Streptomyces strain, whose genome was sequenced, was of particular interest because of its inhibitory activity, which was due to the Type I polyketide aureothin. Moreover, all strains were tested for the production of secreted proteases, with those belonging to the genus Nocardioides having the most active enzymes. Finally, protocols commonly used for ancient DNA studies were applied to evaluate the antiquity of isolated bacterial strains. Altogether these results show how paleomicrobiology might represent an innovative and unexplored source of novel biodiversity and new biotechnological tools.The urban plan of Palermo  has evolved throughout Punic, Roman, Byzantine, Arab, and Norman ages until it stabilized within the borders that correspond to the current historic center. During the 2012 to 2013 excavation campaign, new remains of the Arab settlement, directly implanted above the structures of the Roman age, were found. The materials investigated in this study derived from the so-called Survey No 3, which consists of a rock cavity of subcylindrical shape covered with calcarenite blocks: it was probably used to dispose of garbage during the Arabic age and its content, derived from daily activities, included grape seeds, scales and bones of fish, small animal bones, and charcoals. Radiocarbon dating confirmed the medieval origin of this site. The composition of the bacterial community was characterized through a culture-dependent and a culture-independent approach. Culturable bacteria were isolated under aerobic and anaerobic conditions and the total bacterial community was characterized through metagenomic sequencing. Bacterial isolates were tested for the production of compounds with antibiotic activity: a IMPORTANCE One of the goals of paleomicrobiology is the characterization of the microbial community present in archaeological sites. These analyses can usually provide valuable information about past events, such as occurrence of human and animal infectious diseases, ancient human activities, and environmental changes. However, in this work, investigations about the composition of the bacterial community of an ancient soil sample  were carried out aiming to screen ancient culturable strains with biotechnological potential, such as the ability to produce bioactive molecules and secreted hydrolytic enzymes. Besides showing the biotechnological relevance of paleomicrobiology, this work reports a case of germination of putatively ancient bacterial spores recovered from soil rather than extreme environments. Moreover, in the case of spore-forming species, these results raise questions about the accuracy of techniques usually applied to estimate antiquity of DNA, as they could lead to its underestimation. Panormus as a Phoenician-Punic city) has evolved throughout Punic, Roman, Byzantine, Arab, and Norman ages until it stabilized within the borders that today correspond to the current historic center. Generally speaking, the centrality of this area from the 2nd century BCE to the 3rd century CE was well known so far . In the case of the grape seeds, such a quantitatively large finding is very rare. The medieval origin of this site was confirmed by AMS 14C dating: in fact, calibrated radiocarbon dating of the grape seeds gave results reported in Content analysis and dating indicated that during the High Middle-Ages, the pit described in this work might have been used to dispose rubbish. Indeed, besides soil, samples contained 210 grape seeds, some metal wastes, a fragment of upper bivalve valves (possibly derived from the biogenic limestone), and some animal bone fragments  and presumably the short duration, in terms of use, of this stratum.The morphometric analysis of the grape seeds identified a low level of variability inside the sample and an intermediate position in terms of fine morphology between current wild 4 CFU/g, while that of anaerobic ones was 5 × 107 CFU/g. Isolated colonies were clustered by phenotype and two representatives of each were used for further analyses. Thus, 23 isolates were considered. Sequencing and analysis of genes encoding the 16S rRNA (16S rDNAs) revealed that bacterial isolates were affiliated with the following genera: Arthrobacter, Bacillus, Nocardia, Nocardioides, Paenibacillus, and Streptomyces  and that are generally cultivable in nonselective media (like those used in this work) were not isolated, probably because they could be unviable or in a viable but not-culturable (VBNC) state . Microbial taxonomic profiling showed that the two extraction methods produced similar results regarding the bacterial taxonomic composition (Table S3). Bacterial genera having a relative abundance >0.2% are represented in C) state , 35. ThuAnaerostipes hadrus, Bifidobacterium angulatum, Blautia sp. SC05B48, Castellaniella defragrans, Clostridium perfringens, Escherichia coli, Methylocystis rosea, and Methylocystis sp. SC2. Except for C. perfrigens .To this aim, antiquity of DNA was then investigated through the analysis of deamination patterns. Data showed that DNA of only eight species could be considered ancient (Table S4): rfrigens , none ofrfrigens –44: in tEscherichia coli, Kocuria rhizophila, and Saccharomyces cerevisiae that were chosen because they are a Gram-negative bacterium, a Gram-positive bacterium, and a eukaryote, respectively. Isolates were cultivated on three different media to evaluate the possible influence of nutrients on antimicrobial synthesis. Among them, only those belonging to the genus Streptomyces exerted an antimicrobial activity. In particular, Streptomyces sp. AV8 and Streptomyces sp. AV19 were of particular interest because they produced the largest inhibition halos , and alignment of their 16S rDNA partial sequences (100% identity) suggested that these two isolates belonged to the same species and/or strain.As shown in Streptomyces sp. AV19 grown on R2YE was performed, and methanolic extracts whose antimicrobial activity was verified by microbiological assays against K. rhizophila  were analyzed by means of HPLC/ESI/Q-TOF (High Performance Liquid Chromatography/Electron Spray Ionization/Quadrupole-Time of Flight). In Streptomyces sp. AV19 is reported.Hence, on the basis of these results, a screening of bioactive compounds produced by The main compound found in the extract was the Type I polyketide antibiotic aureothin . The antiSMASH program (version 6.1.1) (Streptomyces sp. AV19 genome (Table S6). The most represented gene cluster type was Polyketide Synthase (PKS), followed by Non-Ribosomal Peptide Synthetase (NRPS). In agreement with the HPLC/MS data, aureothin biosynthetic gene cluster was detected, having a 100% similarity with the Streptomyces thioluteus one (50Streptomyces coelicolor one (53Genomic DNA of n 6.1.1)  was usedus one 5052. Moreoor one 5355.Nocardioides genus  had the most complex patterns  has been described. The isolation of culturable bacteria was performed in both aerobic and anaerobic conditions, yielding to the growth of strains belonging to Arthrobacter, Bacillus, Nocardia, Nocardioides, Paenibacillus, and Streptomyces genera. They belong to either Actinomycetota or Bacillota phyla  (Arthrobacter)  nor fungi, for example molds, were isolated, although normally able to grow in the nonselective media used in this work. However, in the case of fungi, it should be considered that specific and/or selective media for their isolation were not used here and that the adopted cultural conditions might have not supported their growth.In this work the characterization of the bacterial community of an ancient soil retrieved from an Arabic site located in ta phyla  and are tomyces) , 57 or dobacter) . This pea priori be excluded that the site could have been contaminated because of disparate and unpredictable events  after it stopped being used for its original purpose and it was abandoned and eventually forgotten.Metagenomic analysis of the soil sample revealed the existence of a more complex bacterial composition. It is quite possible that bacteria detected by metagenomic analysis, but not isolated, (i) were unviable and only their DNA endured in the soil, or (ii) they could be in a VBNC state, or (iii) they were viable but they did not grow due to the adopted culturing conditions or to inhibition activity by other growing bacteria. Nevertheless, it is not possible to know whether the site has remained constantly isolated along the centuries. In this regard, it cannot Isolation of bacteria potentially able to produce spores or dormant forms might suggest that these isolates derived by germination of ancient spores. Shotgun sequencing and degradation patterns analysis identified a group of eight species with the expected features of DNA isolated from ancient organisms , with n60–64–Bacillus spores have been reported, environmental conditions described thereof do not resemble ours  and grape seeds coming from the stratigraphic unit underwent AMS 14C dating .Two samples, consisting in 500 g of soil collected from the pit located in −1 and 10−2 dilutions were plated on LB agar  and SFM  (One g of sample was resuspended in 1 mL of sterile 0.9% wt/vol NaCl solution and 100 μL of 10to agar) . CFU wer2PO4, 2.5 g/L NaHCO3, 1.5 g/L NH4Cl, 0.1 g/L CaCl2 · 2H2O, 0.1 g/L KCl, 0.1 g/L MgCl2 · 6H2O, 0.005 g/L MnCl2 · 6H2O, 0.001 g/L NaMoO4 · 2H2O, pH 7.0. The medium was sterilized and cooled down under nitrogen flux, then 100 mL of medium were distributed in 10 different sealed vials. The soil sample (100 mg) was suspended in 3 mL of sterile water, injected in the sealed vials and incubated at 28°C.Bacteria belonging to the functional group of Fe(III) reducing bacteria (FeRB) were isolated using an anaerobic medium (FeRid) containing: 1.0 g/L Fe citrate, 1.5 g/L peptone, 0.6 g/L NaH2PO4, 1.0 g/L NH4Cl, 4.5 g/L Na2SO4, 0.06 g/L CaCl2 · 6H2O, 0.06 g/L MgSO4 · 7H2O, 3.0 g/L Na lactate, 3.0 g/L Na acetate, 1.0 g/L yeast extract, 0.1 g/L FeSO4 · 7H2O, 0.3 g/L Na citrate · 2H2O, pH 7.5.Bacteria belonging to the functional group of sulfate reducing bacteria (SRB) were isolated in the same way (at 28°C), but the previous medium was replaced by medium “Postgate C,” which contained: 0.5 g/L KHBoth FeRB and SRB were isolated by injecting 1 mL of culture into new fresh medium for five times in a row, spending 3 months for isolating FeRB and SRB in liquid media. At the end, 100 μL of culture were streaked on the corresponding agar plates for the final isolation.2, 200 μM each dNTPs (Invitrogen), 0.2 μM each universal primer (27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGYTACCTTGTTACGACTT-3′) and 1 U of Taq DNA polymerase Recombinant (Invitrogen). One μL of single colony thermal lysate was used as the template DNA: briefly, a bacterial colony was resuspended in 25 μL of TE buffer  and incubated at 100°C for 5 min; then the lysate was incubated in ice for 5 min and centrifuged ; the resulting supernatant was eventually used as the template DNA. The thermal cycle was 94°C for 3 min, followed by 30 cycles of 94°C for 45 sec, 50°C for 1 min and 72°C for 90 sec, and finally 72°C for 10 min. PCR products were purified using the Purelink Quick Gel Extraction and PCR Purification Combo kit (Invitrogen) and sequenced by BMR Genomics  through Sanger sequencing.Genera of all isolated strains described in this work were determined through sequencing of 16S rDNA. PCRs were performed in a 25 μL volume containing 1× PCR Reaction Buffer (Invitrogen), 3.5 mM MgClStreptomyces sp. AV19, a 16S rDNA-based phylogenetic analysis was performed. The complete 16S rDNA was used as BLAST query, choosing the 16S rRNA sequences (Bacteria and Archaea) database, and limiting the search to the species recognized as whorl-forming Streptomyces (Table S7) (In the case of able S7) . Sequencable S7) . The maxable S7)  with 100able S7) .All molecular laboratory work was conducted in the dedicated ancient DNA (aDNA) facilities at the Laboratory of Anthropology (University of Florence), following strict guidelines to prevent contaminations. Blank controls were processed in parallel with samples for monitoring of potential reagent contamination. Additionally, before proceeding with DNA extraction, the archaeological sample was exposed to UV light for 10 min to sterilize the external surface.Two different silica-based extraction methods were used to increase the chances of recovering ancient microbial DNA from the sample. Here, these two methods are designated D and PS, respectively.