were added to the prepared agarose gel electrophoresis buffer, and deionized water was added to make a volume of 100 mL. Bacillus subtilis BSN314 (which was able to produce the higher rate of lysozyme) was used for the isolation and purification of lysozyme, and Micrococcus luteus was used for the cell lytic/enzymatic activity of the purified lysozyme. These two strains were stored at −80 °C. For activation, the culture was taken, spread on Luria agar (LA) medium and allowed to grow at 37 °C for 24 h. A single colony was selected for further inoculation in 4 mL of Luria broth (LB) medium in a 10 mL shaking tube, and incubated for 24 h at 37 °C and at 220 rpm. To obtain a second generation of cultures, 1 mL of the inoculum was transferred to 100 mL of the LB medium and incubated for another 24 h under the same growth conditions [The previously isolated strain nditions . Bacillus subtilis BSN314 culture was transferred into a 250 mL sterilized flask which contained the 100 mL MOM medium , and incubated at 37 °C, 220 rpm for 24 h. The broth culture was centrifuged at 8000 rpm for 20 min to remove the bacteria, the supernatant was used for following steps. Ammonium sulfate precipitation, dialysis, ultrafiltration, and gel column chromatography methods were designed for this study to obtain the maximum product and high level of lysozyme produced by Bacillus subtilis BSN314. Initially, 1 mL of Bacillus subtilis BSN314 (at 4 °C). A magnetic stirrer was used to increase the saturation of ammonium sulfate in the solution to 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. Then, the solution was centrifuged at 1000 rpm for 30 min under refrigerated conditions, and the precipitate was carefully collected and dissolved in phosphate buffer solution (PB). Protein concentration was determined by the bicinchoninic acid (BCA) method [Isolation of lysozyme was performed according to the method described by Chen et al., 2019) . This me) method ,67 and e019 . Thi) method ,69. The protein solution contained ammonium sulfate, which was removed by the dialysis bag method . A 15 cmIn this method, the lysozyme product was concentrated using an ultrafiltration (UF) 10 KD tube  . The UF For further purification, an AKTA Purifier 25 system  was used to isolate lysozyme. The sample (10 KD) collected from the ultrafiltration (UF) tube was loaded into a HiTrap TM Desalting  gel column chromatography. The column was fully equilibrated with PB solution pH 7.0 to purify the lysozyme ,72. The The total protein content from the purification was measured using the modified colorimetric method ,67 usingThis is a technique used to separate proteins, based on their shape, size, mass, and charge. There are two types: native PAGE and sodium dodecyl sulfate (SDS) PAGE. Native-PAGE is an electrophoretic technique that separates proteins, based on their size and charge. A gel kit purchased from Beijing Zhuangmeng International Bio Genomics Division Technology Co. was used for this analysis. Preparation of the native-PAGE gel began with making a 12% separation glue to which 10% ammonium persulfate was added and immediately poured in the glue. We left a height of about 1.5 cm for sample loading. Then, 75% ethanol was poured between the two plates to remove air and promote the solidification of the separating gel. We prepared a 5% concentrated adhesive, added the appropriate 10% ammonium persulfate, and mixed well. Then, we poured the adhesive and immediately inserted the comb. Prior to loading the samples into the gel hole, it was evenly mixed with 5× non-denatured protein loading buffer, and electrophoresis was performed at a constant voltage of 100 V. The protein obtained from the native-PAGE can be used for the determination of cell lytic/enzymatic activity [w/v) acrylamide separating gel and a 5% (w/v) stacking Quick SDS-PAGE Gel Preparation Kit, according Laemmli’s method [SDS-PAGE is a separation technique that separates proteins on the basis of their mass. SDS-PAGE is used to monitor the purification process and to determine the homogeneity and apparent molecular mass of the purified lysozyme. SDS–PAGE was performed on an electrophoresis apparatus , with a 12%  = 0.077 g/mL), then we discarded the supernatant.BSN314 lysozyme samples were processed and enzymatically digested by the SDS-PAGE gel. Using a scalpel blade to cut the target bands, we cut 3 small pieces, 1 mm in size, and transferred them in a 1.5 mL EP tube 600 μL. Decolorizing solution was added to each tube to decolorize the sample, the samples were washed until transparent, and then we removed the supernatant. Then, 300 μL of 100% ACN ThermoMixer was added, shaken for 5 min until the gel particles turned white, we sucked off the ACN, and freeze-dried the samples for 3 min. For the protein reduction, 300 μL of 10 mM DTT/50 mM NH4HCO3 to each tube. Each tube was shaken and the samples were mixed well and then incubate for 30 min in the dark until the gel block became swollen and transparent (600 mM iodoacetamide (IAA) = 0.111 g/mL). Once the gel particles were again dried by adding 300 μL of 100% ACN Thermo Mixer, and shaken for 5 min until the rubber particles turned white, we sucked off the ACN, and freeze-dried the samples for 3 min. Then, 50–80 μL of 50 mM ammonium bicarbonate solution was transferred to each tube, then 1–2 μg of pancreatin was added, and we squeezed the gel with the help of a glass rod. Prior to digestion, the sample was incubated at 37 °C for more than 6 h. The samples were dried by adding 300 μL of 100% ACN ThermoMixer and shaken for 5 min until the glue particles turned white, we sucked off the ACN, and freeze-dried the samples for 3 min. Then, we added 300 μL 60 mM IAA/50 m NHThe samples were digested with trypsin, which was re-suspended in 10 mM acetic acid, the recommended concentration is 0.25 μg/μL, and the optimal pH of trypsin was 7–8.5. Therefore, after adding trypsin, 1 μL of protein solution was taken to measure the pH. Then, 200 μL of acetonitrile containing 0.1% formic acid (FA) was transferred to each EP tube, shaken for 5 min, and the supernatant was transferred to a clean 1.5 mL EP tube. Then, 30 μL of 0.1% FA was added into the gel, shaken it for 5 min, and 200 μL of 0.1% FA containing acetonitrile was added and the solution was shaken for 5 min. The supernatant was aspirated and both supernatants were combined in a 1.5 mL EP tube. The sample was concentrated by using a centrifugal concentrator at room temperature. 18-resin  to obtain the concentration of, desalting, and obtaining the elution of the peptides for the LC-MS mass analysis [18- resin tip on the pipette, we wet the dry C18-resin by aspirating the wet solution containing 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile and dispensing it to the waste. We repeated this 5 times. Then, we equilibrated the C18-resin for peptide binding by aspirating the equilibrium solution containing 0.1 TFA and dispensing it to the waste. We repeated this 5 times. The sample peptide was bound to the C18-resin by the repeated aspiration and dispensed in the same vial 10 times. Following the binding, next step was to wash away the salts from the protein peptide by aspirating the wash solution, containing 0.1% TFA and dispensing it to the waste. It was repeated 5 times to remove the maximum amount of salts. The elocution of the peptide was achieved by the 5 repetitions, then slowly aspired and dispensed in the same 180 µL elocution buffer vial, containing 0.1% formic acid. Desalination of the sample was carried out using Canalysis . Brieflym/z 350–1500; the product ion scan range: started from m/z 120; the max IT: first level 20ms, second level 45 ms; the resolution was at: level 1 60,000@m/z 200, level 2 15,000@m/z 200; the data-dependent MS/MS: top 20; isolation window: 1.6 Da; collision energy: 27%. The high performance liquid chromatography (HPLC) EASY-nLC™ 1200 combined with mass spectrometry (MS) was applied for the proteomic data acquisition. The digested samples (2 μg) were loaded to the column  with the flow rate of 300 μL/min. The mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in 80% acetonitrile), were used as gradients, as shown in Protemo Discoverer 2.2 software was used for data processing and Uniprot was used for the analysis of the protein library. As a condition, trypsin was digested with a maximum of 2 missing sites; fixed modification: C (+57.021), variable modification M (+15.995) N (+42.011); the mass deviation for MS1 was 10 ppm, MS2 was 0.02 Da; FDR 1%. The antimicrobial activity was determined by using the well diffusion method. The supernatant obtained from the ultrafiltration method (pure lysozyme), was used for the antibacterial assay. Using the freeze-drying method, the supernatant was obtained in the form of powdered metabolites, which were used for further antibacterial activity ,76. Bacillus subtilis 168, Bacillus cereus, and Micrococcus luteus, and two gram-negative species, Pseudomonas aeruginosa and Salmonella Typhimurium were used. These bacterial strains were indigenously obtained from the clinical source. For the susceptibility tests, the standard solution of ciprofloxacin (10 μg/mL) was used. This drug was used as the positive control, deionized water was used as the negative control. The zones