Genetic diversity and evolution of infectious bronchitis virus (IBV) are mainly impacted by mutations in the spike 1 (S1) gene. This study focused on whole genome sequencing of an IBV isolate (IBV/Ck/Can/2558004), which represents strains highly prevalent in Canadian commercial poultry, especially concerning features related to its S1 gene and protein sequences. Based on the phylogeny of the S1 gene, IBV/Ck/Can/2558004 belongs to the GI-17 lineage. According to S1 gene and protein pairwise alignment, IBV/Ck/Can/2558004 had 99.44–99.63% and 98.88–99.25% nucleotide (nt) and deduced amino acid (aa) identities, respectively, with five Canadian Delmarva (DMV/1639) IBVs isolated in 2019, and it also shared 96.63–97.69% and 94.78–97.20% nt and aa similarities with US DMV/1639 IBVs isolated in 2011 and 2019, respectively. Further homology analysis of aa sequences showed the existence of some aa substitutions in the hypervariable regions (HVRs) of the S1 protein of IBV/Ck/Can/2558004 compared to US DMV/1639 isolates; most of these variant aa residues have been subjected to positive selection pressure. Predictive analysis of potential N-glycosylation and phosphorylation motifs showed either loss or acquisition in the S1 glycoprotein of IBV/Ck/Can/2558004 compared to S1 of US DMV/1639 IBV. Furthermore, bioinformatic analysis showed some of the aa changes within the S1 protein of IBV/Ck/Can/2558004 have been predicted to impact the function and structure of the S1 protein, potentially leading to a lower binding affinity of the S1 protein to its relevant ligand . In conclusion, these findings revealed that the DMV/1639 IBV isolates are under continuous evolution among Canadian poultry. Gammacoronavirus, the family Coronaviridae, and the order Nidovirales [Avian infectious bronchitis (IB) represents one of the globally ubiquitous diseases of commercial poultry, characterized by high morbidity and variable mortality, and with subsequent economic losses . IB is iovirales . IBV posovirales ,4. The govirales . A largeovirales .The S glycoprotein is one of the main structural proteins, and it is post-translationally cleaved by proteolysis into S1 and S2 subunits at the furin consensus specific site (RRFRR/HRRR) to enhance virus binding to receptor and entry into the host cell . There aGenetic categorization of IBV variants is commonly performed by phylogenetic analysis of a complete sequence of the S1 gene, encoding the relevant protein. There are seven genotypes (GI-GVII), including 35 genetic lineages according to phylogenetic analysis of the IBV S1 gene . Most ofThe N-glycosylation of viral protein plays a key role in virus virulence and tissue tropism . The glyThe majority of genomic variance can be attributed to single nucleotide polymorphisms (SNPs) , which aThe IBV isolate, IBV/Ck/Can/2558004, was isolated from one clinical sample obtained from the Animal Health Laboratory (AHL), at the University of Guelph, Canada. The examined sample was collected from two-week-old broiler chickens raised in a commercial farm with a capacity of 46,000, situated in Ontario, Canada. For virus isolation and propagation, embryonated chicken-specific pathogen-free (SPF) eggs were purchased from the Canadian Food Inspection Agency (CFIA), Ottawa, Ontario, Canada. The embryonated chicken eggs were kept in the incubator at 37 °C until the embryos were 9 to 11 days old; afterwards, they were inoculated with 200 µL of sample supernatant into the allantoic cavity. Eggs that had embryo deaths after 24 h of incubation were eliminated from further analysis. The allantoic fluid was harvested 2 days following inoculation and then aliquoted and stored at −80 °C. Serial passage of the obtained allantoic fluid was performed in the embryonated eggs two times to increase the virus titer.The viral RNA was extracted from 200 µL of allantoic fluid using a Quick-RNA Viral Kit , according to the manufacturer’s instructions. The concentration of resulted RNA was measured by a Nanodrop1000 spectrophotometer , based on absorbance at a 260/280 nm wavelength. The complementary DNA (cDNA) was converted from 150 ng of the obtained RNA using random primers , as indicated by the manufacturer. To detect and confirm IBV propagation on embryonated eggs, an already established real-time PCR was performed, targeting the IBV-N gene . The RT-https://www.ncbi.nlm.nih.gov/genbank/) (accessed on 1 February 2022). The retrieved reference sequences were considered to be representative for each genotype and lineage as previously described [® software v10.2.6 (https://www.geneious.com/) (accessed on 5 February 2022). The phylogenetic analysis was constructed by maximum-likelihood method using 1000 bootstrap replicates in MEGA X software [The phylogenetic analysis and genetic relatedness were based on the complete nucleotide (nt) sequence of the S1 gene of IBV/Ck/Can/2558004 and 85 reference IBV strains  retrieveescribed  as a toosoftware . Then, tsoftware . MAFFT whttps://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0) (accessed on 14 February 2022). For recognition of potential phosphorylation sites, the NetPhos-3.1 [https://services.healthtech.dtu.dk/service.php?NetPhos-3.1) (accessed on 20 February 2022) was used. The palmitoylation sites were determined by CSS-Palm 4.0 software  (accessed on 20 February 2022), which depends on the fourth generation of the Group-based Prediction System (GPS) algorithm [S1 glycoprotein of IBV/Ck/Can/2558004 and MDL_DMV1639_15-5582 KX529725) isolates were subjected to post-translational modifications analysis. Firstly, the N-glycosylation sites were predicted with the aid of a web server called NetNGlyc-1.0 [9725 isolPhos-3.1  (https:/lgorithm .https://selecton.tau.ac.il/) (accessed on 10 February 2022). For every single site within the protein sequence, the SELECTION server could count the ratio between synonymous (dS) and non-synonymous (dN) substitutions [To find the aa residues under selection pressure, the aa sequence alignment of S1 glycoproteins of eight DMV/1639 IBVs  and IBV/itutions . AdditioInitially, we employed aa sequence alignment for both S1 glycoproteins of IBV/Ck/Can/2558004 and MDL_DMV1639 _15-5582 (KX529725) to list the aa substitutions using MAFFT. To predict the functional outcomes of aa substitutions, PROVEAN, SIFT, Poly-Phen-2, PhD-SNP, and SNPs&GO were performed.http://provean.jcvi.org/index.php) (accessed on 20 February 2022), an online database, expects the deleterious effects of single or numerous aa changes on protein function [https://sift.bii.a-star.edu.sg/) (accessed on 20 February 2022) anticipates the capability of the mutant codons within the protein to affect the function through sequence homology and physical properties of protein residues [http://genetics.bwh.harvard.edu/pph2/) (accessed on 22 February 2022) based on reasonable physical and comparative foresights [https://snps.biofold.org/phd-snp/phd-snp.html) (accessed on 22 February 2022) is a web server that depends on the support vector machines to predict the disease-related effects of aa variations within the protein [https://snps.biofold.org/snps-and-go/snps-and-go.html) (accessed on 22 February 2022) is employed to ratify whether the variant codons have disease effects or not within the tested protein [PROVEAN (function . The varresidues . The proresights . The obt protein . Based o protein .http://mutpred.mutdb.org/) (accessed on 25 February 2022), an online application, depends on a probabilistic paradigm to discover the functional and structural effects of variants within the protein, and thus could be beneficial for the mutants associated with phenotype alteration in the experimental analyses [https://www.ics.uci.edu/~baldig/mutation.html) (accessed on 25 February 2022) relies on support vector machines to expect the effect of each point mutation on the protein stability with 84% accuracy [https://services.healthtech.dtu.dk/service.php?NetSurfP-2.0) (accessed on 25 February 2022) was employed to anticipate the secondary structure, solvent accessibility, and structural changes among each codon site within the examined protein [http://biosig.unimelb.edu.au/duet/) (accessed on 25 February 2022) are collectively implemented in the same web server and they depend on the support vector Machines [http://www.cmbi.ru.nl/hope/input/) (accessed on 27 February 2022) was performed to analyze the impacts of variable aa residues on protein function and structure [To predict the structural effects of aa changes, seven computational tools  were used. MutPred2  (accessed on 1 March 2022) was employed to accomplish the consequences of mutant aa residues on the protein flexibility and stability [For the aim of interatomic interactions analysis, the DynaMut server (tability .http://zhanglab.ccmb.med.umich.edu/I-tasser.) (Accessed on 1 January 2022) was performed to generate their 3D structures. The I-TASSER depends on aa sequence, and the output includes secondary, tertiary, and 3D structures predictions, as well as some protein function-related purposes [http://zhanglab.ccmb.med.umich.edu/COACH/) (accessed on 1 January 2022) was used to recognize the active binding sites against specific ligands. The COACH server acts by two methods named TM-SITE and S-SITE [http://www.cgl.ucsf.edu/chimera/) (accessed on 4 January 2022). The quality of the generated structures was verified using the Ramachandran Plot Server (https://zlab.umassmed.edu/bu/rama/index.pl) (accessed on 4 January 2022), which shows a colored scale graph with three categories .Based on aa sequences of S1 glycoprotein of IBV/Ck/Can/2558004 and MDL_DMV1639 _15-5582 (KX529725) isolate, the I-TASSER server (purposes . After hd S-SITE . The comhttps://pubchem.ncbi.nlm.nih.gov/) (accessed on 11 January 2022) and was utilized as a ligand. Then, the ligand was prepared through minimizing the energy by Avogadro software [Initially, the sialic acid 3D structure (PubChem CID 444885) was retrieved from the PubChem database -M-4b-4c-5a-5b-N-6b-3′UTR). ORF1a and 1b consisted of 11,889 nt and 8034 nt, respectively, and they formed the gene 1. The S glycoprotein was encoded by gene 2, and it had 3501 nt (encoding 1166 aa). Furthermore, the S protein was cleaved post-translationally into the S1 and S2 subunits by cleavage site RRSRR into 536 aa and 625 aa residues, respectively. Gene 3 contained 3 ORFs  that encoded three different-sized proteins . The M protein was encoded by a single ORF consisting of 678 nt (225 aa) and was located in gene 4. while gene 5 included 4b and 4c ORFs that had 94 and 56 aa, respectively. Two ORFs  were observed at gene 6, and they were composed of 198 nt and 249 nt with 65 aa and 82 aa, respectively. A single ORF (1230 nt) was located at gene 7 encoding the N protein with 409 aa. A non-coding region (8 nt) between gene N and 6b has been detected. The length of UTRs at both genomic ends, 5′ and 3′, were 530 nt and 274 nt, respectively.Based on S1 gene homology analysis, IBV/Ck/Can/2558004 had 99.63%, 99.50%, 99.44%, 99.50%, and 99.57% identities when compared to five Canadian DMV/1639 IBVs: IBV/Ck/Can/17-035614, IBV/Ck/Can/18-048430, IBV/Ck/Can/18-048192T, IBV/Ck/Can/17-036989, and IBV/Ck/Can/18-049707 , respect54A and 60S, while six variants were observed in the HVR 2 at positions 104V, 113R, 116H, 117F, 123F, and 136I. HVR 3 included four aa changes at positions 278F, 315S, 339T, and 369N. Alternatively, the IBV/Ck/Can/2558004 S1 protein shared the highest aa similarity (98.88% to 99.25%) in comparison with that of the Canadian DMV/1639 isolates, and it had three aa substitutions. Two of the three aa variants were nearly situated in HVR 1 and HVR 2 at positions 31R and 269I, respectively, while the S1 protein of GA9977/2019 (MK878536) exhibited 97.20% aa identity in comparison with that of IBV/Ck/Can/2558004, where four aa changes were found in and/or near HVR 1, HVR 2, and HVR 3 at positions , , and , respectively.The similarity of the deduced aa sequences of the S1 protein of IBV/Ck/Can/2558004, other Canadian DMV/1639, and USA DMV/1639 IBVs isolated between 2011 and 2019 are presented in A predictive computational analysis was performed to identify and compare post-translational modifications such as N-glycosylation, phosphorylation, and palmitoylation motifs in the S1 glycoprotein of IBV/Ck/Can/2558004 and that of MDL_DMV1639 _15-5582 (KX529725).N-glycosylation motifs were predicted based on a consensus sequence (sequon) Asn-X-Ser/Thr, where Asn refers to Asparagine, X refers to any aa except Proline (Pro), Ser is Serine, and Thr refers to Threonine. Among the S1 glycoprotein of IBV/Ck/Can/2558004, there were 41 sequons (Asn-X-Ser/Thr) where 14 sites were predicted to be N-glycosylated A, whereaThe prospective phosphorylation sites were identified by the NetPhos 3.1 server, which predicts Ser, Thr, or tyrosine (Tyr) phosphorylation sites in the S1 glycoprotein. The conserved potential phosphorylation sites and their positions in the S1 glycoprotein of IBV/Ck/Can/2558004 and MDL_DMV1639 _15-5582 (KX529725) are listed in To predict the palmitoylation sites, CSS-Palm 4.0 software was employed. The prospective palmitoylation sites were conserved and showed the same pattern in the S1 glycoprotein of IBV/Ck/Can/2558004 and that of MDL_DMV1639 _15-5582 (KX529725) .The selection pressure examination was conducted by a web server called SELECTION. It estimates the selection pressure by using a Mechanistic Empirical Combination (MEC) model at certain codons. The MEC model acts by consideration of the different rates of aa changes. The positive selection was observed at different aa residues all over the S1 glycoprotein . The posBecause the highest aa changes in the S1 glycoprotein were detected when comparing IBV/Ck/Can/2558004 to MDL_DMV1639 _15-5582 (KX529725), we therefore considered the later as a wildtype (non-mutant). Further computational analysis was conducted on the wildtype S1 glycoprotein versus (vs.) IBV/Ck/Can/2558004 (mutant one) to study the prospective effects of aa substitutions on protein function, structure, and physico-chemical properties as well as to analyze interatomic interactions and protein–ligand interaction.To detect the functional effect of aa substitutions on the S1 protein of IBV/Ck/Can/2558004, a total of five tools were used . Twelve out of twenty-nine aa substitutions were predicted by the SIFT server to have a damaging effect on the S1 protein, and their tolerance index was 0. The PROVEAN server showed four aa residues had a deleterious effect (PROVEAN score > −2.5). The number