(i) Extraction method D. This method is based on a digestion and purification protocol commonly used to extract highly fragmented DNA molecules from mineralize tissues (– tissues . While t tissues –84. Appr(ii) Extraction method PS. A standard microbial extraction protocol for DNA recovery from soil was applied using the DNeasy PowerSoil Pro kit (Qiagen) and following manufacturer instructions with some modifications. In brief, approximately 200 mg of sediment were resuspended in a solution prepared with 400 μL of 0.5 M EDTA and 100 μL of 20 mg/mL Proteinase K. The sediment suspension was added to a PowerBead Tube containing 750 μL of Solution CD1 and rotated for 4 h at room temperature, followed by bead beating at 3,200 rpm for 10 min. After pelleting, the supernatant was split into two equal aliquots to reduce clogging issues encountered when loading extraction lysates onto a single column  Preparation of DNA library and sequencing. Twenty μL of each extract  were prepared for shotgun metagenome sequencing. Double-stranded Illumina libraries were constructed following a custom double-indexing protocol (protocol  optimizeprotocol . After 1(iv) Bioinformatic analysis. Sequencing data were demultiplexed and sorted according to the individual sample barcodes using Illumina bcl2fastq conversion software. Raw reads were processed for their quality, the adapter sequences were removed, and paired-end reads collapsed using AdapterRemoval (v2)   software Prinseq , Kraken2 Prinseq  was usedhttps://www.ncbi.nlm.nih.gov/refseq/) was created. To avoid spurious classification, we masked reference genomes for low-complexity regions with Dustmasker (https://www.ncbi.nlm.nih.gov/books/NBK569845/). The output of Kraken2 was analyzed through Bracken software to estimate species read abundances  (threshold 1 –requiremapq = 30) (https://github.com/pontussk/PMDtools). For each species aligned, bam files were converted to bed using bedtools (https://bedtools.readthedocs.io/en/latest/) and we estimated the edit distance (-tag NM) that was subsequently used to compute the edit distance algorithm (−Δ%) to further confirm sequence authenticity  database (undances . In orde(-n 0.1) . Alignedenticity . The Braenticity .Streptomyces sp. AV19 was extracted as previously reported  in ‘conservative’ bridging mode  in case of E. coli and K. rhizophila, and in 10 mL of YPD Soft Agar  in case of S. cerevisiae (both media were previously melted and cooled before addition of microorganisms), and poured in Petri dishes. Then, a circular plug from the aforementioned cultures of testers was placed on the surface of solidified target organism cultures. E. coli and K. rhizophila plates were incubated at 37°C for 24 h, while those with S. cerevisiae were incubated at 30°C for 24 h. The production of antibiotics was qualitatively evaluated as the presence of growth inhibition halos surrounding the agar plugs. This assay was performed in triplicate and agar plugs of the corresponding solid media  were used as control.All bacterial isolates were tested to assess the production of bioactive metabolites. The agar plug method was applied and and R2YE  at 30°C Streptomyces sp. AV19 grown on R2YE with MeOH (40 mL), under sonication for 30 min. The methanolic extract was centrifuged  and the supernatant was directly injected or diluted with mobile phase. Water and acetonitrile were of HPLC/MS grade. Formic acid was of analytical quality. The HPLC system was an Agilent 1260 Infinity. A reversed-phase C18 column  with a Phenomenex C18 security guard column (4 mm x 3 mm) was used. The flow rate was 0.4 mL/min and the column temperature was set to 30°C. The eluents were formic acid–water  (phase A) and formic acid–acetonitrile  (phase B). The following gradient was employed: 0 to 10 min, linear gradient from 5% to 95% B; 10 to 15 min, washing and reconditioning of the column to 5% B. Injection volume was 10 μL. The eluate was monitored through MS TIC. Mass spectra were obtained on an Agilent 6540 UHD accurate-mass Q-TOF spectrometer equipped with a Dual AJS ESI source working in positive mode. N2 was employed as desolvation gas at 300°C and a flow rate of 8 L/min. The nebulizer was set to 45 psig. The Sheat gas temperature was set at 400°C and a flow of 12 L/min. A potential of 3.5 kV was used on the capillary for positive ion mode. The fragmentor was set to 175 V. MS spectra were recorded in the 150 to 1000 m/z range. Metabolite identification was accomplished by means of HRMS data and comparison with the Metlin database . Two independent replicates were analyzed.Samples for HPLC/MS analysis were prepared by treating cultures of 2PO4, 3.4 g/L K2HPO4, 0.3 g/L MgSO4 · 7H2O, 0.01 g/L FeSO4 · 7H2O, 0.1 g/L ZnCl2, 0.01 g/L CuSO4 · 7H2O, 0.003 g/L MgCl2 · 4H2O, 0.01 g/L CaCl2, 0.03 g/L NaCl, pH 7) (3) to remove running buffer traces, then they were incubated in activation buffer  for 1 h at 50°C to activate proteases. Gels were stained with a staining solution  overnight under gentle shaking. Finally, gels were treated with a destaining solution  and presence of proteases was assessed as the appearance of clear bands on the stained background.The protocol used for detection of secreted proteases in spent media derived from Salamone et al.  with minl, pH 7)  at 30°C PRJNA877812.Shotgun sequencing reads have been deposited in the Sequence Read Archive (SRA) database and are available under the BioProject with accession number JAOAQK000000000. The version described in this paper is version JAOAQK010000000. The BioProject accession number is PRJNA877812.Draft genome was deposited at DDBJ/ENA/GenBank under the accession


The Greiss and trapping reaction results showed that Azospirillum brasilense (HM053 and FP2), Rhizobium tropici (Br322), and Gluconacetobacter diazotrophicus  produced the highest NO levels 24 h after inoculation, whereas Nitrospirillum amazonense (Y2) and Herbaspirillum seropedicae (SmR1) showed no NO production. In contrast to the literature, in NFbHP–NH4Cl–lactate culture medium with KNO3, NO trapping led to the recovery of a product with a molecular mass ion of 182 Da, namely, 1,2,3,4-naphthotetrazole (NTT), which contained one more nitrogen atom than the usual NAT product with 169 Da. This strategy allows monitoring and tracking NO production in potential biofertilizing bacteria, providing future opportunities to better understand the mechanisms of bacteria–plant interaction and also to manipulate the amount of NO that will sustain the PGPB.Plant growth-promoting bacteria (PGPB) can be incorporated in biofertilizer formulations, which promote plant growth in different ways, such as fixing nitrogen and producing phytohormones and nitric oxide (NO). NO is a free radical involved in the growth and defense responses of plants and bacteria. NO detection is vital for further investigation in different agronomically important bacteria. NO production in the presence of KNO Rhizobacteria that establish positive interactions with roots are referred to as plant growth-promoting bacteria (PGPB) and are promising candidates for building biofertilizers useful for sustainable agriculture practices . PGPB caBradyrhizobium genus [Azospirillum, Gluconacetobacter, Azoarcus, Enterobacter, Herbaspirillum, Burkholderia, and Rhizobium, have been reported as potential biofertilizers [Bradyrhizobium and Rhizobium, are responsible for nitrification–denitrification, where nitric oxide (NO) is a key intermediate. During denitrification, nitrate (NO3−) is reduced to nitrite (NO2−), NO, nitrous oxide (N2O), and molecular nitrogen (N2) through the actions of nitrate reductase (reduces NO3− to NO2−), nitrite reductase (reduces NO2− to NO), nitric oxide reductase (reduces NO to N2O), and nitrous oxide reductase (reduces N2O to N2) [3), to NO2−, with hydroxylamine (NH2OH) as a key intermediate. Nitrification enzymes include ammonia monooxygenase (converts NH3 to NH2OH) and hydroxylamine oxidoreductase (converts NH2OH to NO2−), along with nitrite and NO reductases [Azospirillum genus, can use NO3−, NO2−, or nitrous oxide (N2O) as final electron acceptors [The benefits of biofertilizers originate from the symbiosis of legumes with bacteria, such as the um genus . Other btilizers ,7,8,9,10O to N2) . Conversductases . Studiescceptors NO is a reactive gaseous molecule with many important biological functions. It is present in all living organisms and can act as a signaling molecule at low concentrations (nM levels). In plants, NO signaling controls growth, development, and plant defense responses to pathogens. In bacteria, NO also induces a defense response against pathogens and may protect against oxidative stress ,15. FromAzospirillum brasilense using fluorescence probes, 4,5-diamino fluorescein diacetate (DAF-2D) has been developed for NO detection, quantification, and mechanism elucidation [A. brasilense show evidence of NO mediation of the indole-3-acetic acid (IAA) signaling pathway to increase lateral and adventitious root formation in tomato plants [Common methods for NO detection in bacteria include the Griess method, electron paramagnetic resonance (EPR), electrochemical methods, and fluorescent probes. These methods have been used for NO detection in plants inoculated with PGPB . In studcidation . Inoculao plants ,20.2 and H2O, mainly forming NO3− and NO2−. An indirect method for NO determination involves the spectrophotometric measurement of its decomposition products NO3− and NO2−. This method, known as the Greiss reaction, involves the reduction of NO3− to NO2−. NO2− subsequently reacts with sulfanilamide in an acidic medium to produce a diazonium ion that reacts with N-(1-naphthyl) ethylenediamine to form an azo-chromophore compound that exhibits strong absorbance at 543 nm . DAN detection exhibits NO detection limits in the nanomolar range of 10 nM to 10 μM [To improve NO detection sensitivity, another method was developed based on the diamino aromatic compound 2,3-diaminonaphthalene (DAN) as an indicator of NO formation . DAN is to 10 μM ,22,23 anAgave pacifica plant cells using DAN with high-performance liquid chromatography (HPLC) analysis and fluorescence detection. Furthermore, the DAN method is efficient for NO detection in fungal and bacterial cells, although studies have mainly focused on plant growth-promoting symbionts, including mycorrhizae, nutrient solubilizers, and diazotrophs [Reports have shown an improved understanding of NO production in biological systems based on the DAN method. Wada et al. (2002)  showed tzotrophs ,27,28,292-fixing bacteria can synthesize different amounts of NO in the presence of KNO3 and at different cultivation times and that NO can be detected by fluorescent probes. Bacteria