of inhibition (ZOI), produced by the lysozyme were compared with ZOI (mm) of the standard drug (positive control). Around 3 gram-positive bacteria, including; g and the supernatant removed. A cell suspension equivalent to 0.5 McFarland standard (1.5 × 108 CFU/mL), was prepared in a 5.0 mL sterile physiological saline solution. Confluent lawns were made on fresh MHA and Sabouraud dextrose agar (SDA) plates and allowed to diffuse for 5–10 min. The wells were dug (6 mm), stock solutions of 10 μL purified lysozyme were dispensed into them and allowed to diffuse into the media for 15–20 min. The plates were incubated at 37 °C for 24–48 h [The selective bacterial strain cultures were grown overnight in Mueller–Hinton agar (MHA) and Sabouraud dextrose broth , then centrifuged at 9000×  24–48 h ,76. For A modified dilution method was used for the determination of MIC and MBC. Lysozyme was diluted at different concentrations  in sterile nutrient broth in test tubes. Using a sterile wire loop, a culture of each bacterial strain was inoculated into test tubes containing 1 mL of the various concentrations of lysozyme in the nutrient broth. The tubes were incubated at 37 °C for 24 h and growth was observed by turbidity. At the concentration where no growth/turbidity occurred, a sterile culture swab stick was dipped into the broth from each test tube and was loaned to the agar culture plates. Equal volumes of sterile nutrient broth were added to the test tubes containing broth media and incubated at 37 °C for 24 h . The tubThe AMI was calculated by the following formula;AMI values > 1 indicating extract is more active than the standard drug (antibiotic) while the AMI < 1 indicating extracts are less active than the standard drug ,76It is the ratio between the zone of inhibition by the pure lysozyme to the zone of inhibition of the antibiotic, and multiplied by 100.tibiotic .Bacillus subtilis 168, Bacillus cereus, and Micrococcus luteus) and gram-negative  strains were used for the atomic force microscopy (AFM) analysis. The selected bacterial cells were collected from each of the activated stock cultures in an Eppendorf (1.5 mL) tube, centrifuged at 8000 rpm for 5 min, and the bacterial residues were collected and the supernatant discarded. The centrifugation process was repeated three times to obtain sufficient cells. The effective concentrations of purified lysozyme were prepared (same as used for MIC/MBC) in the deionized water, added to the isolated cells of each strain and allowed to stand in a shaking incubator (100 rpm) for 3 h. In the present research work, the gram-positive (The atomic force microscopy (AFM) analysis was performed according to the method of Usmani et al., (2021)  with somMicrococcus luteus was used as substrate. It was cultured in a Luria broth (LB) medium in a shaker tube. The speed of the shaker tube was set at 200 rpm while it was incubated at 37 °C for 24 h. Following the incubation period (24 h), the culture was added to the centrifuged tubes and was centrifuged at 8000 rpm for 10 min. The residue and supernatant were obtained and the supernatant was discarded. The residue containing the pellets of Micrococcus luteus was poured into a phosphate buffer solution (pH 6.2). Then, 2 mL of this solution was added to a cuvette, and the optical density (OD) was adjusted to 0.8 (OD450).Then, 0.5 mL of the selected bacterial strain (sample) was taken from the fermentation broth culture and placed in the cuvette that already contained 2 mL of Micrococcus luteus and the PB suspension. Now, both were mixed well. Pure liquid Luria broth (LB) medium was used as a control. The optical density (OD) values were measured at 450 nm after the intervals of 15–75 s. All samples were repeated at least 5 times. The lytic activity of the enzyme (lysozyme) was calculated under the conditions of: 25 °C, pH 6.2, and at a wavelength of 450 nm. The difference between the two OD450 values (for the samples and control) was calculated as ∆A450. The calculation was carried out by the unit of activity corresponding to the amount of enzyme required to decrease the absorbance by 0.001 per minute. The enzyme activity was calculated by the following formula:The lysozyme assay or the cell lytic activity was assessed by the turbidimetric method ,3,67,68.solution . Bacillus subtilis BSN314 could play an important role in the food industry. It can be used as a dietary supplement to boost immunity against pathogens and could also be used as a natural preservative in the food industry. Future gene identification and expression studies may lead to improve the importance of lysozyme biosynthesized from Bacillus subtilis BSN314. Lysozyme is presented as a natural antimicrobial agent against pathogens. In the present study, the lysozyme was purified. The observations of the results showed that the purified lysozyme exhibited a high potential against some pathogenic bacteria. In this study, two components were identified, namely A0A1Q9G213  and A0A1Q9FRD3 . It was concluded that these components may be responsible for enhancing the bactericidal activity of lysozyme. This purified lysozyme could be used to control microbial infections in the host by killing the microbes and strengthening the host’s defense mechanisms against microbial pathogens. This could contribute to the development of a strategy of biological control of bacterial infections in humans and animals. Further research is needed to identify the antibacterial mechanism of the purified components of lysozyme. Due to its strong bactericidal potential, the lysozyme purified from


Rht13 encodes an autoactive NB-LRR gene rather than a component of gibberellin signaling or metabolism. The autoactive Rht13 allele (Rht-B13b) causes up-regulation of pathogenesis-related genes and affects cell wall properties. Rht-B13b reduces height to a comparable degree as conventional dwarfing genes and offers an additional benefit of increased stem strength. This discovery reveals an unexpected class of reduced height gene in wheat and opens up opportunities to use autoactive NB-LRR genes to reduce height in a range of crop species.Conventional dwarfing genes increased wheat yields by disrupting plant hormone (gibberellin) signaling. Alternative wheat-dwarfing genes, suitable for use in additional environmental conditions, have been shown to encode components of gibberellin metabolism. Here, we found that the alternative dwarfing gene  Rht-B1b and Rht-D1b have disadvantages for seedling emergence. Use of the GA-sensitive semidwarfing gene Rht13 avoids this pleiotropic effect. Here, we show that Rht13 encodes a nucleotide-binding site/leucine-rich repeat (NB-LRR) gene. A point mutation in the semidwarf Rht-B13b allele autoactivates the NB-LRR gene and causes a height reduction comparable with Rht-B1b and Rht-D1b in diverse genetic backgrounds. The autoactive Rht-B13b allele leads to transcriptional up-regulation of pathogenesis-related genes including class III peroxidases associated with cell wall remodeling. Rht13 represents a new class of reduced height (Rht) gene, unlike other Rht genes, which encode components of the GA signaling or metabolic pathways. This discovery opens avenues to use autoactive NB-LRR genes as semidwarfing genes in a range of crop species, and to apply Rht13 in wheat breeding programs using a perfect genetic marker.Semidwarfing genes have greatly increased wheat yields globally, yet the widely used gibberellin (GA)-insensitive genes  Rht-B1b or Rht-D1b which encode negative regulators of gibberellin (GA) signaling  resulting in the removal of GA12 precursors from the GA biosynthesis pathway, a reduction of bioactive GA1 and reduced plant height    , 12 and Rht13 is another promising alternative dwarfing gene that reduces final plant height without affecting seedling growth  gene at the Rht13 locus on chromosome 7BL. Autoactivation of Rht13 leads to the up-regulation of pathogenesis-related (PR) genes, including class III peroxidases, which may catalyze the cross-linking of cell wall compounds to limit cell elongation and hence reduce height.g growth , 16. Theitions 16. Geneticosome 7B , but theRht13 semidwarfing gene was originally identified as an induced mutant in the Magnif background (Rht13 caused a 30 to 35% height reduction in both greenhouse and field conditions (birdcage) (The ckground . We carrirdcage) . A compairdcage) C and D. Rht13 was mapped to the long arm of chromosome 7B and genetically linked to simple sequence repeats (SSR) marker gwm577 . Additional DNA markers were added to the genetic interval after parental lines were screened with the 9K and 90K wheat single nucleotide polymorphism (SNP) arrays . In total, 33 DNA markers were added to the genetic interval. BAC sequence-derived markers 7J15.144I10_2_2 and 127M17.134P08_3 flanked the Rht13 locus on the proximal and the distal sides, respectively, and defined a genetic interval of approx. 0.1 cM  arrays , 24. In . 