of affecting ones expected with the aid of PolyPhen-2 was five. The damaging variants identified by poly-Phen-2 were five; four of them were “probably damaging” (score 0.980–1.00) and only one was “possibly damaging” (score 0.919). The disease -associated variant aa residues predicted by PhD-SNP, SNAP-2, and SNPs&GO were nine, five, and one in number, respectively.A total of eight variant aa residues were collectively anticipated by three or more tools to have damaging effects and were therefore functionally significant. These variants were taken into account in the following analyses.Eight variants  that had been expected to cause a negative effect on protein function were then subjected to structural impact analysis. For this, seven tools  were used.MutPred2 exhibited four mutants to be pathogenic, while Mupro predicted three variants to increase the stability of the protein structure, and the rest  had a decreasing effect. NetSurfP-2.0 showed that the residue is either buried or exposed among the protein structure. It was shown that one mutant (T269I) shifted from exposed to buried in comparison with the wildtype S1 structure. Additionally, DUET predicted five mutants with destabilizing effects, and the others had stabilizing effects. In terms of SDM, four showed stabilizing effects, whereas mCSM revealed seven aa residues to be destabilizing. The HOPE server was used to determine the impact of physico-chemical characters, solvent accessibility, the interaction between the molecules, and effects on function and structure. Seven mutants were anticipated to induce a change in protein size, and three mutations revealed a modification in the charge. In the case of solvent accessibility, four mutations induced a variation in the hydrophobicity while two lost their hydrophobicity. Glycine residues at positions 116 and 520 of the wildtype structure were flexible enough to make torsion angles but replacing glycine with other aa residues caused the local backbone to be in an incorrect conformation, thus disrupting the local structure in the mutant S1 protein.The atomic interactions of the native and variant aa residues of the S1 protein were analyzed and illustrated by the DynaMut server . It has To generate the 3D structures of the wildtype and mutant S1 protein of DMV/1639 IBVs, the I-TASSER server was employed. The generated models were topped in a certain arrangement and classified by the TM-score, which degrees the distance of deviation (in Angstrom) between the residual position of the model and wildtype structure. For both of the modeled sequences utilized in this study, the TM-score output exceeded 0.5, thus indicating that both 3D structure models were biologically significant and had a convenient structural topology ; Table 1It has been shown that sialic acid residues on cell surfaces have a major role in initiating IBV infection, particularly in the early stages ,18,19. TOver the last few years, DMV/1639 became the most prevalent strain of IBV in the Eastern part of Canada. The IBV S1 gene plays a major role in virus evolution and the emergence of variants. Our study was conducted to analyze the whole genome sequence of an IBV isolate (IBV/Ck/Can/2558004) and to identify the evolutionary trends within the S1 gene, with potential functional and structural impacts within the encoded protein. In the present study, the nt sequence of the S1 gene of IBV/Ck/Can/2558004 was almost identical (above 99%) to that of Canadian DMV/1639 IBV strains isolated in 2019  and was Our results revealed that IBV/Ck/Can/2558004 had 27, 710 nt long, consisting of 13 ORFs organized as 5′UTR-1a-1b-S-3a-3b-3c(E)-M-4b-4c-5a-5b-N-6b-3′UTR, and this agreed with previously characterized IBV isolates in some parts of the world ,75. AddiIn the current work, the S1 cleavage site of IBV/Ck/Can/2558004 was RRSRR, and was thus in accordance with a similar study . The homWith regards to the alignment of aa sequences in the S1 protein, IBV/Ck/Can/2558004 had 29 aa substitutions that represented 5.22% aa dissimilarity when aligned with that of the US DMV/1639 (MDL_DMV1639_15-5582), while it shared only 2.8% and 0.75 to 1.12% aa difference compared to the other US DMV/1639 (GA9977/2019) and the five Canadian DMV/1639 IBVs, respectively. Interestingly, it has been observed that a reasonable numbers of these aa substitutions were located within and/or near the three HVRs of the S1 subunit, thus reflecting a continuous change of neutralizing sites and serotype-specific epitopes with subsequent lower protective efficacy of homologous IB vaccines. These observations agreed with other previous studies, which indicated that the different serotypes within the genetically divergent IBVs are generally associated with changes in the HVRs of the S1 subunit ,80. AnotFor N-glycosylation sites, our findings showed a lack of 3 N-glycosylation sites within the S1 protein of IBV/Ck/Can/2558004 compared to that of US DMV/1639 (MDL_DMV1639_15-5582). One of the three lost N-glycosylation sites was detected at position 267 because the consensus N-glycosylated sequence (Asn-X-Ser/Thr) was lost because the aa residue I substituted T at position 269. These changes in the N-glycosylation sites may have some functional consequences in the S1 protein. Similar analyses showed that the differences in N-glycosylation motifs of viral proteins could influence the receptor binding capacity and therefore minimize the recognition capacity by host innate immune responses and antibodies, ultimately affecting viral infectivity and replication ,32,33. PIt has been shown that virus evolution is associated with two essential steps: the appearance of genetic variations and selection . Here, wWe employed further bioinformatic tools to predict if aa changes had an influence on the function, structure, and protein–ligand interaction in the S1 glycoprotein of IBV/Ck/Can/2558004 compared to that of US DMV/1639. Occasionally, the bioinformatic analysis could output negative results; however, we overcame this bias by using several tools. Initially, seven tools were used to determine the functional impacts. Eight variant aa residues  out of the substituted ones (29 aa) were unanimously detected by four tools to functionally affect the S1 subunit of IBV/Ck/Can/2558004. The eight filtered variants were then involved in the following analyses. It was shown that two mutants (G116H and T269I) were collectively detected by all tools to structurally impact the S1 subunit; however, the other mutants were predicted to influence the protein structure by some tools. The functional and structural consequences within the S1 subunit of IBV/Ck/Can/2558004 may alter the binding affinity to its natural receptors . Therefore, we performed molecular docking to identify the interactions between S1 glycoprotein and its relevant ligand. It was notable that the binding energy of the S1 glycoprotein of IBV/Ck/Can/2558004 to the sialic acid was lower compared to that of US DMV/1639. The possible explanation for this scenario would be that the S1 subunit contains two separate folded domains: the N-terminal domain (NTD) (aa 21 to 237) and the C-terminal domain (CTD) (aa 269 to 414), which act as the receptor binding domain . DependiThe forementioned in silico findings reflect