that form associations with grasses and soybeans were selected; the Greiss reaction and DAN chemical trap were used to quantify and monitor the synthesized NO levels, respectively. Fluorescent probes have provided abundant information regarding the location and mechanism of reactive species produced in biological systems due to their detection sensitivity. However, few studies have investigated NO detection in diazotrophic biological systems, including bacteria and cyanobacteria ,30,31. THerein, an improved DAN chemical trap and the Griess method were applied as facile and inexpensive methods to identify PGPB that produce NO. This is an important advancement because only a few species and strains have been evaluated for NO production to date. The method developed in our study will help identify strains that can be potentially used as biofertilizers for important agricultural plants. In addition, the methods can be applied in future studies to monitor and manipulate NO production on plant–bacteria interactions. A. brasilense (FP2); A. brasilense (HM053); Herbaspirillum seropedicae (SmR1); Nitrospirillum amazonense (Y2); Rhizobium tropici (BR322); Agrobacterium fabrum (L40); Gluconacetobacter diazotrophicus ; and Agrobacterium fabrum (R5). The bacteria were grown in 96-well plates containing liquid NFbHP–NH4Cl–lactate [3 at 30 °C with shaking (140 rpm). After 24, 48, and 72 h of growth, the bacteria were collected and centrifuged, and then the supernatant was used for NO measurements.Eight strains of bacteria were used: –lactate  supplemeBacterial NO production was quantified using the Griess method  and moni−1 of NO/µg mL−1 of total protein.The Griess method was performed according to the procedures described in the literature , and sam4Cl–lactate medium with 10 mM KNO3 and 80 µg mL−1 DAN with bacteria; (2) NFbHP–NH4Cl–lactate medium with 10 mM KNO3 and 80 µg mL−1 DAN without bacteria; and (3) NFbHP–NH4Cl–lactate medium with 10 mM KNO3 without DAN and bacteria. Conditions 2 and 3 were used as controls. After incubation, the bacteria were centrifuged and the supernatant was deposited in a 96-well ELISA plate (suitable for fluorescence) and analyzed using SpectraMax Paradigm Multi-Mode Microplate Reader  with excitation and emission at 365 and 465 nm, respectively. For fluorescence detection, the bacteria were grown under the same conditions described above, except with the addition of the NO trap DAN. The following incubation conditions were used: (1) NFbHP–NH4Cl–lactate medium; (3) DAN 0.005 mg mL−1; (4) DAN 0.005 mg mL−1 with DEA NONOate 0.005 mg mL−1; (5) DAN 0.005 mg mL−1 with DEA NONOate 0.0125 mg mL−1; and (6) DAN 0.005 mg mL−1 with DEA NONOate 0.025 mg mL−1. Conditions 1 and 2 were used as controls. Analyses were performed using a 96-well ELISA plate and SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices) equipment with excitation and emission wavelengths of 365 and 465 nm, respectively.DAN fluorescence analyses were performed using diethylamine NONOate diethylammonium salt (DEA NONOate), a NO generator. The following conditions were applied: (1) no sample; (2) water and NFbHP–NHThis procedure was based on detecting NAT complex formed in bacteria using gas chromatography–mass spectrometry (GC-MS) and Varian HPLC. NAT was extracted with toluene by adding 1 mL toluene to a 1 mL bacterial sample. The phases were separated, and the toluene phase was inserted in a GC-MS injection vial.−1 and then to 280 °C at a rate of 15 °C min−1, with He as the carrier gas. The injector, interface, and source temperatures were 240 °C, 250 °C, and 250 °C, respectively. The injection volume was 1 μL, with a split of 20 mL min−1. For GC-MS NAT detection, a Perkin Elmer Claris SQ 8T instrument with an Elite-5 ms Perkin Elmer column (30 m × 0.25 mm I.D. × 0.25 µm) was used. The oven was programmed from an initial temperature of 60 °C rising to 240 °C at a rate of 3 °C minHPLC was used to evaluate the products formed in the DEA NONOate experiment, with method development featuring an acetonitrile/water elution system with 30–90%, with an acetonitrile gradient for the first 10 min, followed by 30–90% acetonitrile for an additional 2 min, and the remaining with 30% acetonitrile until 15 min, at a flux rate of 1 mL/min. A C18 Luna Phenomenex column  was used with an injection volume of 20 µL sample and a standard injection volume. NO production by bacterial strains was evaluated in a 10 × 3 factorial scheme, with 8 bacterial strains at 3 time points after inoculation . The fluorescence data obtained from NAT production by the different bacterial strains were also evaluated in a 10 × 3 factorial scheme, including the 8 bacterial strains and 2 controls at 3 time points after inoculation . Data were analyzed using analysis of variance (ANOVA), and means were compared using Tukey’s test at a 5% probability.3. NO is an important messenger in biological systems, and we aimed to determine its production in different bacterial strains using a Greiss assay and a fluorescent probe to quantify and monitor the NO generation processes in the presence of KNOA. brasilense HM053 and FP2 synthesized the highest amounts of NO , followed by R. tropici Br 322 (0.0521 µg mL−1) and G. diazotrophicus Pal 5  and H. seropedicae (SmR1) did not produce NO at any time point, as indicated by the Griess and fluorescent assays (A. fabrum (R5) showed a low (0.0036 µg mL−1 NO) production at 24 h of growth and no NO production at other times, similar to the fluorescence results  strain, which did not produce NO after 24 h. However, after 48 and 72 h, L40 showed NO concentrations of 0.0239 and 0.0058 µg mL−1, respectively  and G. diazotrophicus Pal 5 (2.9 × 109 a.u.), followed by those of A. brasilense FP2 (6.9 × 108 a.u.) and HM053 , N. amazonense (Y2), H. seropedicae (SmR1), and A. fabrum (R5) did not show significant NAT formation over time, whereas A. fabrum (R5) showed low fluorescence at 24 h after inoculation  instead of NAT, given the presence of reactive nitrogen species (RNS) in the medium , where its decomposition is nearly instantaneous at pH 5.0. Fluorescence analysis was performed with excitation and emission at 365 and 465 nm, respectively, showing product formation in water and culture medium  medium supplemented with 12 mM KNO3 showed initial rates of NO production of 10 nmol min−1 per mg of cell protein and a maximum concentration of 200–300 µmol L−1 [The DAN assay has already been demonstrated as a plausible model for NO production monitoring in hia coli ,42; howeµmol L−1 ,42. A. brasilense HM053 and FP2, R. tropici Br322, and G. diazotrophicus Pal5 to synthesize NO in NFbHP–NH4Cl–lactate medium supplemented with 10 mM KNO3. This study is the first to establish DAN fluorescent probes as an inexpensive and reliable NO detection method that can be applied in different rhizobacteria species and in studies that aim to understand the mechanisms associated with plant–bacteria interactions. In this study, the main goal was to show that the Griess and DAN assays were efficient for NO detection; here, we confirmed the ability of A. brasilense Sp245, which can produce NO by aerobic denitrification in an OAB medium containing NO3. The NO concentration was approximately 25-fold higher in NO3−-containing media (120 nmol NO per gram of bacteria) than in OAB with NH4+ (4.2 nmol NO per gram of bacteria) as detected by EPR spectroscopy [3− is the main source of NO production by A. brasilense. In our work, NFbHP–NH4Cl medium supplemented with KNO3, an extra N source, was used to grow the bacteria strains; after 24 h to 72 h, NO quantification was performed using the Griess assay. NO concentrations ranged from 0.1435 for A. brasilense HM053 to 0.0036 for A. fabrum R5, normalized to total protein content.Denitrification can also occur under fully aerobic conditions , as showtroscopy . This suA. brasilense plasmids  [Moreover, genes associated with denitrifying activity have been identified in s genes) ,45. Mores genes) .A. brasilense and concluded that the bacterially-derived NO is involved in the induction of lateral roots in tomato plants. Important functions have been attributed to NO in PGPB. Molina-Faveiro et al. 2008)  and Creu08  and CA. brasilense is potentially involved in biofilm formation, which is important for root colonization and growth; moreover, considerable quantities of endogenous NO can be formed by A. brasilense by different metabolism using two N sources, such as NH4Cl and KNO3. Thus, a correlation between the production of NO3− and the two N sources used was concluded [A. brasilense are associated with NO-promoted activation of IAA signaling pathways [A. brasilense, R. tropici, and G. diazotrophicus may participate [NO produced by oncluded . Howeverpathways ,49,50. Pticipate ,52. Thesticipate , which aticipate .Rhizobium genus, NO production occurs during the symbiosis process between Rhizobium and legumes, from the initial interaction to nitrogen fixation nodule formation [For the ormation . Signoreormation  studied A. brasilense, the H. seropedicae (SmR1) genome was completely sequenced, and no genes encoding denitrification enzymes, such as nitrous oxide reductase, [H. seropedicae. In contrast to NO-producing bacteria such as ductase,  were ideductase,  also fouN. amazonense, Kloos et al. (2001) [Regarding . (2001)  evaluate2), NO3−, NO2−, and others [2-fixing bacteria were cultivated in NFbHP–NH4Cl–lactate medium supplemented with KNO3 under aerobic conditions, transient NO formation occurs during denitrification through the action of nitrite reductases [In general, the synthesis of RNS in NO-producing bacteria decreased or did not occur after 24 h of incubation, as indicated by the detection of the reaction product with DAN. Moreover, NO synthesis as a signaling molecule appears to be a rapid transient process, as it is rapidly converted into important biological derivatives, including nitrogen dioxide  [Neisseria subflava was optimal with glucose as an electron donor for NO2− reduction to a nitrosating species. Herein, NFbHP–NH4Cl–lactate medium was used with lactate as the carbon source. Thus, lactate decarboxylation can provide the electrons necessary for the reduction of nitrogen by-products from the KNO3 and NH4Cl reactions.The etection . Ji and etection ,42 demone (1990)  demonstr4Cl–lactate medium and the same medium with KNO3, DAN was converted into a product with a molecular mass of 182 Da, NTT. Future work will focus on the structural elucidation of this product to help further understand the mechanism by which DAN acts as a NO probe in bacterial systems and its application for monitoring processes related to KNO3 metabolism by N2-fixing bacteria.Through bacterial metabolism and the generation of NO via DEA NONOate in NFbHP–NH3 by indirect detection . Notably, in the absence of KNO3, NO was not detected.In this study, we present results that show the production of NO metabolized by bacteria treated with a source of KNOOur study helps elucidate the NO production mechanism in bacteria because this molecule is an essential component in the agribusiness sector, given its role in promoting plant growth through the BNF mechanism. However, more refined future experiments are needed, such as isotopic labeling and elucidation of the product formed in chemical trapping.A. brasilense HM053 and FP2, R. tropici Br322, and G. diazotrophicus Pal5 can produce detectable NO concentrations in NFbHP–NH4Cl–lactate medium containing KNO3. After 24 h of growth, NO concentrations ranged from 0.1435 µg mL−1 (A. brasilense HM053) to 0.0036 µg mL−1 (A. fabrum R5), as determined using the Griess assay. After 48 and 72 h of growth, these strains showed decreased or no production of NO, similar to R5. Azospirillum sp. L40 differed from the other strains, not producing detectable amounts of NO at 24 h of growth, but started forming NO after 48 and 72 h. Overall, the Griess and DAN results are mutually consistent, confirming the applicability and reliability of DAN for NO detection. The results presented herein are consistent with our hypothesis that 4Cl–lactate as a culture medium added with KNO3 results in the recovery of a product different from NAT  with or without bacterial metabolism. This product ) has a molecular mass of 182 Da and may assist in the understanding of mechanisms associated with the action of DAN and in the elucidation of KNO3 metabolic pathways as a nitrogen source for NO generation in diazotrophic bacteria. The approaches used herein may contribute to further understanding of processes associated with NO production by bacteria and be applied in future projects to better understand the role of NO in cross-talk between plants and PGPB. The ability to synthesize NO is linked to oxidative stress resistance, which can amplify BNF and promote plant growth. DAN probing of NO is an efficient method for detecting NO production in bacterial systems. Moreover, using NFbHP–NH