0.1 cM .Rht13 region defined by flanking markers 7J15.144I10_2_2 and 127M17.134P08_3 corresponded to a 1.93-Mb interval on chromosome 7B in Chinese Spring RefSeqv1.0. To identify candidate SNPs in the interval, we generated an additional population from a Magnif x Magnif M cross and selected four short and two tall F3:F4 lines that were homozygous at Rht13. For each of these lines, we isolated chromosome 7B by flow sorting and then sequenced the chromosome using Illumina short-reads. We attempted to identify SNPs within the mapping interval by mapping this chrom-seq data to the RefSeqv1.0 genome sequence ; therefore, we tested whether CDC Stanley would be a more appropriate reference sequence. Using CDC Stanley as the reference, the flanking markers spanned 1.86 Mb on chromosome 7B (3:F4 lines derived from Magnif x Magnif M cross) and the reference sequence, suggesting some divergence between CDC Stanley and Magnif.The sequence , but we sequence . We founosome 7B B. WithinRht13, we carried out RNA-seq on developing peduncle tissues from four fixed short and four fixed tall F3:F4 lines from the same Magnif x Magnif M population that was used for chrom-seq. We found that one transcript within the interval from our de novo annotation was more highly expressed in Magnif M than Magnif samples .We identified 12 SNPs and 1 INDEL between the tall and short fixed lines in the mapping interval C. To ideNB-LRR gene at the Rht13 locus, resulting in the same amino acid change (S240F) as found in Magnif M. Examination of mega base-scale haplotypes  was 100% identical between these cultivars before Ns in the contig interrupted the alignment at both ends. The KASP marker developed for the mutation segregated within progeny derived from Cadenza0453. Homozygous mutant plants (Rht-B13b) were on average 16.7 cm shorter than homozygous wild-type plants (Rht-B13a) at maturity in the Cadenza0453 background . This difference in height was reflected in shorter peduncle and internode lengths, except for the first internode .The expressed transcript with an amino acid change was predicted to encode an NB-LRR protein F. The muplotypes  did not Rht-B13b) into Fielder , although there was variation in the degree of dwarfism, which did not relate to the copy number or expression levels .To confirm that the amino acid change caused the reduction in height, we transformed the mutant allele from Magnif M ( Fielder C–E. Fielassembly ). We fouRht-B13b in breeding programs, we generated sister lines for Rht13 in three Australian elite backgrounds, alongside Rht-B1b (in EGA Gregory) or Rht-D1b (in Espada and Magenta) dwarfing alleles for comparison. We found that Rht-B13b stems elongated earlier than Rht-B1b or Rht-D1b stems, but final lengths were shorter than the tall sister lines due to an earlier arrest in growth . Comparing Rht-B13b with tall plants lacking conventional dwarfing genes, the reductions in heights are larger in Magenta and EGA Gregory than Espada. Taken together, our results (Rht-B13b is effective at reducing height in a range of genetic backgrounds including lines from the United Kingdom (Cadenza), Australia , Argentina (Magnif), and the United States (Fielder).To assess the potential for use of n growth A–C. Thisb plants D–F. No d results , 3 and 4Lycopersicon esculentum) NB-LRR protein I-2  and mutant Rht13 gene (Rht-B13b) in tobacco leaves. We found that the Rht-B13b allele induced more cell death 5 d post inoculation than the Rht-B13a allele  compared with Magnif (Rht-B13a) , similar to observations in Cadenza  than the qPCR data (PR4 up-regulation was only borderline significant (P = 0.05). The up-regulation of PR genes was consistent with up-regulation of defense response-associated genes in the Magnif M plants compared with Magnif, identified by gene ontology (GO) term enrichment . Overall, we found that more genes were up-regulated  than down-regulated (726 genes) in Magnif M compared with Magnif . Up-regulated genes were enriched for GO terms including defense responses, cell wall organization, regulation of hydrogen peroxide metabolic processes, and salicylic acid biosynthetic processes. We did not detect any enrichment for genes related to GA signaling or biosynthesis. Down-regulated genes were associated with flavonoid biosynthetic processes, responses to cytokinin and photosynthesis .To further explore the pathways through which gene see . We conf Cadenza C–H. The PCR data C–H; howeRht-B13b) compared with Magnif (Rht-B13a) in the peduncle . Furthermore, many of the class III peroxidase genes were very strongly up-regulated (11/28 are up-regulated >10-fold).We further hypothesized that the autoactivation of defense responses in the mutant line will cause the production of reactive oxygen species, which can promote cross-linking and cell wall stiffening leading to less growth , 33. To Rht-B13b) peduncles had lower hydrogen peroxide content than Magnif (Rht-B13a) , consistent with the up-regulation of class III peroxidases in the mutant (Rht-B13b) and Magnif (Rht-B13a) peduncles , and 0/15 Cu/Zn superoxide dismutase genes were differentially expressed, P > 0.05). To test whether the changes to class III peroxidase gene expression and hydrogen peroxide content influence cell wall mechanical properties, we used a three-point bend test to measure peduncle strength and rigidity. We found that the Magnif M (Rht-B13b) peduncles were stronger and more rigid than Magnif (Rht-B13a) peduncles . The Magnif M (Rht-B13b) peduncles had shorter cell lengths in their epidermis, with cell lengths of approximately 2/3 of wild type, suggesting a lower level of cell expansion (Rht-B13b) peduncles had much lower staining of lignin in and around vascular bundles than the Magnif (Rht-B13a)  G.Rht genes in wheat is that Rht13 is not directly involved in GA signaling or metabolism, as is the case for conventional dwarfing genes Rht-B1b and Rht-D1b  we observed upon expressing Rht-B13b in N. benthamiana leaves.A striking difference to other reported  Rht-D1b  and the b , which is mainly associated with reduction in peduncle growth. Therefore, the gene is well suited to water-limiting environments that require deeper planting to access available moisture and rapid leaf area development to lower evaporative losses from the soil surface. We found that Rht-B13b increased bending strength, which may further decrease lodging and reduce yield losses compared with conventional Rht genes. In previous work, Rht-B13b was reported to increase yield by 18 to 21% in recombinant inbred and near-isogenic lines grown in Australia  was identified on chromosome 7B, for which the peak SNP marker maps only 10 Mb away from the location of Rht13 , heterologous N. benthamiana expression, qPCR, hydrogen peroxide, cell size, and stem property measurements.All methodological information is available in Appendix 01 (PDF)Click here for additional data file.


TP53 (or p53) is widely accepted to be a tumor suppressor. Upon various cellular stresses, p53 mediates cell cycle arrest and apoptosis to maintain genomic stability. p53 is also discovered to suppress tumor growth through regulating metabolism and ferroptosis. However, p53 is always lost or mutated in human and the loss or mutation of p53 is related to a high risk of tumors. Although the link between p53 and cancer has been well established, how the different p53 status of tumor cells help themselves evade immune response remains largely elusive. Understanding the molecular mechanisms of different status of p53 and tumor immune evasion can help optimize the currently used therapies. In this context, we discussed the how the antigen presentation and tumor antigen expression mode altered and described how the tumor cells shape a suppressive tumor immune microenvironment to facilitate its proliferation and metastasis. Genome instability is one of the hallmarks of cancer . TP53 o to maint+ T cells, CD8+ T cells, and natural killer (NK) cells. The immune response to cancer is thought to be activated in a tumor genome-dependent manner  molecules expressed on cell surface present peptides to T cells to motivate immune responses. MHC molecules can be divided into two major classes. MHC Class I mainly presents peptides came from intracellular proteins , which p network . Under mvia repressing the myelocytomatosis (Myc) oncogene transcription  one signaling pathway . The derepression of ERV triggered ERV-dsRNA-interferon (IFN) pathway followed by activation of antigen processing and presenting genes, including B2M, HLA-A, HLA-B, and HLA-C which encode MHC class I molecules . Besides pathway .via proteasome. By detecting the secretion of IFNγ, tumor necrosis factor (TNF)-α, and the proportion of CD69+ T cells, it was found that p53-bearing destabilizing mutations, such as R175H and Y220C mutations, are recognized more efficiently and activated more p53 target T cells than G245S mutation  after allogeneic hematopoietic cell transplantation. The p53 deletion caused the downregulation of MHC II and tumor necrosis factor related apoptosis-inducing ligand receptor one and receptor 2 (TRAIL-R1/2) which induced immune evasion of AML .Over the past few centuries, researchers came to realize tumors are more than a group of malignant proliferating cells, but a complex “organ” including tumor cells, stromal cells, immune cells, extracellular matrix, vessels, cytokines, chemokines, as well as other metabolic products . It has Cytokines are the main proteins produced and secreted by many different cell types. They mediate immune system responses  and commvia interacting with TANK binding protein kinase 1 (TBK1) (Type I IFN (IFN I) is an important cytokine whose response plays significant roles in antiviral innate immune and anti-tumor adaptive immune . One of 1 (TBK1) , consequInterleukin-6 (IL-6) is an important trigger of tumor-promoting inflammation. Tumor cells exposed to IL-6 activate the oncogenic STAT3 transcription factor to promote epithelial-to-mesenchymal transition (EMT), which is the first step for tumor cell migration. MiR-34a, activated by p53, is a major inhibitor through targeting IL-6R . In studTransforming growth factor-β (TGF-β) is an immunosuppressive cytokine that has both positive and negative roles in tumor formation . The canvia switching the viral E2-associated factor 4 (E2F-4)/p107 complex to Smad/E2F-4 corepressor, which represses transcription and translation of p53  and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), have attracted much attention since their discoverers won the 2018 Nobel Prize in Physiology or Medicine. Immune Checkpoints are inhibitory pathways to maintain the persistence of immune response and the stability of the internal environment based on self-tolerance . CurrentIt is discovered that PD-L1, PD-L2, and CTLA-4 expression is significantly increased in patients with TP53 mutations in both clinical samples and mouse models of hematologic neoplasms . Similar+ T cells to counteract the effects of PD-1 inhibitors  refers to immunological components with tumors . Various+ T cells increased in a p53-dependent manner  . Further+ cells through upregulating the expression of CXCR3/CCR2-associated chemokines and macrophage colony-stimulating factor (M-CSF) (Myeloid-derived suppressor cell (MDSC) is another important member of the suppressive immune microenvironment, which produces reactive oxygen species and other cytokines to inhibit T cell mediated immune response . p53 med (M-CSF) . The des (M-CSF) . The dys (M-CSF) .In vitro co-cultures of M0 macrophages with H358 (a p53-null cell line) exosomes demonstrated that exosome-induced M2 polarization may be p53 independent (TAMs are major tumor-infiltrating cells mediated a variety of cellular activities such as tumor cytotoxicity , angiogeependent . Besidesependent . Equallyependent .In recent years, immunotherapy including ICIs, cancer vaccination, and adoptive cell therapy has revolutionized the treatment of cancer. As introduced above, the p53 mutation or deletion play a central role in tumor immune evasion, so reactivating wt-p53 or restoring tumor suppressive function of mutp53 are promising anti-tumor immunotherapy strategies.Scientists has appreciated the antigenic character of p53 since 1990s and developed p53-based vaccines . A novelTargeting p53-MDM2 pathway is another strategy. Combination of reactivation of wt-p53 and ICIs is also proved to have a better anti-tumor effect. Wang and co-workers demonstrated that HDM201, a potent and selective second-generation MDM2 inhibitor, could trigger adaptive immunity and develop durable, antigen-specific memory T cells in a p53-dependent manner. Combination of HDM201 and PD-1/PD-L1 blockade is more efficient for complete tumor regressions . SimilarIt isn’t difficult to find from the previous studies that p53 has a strong connection with TIME . The p53p53 is a star molecular which mediates multiple cellular activities of tumors and attracts much attention since its discovery. Scientists gradually realized the better treatment is to recover normal immune response, not only to enhance immune response. Owing to the essential role of p53 mutation and deletion in tumor immune evasion, reactivation of expression and function of p53 to reshape TIME and restore anti-tumor immunity may be efficient treatment for anti-tumor. Although scientists have spent almost 30 years to develop p53-based therapies, p53 is still a mystery protein attracting our attention. Great expectations of targeting p53 and unstable efficacy urge us to have a deeper understanding of it.


A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS). G4-containing RNP granules are identified in both prokaryotes and eukaryotes, but extensive studies have been performed in eukaryotes. We have been involved in analyses of the roles of G4-containing RNAs recognized by two G4-RNA-binding proteins, TDP-43 and FUS, which both are the amyotrophic lateral sclerosis (ALS) causative gene products. These RNA-binding proteins play the essential roles in both G4 recognition and LLPS, but they also carry the risk of agglutination. The biological significance of G4-binding proteins is controlled through unique 3D structure of G4, of which the risk of conformational stability is influenced by environmental conditions such as monovalent metals and guanine oxidation. The G4 e genome . Such G4n genome . Some ofn genome   gene, TDP-43, FUS, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP A2/B1, hnRNPA3, EWSR1 (Ewing’s sarcoma RNA binding protein 1), and TIA1 (T cell-restricted intracellular antigen-1)  and MALAT1 (metastasis associated in lung adenocarcinoma transcript 1/NEAT2) contain abundant G4 motifs  . Surpristigen-1) . For ins4 motifs . More in4 motifs . RNP gra4 motifs . RNP gra4 motifs . Dysregu4 motifs .The G4-binding proteins often carry one or more intrinsically disordered regions (IDRs) . In geneIntrinsically disordered proteins drive protein clustering and subsequent LLPS using their IDRs . These GThe best characterized ALS-linked G4-RNA binding proteins, TDP-43 and FUS, share structural and functional similarities, including participation in ALS pathogenesis . RNP graThe effects of amino acid substitution mutations from ALS patients are common to these two proteins, and TDP-43 and FUS mutant proteins show reduced interaction with G4.  Figure . ALS-linThe assembly of RNP granules by G4-RNA and G4-RNA binding proteins control many biological processes, including mRNA transport from soma to distal neurites for local translation. However, there are several risks associated with the molecular properties of G4-RNA and G4-RNA binding proteins that lead to perturbation of the interplay between G4-RNA and G4-RNA binding protein including the pathogenesis of ALS. Several factors are shown below :ALS is characterized by the accumulation of protein aggregates in motor neurons . HoweverOxidative stress is a risk factor of neurodegeneration and also a therapeutic target . Mutatio+ is known as a factor that interferes with the uptake of Na+ and K+, which stabilizes the G-quadruplex structure in vitro. Although Na+ and K+ are the physiologically relevant monovalent ions in the context of G4 structures, however, Li+ with smaller ionic radius than Na+ and K+ causes destabilization of G4 conformation (+ has not been observed, administration of lithium to ALS patients resulted in almost 70% of adverse events, including death (G-quartets are stabilized in the presence of cations, but different cations result in different structural properties . Li+ is ormation . Althougng death .G4-associated regulations are involved in varieties of biological systems, including replication, recombination, telomere regulation, transcriptional regulation, RNA processing, RNA stability, translation, RNA transport, and stress responses. Although many of them have long been overlooked without experimental confirmation, but the advent of a new age arrived to reveal molecular details underlying the interaction of G4-RNAs and G4-binding proteins. The dysregulation of G4-RNAs and G4-binding proteins has been identified as neurodegenerative diseases in human. Risk factors in G4 and G4 binding proteins are involved in the pathogenic mechanism of ALS (for instance, E. coli. It may provide a platform for analyzing the formation of complex structures in G4.As an extension of this line research, we have started to identify the role of G4 in the most well-characterized prokaryote


Marburg virus (MARV) is one of the most harmful zoonotic viruses with deadly effects on both humans and nonhuman primates. Because of its severe outbreaks with a high rate of fatality, the world health organization put it as a risk group 4 pathogen and focused on the urgent need for the development of effective solutions against that virus. However, up to date, there is no effective vaccine against MARV in the market. In the current study, the complete proteome of MARV (seven proteins) was analyzed for the antigenicity score and the virulence or physiological role of each protein where we nominated envelope glycoprotein (Gp), Transcriptional activator (VP30), and membrane-associated protein (VP24) as the candidates for epitope prediction. Following that, a vaccine construct was designed based on CTL, HTL, and BCL epitopes of the selected protein candidates and to finalize the vaccine construct, several amino acid linkers, β-defensin adjuvant, and PADRE peptides were incorporated. The generated potential vaccine was assessed computationally for several properties such as antigenicity, allergenicity, stability, and other structural features where the outcomes of these assessments nominated this potential vaccine to be validated for its binding affinity with two molecular targets TLR-8 and TLR-4. The binding score and the stability of the vaccine-receptor complex, which was deeply studied through molecular docking-coupled dynamics simulation, supported