continuous changes in the S1 gene and its encoded protein of IBV/Ck/Can/2558004; subsequently, this may affect the virus pathogenesis and also lead to the existence of dozens of serotypes, which are problematic with regard to vaccination efficiency and could result in a high prevalence of Canadian DMV/1639 among Canadian poultry operations. The data obtained by the Animal Health Lab  from 2015 to 2016 supported our findings, in which the prevalence of DMV/1639 had reached to just below 25% among the detected IBV strains in the country in 2016. In 2017 and 2018, there was a substantial increase in the prevalence of DMV/1639, representing around 45% of all IBV strains in Canada, which dominated all other strains . FurtherIn conclusion, our results elucidated that the S1 glycoprotein of Canadian DMV/1639 has experienced remarkable evolutionary changes in the form of mutations and selection pressure, which lead to functional and structural impacts within this protein. It is important to note that the S1 glycoprotein possesses a significant role in cell binding and receptor identification, and genetic diversity is frequently observed in this protein of IBV. Therefore, by identifying these properties, we will be able to better understand the epidemiology, the molecular mechanisms of infection, and virus evolution. Additionally, the continuous evolution of the S1 glycoprotein of Canadian DMV/1639 indicates the significance of constant monitoring of circulating IBV strains and the necessity of developing effective vaccines that counteract the emergence of new variants. The approach of computational analysis is user-friendly, reliable, accessible, and essential to predict multiple events within a gene. However, validation of these predictions requires proper laboratory and in vivo experiments.


DRAXIN plays an important role in the malignant process of tumors, but the mechanism of its involvement in glioma has not yet been revealed. The main aim of this study is to explore the relationship between DRAXIN and the prognosis and pathogenesis of glioma through a large quality of data analysis. Firstly, thousands of tissue samples with clinical information were collected based on various public databases. Then, a series of bioinformatics analyses were performed to mine data from information of glioma samples extracted from several reputable databases to reveal the key role of DRAXIN in glioma development and progression, with the confirmation of basic experiments. Our results showed that high expression of the oncogene DRAXIN in tumor tissue and cells could be used as an independent risk factor for poor prognosis in glioma patients and was strongly associated with clinical risk features. The reverse transcription-quantitative PCR technique was then utilized to validate the DRAXIN expression results we obtained. In addition, co-expression analysis identified, respectively, top 10 genes that were closely associated with DRAXIN positively or negatively. Finally, in vitro experiments demonstrated that knockdown of DRAXIN significantly inhibited proliferation and invasion of glioma cell. To sum up, this is the first report of DRAXIN being highly expressed in gliomas and leading to poor prognosis of glioma patients. DRAXIN may not only benefit to explore the pathogenesis of gliomas, but also serve as a novel biological target for the treatment of glioma.An increasing number of evidences have shown that the carcinogenic effect of  Gliomas are malignant tumor of neuroepithelial origin due to the interaction of congenital genetic risk factors and environmental carcinogenic factors, which frequently occur in brain and spinal cord glial cells. The annual incidence rate of glioma is 5.26 per 100,000 or 17,000 new cases diagnosed each year  is involved in tumorigenesis and multiple signaling pathways. Disregulated expression of EGFR occurs in approximately 40% of glioblastoma patients, and its mutation, EGFRvIII, reaches 20 to 30% of glioblastoma , encoded by the DRAXIN expression is much higher in glioma tissues and cell lines compared to normal samples. KM and ROC curve–exhibited DRAXIN was correlated with shorter OS, and univariate and multivariate regression analysis claimed that DRAXIN was an independent prognostic factor for glioma. We performed Pearson correlation coefficient to prove the positive relationship of DRAXIN and malignant clinical features. Furthermore, GSEA revealed potential signaling pathways that DRAXIN might participate in. At last, individually ten most-related genes and four candidate drugs for glioma were identified. It is believed that this study will provide DRAXIN as an effective biological target for the prognosis of glioma and may provide new research directions for the drug treatment of glioma.Firstly, we demonstrated that http://gepia.cancer-pku.cn/) is a database established by Peking University that contains massive RNA sequencing data from human tumor tissues and mutually matched normal tissues, creating new opportunities for data mining and a deeper understanding of gene function  is an international public repository containing high-throughput microarray and next-generation sequence functional genomic datasets, containing data from multiple pathological and normal samples  database is dedicated to GBM-related studies. Here, we obtained the characteristic annotation of DRAXIN in glioma tissues by ISH/FISH/HE for its expression level in gliomas.GEPIA  (http://www.cgga.org.cn/) is a database of gene sequencing and clinical characteristics of a large number of glioma patient samples established by Beijing Tiantan Hospital, Capital Medical University  (DRAXIN expression. GSEA (version 4.0.3) jar software was used for enrichment analysis. The number of enrichment was set to 1000 times, and the KEGG cell signaling pathway was selected as the gene database. Normal P < 0.05 and FDR < 0.25 were regarded as significantly enriched.GSEA  can be used for data analysis of whole genome expression profiles. It is used to compare genes with predefined gene sets  were obtained from patients in the clinical department of Henan Provincial People’s Hospital, and patients signed a written informed consent. Collected samples were stored in liquid nitrogen environment, and then transferred to − 80 °C freezer for use. This study was approved by the Ethics Committee of Henan Provincial People’s Hospital.http://www.xrshbio.com/). All cells were cultured in an incubator at 37 °C and 5% CO2 using DMEM medium  containing 10% fetal bovine serum . U251 cells with a more stable phenotype were selected for the in vitro experiments. U251 cells were cultured in 6-well plates and when the cells were fused to 70–80%, the cells were intervened with siRNA purchased by Genepharma. The siRNA specifically targeting DRAXIN (siDRAXIN) was Sense 5′-GCCCUCUGCAAAGAAGAAATT-3′, Antisense 5′-UUUCUUCUUUGCAGAGGGCTT-3′. The siRNA used as negative control (siNC) was Sense 5′-UUCUCCGAACGUGUCACGUTT-3′, and Antisense5′-ACGUGACACGUUCGGAGAATT-3′. After incubated for 48 h, U251 cells were applied to further experiments.Human glioma cell lines  and human astrocytes (HA) were purchased from Shanghai Shunran HAKATA Cell Bank . Subsequently, the RNA solubility was determined using an ultra-micro spectrophotometer . The NovoStart SYBR qPCR SuperMix Plus  kit was used to perform RT-qPCR. GADPH was used as an internal reference. The primer sequences used for DRAXIN were 5′-CGACTGGACCGATTATGAAGAC-3′ (F) and 5′-CGGCTGGTGATGTTTCGTTAC-3′ (R). “2−ΔΔCT” or “2−ΔCT” methods were individually applied to analyze the DRAXIN expression of glioma cells and tissues. The unpaired t test was used for the statistical difference between the two groups. When the p value was < 0.05, the difference between the two groups was statistically significant.RT-qPCR was used to detect the expression of U251 cells treated with siRNA were cultured in the 96-well plate with a density of 2000 cells per well. At the set time point, the working solution of Cell Counting Kit-8 was added into the 96 well plate. After continuous incubation at 37 ℃ for 4 h, the absorbance value of U251 cells was detected.Transfected cells were cultured at a density of 500 cells per well in 6-well plates and continued to be cultured under complete medium conditions for 14 days. The next step, colony-forming cells were washed with PBS and fixed using paraformaldehyde for 30 min. Crystal violet solution was used to stain cell colonies, and finally the number of colonies was counted and statistically analyzed.Cells were treated with siRNA and cultured in transwell chambers at a density of 1000 per well in the culture medium with 5% FBS. Then, the medium with 20% FBS was added in the lower chamber of the 24-well plate. After 48 h of incubation, cells invading the lower sidewalls of the transwell chambers were fixed and stained. The number of invading cells was recorded and used for statistical analysis.DRAXIN  was used to incubate the samples overnight at 4 °C. Further incubation with secondary antibody, samples were dyed via Mouse/Rabbit enhanced polymer detection system , and then nuclei in samples were stained with hematoxylin. Finally, the protein expression of DRAXIN was photographed by a microscope and analyzed by ImagePro-Plus software (version 6.0).Normal brain tissues and glioma tissues with different grades from laboratory were used for immunohistochemical staining. First, samples were dewaxed using xylene and dehydrated using gradient ethanol. After antigen repair and endogenous peroxidase blockade, the samples were sealed by 10% BSA solution. Next, specific antibody targeting DRAXIN in glioma and non-tumor brain tissues. Cox regression and Kaplan–Meier method were used to examine the relationship between the expression level of DRAXIN and the patient’s OS, and to draw survival curves and ROC curve. Univariate Cox analysis and multivariate Cox analysis based on statistical data sheet was performed. Use Wilcox or Kruskal test to detect the relationship between clinically relevant information and DRAXIN in patients with glioma expression, p < 0.05 is statistically significant.The R software (v.4.0.3) was frequently used to analyze the above multiple sets of data and perform statistical analysis. The Wilcox test was used to determine the expression of In total, the 748 cases of glioma transcriptomic data from the CGGA database were collected in the present study. These data include general clinical information of glioma patients, such as gender, age, pathological type, and WHO classification of glioma, in addition to PRS  type, IDH (isocitrate dehydrogenase) mutation and some data on the missing status of the 1p19q co-deletion. Detailed clinical information is shown in Table DRAXIN in a variety of tumors using the GEPIA database, and the results showed that DRAXIN was significantly highly expressed in GBM . In the end, univariate and multivariate logistic regression analysis exhibited three independent risk factors  for glioma, the expression of DRAXIN, PRS grade and high-grade glioma leading to poor prognosis . Based on the above data, high expression of DRAXIN could be used to indicate a poor prognosis for glioma patients, and it had a certain clinical diagnostic value.To further explore whether the high expression of sis Fig. A. In addsis Fig. B–D. Moresis Fig. I, J. IDHDRAXIN was also estimated via correlation analysis. The data of glioma was screened for the complete information including gender, age, pathological type, and WHO grade of glioma, PRS  type, IDH (isocitrate dehydrogenase) mutation and the status of the 1p19q codon. After Wilcox and Kruskal tests, it was concluded that the increased expression of DRAXIN was positively correlated with higher WHO grade and recurrent and secondary glioma  and 96 h (p < 0.0001)  . Both experiments indicated that knockdown of DRAXIN significantly inhibited the proliferation ability of glioma cells. Furthermore, the number of invading cells in the transwell assay (p < 0.05) and the relative cell migration distance in the wound healing assay (p < 0.01) were both significantly lower in the siDRAXIN group than in the siNC group (Fig. DRAXIN does promote cell proliferation capacity and invasive ability in glioma.To verify the role of DRAXIN is commonly recognized as an axon guidance cue for thalamocortical development (Shinmyo et al. DRAXIN is reported to be a key factor in the progress of lung adenosquamous carcinoma via the regulation of cell proliferation and apoptosis (Sato et al. DRAXIN in glioma has not been reported. The aim of this study was to reveal the function of DRAXIN in glioma from multiple perspectives and its impact on patient treatment and prognosis.DRAXIN in glioma was improved by validation using GEPIA, CGGA database, IVY-GAP database and basic experimental RT-qPCR and IHC. High expression of oncogenes is often closely associated with poor prognosis and malignant features of tumors (Li et al. DRAXIN expression was found positively correlated with glioma WHO Grade, 1p19q non-co-deficiency, GBM and recurrent GBM. Interestingly, these factors are reported to act as independent risk factors for glioma. (Ducray et al. DRAXIN expression were still significantly associated with poor prognosis. This result strongly suggests that DRAXIN is a novel prognostic factor for glioma beyond 1p19q co-deletion and IDH mutant.The rigor of the analysis for aberrant expression of DRAXIN that might participate in leading to the carcinogenesis of glioma. Enrichment results showed cell cycle, DNA replication, pyrimidine metabolism, VEGF signaling pathway, focal adhesion, and Toll-like receptor signaling pathway, which had influential functions in the development of glioma. First of all, the cell cycle signaling pathway exists throughout the cell division cycle and is activated in almost all tumors (Liu et al. DRAXIN may partake in migration and tumor immunity of glioma. From the above conclusions, it can be inferred that the mechanism of DRAXIN in the poor prognosis of glioma depends on synergy of multiple pathways.To further discover the potential mechanism, GSEA analysis was performed to reveal the possible signaling pathways of DRAXIN were shown separately, among which, CDCA8 gene is the one with the strongest positive association to DRAXIN. According to previous studies, KIF23, a member of the kinesin family positively associated with CDCA8, is involved in the regulation of cytoplasmic division, and its high expression is associated with poor prognosis of gliomas (Gao et al. DRAXIN standing