The cutaneous lipidomics of consumers of water A vs. water B were statistically different (p < 0.05). Twenty cutaneous lipids were necessary to predict the water type previously consumed (AUC ~70). Our study suggests that drinking oligo-mineral water may change skin biology and may influence the cutaneous barrier, so future dermatological clinical trials should also account for the water type consumed to avoid potential confounders.Despite the well-known cutaneous beneficial effect of thermal water on the skin, no data exist regarding the potential biological effect of orally consumed water on healthy skin. Thus, in this single-center, double-blind, randomized controlled clinical trial conducted on age and menstrual cycle timing-matched healthy female volunteers (24 + 24) consuming water A  or water B  for 1 month (T1), the cutaneous lipidomics were compared. Interestingly, only water A consumers had a statistically significant ( Ablutions in thermal water are an ancient treatment for several dermatological disorders refractory to the poor topical pharmacopeia that shamans, healers, alchemists, surgeons and doctors worldwide adopted ,2,3,4,5.Preliminary in vitro studies on keratinocyte cultures suggested antioxidant , anti-anWater can also be classified using the fixed residue (fd) of mineralized, oligo-mineral, mineral or medium-mineral, and highly mineralized , randomized controlled clinical trial conducted on healthy volunteers. The study includes age and menstrual cycle timing-matched female subjects who blindly consumed water A or water B, both marketed as food and not supplements. The enrolled subjects were asked to drink 2 L of water of the assigned type (water A or water B) per day.®.At the baseline (T0) and after 1 month (T1), patients (a) were clinically evaluated independently by two board-certified dermatologists (>5 years of experience), (b) performed complete blood count (CBC), creatinine and physical (macroscopic) chemical urine tests, and (c) collected a skin stripping with Patch SebutapeThe study fulfilled the Helsinki Declaration and the General Data Protection Regulation 2016/679, so it was approved by the Institutional Review Board of San Raphael Hospital . All participants signed an approved informed consent form.Waters were classified in accord with the Marotta & Sicca classification of Italian mineral waters that remains the parameters required by Italian law to market mineral waters (see D.Lgs. 25.01.1992 n°105 and D.Lgs. 176/2011) [Notably, chemical analyses of water A  and B  are reported in We enrolled (a) female subjects, (b) aged between 30 and 50 years, (c) in good health  code in compliance with the definition of the World Health Organization: “a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity.”), (d) omnivores ,21,22,23Conversely, subjects were excluded if they were/had (a) males; (b) in a different age range (<30 and >50 years of age) or with a Body Mass Index (BMI) < 19 or >30; (c) active or latent inflammatory, oncological and rheumatological diseases; (d) menopausal/gynecological disorders ; (e) chronic dermatoses ; (f) allergic disorders , board-certified dermatologists (G.D. and P.P.). Clinical and dermatoscopic evaluations were performed at T0 and T1.Dermatologists also administered the Skin Satisfaction Questionnaire (SSQ) at T0 and T1 to the enrolled subjects .®  placed on the cleansed (micellar water + clorexidin 2%) glabella region for 30 min, and after removal, it was stocked in a 13 mL vial. Then, we added 5 mL dichloromethane:methanol (2:1), and the vial was shook for 15 s with a vortex. We performed a sonication for 5 min and a centrifugation at 4000 rpm for 10 min at 15 °C on the solution of the organic phase, which was transferred to an Eppendorf tube and concentrated under nitrogen flow until the extract was completely dry. The residue was centrifuged for 5 min at 4000 rpm at 15 °C, and the supernatant was collected and finally analyzed. At the same time as the analysis of the samples, QC samples obtained by taking and combining 15 µL of each extracted sample were prepared and analyzed.The cutaneous sample was obtained with Patch SebutapeAll samples were analyzed at UNITECH OMICs  using an ExionLC™ AD system  connected to a TripleTOF™ 6600 System  equipped with a Turbo V™ Ion Source with an ESI Probe. The software used was SCIEX OS version 3.1 .® EVO C18—2.1 × 100 mm, 1.7 μm  equipped with a precolumn was achieved using, as mobile phase A, water/acetonitrile (60:40) and, as mobile phase B, 2-propanol/acetonitrile (90:10), both containing 10 mM ammonium acetate and 0.1% of formic acid. The flow rate was 0.4 mL/min, and the column temperature was 45 °C. The elution gradient was set as 0–2 min (45% B), 2–12 min (45–97% B), 12–17 min (97% B), 17–17.10 min (97–45% B) and 17.10–20 min (45% B). The sample injection volume was 5 μL. Details are carefully reported in The chromatographic separation on a Kinetexm/z range of 140–2000 Da in positive and negative polarity, operating in IDA mode . The collision energy was set at 35 (CES 15).MS spectra of the skin was collected over an The False Discovery Rate (FDR) was calculated with Tibshirani and Storey’s q-value, with a = 0.05 or b = 0.10. The True Discovery Rate (TDR) was estimated from 1000 series of simulations in R. The CV (coefficient of variation) for the simulation was set at 0.35. The importance of the single metabolite depends on the ratio between the log ((FC))/CV, in which FC increases or decreases in the single metabolite. We chose an FDR of 0.83, hypothesizing that 200 metabolites of concentrations of at least 1.5 times would truly differ. Thus, our sample size calculation suggested a minimum of 20 subjects for the cases and controls that we increased 20%, arriving at an enrollment of 48 (24 + 24) subjects.t-test for paired samples was applied to compute the mean differences before and after drinking Water type A or B.Clinical variables’ distribution was assessed for normality by applying the Kolmogorov–Smirnov test. Data were computed as means ± standard deviations for continuous variables, and they were expressed as percentages in the case of categorical parameters. Student’s m/z (parent ion) found and determined in high resolution of these IDs, those who, in the QC samples alone, had a CV%  lower than 40%, with a number of identifications equal to thirty (n = 30). After this first processing of the samples under analysis, only the certain identifications (IDs) were considered by comparison with the LipidBlast database and with the acquired MS/MS spectrum.All samples were processed with MS-DIAL software ver. 4.24 by setting the LipidBlast integrated database (version 68) . The processing of the files was set by applying the normalization that supports the LOWESS algorithm and the “Blank Filter” option that filters the peaks present in the “Blank Sebutape” samples. Following this normalization, the program returned a number of identifications (IDs) based on the value of the Data for skin were obtained separately and analyzed using R version 4.0.3 . Each dataset was cleaned and underwent quality control to ensure there were no outliers. Samples that had an “nd”, signifying not detected, were changed to 0.Principal component analysis was performed on the datasets using the function prcomp, with the options “center = TRUE” and “scale = TRUE”, from R package stats version 4.0.3. Plots were generated using R package ggplot2 version 3.3.6, while heatmaps were generated using pheatmap version 1.0.12.p.adjust function using the Benjamini–Hochberg method. Samples with a PAdj ≤ 0.05 were considered significant; meanwhile, when there was a lack of any significant lipids, a p value ≤ 0.05 was used instead.Differential abundance analysis was performed using the wilcox.test function from R package stats version 4.0.3. Given that we paired samples, Time 0 and Time 1 for each sample pair, in comparisons with paired samples, we used the option “paired = TRUE”; otherwise, comparisons between different groups used “paired = FALSE”. The correction for multiple testing was performed using the Using the cv.glmnet function with “family = ‘binomial’” from R package glmnet version 4.1-2, a classifier using k-fold cross-validation was built to attempt to differentiate between the different types of water used. The area under the curve was calculated and used as the metric to determine the model that has the highest accuracy.2 (64.6 ± 8.1 kg), with no difference between the two considered groups (p < 0.05).In the present clinical trial, 48 (24 + 24) female subjects were enrolled, and their mean age was 44.7 ± 2.6 years of age. No age differences were detected between Water A and Water B drinkers. The average BMI of the enrolled patients was 24.3 ± 2.6 kg/mp > 0.05); interestingly, subjects that consumed Water A manifested a statistically significant decrease of SSQ from T0 to T1.SSQ globally improved in both groups, but in a non-statistically significant manner (p < 0.001): 54 Glycerolipids (18 Diacylglycerols (DG), 12 EtherDG, 24 Triacylglycerols (TG)), 8 Glycerophospholipids (5 Lysophophatidylcholines (LPC), 3 Phosphatidylcholines (PC)), 3 Sphingolipids, 1 Hexosylceramide hydroxyfatty acid-sphingosines (HexCer_HS), 1 Sphingosine (Sph), 1 Sphinganine (DHSph) and 1 Fatty acyls (1 Acylcarnitine (CAR)). Overall, 8 glycerolipids decreased (6 TG and 2 DG), and the others increased. For further details, see Interestingly, after 1 month, only patients that consumed Water A displayed a change in cutaneous lipidomics. In total, 66 lipids registered a statistically significant change  the present clinical trial also has some limitations, such as the inclusion of solely female subjects between 30 to 50 years of age. Thus, future studies should also validate these results in different ages, males and even diets.Water orally consumed has a remarkable effect on the skin lipidomics in healthy subjects, so especially oligo-mineral water should be regarded as a complementary therapy in dermatological disorders. Furthermore, clinical trials evaluating nutritional supplements or diets in dermatology should account also for the water type consumed. Thus, water has to be regarded as a supplement for its biological effect on skin.