the selection of our designed vaccine as a putative solution for MARV that should be validated through future wet-lab experiments. Here, we describe the computational approach for designing and analysis of this potential vaccine. Filoviridae family. It was first discovered in West Germany in the year of 1967 (Rousettus aegyptiacus) acts as a reservoir for MARV where primary human infection occurs through exposure to the viral reservoir, then transmission between people occurs via body fluids (Marburg virus (MARV) is a notorious pathogen that belongs to y fluids . MARV pay fluids . Since iy fluids  and 90% y fluids  respectiThe MARV proteome and genome analysis showed seven basic proteins in addition to a negative-stranded linear RNA genome that sized approximately 19 kb . Most MAThe world health organization has put MARV on a list of an urgent need to find a solution for this deadly virus where the severe outbreaks and the high fatality rate have increased the importance of this call. Consequently, several techniques including vaccines based on viral vectors such as vesicular stomatitis virus , AdenoviMoraxella catarrhalis  standard proteome which had the proteome Id (UP000008239). The analysis of the whole proteome was performed to identify proteins with an antigenicity score that exceeds 0.5 besides a necessary physiological role or virulent role for MARV to allow nominating them as the vaccine candidates of our study. Vaxigen v2.0 was used to calculate the antigenicity score . Upon usB cell epitopes have been predicted by bepipred linear epitope prediction method  which devia NetMHCpan EL 4.0 prediction tool, the server recommended prediction method, and the binding prediction was performed against the reference set of HLA alleles as they represent the common binding specificities in addition to having population coverage of more than 97% (via INF prediction server (https://webs.iiitd.edu.in/raghava/il4pred/), and IL-10 induction ability was estimated through the webserver (https://webs.iiitd.edu.in/raghava/il10pred/). Finally, the allergenicity and toxicity for the selected epitopes were predicted using AlgPred 2.0 and ToxinPred webservers respectively.This stage started with the submission of filtered protein candidates to the Immune Epitope Database (IEBD) . MHC-I bthan 97% . Regardithan 97% . Then, tn server . In addiThe final stage of epitopes prioritization before reaching the step of multitope vaccine construction was the analysis of conservancy through multiple sequence alignment and a second stage of binding assessment through molecular docking analysis. In order to run the docking study for the single epitopes, the epitope 3D structure was predicted using PEP FOLD 3 webserver . For MHCvia VaxiJen v 2.0  . Following that, the vaccine’s epitopes are digested by antigen-presenting cells (APC) and presented to T cells . Hence, nerate/) .http://www.imods.chaconlab.org/)  . This emab.org/) . Furthervia sufficient chloride/potassium ions. Models were minimized for 0.05 ns under steepest-descent algorithm . For this purpose, JCAT server  and a large antigenicity score. We selected HLA-A*11:01 and HLA-DRB1*04:01 as representative alleles to analyze the binding affinity of the filtered MHC-I and MHC-II epitopes respectively. After selecting the most promising B and T cell epitopes from VP24, VP30, and envelope glycoproteins, we moved to design the multitope vaccine depending on six CTL and six HTL epitopes (two epitopes for each protein) in addition to six BCL epitopes (three from VP30 and three from envelope glycoprotein), these epitopes were linked together using GGGS, GPGPG, and KK linkers respectively. In order to finalize the construct, we incorporated PADRE peptide sequence and beta-defensin adjuvant to reach the final sequence of our multitope vaccine which was composed of 349 amino acids and sequenced as the following:“EAAAKGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKKEAAAKAKFVAAWTLKAAAGGGSIITRVNMGFGGGSTVKWGNFIFGGGSSSISVQASYGGGSSVQASYDHFGGGSRTFSLINRHGGGSTTRPPIYFRGPGPGEWLLLEVTSAIHISPGPGPGSEWLLLEVTSAIHISGPGPGTRPPIYFRKKRSIFWGPGPGTIYFLISLILIQSIKGPGPGTNRELLLLMARKMLPGPGPGLDNLTNRELLLLMARKKNLSKPPPPPKDMCKKPCTDPACNRDHDLDKKASNSQPQDVDSVKKTGVPPKNVEYTEGEEAKKKAKFVAAWTLKAAAGGGS”This was followed by analyzing this final design for allergenicity, the SVM method that depends on amino acid composition predicted our designed vaccine to be non-allergen, Moreover, toxicity analysis predicted our designed vaccine to be non-toxic. Furthermore, the analysis of the antigenicity predicted our multitope construct to be antigenic with an antigenicity score of 0.61. The final design was found to have a SOLpro score of 0.993 indicating it to be soluble upon overexpression (a score that exceeds 0.5 indicating solubility upon overexpression) and did not significantly resemble human protein sequences when analyzed through Blastp . In addition, ProtParam online tools were used to further analyze the other physicochemical characteristics of this multitope vaccine as shown in Firstly, the 3D model of the proposed vaccine was predicted by 3Dpro webserver. Following that, the predicted model was validated before and after structure refinement on GlaxyRefine webserver. In order to perform this structural validation, we run a Ramachandran plot analysis and calculated the Z-score for the models. Regarding the primary structure, 88.5%, 11.1%, and 0.4% of residues were located in favored, allowed, and outlier regions, respectively with a Z-score of -3.68. Moving to the refined model, 93.3%, 6.3%, and 0.4% of residues were located in favored, allowed and outlier regions, respectively and the Z-score was -4.29. The refined model and its structural validation scores are shown in Brucella Lumazine Synthase, which acts as an agonist for TLR-4 . The latter depicted 1773 Å2 and 774 Å2 interface area for the epitope in regard to 1709 Å2 and 748 Å2 for TLR-4 protomer B and protomer A, respectively. Concerning the epitope/TLR-8 docked complex, a differential ligand orientation was depicted in relation to the binding pocket of the TLR-8 target. The N-terminal of the docked epitope showed relevant insertion into the TRL-8 protomer B binding pocket with depicted proximity towards two target’s loops (260-268 and 433-482 residue ranges) protruding inside the binding cavity (2 for epitope: protomer B and 256:234 Å2 for epitope: protomer A) as compared to the TLR-4 system. However, the respective TLR-8 protomer B and A interface areas were higher for the earlier protomer within a similar differential fashion to those depicted for the epitope/TLR-4 model.ClusPro 2.0 server was employed to validate the binding process between our designed potential vaccine and its relevant receptors. The predicted binding energy values were -1458 and -1386 kCal/mol for TLR-4 and TLR-8, respectively. In order to validate these values, or TLR-4 , was docEvaluation of the nature of epitope/TLR binding interactions showed interesting findings. Higher polar interaction patterns were depicted with the epitope/TLR-4 interface as compared to the TLR-8 complex. A total of three salt bridges and 21 hydrogen bond pairs were assigned for the docked epitope and TLR-4 binding site  for the simulated epitope/TLR-4 model showed typical dynamic behaviors . Rapid equilibration and steady Cα RMSDs were maintained for more than half the simulation runs (> 70 ns) regarding the epitope, TLR-4, and their respective combined complex. Across each respective equilibration plateau, the epitope’s average Cα RMSD values were higher than those of the corresponding bound TLR-4 protein target (24.27 ± 1.11 Å versus 8.73 ± 0.71 Å). Regarding the other simulated model, epitope/TLR-8, significant Cα RMSD fluctuations were depicted for both the simulated epitope and bound target (Cα RMSD fluctuations were depicted for the ligand epitope as compared to its bound TLR-8 target (average 26.24 ± 2.09 Å versus 8.71 ± 1.42 Å) following respective thermodynamic equilibrations. Interestingly, both the ligand and target proteins were converged down to Cα RMSD values being comparable to their corresponding protein within the other epitope/TLR model near the end of the molecular dynamics simulation (100 ns).The simulated multiepitope vaccine models showed differential thermodynamic stability profiles in regard to the kind of the bounded TLR target. The alpha-carbon RMSD trajectories (Cα RMSF trajectories were monitored across the entire 100 ns simulation runs (Cα RMSFs) in regard to their central core ones. The latter Cα RMSF fluctuations were of higher values for the simulated epitopes in regards to their bounded TLR protein targets. Interestingly, both TLR-4 protomers were assigned with comparatively lower mobility/fluctuation profiles as compared to those of TLR-8 ones (3.62 ± 0.80 Å versus 4.64 ± 1.32 Å). Similarly, higher stability/immobility patterns were also depicted for the TLR-4 bounded epitope in relation to that of the TLR-8 model (6.44 ± 3.10 Å versus 10.91 ± 4.94 Å). It is worth mentioning that the simulated epitopes of both models showed the most recognized flexibility/mobility trends for their respective C-terminal residues as well as vicinal amino acids (high numbered amino acid sequencing).For gaining more insights regarding the