in glioma. On the other hand, ETNPPL, a gene inversely associated with DRAXIN, is also negatively associated with the grade of glioma as its expression is not detected in glioblastomas. Overexpression of ETNPPL protein can exhibit tumor suppressive effect and inhibit the proliferation of glioma stem cells (Leventoux et al. KIF2C (Bie et al. DLGAP5 (Zhou et al. CCNB1 (Yang et al. . The above biomarkers show a coordinated association with glioma progression, indicating that their co-expressed gene DRAXIN is inevitably functional in the development of glioma.As itself, genes with similar functions or variations are somewhat linked and will have a tendency to co-express, which can be used to screen genes with similar sequences (Cheng et al. DRAXIN in glioma using in vitro experiments. As one of the most fundamental features of gliomas, abnormal proliferation can lead to continuous tumor progression, crowding out the viability of normal brain tissue and leading to immature differentiation of neoplastic cells, further aggravating the malignancy of gliomas (Caglayan et al. DRAXIN was demonstrated in this experiment to significantly inhibit the proliferative capacity of U251 cells. Furthermore, the aggressiveness of glioma is a key indicator of its malignancy. Invasion into normal tissues will lead to unclear margins of glioma, making it difficult to remove surgically and laying the hidden danger of tumor recurrence (Tong et al. DRAXIN could promote the invasion of glioma cells, as other oncogenes have been reported (Zhang and Ma DRAXIN as a key pathogenic factor in glioma from multiple dimensions, suggesting that DRAXIN can be targeted for intervention to improve the prognosis of glioma patients.This study not only contains the predicted results from the above bioinformatics, but also validates the exact function of DRAXIN and the prognosis of glioma for the first time. However, there are some shortcomings in this study worth noting. Foremost, due to different medical environments and data processing methods, clinical information is collected based on different standards, resulting in unsatisfactory consistency of the data profiles. However, we collected data from several public databases with a large amount of glioma samples, to the best elimination of data bias. Secondly, the statistical results may be affected by the lack of normal samples in the database, which has a large gap with the tumor sample size. To compensate for these shortcomings, we used RT-qPCR and IHC to verify the differences in DRAXIN expression at the tissue level and cellular level. And numerous analysis methods also make a guarantee for accurate results.In summary, we have studied a large amount of high-throughput sequencing data based on several databases, which reveals the association between DRAXIN was found to be highly expressed in gliomas as an oncogene leading to shorter survival and poor prognosis of patients. In addition, DRAXIN may be involved in the cell cycle and VEGF pathway to affect the progression of gliomas. Moreover, the role of DRAXIN in promoting glioma proliferation and invasion has also been demonstrated. This study provides a molecular basis for subsequent studies on DRAXIN as a potential biomarker to predict prognosis of glioma patients and also a therapeutic target for glioma therapy.Collectively, for the first time,


Bound to its high affinity receptor on mast cells and basophils, the IgE antibody molecule plays an integral role in the allergic reaction. Through interactions with the allergen, it provides the sensitivity and specificity parameters for cell activation and mediator release that produce allergic symptoms. Advancements in human hybridoma technologies allow for the generation and molecular definition of naturally occurring allergen-specific human IgE monoclonal antibodies.2 conformational epitope on Der p 2.A high-resolution structure of dust mite allergen Der p 2 in complex with Fab of the human IgE mAb 2F10 was recently determined using X-ray crystallography. The structure reveals the fine molecular details of IgE 2F10 binding its 750 ÅThis review provides an overview of this major milestone in allergy, the first atomic resolution structure of an authentic human IgE epitope. The molecular insights that IgE epitopes provide will allow for structure-based design approaches to the development of novel diagnostics, antibody therapeutics, and immunotherapies. At the center of the pathogenesis of allergic diseases is the IgE antibody molecule. In sensitized individuals, re-exposure to the allergen results in IgE engagement, causing Fcε receptor cross-linking and activation of mast cells and basophils. This triggers the release of mediators into the local tissue, culminating in the broad array of immediate symptoms associated with allergy and contributes to chronic allergic inflammation. To date, studies of the human IgE molecule, and its targeted epitopes on allergens, have been very limited. Most of our knowledge of this process has come from studies using allergic patient sera and surrogate monoclonal antibodies. Because serum contains a mixture of many antibodies, having many specificities, directed toward many different epitopes, and having heterogenous affinities, the ability to study the molecular interaction of human IgE with its target allergen by this method is limited. The ideal way to study this process is to use naturally occurring human IgE monoclonal antibodies (mAbs), that is, those antibodies which occur naturally as part of the human immunoglobulin repertoire, isolated from allergic patients. Unfortunately, until very recently, no such antibodies had ever been made. The field of molecular allergy, however, has worked for many decades on defining the allergen; making, and defining hundreds of the most common disease associated allergens. This is an invaluable asset, now allowing for the rapid identification and characterization of allergen-specific human IgE molecules.To have the ability to use naturally occurring human IgE mAbs (hIgE mAb) to study the epitopes at the center of allergy pathogenesis has been desired for many decades. There are three principal reasons why these antibodies have not been developed. The first reason is that techniques for efficiently making full-length  human mAb were not in place until recently. The second reason is that the frequency of IgE producing memory B cells in peripheral blood is exceedingly low , even inAdvances in hybridoma technologies have allowed for the first time the identification, amplification, and ultimately the generation of stable cell lines, human hybridomas, from the very rare population of IgE expressing B cells present in peripheral blood. With the ability to generate full-length naturally occurring hIgE mAb, we can now proceed with studies of the most basic, and critical, molecular interactions that make up the heart of allergy pathogenesis and disease. In addition, the antibody and allergen tools/reagents, and the many insights that they provide, will allow for the design and development of novel diagnostics, therapeutics, and immunotherapies.Despite IgE causing an abundance of human disease, it was not until 1967 that the “reagin” molecule was discovered . The IgEWhen common staining methods