The synthesized Ag/GN@PPy–Ni(OH)2 nanocomposites were characterized for their morphological characteristics by field emission scanning electron microscopy (FESEM), while the structural investigations were done by X-ray diffraction and X-ray photoelectron spectroscopy (XPS). The FESEM studies showed Ni(OH)2 flakes and silver particles attached over the surface of PPy globules, along with the presence of GN sheets and spherical silver particles. The structural analysis also showed the presence of constituents, i.e., Ag, Ni(OH)2, PPy, GN, and their interaction, therefore vouching that the synthesis protocol is efficacious. The electrochemical (EC) investigations were done in potassium hydroxide (1 M KOH) using a three electrode setup. The quaternary Ag/GN@PPy–Ni(OH)2 nanocomposite electrode showed the highest specific capacity of 237.25 C g−1. The highest electrochemical performance of the quaternary nanocomposite is associated with the synergistic/additional effect of PPy, Ni(OH)2, GN, and Ag. The assembled supercapattery with Ag/GN@PPy-Ni(OH)2 as a positive and activated carbon (AC) as a negative electrode displayed eminent energy density of 43.26 Wh kg−1 with the associated power density of 750.00 W kg−1 at a current density of 1.0 A g−1. The cyclic stability of the supercapattery (Ag/GN@PPy–Ni(OH)2//AC), comprising a battery-type electrode, displayed a high cyclic stability of 108.37% after 5500 cycles.In this work, silver (Ag) anchored over graphene (GN) wrapped polypyrrole (PPy)@ nickel hydroxide (Ni(OH) Due to the rapid industrialization and depletion of non-renewable fuel resources, there is a huge demand to explore newer alternatives and greener energy sources ,2,3. SolFuel cells and batteries are commonly used storage devices, but their major limitations are their low power supply, where a large supply is needed . Superca2 electrode displayed a specific capacitance of 142.30 F g−1 while retaining the cyclic stability of 98.11% at a scan rate of 100 mV s−1. The developed hydrogel composite of polyaniline/GN/Fe2O3 by Gupta et al. [−1 at 0.25 A g−1. Similarly, composite of GN, polypyrrole (PPy) and Cu2O–Cu(OH)2 by Asen et al. [−1 at a current density of 10 A g−1. Among conducting polymers, PPy has many advantages owing to its reversible electrochemical doping/dedoping, low cost, and long shelf life [2 is exciting due to its well-defined redox behavior, layered structure, and cost-effectiveness. Liu et al. [2 nanoparticles and GN sheets delivered a specific capacitance of 1717 F g−1 at 0.5 A g−1. Similarly, a polyaniline composite with Ni(OH)2 has been reported to possess a specific capacitance of 113.8 F g−1 [2 is expected to exhibit eminent electrochemical characteristics owing to the synergism between the constituents. In our previous reports, it was observed that the addition of Ag nanoparticles to conducting polymer composites enhances electrochemical conductivity [2 can give a new class of quaternary nanocomposite with much-enhanced electrochemical features.The electrochemical evaluation of supercapattery depends on the electrode material. Carbon-based materials and conducting polymers have been widely employed in combination with metal oxides, sulfides, hydroxides, etc., as electrodes have shown excellent electrochemical phenomena ,9,10. Paa et al.  demonstrn et al.  showed aelf life . Among mu et al.  showed t2 was synthesized. The characterization of the prepared quaternary nanocomposites was done by diverse analytical techniques such as XPS, EDAX, XRD, and FESEM. The standard three-electrode cell was used for electrochemical findings, such as galvanostatic charge–discharge and cyclic voltammetry. Finally, the evaluation of the final fabricated device comprising a battery-type quaternary nanocomposite, Ag/GN@PPy-Ni(OH)2 as a negative electrode and AC as a positive electrode was tested in a two electrodes system by means of CV and GCD.A simplistic synthesis of novel quaternary nanocomposites of silver anchored on GN-wrapped PPy–Ni(OH)w ~534,000; purity 98%), FeCl3 , Ni(NO3)2·6H2O , AC (M.W. 12.01), graphite flakes (M.W. 12.01), silver nitrate , ascorbic acid nitrate , H2SO4 , H3PO4 (85%) sodium dodecylbenzenesulfonate (SDBS) (M.W. 348.48), and acetylene black were bought from Sigma-Aldrich. Potassium permanganate , N-methyl-2-pyrrolidone (NMP) (M.W. 99.13), HCl , and ethanol  were purchased from Otto chemicals . De-ionized water was used throughout the experiments.Pyrrole monomer , Polyvinylidene fluoride  was added to the pyrrole dispersion under vigorous stirring. The solution soon turned dark black, thereby confirming the polymerization. Thus, the formed PPy settled down after centrifuging the mixture solution. Afterwards, repeated washing was done with a mixture of water and ethanol and finally dried in an air oven at 80 °C. In order to prepare Ag/GN@PPy-Ni(OH)2, 0.20 g of PPy and 0.20 g of Ni(OH)2 were mixed with 10 mL of GO solution (4 mg mL−1) and to AgNO3 (20 mg), followed by the addition of a 15 mL NH3 (6%) solution under stirring conditions. The whole reaction mixture was transferred into a Teflon-lined hydrothermal rector and subjected to a temperature of 150 °C for 5 h. Thus, the formed nanocomposite, Ag/GN@PPy–Ni(OH)2, was centrifuged in order to separate it from other impurities. After separating the synthesized material, washing was done with water and ethanol to ensure the complete removal of any impurities and finally dried in an air oven at 80 °C. The binary PPy–Ni(OH)2 was synthesized by mixing 0.20 g of each PPy and Ni(OH)2 in ethanol under stirring, followed by the evaporation of ethanol. While the ternary GN@PPy–Ni(OH)2 was produced by setting the hydrothermal reaction following the same conditions and procedure as mentioned above for the quaternary nanocomposite but without AgNO3.Ni(OH)lsewhere . The synlsewhere . Pure PPKα source, was used to explore the structure of the formed crystals and the identification of the phases of Ag/GN@PPy–Ni(OH)2 nanocomposites at 2θ° through a scan range of 5° to 70°. A highly evacuated (∼10−9 m bar) chamber, comprised of monochromatic Al-Kα radiation, was used to perform the XPS study.The FESEM and EDAX techniques were conducted for the topography, elemental analysis, and mapping. The XRD machine, along with Cu-2 and AC. Chemically cleaned nickel foam of area 1 × 1 cm2 was used to coat active material to get the electrodes for the electrochemical measurements. The nanocomposite of Ag/GN@PPy–Ni(OH)2 at 75 wt. %, AC at 15 wt. %, and PVdF at 10 wt. % were mixed in NMP to prepare the slurry for the active material. The homogeneity of the solution was ensured after 12 h of magnetic stirring at ambient temperature. After a uniform drop coating of the slurry on the 1 × 1 cm2 nickel foam, it was left for drying in an oven at 80 °C for a period of a minimum 12 h. A similar method was implemented on GN@PPy–Ni(OH)2, PPy–Ni(OH)2 and PPy to prepare their working electrodes. The prepared electrodes were then brought to mass loading of ~5.00 ± 0.05 mg cm−2 of the active material. The electrode fabrication procedure was done with great accuracy to obtain consistent mass loading.The positive and negative electrodes of the supercapattery were fabricated from quaternary nanocomposite of Ag/GN@PPy–Ni(OH)−1.A working potential between 0–0.5 V versus reference (Ag/AgCl) was implemented to perform the cyclic voltammetry. To study the galvanostatic-charge discharge, a potential of 0.5 V versus Ag/AgCl was used through the electrochemical workstation (Gamry Interface 1010E) at a current density range of 4.0–10.0 A g2, GN@PPy–Ni(OH)2 and Ag/GN@PPy–Ni(OH)2 nanocomposite. Pure PPy showed loosely interconnected globular morphology with globules interconnected to form large lumps and long chains of micrometer length. The Ni(OH)2 flakes and particles were attached over the surface of PPy globules. It can be seen that Ni(OH)2 is attached as agglomerates, thereby covering PPy globules or a few clusters of Ni(OH)2 flaked/particles that can also be seen protruding out of PPy globules in other areas. The GN@PPy–Ni(OH)2 and Ag/GN@PPy–Ni(OH)2, in addition to the above features, show the presence of GN sheets and GN along with Ag spherical particles, respectively. The GN sheets can be seen distributed throughout the composite and the other constituents attached onto the sheets. The TEM images of Ag/GN@PPy–Ni(OH)2 nanocomposite at different magnifications can be seen in 2, is shown in 2 could be observed from the individual elemental mapping analysis, as well as in the compounded image, which further vouched for the efficiency of the synthesis procedure rocedure . The ima2, GN@PPy–Ni(OH)2, and Ag/GN@PPy–Ni(OH)2 nanocomposites, as shown in θ°) is associated to the scattering in PPy chains at interplanar spacings [2 shows the hump of PPy, along with the peaks corresponding to Ni(OH)2 at 2θ° of 18,80° 33,10° 38.40°, 52.17°, 59.26°, and 62.87 (JCPDS 14-0117), which is in good agreement with the reports of Li et al. [2, a similar diffractogram was observed, as in the case of PPy–Ni(OH)2. The non-observance of graphitic 002 planes in the binary and quaternary nanocomposites’ XRD spectra suggests a few layered GN present without any graphitic impurities [2, in addition to the peaks of PPy and Ni(OH)2, show peaks at 2θ degrees of 38.71°, 48.18°, 67.44° [θ° angle of 38.40°, which might be due to the similar peak positioning of Ni(OH)2 and Ag.X−ray powder diffraction analysis was done to determine the structural and crystallinity characteristics of PPy, Ppy–Ni(OH)spacings . PPy–Niectively . The O1sectively ,25. The ectively . Similar species . The Ni omposite .