simulated protein global stability and residue-wise flexibility contributions, the ion runs Figure 7-3 Kcal/mol.A2 gradient via MOE2019.01 package before being visually analyzed via PyMol software. Interestingly, the simulated epitope managed to be confined within the binding sites of both investigated TRLs, while exhibiting stable binding complex states .Subsequent conformational analysis was proceeded to grasp the main time-evolution conformational changes within the different epitope/TLR models. This was performed through comparative conformational analysis of simulated models at the initial and final dynamic trajectories . Extractx states Figure 8totalG) showed significant affinity of the designed vaccine with higher preferentiality  towards TLR-4 as well as its protomer B in regard to TLR-8 and cognate protein, respectively , Coulomb’s electrostatic energies (ΔelectrostaticG), polar solvation (ΔsolvationG), and non-polar solvation (ΔSASAG) energy contributions. Notably, the ΔelectrostaticG within both vaccine/TLR models depicted superior energy contribution for the ΔelectrostaticG interactions over the hydrophobic potentials reaching up to several folds. Within both simulated models, minimal polar solvation energy was assigned for protomer A in regard to protomer B, being of particular differential values for the vaccine in bound with TLR-8 target. On the contrarily, higher apolar solvation energy term contributions were assigned for the TLR’s protomer B over those of protomer A, showing the greatest value for the TRL-4 complex system.Evaluation of the total ligand/TLR free-binding energies ΔGtotal showed selectrostaticGprotein-protein binding interactions between both TLR models as well as cognate protomers, hydrogen bond analysis was conducted across the simulated trajectories  were assigned for the vaccine-TLR residue pairs; Gly21-Arg496 (29.11%), Arg22-Asp490 (49.01%), Cys23-Ser518 (38.12%), Lys88-Glu608 (46.14%), Gly97-Glu485 (28.22%), Tyr107-Glu605 (24.75%), Tyr114-Asp502 (35.46%), Ser116-Gln484 (26.24%), Arg125-Asp550 (60.89%), Asn131-Glu603 (27.23%), Arg132-Glu603 (55.45%), and Arg140-His426 (60.89%). Concerning the inbound TLR-8 model, the following vaccine-TLR residue’s hydrogen bond pairs were correlated to significant occupancies; Glu1-Glu129 (26.63%), Glu1-Glu150 (22.57%), Lys5-Glu133 (47.52%), Arg19-Asp462 (40.69%), Glu32-Lys476 (33.17%), Thr40-Asp645 (20.99%), Arg41-Asp536 (73.76%), Arg41-Asp560 (48.02%), Lys44-Glu612 (50.10%), Arg47-Glu668 (67.33%), Arg48-Glu612 (99.56%), Lys50-Glu768 (42.08%), and Lys88-Glu691 (64.65%). It worth noting that the residue pairs of TLR-8 model were exclusively related to the protomer B rather than protomer A target protein.Total bindingG was further decomposed into the protein’s residue-wise energy contributions , Lys88 (-373.25 kJ/mol), Arg 125 (-359.65 kJ/mol), Arg132 (-353.73 kJ/mol), and Arg140 (-315.34 kJ/mol) for epitope/TLR-4 model. on the other hand, Lys5 (-192.88 kJ/mol), Arg19 (-200.52 kJ/mol), Arg41 (-198.69 kJ/mol), Lys44 (-206.76 kJ/mol), Arg47 (-207.69 kJ/mol), Arg48 (-207.42 kJ/mol), Lys50 (-183.53 kJ/mol), and Lys88 (-165.52 kJ/mol) were assigned of highest energy binding contributions within epitope/TLR-8 complex system.To further highlight the significance of the above-described hydrogen bond-residue pairs within the multiepitope vaccine/TLR complex stability, the ΔThe generated immune response as a result of the proposed vaccine successive injections is shown in Finally, we reached the last stage where the computational analysis included a reverse translation and codon optimization of our multitope vaccine using JCat server for this purpose. The analysis of the amino acid sequence of our vaccine showed GC content of 52.9% indicating an accepted value as the accepted range ranged from 30 to 70%. Moreover, calculating Codon Adaptation Index (CAI) indicated a high probability of protein expression with a value of 1, when transferring these experiments to the wet lab, the CAI value ranges from 0 to 1 with the accepted range ranges between 0.8 and 1.in silico vaccine prediction that in turn has been extended to involve bacteria, viruses, fungi, and even cancer  into the TLR-8 binding pocket could have hindered the epitope/TLR-4-like transverse orientation across the TLR-8’s inner pocket. The binding of the designed vaccine at the lateral interface of TLR-8 was also depicted through several reported studies. Three research groups showed significant affinity of a novel engineered anti-SARS-CoV-2 vaccines towards TLR-8 target  arose during the multiepitope vaccine ligand binding at TLR-8 binding site. Generally, solvation energies confer significant forces of repulsive against the ligand-binding process since such processes rely on solvent-displacement. Despite the depicted shift, the polar solvation energy terms were shown higher within the multiepitope vaccine/TLR-8 model as compared to those of the TLR-4 one. This could confer the preferentiality of the vaccine’s transverse orientation at TLR-4 as compared to TLR-8, while as further signify the TLR-8 protruding loops that would impose great challenge against vaccine/TLR-8 proper anchoring. This was highly rationalized since several reported in-silico studies, including our presented data, showed a significant docking of their designed vaccines towards the TLR-8 lateral interface while depicting relevant thermodynamic stability throughout variable molecular dynamics simulation time runs  have managed to overcompensate. This benched assistance allowed the vaccine/TLR-8 complex to exhibit an overall binding profile being comparable to that of the epitope/TLR-4 model. The latter highlights the significance of the vaccine’s ime runs –86.via the TLR residues rather than those of the vaccines. This could be reasoned for the high-ordered water molecules along the hydrophobic surface of the multiepitope vaccine/TLR binding site. On the other hand, the total non-polar interactions  were shown to be higher at the TLR-4, particularly for the protomer B, conferring it respective larger binding surface area as well as higher hydrophobic potentialities towards vaccine anchoring. This speculation could be rationalized since accumulated evidence has considered the investigated TLRs’ binding sites to be extended and of more hydrophobic nature  with essential physiological and pathological roles as vaccine candidates. Utilization of The original contributions presented in the study are included in the article/MAS, WKA, AMR, MA, MAE, and KMD: conceptualization, methodology, and original draft preparation. SA, EF, SMAS, SSE., and RAE: writing— review, and editing. AAB, SSE, and RMD: Funding. MAS and MAE: supervision and project administration. All authors contributed to the article and approved the submitted versionThis research was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University (KAU), Jeddah, Saudi Arabia, under grant number (RG-1-166-43).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Graphene derivatives doped with nitrogen have alreadybeen identifiedas active non-noble metal materials for oxygen reduction reaction(ORR) in PEM and alkaline fuel cells. However, an efficient and scalablemethod to prepare active, stable, and high-surface-area non-noblemetal catalysts remains a challenge. Therefore, an efficient, potentiallyscalable strategy to improve the specific surface area of N-dopedgraphene derivatives needs to be developed. Here, we report a novel,rapid, and scalable electrical induction heating method for the preparationof N-doped heat-treated graphene oxide derivatives (N-htGOD) witha high specific surface area. The application of the induction heatingmethod has been shown to shorten the reaction time and improve theenergy efficiency of the process. The materials synthesized by inductionheating exhibited very high specific surface area and showed improvedORR activity compared to the conventional synthesis method. Moreover,we demonstrated that the temperature program of induction heatingcould fine-tune the concentration of nitrogen functionalities. Inparticular, the graphitic-N configuration increases with increasingfinal temperature, in parallel with the increasing ORR activity. Thepresented results will contribute to the understanding and developmentof nonmetal N-htGOD for energy storage and conversion applications. The protonexchange membrane (PEM) fuel cell is the most mature among fuel celltechnologies. However, active, selective, and stable electrode materialsare required to achieve the desired performance of the PEM fuel cell.Nowadays, platinum-based electrode materials meet all these conditions,but platinum is a scarce, expensive metal and is susceptible to COpoisoning that needs to be replaced by a non-noble metal alternative.Therefore, an efficient non-noble metal material that lowers the costand meets the energy performance of the PEM fuel cell is of greatimportance.4The increasing demand for economic andenvironmentally friendlypower generation has led to the development of energy storage andconversion devices such as batteries, supercapacitors, and fuel cells.7Among non-noble metalmaterials, the class of heteroatom-dopednanocarbons, N-doped graphene derivatives, have great potential aselectrocatalysts for the oxygen reduction