are employed to measure IgE B cells using flow cytometry, nonspecific surface-bound IgE may obscure the analysis because non-IgE-expressing cells stain positively due to IgE bound to CD23, present on all B cells , 11. StuSince the technologies to make naturally occurring human IgE have not existed previously, investigators have used alternate strategies to gain insights into to human allergen specific IgE antibody response. Recombinant chimeric human mAb have been generated by expressing allergen-specific murine variable gene sequences with human IgE constant region –17. ChriNaturally occurring human allergen-specific IgG mAb have been isolated from individuals receiving immunotherapy –22. ThesTo date, two techniques have been recently published that successfully capture the naturally occurring IgE antibody, using human hybridoma and single cell RNA sequencing technologies •, 27•. CHistorically, it has been easy to make murine mAb using hybridoma technologies, but extremely difficult to make hybridomas to selected targets using human peripheral blood B cells. Improvements in the efficiency and versatility of human hybridoma generation have enabled us to generate hundreds of naturally occurring human IgG mAb using hybridoma technology –32. HoweSeveral specific advancements allowed IgE secreting hybridomas to be generated. First, and most importantly, IgE encoding B cells were expanded in primary culture in only 7 days. IgE encoding memory B cells do not expand sufficiently using Epstein–Barr virus (EBV) transformation , 34. ThiWe have generated > 600 naturally occurring human IgE secreting hybridomas. Using patient serum IgE profiles and skin prick testing results, obtained by their allergist, we can identify subjects which not only have high titers of circulating IgE, but also circulating IgE encoding memory B cells. Hamilton et al. described the relationship between total serum IgE and allergen-specific IgE concentrations for several important allergens using 18,950 paired readings . This inA very important concept at the heart of allergic disease is the antigenic site (antigenic region) where IgE binds. For cross-linking of Fcε receptors to occur with a native monomeric allergen protein, two different IgE antibodies must bind simultaneously to two different antigenic sites on the allergen –39. In con, koff, KD) of IgE-allergen protein interactions can only be measured using monoclonal preparations of antibody. Using murine IgG antibodies expressed as chimeric human IgE constructs, to act as a surrogate of human IgE antibodies, it was found that higher affinities possess more functional potency [An additional consideration that IgE antibody molecules provide, which influence the strength of receptor cross-linking and the downstream effects of cell activation, is their affinity of binding . The kin potency . Taken t potency –45. Cons potency .Allergen-IgE interactions are key for the development of allergic disease. The interactions occur either at the level of facilitated antigen presentation by dendritic cells, or at the level of induction of an allergic response, by cross-linking IgE bound to basophils and mast cells, which leads to induction of mediator release. Therefore, mapping of IgE antibody binding sites or epitopes on allergens provides insight into the IgE antibody repertoire and the allergic response. Epitope mapping allows the specificity and cross-reactivity of homologous allergens to be elucidated, which has its implications for diagnosis and immunotherapy. Finally, by locating IgE antibody binding epitopes, hypoallergens with reduced capacity to bind IgE can be designed, with the goal to reduce side effects resulting from increasing allergen doses during immunotherapy. The recent availability of human IgE monoclonal antibodies has opened new possibilities for precise epitope mapping using X-ray crystallography.To provide a historical perspective, several approaches to map IgE epitopes are reviewed, based on literature published recently Table . These aSimilarly, a global microarray platform representing all possible linear overlapping 16-mer epitopes  of 731 allergens defined by the World Health Organization and the International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee was generated •. The plSome of the peptides identified in these studies have been reported to be useful to assess the epitope-recognition profiles of antibody repertoires for different isotypes , 61, forLimitations of these approaches are the lack of posttranslational modifications in the peptides and the focus on only linear epitopes by design. Linear epitopes are consecutive amino acids in the allergen sequence recognized by IgE.A reduction of IgE antibody binding to modified allergen molecules in dot blots or enzyme-linked immunosorbent assays (ELISA) , 69.Relative epitope mapping using an antibody of known epitope specificity for capture of the allergen and the IgE for detection . Based oRaising rabbit antisera against surface-exposed allergen derived peptides. Blocking of binding of allergic subjects’ IgE to the complete folded allergen by the antisera indicates the presence of a conformational epitope .Mimotopes or peptides that mimic IgE epitopes have been identified by phage display technology , 74. PepCreating hybrid molecules or chimeras, by grafting an area containing conformational or linear epitopes into a homologous nonallergenic molecule. Conformational IgE epitopes have been localized on surface patches of the allergen by comparing IgE binding to the chimeras , 76•.Using NMR-based hydrogen/deuterium exchange to identify areas containing conformational epitopes. Three conformational epitopes for murine mAb were identified in the lipid transfer protein Art v 3. Variants with mutated epitopes showed reduced binding of IgE from serum of allergic patients, which indicated the importance of the targeted residues for IgE recognition .X-ray crystallography of an allergen with an IgG mAb construct as surrogate of human IgE, followed by site-directed mutagenesis analysis .While linear epitopes are common in food allergens due to their processing in the digestive tract and/or before ingestion , conformational or discontinuous epitopes also exist in foods , 63, andWith the advent of hIgE mAb, direct epitope mapping can be performed. Four hIgE mAb were isolated that are specific for the mite allergen Der p 2. Immunoassays showed that three of them  overlapped with a murine IgG mAb (α-DpX), whereas a fourth (2F10) bound at a more distal position in the molecule. An NMR approach using methyl labeled Der p 2 identified two areas containing the epitopes for the four hIgE mAb . Additio2, which is lower than average for currently known allergen-antibody complexes [X-ray crystallography provides precise structural detail of the interactions between allergens and antibodies. Mapping of an epitope requires crystallization of the allergen with a fragment of an antibody , as the whole antibody is conformationally flexible and therefore difficult to crystallize. Currently, only one X-ray crystal structure of a complex of an allergen Der p 2) with a human IgE mAb construct that has the correct pairing of the heavy and light chains has been reported (PDB code: 7MLH) ••. Previ with