−1 in a potential range of 0–5.0 V. It is evident from the CV that the current density gradually increased as the scan rate increased, showing good electrochemical performance. However, there is a slight shift in redox peak potential at higher scan rates which is associated with the sluggish movement of 2 at various scan rates. The addition of nickel hydroxide particles to PPy improved the electrochemical behavior of conducting polymer [2 have been presented in 2 in GN provides the conducting plate form to PPy–Ni(OH)2, which promotes the electrochemical performance of ternary composite [2 particles helps to increase the active sites of GN@PPy–Ni(OH)2 nanocomposite. It is well known that a decoration of highly conductive noble metals such as Ag nanoparticles to a nanocomposite material boosts the electrochemical conductivity by providing the pathways to the shuttling of electrons [2. It is clear from the cyclic voltammetry studies that the addition of suitable constituents to the polypyrrole helps to increase the electrochemical performance due to the synergistic effect of all contributing materials in a nanocomposite. −1. A noticeable increase in the current density could be observed, thereby providing evidence of the usefulness of the chosen synthesis strategy for the prepared nanocomposite materials. The specific capacity 2, GN@PPy–Ni(OH)2, Ag/GN@PPy–Ni(OH)2) was calculated using the following relation (1) [−1) represents the scan rate of cyclic voltammogram, m (g) represents the active mass of the material, and the integral part of the relation is the area under the redox peak of CV. The specific capacity values computed at a scan rate of 3 mV s−1 for PPy, PPy–Ni(OH)2, GN@PPy–Ni(OH)2, and Ag/GN@PPy–Ni(OH)2 are 134.01, 155.65, 175.20, and 237.25 C g−1, respectively.The electrochemical characterization of pure PPy and its variants was performed in three electrodes cell using Ag/AgCl as the reference and the platinum wire as a counter, along with a working electrode fabricated by the mechanical coating of active material on nickel foam. Potassium hydroxide was used as an electrolyte (1 M) in all electrochemical measurements. t of OH− . Figure  polymer . The eleomposite . The aggomposite . The intlectrons . Figure tion (1) ;(1)QS=1−1 in pure form. The PPy was merged with nickel hydroxide, GN, and silver nanoparticles to form binary, ternary, and quaternary nanocomposites for enhanced electrochemical performance for energy storage. The FESEM micrographs, along with the GCD results, provide insight as to how PPy and GN enhance the EC performance and contribute to the phenomena of de−aggregation which in turn, exposed the active sites of the nanocomposite materials.The PPy material is considered highly useful for supercapattery because it has superior redox chemistry, large electrical conductivity, and a highly accessible surface area with a specific capacity of 125.85 C g2, GN@PPy–Ni(OH)2, and Ag/GN@PPy–Ni(OH)2 taken in 1 M KOH and galvanostatic current densities between 4.0–10.0 A g−1 in 2, as well as GN.The discharge curves of PPy, PPy–Ni(OH)I (current) is measured in A, Δt (the discharge time) in s and m  in g. The −1 were 125.85, 180.48, 228.80, and 268.00 C g−1 for PPy, PPy–Ni(OH)2, GN@PPy–Ni(OH)2, and Ag/GN@PPy–Ni(OH)2, respectively.The sion (2) ;(2)Qs=I2 was used as a positive and AC was sued as a negative electrode to build a supercapattery (Ag/GN@PPy–Ni(OH)2//AC). The 1 M KOH was used as an electrolyte in ambient conditions for the EC analysis of the constructed device. The schematic of the supercapattery is shown in 2, AC) were tested through separate CVs using a three-electrode cell system before the construction of the device. The potential window for capacitor and battery grade material was −1.0−0 V and 0−0.5 V, respectively ectively b. Thus, −1 is revealed in The display of these scan rates ranging from 3–100 mV s−1 at 1.5 V, a fixed potential. The faradaic reactions during the charging and discharging of the device were evident from the nonlinear GCD curves. An extremely reversible EC trend was seen in the constructed device.−1 at 1.0 A g−1 by using Equation (1) (I × ∆t)/m). Nevertheless, the −1.The estimated specific capacity of the device was 211.00 C gE, and power density, P, which were calculated by using Equations (3) and (4) [V, −1 and −1 and 5250.00 W kg−1, respectively, at a fixed current density of 7.0 A g−1. A decrease from 5250.00 to 750.00 W kg−1 in power density was detected when the energy density was changed from 43.96 to 20.53 Wh kg−1 with a corresponding current density 7.0 to 1.0 A g−1, respectively. The obtained values were higher than those already reported for nanocomposites electrode materials as shown in The Ragone plot shown in the inset of  and (4) ;(3)E2 with GO and Ag precursor and subsequently subjecting it to hydrothermal conditions to get silver decorated and GN wrapped PPy–Ni(OH)2 nanocomposite. The morphological analysis showed PPy globules or clusters of Ni(OH)2 laked/particles protruding out of PPy globules, along with the GN plus Ag spherical particles. The observance of C, N, O, Ni, and Ag in Ag/GN@PPy–Ni(OH)2 from EDAX analysis and their uniform distribution from elemental analysis suggests the efficiency of the synthesis procedure. The structural analysis by XRD and XPS suggests a few layered GN structures and the interaction between the constituents. The electrochemical analysis was done in 1 M KOH using a standard three electrodes cell system, which showed that the quaternary Ag/GN@PPy–Ni(OH)2 possessed the highest specific capacity of 237.25 C g−1 at 3 mV s−1. The synergistic/additional effect of PPy, Ni(OH)2, GN, and Ag accounts for the highest electrochemical characteristics of the quaternary composite. The supercapattery assembly of Ag/GN@PPy–Ni(OH)2 as a positive and AC negative electrode showed high energy density E = 43.26 Wh kg−1 with the associated power density P = 750.00 W kg−1 at a current density of 1.0 A g−1. The remarkably high energy density of the assembled (Ag/GN@PPy–Ni(OH)2//AC) supercapattery showed high cyclic stability after 5500 cycles with a final retention capacity of 108.38% compared to its initial value. The high value of specific capacitance, energy density, and power density, as well as excellent cyclic stability, paves the way for the application of Ag/GN@PPy–Ni(OH)2 for high-performance supercapattery.In summary, Ag/GN@PPy–Ni(OH)


Escherichia coli, the highly conserved tubulin homolog FtsZ polymerizes and assembles into a ring-like structure, called the Z-ring, at the site of septation. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins, predominantly FtsA in E. coli. FtsA shares structural homology with actin and, like actin, hydrolyzes ATP. Yeast actin detects nucleotide occupancy through a sensor region adjacent to the nucleotide binding site and adopts distinct conformations in monomeric and filamentous actin. Bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and polymerize. Here, we show that several amino acid residues proximal to the nucleotide binding site in FtsA are critical for function in vitro and in vivo. Each of these residues are important for ATP hydrolysis, phospholipid (PL) binding, ATP-dependent vesicle remodeling, and recruitment to the divisome in vivo, to varying degrees. Notably, we observed that Ser 84 and Glu 14 are essential for ATP-dependent vesicle remodeling and magnesium-dependent membrane release of FtsA from vesicles in vitro, and these defects likely underlie the loss of function by FtsA(E14R) and FtsA(S84L) in vivo. Finally, we demonstrate that FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is defective for rapid ATP hydrolysis and ATP-dependent remodeling of PL vesicles in vitro. Together, our results show that loss of nucleotide-dependent activities by FtsA, such as ATP hydrolysis, membrane binding and release, and, most importantly, ATP-dependent PL remodeling, lead to failed Z-ring assembly and division defects in cells.During cell division in  Escherichia coli, the process of division and separation begins with the establishment of a network of cytoskeletal proteins, called the divisome, and the formation of a Z-ring. FtsZ polymers, containing protomers of the conserved, tubulin-like protein FtsZ, coalesce at midcell on the interior of the cell membrane and are recruited there by interactions with FtsA, an actin homolog, and ZipA to form the Z-ring , has been reported to require adenine nucleotide for FtsZ polymer destabilization  and MCA12 (ftsA12) , but the cells grow robustly at the permissive temperature (30°C). To compare function of FtsA mutant proteins by a complementation assay, cultures