reaction (ORR), which isconsidered the bottleneck of the PEM fuel cell. N-Doped graphene derivativesare environmentally friendly, low cost, tunable, and functionalizable,which makes them perfect candidates for electrode materials for low-temperaturefuel cells.7 Most of mentioned methods are time consumingand furnish low quantities of the final product, which is problematicin terms of future commercialization. However, if graphene oxide (GO)is used as a precursor for the synthesis of N-doped graphene derivatives,the process can be used with subsequent heat treatment for largerscale applications. However, depending on the GO synthesis conditionsand GO heat treatment method, the complete restoration of the π-conjugated structure and exfoliation of N-doped heat-treated grapheneoxide derivatives (N-htGOD) may not be fully achieved, resulting ina lower conductivity and lower specific surface area. Because highspecific surface area and high electrical conductivity are of paramountimportance for the electrochemical performance of N-htGOD as an electrodematerial, efficient methods need to be developed.8The methods used for the synthesis of N-doped graphene derivativesare diverse and can be classified as direct  or post-treatmentsynthesis  methods.14 ultrasonic heating,16 intense pulsed light,17 infrared,18 and microwaveheating.20 In these protocols, the oxygen functionalitiesof GO are thermally decomposed, the nanostructure is exfoliated, andat the same time, the carbon nanostructure is N-doped, when a suitablenitrogen source is used.7 Depending onthe post-treatment protocol and its parameters, the N-configurationscan be doped into the structure in four different configurations,namely, pyridinic-N, pyrrolic-N, graphitic-N, and oxidized-N.24 Despite numerous research efforts to identify the active sites forORR, there are conflicting claims about which N configuration is themost active. Some reports claim that ORR activity correlates withpyridinic-N,30 others claim that it is graphitic-N,31 whereas some claim that both functionalities contribute to ORR activity.32To date, several different heatingprotocols have been reportedfor this process, e.g., Joule or resistive heating,40Despite the influence of N configurationon ORR, many literaturereports support the assumption that the superior ORR activity of N-htGODis due to trace metal impurities.41 we showedthatthe aspect ratio of the graphene derivative, the nitrogen concentration,the specific surface area, and inherent metal impurities play an importantrole in enhancing the ORR activity. To further investigate the effectsof surface area on N-htGOD materials, we developed a novel post-treatmentprotocol to generate higher-surface-area materials. A new one-potnitrogen doping and heat-treatment method based on electrical inductionheating was developed. In the present work, GO derivatives are heatedwith a heating rate up to 420 K/min in a graphite holder using a high-frequencyelectromagnetic field generated by an inductor. Is it to be notedthat faster Joule heating procedures were also developed, utilizingthe direct current discharge, with heating rates of 0.2–1 K/s,but they usually require complex instrumentation, are not scalableand the final temperature is difficult to control.14 All the synthesizedN-htGOD, namely, N-doped heat-treated graphene oxide (N-htGO) andN-doped heat-treated graphene oxide nanoribbons (N-htGONRs), weresubjected to a thorough morphological analysis and chemical and physicalcharacterization as well as extensive electrochemical testing forORR activity and stability in alkaline and acidic electrolytes.In our previous study,4 , H3PO4 , H2SO4 , HCl ,HF , HNO3 , KMnO4 , H2O2  and , 2-propanol , Nafion  perfluorinatedresin solution were used as received. All dissolutions were performedwith ultrapure water obtained from a Milli-Q system (Millipore) witha resistivity 18.2 MΩ cm.Graphite , multiwalledcarbon nanotubes (MWCNT) (NanoTechLabs M-grade MWCNT), KOH , HClO42 where a mixture of concentrated H2SO4/H3PO4 (vol. ratio = 900 mL:100 mL) was added to 20g of graphite  or 15 g of MWCNT (NanoTechLabsM-grade MWCNT) and then KMnO4 was added in aliquots with stirring, resulting in a mild exotherm to 35–40 °C.The reaction mixture was then stirred with a mechanical stirrer atroom temperature in a 3 L beaker for 10 days. The reaction mixturewas then poured onto ice (1500 mL), and 30 vol% H2O2 (approximately 15 mL) was added dropwise until the colorchanged from purple to yellow. Next, the mixture was transferred to1 L plastic centrifuge bottles, diluted with ultrapure water, andcentrifuged at 10 500 rpm for 30 min in a centrifuge . The supernatant was decanted, and theremaining solid was redispersed for 2 h in 5 vol% HCl ultrapure watersolution. The last cleaning step comprised redispersing and soakingthe GO/GONR suspension in ultrapure water until the next day. Thiswas followed by centrifugation at 10 500 rpm for 1 h to discardthe supernatant. This last cleaning step was repeated four times intotal. After the final supernatant was discarded, GO/GONR was redispersedin ultrapure water. In the case of GO, the suspension was treatedwith a homogenizer  for 1 h at max rpmsetting to exfoliate the product. In the case of GONR suspension,it was treated only with an ultrasonic bath for 15 min. Finally, the GO/GONR were freeze-dried.For GO and GONR synthesis, we utilized of slightlymodified and improved Hummers method,3 atmosphere ata constant flow rate of 30 mL/min in an aluminum oxide vessel in aquartz tube. The heat-treatment protocol consisted of three phases:(i) heating at 10 K/min from room temperature to the set point temperature(800 °C), (ii) maintaining the temperature at 800 °C for10 min, and (iii) then cooling at average 5 K/min from the set point(800 °C) to room temperature.Heat treatment was performed in an NH3 atmosphere ata constant flow rate of 30 mL/min in a graphite (99.9 wt%) crucibleinside a Pyrex glass reactor using the induction heat-treatment protocol:(i) heating from room temperature to 800 or 1200 °C (heatingrate of 420 K/min), (ii) holding at 800 or 1200 °C for 2 min,and (iii) cooling from 800 or 1200 °C to room temperature (averagecooling rate of 50 K/min). The heat was generated by an HTG-2400 inductor(Induktio) operating at a frequency of 250 Hz and an output powerof 1700 W.Heat treatment was performed in an NHThe catalyst ink was prepared by mixing the sample (N-htGO or N-htGONR),ultrapure water, 2-propanol (IPA), and Nafion (5 wt% water resin)in a ratio of 4.5 mg:0.8 mL:0.3 mL:30.0 μL, respectively. Themixture was then homogeneously dispersed with a horn sonicator (37.5W). Finally, an aliquot of 25 μL was applied to a glassy carbondisk electrode (diameter = 5.5 mm) on an inverse rotating ring diskelectrode (RRDE), equipped with a gold ring electrode at a rotationof 300 rpm at room temperature for 45 min.2 and TiO2 on Ti support, Specialist Casting) as the counterelectrode, and rotating ring disk electrode (RRDE) (Pine Research)with glassy carbon disk and gold ring electrode as the working electrode.RRDE experiments were performed at 1600 rpm on a WaveVortex 10 (PineResearch) rotation control unit. For all electrochemical experiments,0.1 M KOH or 0.1 M HClO4 aqueous electrolytes saturatedwith Ar or O2 were used at scan rate of 20 mV/s. Electrolyteresistance was determined by the positive feedback method before eachmeasurement and total solution resistance compensation (90%) was performed.All potentials in this work refer to the RHE (V vs. RHE). Before eachmeasurement, the potential of the Ag/AgCl (3 M NaCl) RE was checkedby measuring the opening circuit potential of the reference electrodewith respect to a platinum wire, both immersed in an H2-saturated solution .We compared the ORR activity of the N-htGOD by the onset potential(Eonset), determined by the tangentialmethod.43Cyclic voltammetry (CV) measurements wereperformed with PGSTAT30 (Autolab) potentiostat/galvanostat with thethree-electrode system using a reference electrode (Basi ResearchProducts RE 5B) Ag/AgCl in 3 M NaCl, mixed metal oxide  was determined using the following equation:id is thedisk current, ir is the ring current,and Nc (0.38) is the collection efficiency. Nc was determined in 10 mM Fe(CN)63–/Fe(CN)64– at 1600rpm in 0.1 M HClO4 solution.The number of transferredelectrons per reduced oxygen molecule on the RRDE (Microstructure characterizationof N-htGOD was performed using a SEM (Zeiss ULTRA plus). SEM imageswere acquired at 2 kV with secondary electron detector (SE2) at aworking distance of 5.5 mm.2 gas adsorption at 77 K.Prior to BET analysis, N-htGOD were degassed under a vacuum (10 μmHg)at 120 °C for 2 h. Subsequently, the BET specific surface areawas determine on the ASAP 2020 Micromeritics instrument with Nα X-ray source. Survey spectra were measuredusing a pass energy of 160 eV with a step of 1 eV/s. High-resolutionspectra were measured using a pass energy of 20 eV at a step of 0.1eV/s. The data were acquired using the ESCApe 1.4 software. Fitting of high-resolution XPS spectra was performed usingCasaXPS software, with Shirley background subtraction using 30–70%Gaussian–Lorentzian peak shapes, except for the oxidized-Nconfiguration where additional asymmetric peak tailing was used. Theoxidized-N configuration was fitted with an asymmetric peak shape,using 1.2–1.6 eV constrain for full width at the half-maximum. The binding energy was corrected using the C–C/C–Hpeak in the C 1s spectra at 284.5 eV.XPS measurements were performed using a Supra plus spectrometer  equipped with a hemispherical analyzer and a monochromaticAl KTable S1.44Raman spectroscopy was performedusing the Raman/AFM WITec Alpha 300 RAS with a 532 nm laser lightwith a power of 15 mW and 30 s integration time. Peak fitting of Ramanspectra was performed with the Lorentzian peak shape, baseline constrainat 0 intensity and peak at center constrains shown in MassHunter 4.4 software.Trace element analysis of electrocatalysts was determined using anICP-MS Agilent Technologies 7900CE, with a micro nebulizer, quartzspray chamber, and quadrupole mass analyzer in the flow of high-purityargon (5.0) gas at a flow rate of 15 L/min. The data were processedand analyzed using 2SO4 , 3 mL HNO3 , 2 mL of HClO4 , and1 mL HF  were added. The mixture was heatedaccording to the temperature program: to 150 °C for 15 min, 220for 20 min, 230 for 15 min, and then cooled in a microwave digestionsystem (Milestone Ethos UP). Next, the mixture was transferred toa 50 mL flask and diluted to the mark with ultrapure water. Finally,the as-prepared solution was filtrated through a 0.45 μm filter,diluted by a factor of 100 with 1% HNO3 solution, and injected into the ICP-MS instrument.Approximately 50 mg of the catalyst powder was placed into a 50mL autoclave and 4 mL of H45 on powder N-htGODcompressed  at 8 tons.We performed four-point probe ex situ conductivity(Ossila) measurements46 In the next step, the preparedGO and GONR were heat treated up to 800 or 1200 °C in a NH3 atmosphere using two different heating methods, i.e., conventionalfurnace heating  and novel electricalinduction heating  theintensity ratio of the 2D/G peak is higher for N-htGO_I (0.16) andN-htGONR_I (0.12) relative to N-htGO_F (0.08) and N-htGONR_F (0.09).One can also speculate from the shape of the gas adsorption isotherms,pore volume and pore area shown in 48The N-htGO and N-htGONR preparedby two heating procedures werefurther characterized and compared morphologically, chemically, andelectrochemically. N-htGO_F/N-htGONR_F indicates N-htGO and N-htGONRprepared with the furnace, and N-htGO_I/N-htGONR_I for N-htGO andN-htGONR that were prepared with the induction heating setup. Themorphology of the two N-htGO is shown in  spectra S2 theinFigures S2 and S3, respectively. The atomic concentrationsof O and N for different N-htGOD were determined using high-resolutionXPS spectra and are presented in 1s spectrafitting was performed to determine the nitrogen functionalities indifferent N-htGOD. The results presented in Because the surface chemical composition of thesematerials isvital for electrocatalysis applications, XPS and Raman spectroscopymeasurements were performed to determine the elemental composition,nitrogen functionalities, and defectiveness of graphene derivatives.The Raman spectra and XPS survey are shown in Table S2. Main impuritiesdetected in ppm were K, Mn, and Fe. The Mn and K impurities are introducedduring the oxidation step of graphite or MWCNT with KMnO4, and the Fe is an inherent impurity present in the starting graphitematerial or MWCNT. As we have shown in our previous work,41 the presence of Fe affects the ORR activityof the graphene derivatives in KOH, and thus, starting materials withlow concentrations of inherent Fe were used in the present study.Next, the ICP-MS analysis of N-htGOD was performedto determinethe composition of trace metal impurities. The impurity concentrationsare given in 4 and alkaline0.1 M KOH electrolytes. 4 and0.1 M KOH, respectively. ORR cyclic voltammograms were compared, inparticular the Eonset for reduction.The above-mentioned chemical composition showedthat the N-htGOD prepared by two different heating procedures arevery similar. On the other hand, the morphological data clearly showthat the materials prepared by the induction heating method are moreexfoliated and therefore have a considerably larger specific surfacearea. The latter should lead to a larger number of accessible activesites. On this basis, the materials were tested for ORR activity,selectivity, and stability in acidic 0.1 M HClOEonset values ofthe N-htGOD preparedby induction heating are considerably higher in both electrolytes, 0.186 V (N-htGO_F), 0.512 V (N-htGONR_I) and 0.477V (N-htGONR_F) vs. RHE. A similar trend with higher Eonset was determined in 0.1 M KOH. The Eonset values were 0.795 V (N-htGO_I), 0.726 V (N-htGO_F),0.841 V (N-htGONR_I), and 0.737 V (N-htGONR_F) vs. RHE. Moreover,as shown previously,41 the trend of increasingaspect ratio of graphene derivative corresponds with increasing electrocatalyticactivity for ORR. In this respect, quasi-1D N-htGONR always performedbetter than 2D N-htGO.The trolytes1. In 0.1e– reduction to peroxide or 4e– directreduction to water. For fuel cell applications, 4e– reduction is desirable because of better energyefficiency.49 The N-htGOD prepared by inductionheating in 0.1 M HClO4 showed better selectivity for thedirect 4e– pathway than that preparedby furnace heating  showsno distinctive morphological difference. However, chemical, and electrochemicalcharacterization interestingly show a trend  and the other set at1200 °C (N-htGO_1200 and N-htGONR_1200). A SEM morphologicalcomparison of both sets S4 shows1s XPS spectra  varieswith the final temperature, whereas there is no significant differencein overall nitrogen concentrations. The materials synthesized at 1200°C showed a lower concentration of pyridine-N and pyrrole-N configurationand a higher concentration of graphitic-N. On the other hand, materialssynthesized at 800 °C showed a higher concentration of pyridine-Nand pyrrole-N configuration and a lower concentration of graphitic-N.When N-configuration was correlated to the electrochemical results,it was evident that nitrogen configuration strongly impacted the electrochemicalactivity , which is anindicator of restored graphene structure thus improving the conductivityof the prepared electrocatalyst. To quantify the electrical conductivityof N-htGO_800, N-htGO_1200, and N-htGONR_800 and N-htGONR_1200, weperformed ex situ four-point probe measurements. The results showedimproved electrical conductivity for the materials heat treated at1200 °C (Table S4). Improved electricalconductivity is also an important property for effective electrocatalysis.To fully understand the main factor for the improved activity of thepresented electrocatalyst, we need to perform additional systematicstudies on defined surfaces, which would give a better insight intothe electrocatalytic properties. Nonetheless, the present resultsshow that the specific surface area, the N-configuration, and theaspect ratio should be considered when designing a non-noble metalelectrocatalyst for ORR.In view of the present results, we believe the electrocatalyticperformance is not related to only one parameter but is a sum of severalparameters. We have shown in Eonset potential (Tables S5 and S6). The best performing material N-htGONR_1200 showedhigh Eonset in 0.1 M KOH and 0.1 M HClO4. This was attributed to the synergistic catalytic effectof the specific surface area, the N-configuration, and the aspectratioFinally, we also compared the ORR activityof N-htGOD with the We have synthesized and characterized quasi1D N-doped heat-treatedgraphene oxide derivatives (N-htGONR) and 2D analogs (N-htGO) usinginduction and furnace heating methods at different temperatures. Inaddition, to rule out the influence of trace metal impurities on ORRperformance, we treated the same materials with different heat-treatmentprotocols.e– reductionin acidic electrolytes than the N-htGOD prepared by the conventionalfurnace method. However, the stability of the inductively heated N-htGODdecreased with a larger surface area. When comparing the ORR performancebetween N-htGOD heat treated at different final temperatures, it wasalso found that the increase in the concentration of graphitic-N couldbe responsible for the increased ORR activity. In summary, the presentresults indicate that the specific surface area, the N-configuration,and the aspect ratio should be considered in the development of newnon-noble metal electrocatalysts for ORR. Moreover, the results indicatethat the electrical induction heating protocol is energy and timeefficient and can be scaled up industrially. This is a step towarda low-cost active non-noble metal catalyst for the ORR reaction inPEM and alkaline fuel cells.The application of this method reduces the need forlarge furnaces,shortens the reaction time, and improves the energy efficiency ofthe heat treatment process. The main advantage of the induction heatingmethod compared to the furnace method is the faster heating rate,which produces graphene derivatives with a larger surface area thatexhibits improved ORR activity in alkaline and acid electrolytes.We have confirmed that a higher aspect ratio improves ORR activity,with quasi-1D N-htGONR exhibiting higher activity than 2D N-htGO.In addition, the RRDE experiments showed that the inductively nitrogen-dopedheated-treated graphene oxide derivatives (N-htGOD) exhibited betterselectivity for the 4