of MCA12 and MCA27 cells containing a control vector (pQE-9) or an expression vector containing wild type ftsA or ftsA variants were cultured, spotted onto LB agar plates containing ampicillin, and then grown overnight at 30 and 42°C. As expected, cells containing pQE-FtsA, containing the wild type ftsA gene, grew at the restrictive temperature, whereas cells containing the control vector did not , FtsA(S84L), FtsA(A188V), and FtsA(D210A) (ftsA(A188V) is not sufficient to alleviate the temperature sensitive phenotype and suggest that the underlying defect may be related to a specific loss of FtsA function. Mutation of Ser 84 also led to loss of function, suggesting that this residue may provide an important contact or activity for FtsA. As expected, neither FtsA(E14R) nor FtsA(D210A) supported growth at the restrictive temperature in either strain background, consistent with previous reports of loss of function associated with mutation of these amino acids  . MCA27 c did not . Cells wA(D210A) . FtsA, FtsA(S84L), FtsA(A188V), and FtsA(Y375A) were purified as described for wild type FtsA  as temperature increased and under the conditions tested , which were added in excess to reactions to supplement copurifying PL’s  and FtsA(S84L) were also released from PL’s when treated with EDTA but observed that both proteins remained associated with PL’s under all conditions tested  and FtsA(E14R) are partially defective for ATP hydrolysis , showings tested . This suin vitro  with and without ATP and supplemental SUV’s to detect PL tubulation. We observed ATP-stimulated tubule formation with wild FtsA, FtsA(Y375A) and, to a lesser extent, FtsA(A188V), but failed to detect tubulation by FtsA(E14R) or FtsA(S84L) . The tubFtsA binds to FtsZ polymers, recruits the polymers to PL’s, and promotes destabilization of polymerized FtsZ, releasing it into the soluble fraction while FtsA remains associated with PL’s . To detein vitro.Next, to determine if the failure to destabilize FtsZ polymers resulted from an inability of FtsA mutant proteins to interact with FtsZ, we performed a PL recruitment assay. In this assay, FtsZ polymers, assembled with GTP, were incubated with FtsA, ATP, and SUV’s, and then the SUV’s and bound proteins were collected by low-speed centrifugation and analyzed by SDS-PAGE. We observed that all FtsA mutant proteins were capable of recruiting FtsZ to varying degrees, including FtsA(S84L) and FtsA(A188V), and, both to a lesser extent, FtsA(E14R) and FtsA(Y375A) . Togethein vivo in temperature-sensitive growth assays  and Gfp-FtsA(E14R) demonstrate any visible membrane localization in vivo. We analyzed large populations of cells for Gfp-FtsA (wild type or mutant protein) localization and also stained the cells with the membrane dye FM 4−64. We failed to observe colocalization of either Gfp-FtsA(E14R) or Gfp-FtsA(S84L) with the bacterial membrane, and instead observed that both proteins were cytoplasmic, similar to Gfp without FtsA  and FtsA(E14R) are defective for releasing from SUV’s out FtsA , and, asE. coli FtsA  supported growth at the restrictive temperature at a level similar to cells carrying the wild type ftsA expression plasmid . We expect that the variants constructed here are stably expressed, since fluorescence can be tracked when each variant is expressed as a Gfp fusion protein and Z-ring localization is quantified . Consistent with this, we previously reported that a truncated FtsA variant, without the C-terminal membrane targeting sequence (MTS), forms stable polymers in vitro, and the stable FtsA polymers bind to stable FtsZ polymers   , 6A. As strains) . This renterface , it is lgulation . Importagulation .in vitro , and pQE9-FtsA , pQE9-ftsA(S84L), pQE9-ftsA(A188V), pQE9-ftsA(Y375A), or pQE9-ftsA(D210A) in temperature sensitive strains MCA27 [ftsA(S195P)] or MCA12 [ftsA(A188V)] were grown overnight in Lennox broth supplemented with 100 μg ml–1 ampicillin at 30°C. The following day, cultures were diluted into fresh LB with ampicillin to an OD600 of 0.05 and grown to an OD600 of 0.4 at 30°C shaking (250 rpm). Cultures were diluted (2-log) into LB containing ampicillin and spotted (5 μl) onto LB agar plates with ampicillin and incubated at 30°C (permissive) or 42°C (restrictive) for 16 h. Leaky expression of FtsA from the T5 promoter of pQE-ftsA is sufficient to support growth without inducer.FtsA wild type and mutant proteins FtsA(E14R), FtsA(S84L), FtsA(A188V), FtsA(D210A), and FtsA(Y375A) were expressed in cells induced with isopropyl β-d-1-thiogalactopyranoside (0.5 mM), and cultures were grown at 16°C for 20 h . FtsA wi2 in the presence or absence of small unilamellar vesicles (SUV’s) (250 μg ml–1) with or without ATP (4 mM), or in the presence or absence of 25 mM EDTA where indicated, for 10 min at 30°C followed by 21,000 x g centrifugation for 15 min at 23°C. SUV’s were prepared from E. coli total membrane PL extracts (Avanti Polar Lipids) as described (–1) (Invitrogen). Fluorescence intensity was measured with an Agilent Eclipse spectrofluorometer using an excitation wavelength of 565 nm and an emission wavelength of 745 nm (slit widths 5/10 nm). Concentration was determined by comparing to a PL standard curve.Phospholipid recruitment assays (25 μl) with FtsA wild type or mutant proteins containing substitutions (E14R), (A188V), (S84L), or (Y375A) were performed by incubating FtsA wild type or mutant proteins (1 μM) in 50 mM Tris pH 7.5, 150 mM KCl and 10 mM MgClescribed . Superna–1) and ATP (4 mM). Reactions were incubated for an additional 10 min at 30°C, and then phospholipid vesicles were collected by low-speed centrifugation at 21,000 × g for 15 min. Supernatants and pellets were analyzed by SDS-PAGE and coomassie staining. Percent of protein in the supernatant fraction was determined by densitometry using NIH ImageJ.Recruitment assays with FtsA and FtsZ were performed by incubating FtsZ (3 μM) with GTP (2 mM), in reaction buffer for 2 min, and then adding to a reaction containing FtsA wild type or mutant protein (2.5 μM), as indicated, pre-assembled with SUV’s  containing reaction buffer and FtsA, FtsA(E14R), FtsA(S84L), FtsA(A188V), or FtsA(Y375A) (each at 1 μM) were monitored at 23°C for 5 min to collect a baseline then ATP (4 mM) was added, where indicated, and reactions were monitored for an additional 15 min at on an Agilent Eclipse spectrofluorometer 450 nm with 5 nm slit widths.2, ATP (4 mM) and FtsA (1 μM), or FtsA mutants (1 μM). Where indicated, SUV’s (250 μg ml–1) were added to reactions.ATP hydrolysis was measured by monitoring the amount of inorganic phosphate released at indicated temperatures in reactions (25 μl) using malachite green reagent (Enzo Life Sciences) for phosphate detection and comparing to a phosphate standard curve. ATP hydrolysis assays were performed at 37°C unless otherwise indicated in reaction buffer containing 50 mM Tris (pH 7.5), 150 mM KCl, and 10 mM MgCl–1), where indicated, were incubated for 10 min at 23°C, applied to a 300-mesh carbon/formvar coated grid, fixed with glutaraldehyde (2.5%) and negatively stained with uranyl acetate (2%). Samples were imaged by transmission electron microscopy (TEM) using a JEM-2100 80 Kev instrument.Reaction buffer containing either FtsA, FtsA(E14R), FtsA(S84L), FtsA(A188V), or FtsA(Y375A) (each 4 μM), with or without ATP (4 mM) and SUV’s  and acetate kinase (25 μg ml–1). Where indicated, FtsA or FtsA mutant proteins (3 μM) and ATP (4 mM) were added. Reactions were incubated for 5 min at 23°C and then centrifuged at 160,000 x g for 30 min in a Beckman TLA 120.1 rotor. Pellets and supernatants were resuspended in equal volumes, analyzed by SDS-PAGE and Coomassie staining, and quantified by densitometry using NIH ImageJ.To collect FtsZ polymers by ultracentrifugation, reaction mixtures (25 μl) with FtsZ (3 μM) were prepared in reaction buffer containing 50 mM MES (pH 6.5), 100 mM KCl, and 10 mM MgClaraECP containing the plasmid pSEB293 (–1) without arabinose  at 30°C and then streaked onto plates of the same composition and grown for 5 h at 30°C. Cells were collected and resuspended in 1x phosphate buffered saline (PBS) and, where indicated, incubated for 10 min in the dark at room temperature with FM 4−64FX (3 μg ml–1) (ThermoFisher). Cells were applied to a 5% agarose pad containing M9 minimal media with glucose (0.4%), and then a coverslip was added. Samples were visualized with a Zeiss LSM 700 confocal fluorescence microscope with excitation at 488 nm and emission at 555 nm for Gfp and 565 nm and 744 nm for FM 4−64FX, respectively. Where indicated, a Nomarski prism was used to acquire differential interference contrast (DIC) images. All images were captured on an AxioCam digital camera with ZEN 2012 software. Cell lengths were measured using NIH ImageJ.Cultures of MG1655  pSEB293  encodingThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JM, ES, and JC designed the study. JM, CF, ES, KN, CT, and BP performed experiments. JM, CF, CT, and BP edited the manuscript. JM and JC wrote the manuscript. JC secured funding for the study. All authors contributed to the article and approved the submitted version.


However, the characteristics and functions of mRNA m1A modification in primary neurons and oxygen glucose deprivation/reoxygenation (OGD/R) induced remain unclear. We first constructed a mouse cortical neuron OGD/R model and then used methylated RNA immunoprecipitation (MeRIP) and sequencing technology to demonstrate that m1A modification is abundant in neuron mRNAs and dynamically regulated during OGD/R induction. Our study suggests that Trmt10c, Alkbh3, and Ythdf3 may be m1A-regulating enzymes in neurons during OGD/R induction. The level and pattern of m1A modification change significantly during OGD/R induction, and differential methylation is closely associated with the nervous system. Our findings show that m1A peaks in cortical neurons aggregate at both the 5’ and 3’ untranslated regions. m1A modification can regulate gene expression, and peaks in different regions have different effects on gene expression. By analysing m1A-seq and RNA-seq data, we show a positive correlation between differentially methylated m1A peaks and gene expression. The correlation was verified by using qRT-PCR and MeRIP-RT-PCR. Moreover, we selected human tissue samples from Parkinson’s disease (PD) and Alzheimer’s disease (AD) patients from the Gene Expression Comprehensive (GEO) database to analyse the selected differentially expressed genes (DEGs) and differential methylation modification regulatory enzymes, respectively, and found similar differential expression results. We highlight the potential relationship between m1A modification and neuronal apoptosis following OGD/R induction. Furthermore, by mapping mouse cortical neurons and OGD/R-induced modification characteristics, we reveal the important role of m1A modification in OGD/R and gene expression regulation, providing new ideas for research on neurological damage.N Previouficiency –8. Recenficiency . Under tficiency , 11. Furnditions , 11, sugnditions  and is cnditions , indicatnditions .Ischaemia-reperfusion injury (IRI) is an important pathophysiological process of a variety of neurological diseases , 16. TheIn the present study, we obtained high-quality primary mouse cortical neurons and constructed a neuronal OGD/R model. Using m1A antibody immunoprecipitation sequencing (MeRIP-seq) technology , 11, we On the sixth day of cell culture, immunofluorescence was used to determine the purity of the cultured neurons. β-III Tubulin is a neuronal skeleton protein that is used to mark immature neurons  Fig. 1A1A. Throup ≤ 10−10, fc ≥ 2)   Fig. . Then, a16) Fig. , the res16) Fig. . In the 16) Fig. .Fig. 3OGWe also observed the distribution of methylation modification peaks on each chromosome Fig.  and founBecause OGD/R can induce different m1A modification patterns in neurons, we used qRT-PCR to analyse the expression of some writer, eraser and reader genes reported thus far. We observed that the expression of the writers Trmt6/Trmt61a and Trmt10c, the eraser Alkbh3, and the reader Ythdf3 significantly changed during OGD/R induction Fig. , demonstp ≤ 0.00001) to define differential modification peaks and identified 692 methylation peaks that were upregulated and 167 methylation sites that were downregulated  . Therefore, of the 303 methylation peaks with upregulated methylation levels, 224 mRNAs had upregulated expression and were designated hyper-up, while 2 mRNAs had downregulated expression and were designated as hyper-down Among the downregulated peaks, 14 mRNAs had upregulated expression and were designated hypo-up, while 1 mRNA had downregulated expression and was designated hypo-down. Nid2, Gfap, Dlk1, Flnc, Lox, Ajuba, Vasn, Arsi, Vim, and Serinc2 were the top ten genes with the most significant upregulated differential expression. Erdr1, Nrgn, Rab3b, Kifc2, Sema3e, Syp, Dnm1, Tubb4a, Tenm1, and Adcy1 were the top ten genes with the most significant downregulated differential expression  Fig.  and alsoFinally, we selected human tissue samples from individuals with PD (GSE19587) from the Gene Expression Omnibus (GEO) database to analyse the selected DEGs and observed that the gene expression of Csf1, Cyp1b1, Egfr, Lox, and Tubb4a was consistent with that observed in our present study Fig.. We alsoStudies in recent years have proven that abundant m1A modifications exist on the mRNA of eukaryotic cells. m1A modifications, which are enriched in the 5′ UTR and near the start codon, can improve protein translation efficiency –11, and Similar to previous studies, the results of our present study confirmed some typical modification characteristics of m1A methylation in mRNA. m1A modification is abundant in the mRNA of neurons, and these peaks were concentrated in the 5′ UTR, near the start codon, and in the 3′ UTR, near the stop codon, showing a “bimodal” distribution Fig. . After OMany studies have shown a correlation between the post-transcriptional modification of genes and gene expression. m6A modification in clear cell renal cell carcinoma (ccRCC) can promote the expression of cancer-related genes , and theThe relationship of m1A modification in mRNAs to apoptosis is currently unclear. A gastrointestinal tumor-based bioinformatics analysis revealed that the canonical apoptosis-related pathways ErbB and mTOR signalling were identified as pathways regulated by m1A-related enzymes. After ALKBH3 gene knockout, the expression of the identified Hub genes ErbB2 and AKT1S1 were decreased, indicating that m1A modification in mRNA is involved in the regulation of apoptosis . In addiRecent studies have shown that neurodegenerative diseases such as AD and PD are associated with cerebral IRI , 42. TheSince the m1A modification of mRNA in mice and humans is highly conserved  and m1A However, due to the characteristics of m1A [m1A can rearrange to m6A under alkaline conditions (Dimroth rearrangement) , there iIn summary, we performed transcriptome-wide profiling of m1A in the mRNA of mouse cortical neurons and an OGD/R induction model, and the findings provide a better understanding of neuronal mRNA post-transcriptional modification and confirm the significance of mRNA m1A in neurological diseases. The results of the present study offer new ideas for understanding the mechanism of neuronal oxidative damage and nerve damage interventions.The C57BL/6 mice used in the present study were purchased from the Laboratory Animal Centre, Academy of Military Medical Science . All experiments were performed in adherence with the National Institutes of Health Guidelines on Laboratory Animals and were approved by the Ethics Committee of Qilu Hospital, Shandong University .3), followed by incubation in 2 mg/ml papain  and 20 units/ml DNase for 15 min at 37 °C . Subsequently, the cells were dispersed in DMEM containing 10% fetal bovine serum . The cell density was adjusted to 50,000 cells/cm2 by counting, and the cells were plated in a Petri dish coated with 0.01% poly-D-lysine  in a 37 °C incubator under an atmosphere with 5% CO2. After 4 h, the previous medium was replaced with neurobasal medium  containing 1% B27 additive , 2 mM L-glutamine  and 1% penicillin/streptomycin  at 37 °C under an atmosphere with 5% CO2 for cultivation in an incubator, with the culture medium replaced every three days.Cortical neurons were isolated and cultivated as described in our previous studies , 60. PriOn the 6th day of cell culture, the cells were fixed with 4% paraformaldehyde for 10–15 min, and 0.1% Triton X-100 was loaded into each well to react for 5 min. To reduce nonspecific binding, 10% goat serum  was added for 2 h at room temperature. Then the primary antibody β-III tubulin  was added to the anti-labelled neurons and axons, and incubated at 4°C overnight. Then the corresponding secondary antibodies were incubated at room temperature for 1 h. After being washed with PBS, the cells were incubated with DAPI  or Hoechst 33342  to label nuclei for 10 min, and images were obtained under a fluorescence microscope or confocal microscope. Under the microscope, the ratio of β-III tubulin-positive to DAPI cells was counted in three random fields to determine the purity of the extracted neurons.2) to simulate the reperfusion state.OGD/R model construction was performed as previously described by our lab and others , 61. On According to the instructions provided by the manufacturer of a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay kit , the cells were fixed with 4% paraformaldehyde for 15 min after the cell culture medium was discarded during OGD/R induction. Then, the cells were treated with 0.1% Triton X-100 for 5 min and washed with PBS. Then, the prepared TUNEL reaction solution, containing 50 µl of enzyme solution and 450 µl of label solution mixture, was added to the cells. Subsequently, the solutions were allowed to react for 1 h before being mixed with 100 µl of a DAPI-containing nuclear reaction solution for 10 min. Finally, the cells were observed under a fluorescence microscope. Three fields of vision were randomly selected in each well, and the percentage of positive cells was obtained with the following formula: number of blue and green double-labelled cells/number of blue-labelled cells × 100%. The experiment was performed with three independent biological replicates.g at 4 °C for 10 min. Then, the supernatant was collected, and a PierceTM BCA Protein Assay Kit  was used to determine the protein concentration. Finally, the protein was denatured at 100 °C for 10 min. Subsequently, 25 µg of protein was separated by 12% SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% skim milk for 1 h and then incubated with primary antibodies on a shaker at 4 °C overnight. The antibodies used in the present study included those against β-actin , caspase-3 and cleaved caspase-3 , Bax , Trmt6 , Trmt10c , Alkbh3 , Ythdf3 , Bag3 , M-CSF , Egfr , Lox-1 , Nid2 , Rab3b  and Tubb4a  which were diluted in Tris-buffered saline containing 0.1% Tween (TBST). After washing the membranes in TBST solution, the membranes were incubated with horseradish peroxidase-linked anti-rabbit IgG  for 1 h. Finally, a horseradish peroxidase mixture was added to expose the bands on an exposure instrument, and ImageJ was used for greyscale value analysis.After the cells were washed with PBS, RIPA lysis buffer  containing protease inhibitors was added to fully lyse the cells at 4 °C for 30 min, which was followed by centrifugation at 13,000 × Following the manufacturer’s instructions, total RNA was immediately extracted with TRIzol when the OGD/R induction process was completed, and chloroform extraction and isopropanol purification were performed to separate and precipitate the RNA. RNA concentration was evaluated with a NanoDrop ND-1000 instrument. The ratio of OD260/OD280 is an indicator of RNA purity, and when the OD260/OD280 value ranged from 1.8 to 2.1, RNA was considered to be non-contaminated  in IPP buffer and extracted with TRIzol reagent according to the manufacturer’s instructions. The m1A-to-m6A rearrangement was induced by incubating the input and immunoprecipitation fragments in alkaline buffer  for 1 h. The purified RNA was used to generate the RNA-seq library with the NEBNext® Ultra™ RNA Library Prep Kit   in the RNA library compared to a reference genome, respectively.We used a commercially available m1A antibody  to co-immunoprecipitate methylated mRNA fragments according to previously described protocols , 13. Afthttps://david.ncifcrf.gov/) for annotation and clustering. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was also performed with the web tools database (https://www.genome.jp/kegg/) and with data described elsewhere [Gene ontology (GO) analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources web tools  was used following the manufacturer’s instructions to determine the viability of the cells in the 96-well plate after the cells were induced by OGD/R. To this end, 10 μl of CCK-8 and 90 μl of neurobasal medium were mixed and added to each well, and the cells were then incubated at 37 °C for 4 h in the dark. Subsequently, the absorbance at 450 nm was measured using a microplate reader. Each sample was assayed in triplicate.The mitochondrial membrane potential was determined with a mitochondrial membrane potential assay kit with JC-1  according to the manufacturer’s instructions. In brief, neurons were induced by OGD/R, after which the cells were washed twice with PBS. Then, we added 1 ml of JC-1 staining working solution for 30 min, after which the cells were washed twice with JC-1 staining buffer. Finally, we observed the cells through a fluorescence microscope.P < 0.05 was considered to indicate a significant difference.For each assay, three independent biological and technical replicates were performed. The data are presented as the means ± standard deviation (SD) of independent experiments. GraphPad Prism 6 (version) was used to analyse and compare all groups using Student’s t-test, unless otherwise noted. Supplementary TablesSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure and Table LegendsOriginal Data File