a homplexes ••. Like omplexes ••. This omplexes ••. Der pomplexes , 83, 84.omplexes ••. MutatThe crystal structure of the Der p 2–2F10 Fab complex also shows that Der p 2 may form dimers Fig. a. The diThe epitope-containing area recognized by hIgE mAb 2F10 was identified by NMR, in conjunction with the binding areas for three other hIgE mAb  . In addiInhalants—Der p 1, Der p 2, Fel d 1, Can f 1, Can f 6Foods—Ara h 1, Ara h 2, Ara h 3, Ara h 6, Gal d 1, Gal d 2, alpha-gal, Jug r 1, Ana o 3The hIgE mAb technology platform has made available panels of antibodies to inhaled and food allergens which provide tools for research and diagnostic applications. Currently, the hIgE mAb include a diverse array of subject derived antibodies to:This is a remarkable collection of reagents. Typically, serum IgE levels are considered high at ImmunoCAP values of class 6 or ~ 100 kUA/L specific IgE antibody. The concentrations of hIgE mAb are several orders of magnitude greater and range from 10,000 to > 500,000 kUA/L IgE. These IgE antibody levels exceed those of even the most highly allergic patients. Several hIgE mAb  recognize non-overlapping epitopes on the allergen which is useful for mechanistic studies. Structural studies of the hIgE mAb/allergen complexes for all these allergens will enable the IgE epitope(s) to be localized. The recent studies with hIgE mAb 2F10 and Der p 2 can be extended to other allergens to define the repertoire of IgE epitopes on these molecules and to answer a fundamental question that has eluded allergists for many years “What do IgE antibodies recognize and what constitutes an ‘allergenic’ epitope?” This is especially relevant for food allergens where there is increasing debate about the role of linear or conformational epitopes in the etiology of food allergy and in the development of tolerance to food allergens. Determining the structure of hIgE mAb complexes with peanut, milk and egg allergens will provide definitive data on the epitopes involved.The hIgE mAb have been validated for allergen specificity by ELISA, by immunoblotting using purified allergens and for antibody reactivity by ELISA, ImmunoCAP, and total IgE assays (unpublished data). The biologic activity of hIgE mAb to Der p 2 has been demonstrated in a mouse model of anaphylaxis ••. The bThe momentum in in vitro allergy diagnostics is firmly in the realm of component resolved or allergen molecules as improved alternatives to allergenic extracts. Measuring IgE antibodies to Ara h 2 has greater diagnostic efficacy than using peanut extract . Molecul2. Having an atomic resolution structure of the interface between antibody and allergen allows for the creation of mutant allergens that are no longer recognized by the IgE mAb, a technique often referred to as structure-based antigen design. Mutation of the 2F10 epitope on Der p 2 established that residues Asp59, Leu61, and Lys100 are especially important for IgE antibody binding and function, also confirmed by in vivo studies. The ability to now study human IgE mAb hereby marks a major milestone in the allergy field, where authentic epitopes can be determined, and structure-based design of hypoallergens can be pursued. These human IgE mAb will contribute to analyze the human IgE antibody repertoire and associated epitopes, and lead to the development of new molecules for diagnosis and therapy of allergic diseases.Allergic diseases are a growing global health concern, with food allergy leading the way. At the helm, IgE antibodies bound to their high affinity receptor on mast cells and basophils that initiate cell activation and mediator release upon allergen recognition. Most of our knowledge of these allergen-specific IgE antibody molecules has come from studies using IgE found in patient serum. Due to the polyclonal nature and exceedingly low concentration of IgE in human serum, the ability to study the fine molecular details of the antibody-allergen interaction is limited. Alternate antibody approaches have been employed, but until recently no naturally occurring allergen specific human IgE monoclonal antibodies have existed. Thanks to advances in human hybridoma technology, IgE mAb can now be generated from patients with allergy. The specificities of the IgE mAb generated using this technology reflect the IgE sensitizations and allergic diseases of the patient from which they were derived. These unique antibody tools can be used to obtain highly detailed insights into their specific interactions with their disease-causing allergen. IgE mAb secreted from human hybridomas are full-length, including the Fc, as they are expressed from the patient’s B cell’s nucleus. IgE mAb variable gene sequences can be amplified and expressed as isotype switch variant immunoglobulins or as molecular Fabs for use in structural studies. Using the IgE variable gene sequences of Der p 2 specific antibody 2F10, the first authentic IgE epitope was determined using X ray crystallography. IgE mAb 2F10 was found to bind a conformational epitope of approximately 750 Å


Chouguiyu exhibited a good inhibition effect on ACE, and the inhibition rate was significantly enhanced after fermentation for 8 days. The ACE inhibitory peptides were further identified, followed by their inhibition and formation mechanisms using microbiome technology and molecular docking. A total of 356 ACE inhibitory peptides were predicted using in silico, and most ACE inhibitory peptides increased after fermentation. These peptides could be hydrolyzed from 94 kinds of precursor proteins, mainly including muscle-type creatine kinase, nebulin, and troponin I. P1 (VEIINARA), P2 (FAVMVKG), P4 (EITWSDDKK), P7 (DFDDIQK), P8 (IGDDPKF), P9 (INDDPKIL), and P10 (GVDNPGHPFI) were selected as the core ACE inhibitory peptides according to their abundance and docking energy. The salt bridge and conventional hydrogen bond connecting unsaturated oxygen atoms in the peptides contributed most to the ACE inhibition. The cleavage proteases from the microbial genera in Chouguiyu for preparing these 7 core ACE inhibitory peptides were further analyzed by hydrolysis prediction and Pearson's correlation. The correlation network showed that P7, P8, and P9 were mainly produced by the proteases from LAB including Lactococcus, Enterococcus, Vagococcus, Peptostreptococcus, and Streptococcus, while P1, P2, P4, and P10 were mainly Produced by Aeromonas, Bacillus, Escherichia, and Psychrobacter. This study is helpful in isolating the proteases and microbial strains to directionally produce the responding ACE inhibitory peptides.Angiotensin I converting enzyme (ACE) inhibitory peptides from fermented foods exhibit great potential to alleviate hypertension. In this study, the peptide extract from  Hypertension is a chronic health problem closely related to many diseases, such as stroke, heart attack, and heart failure . AngioteChouguiyu is a typical fermented fish with rich peptides. Most research has focused on the formation mechanism of its distinctive flavor and taste  in Anhui, China according to the previous study , 4 days (D4 group), and 8 days (D8 group) for further study. The reagents used for liquid chromatogram tandem mass were of mass pure grade, and all other reagents were of analytical grade.us study . After fChouguiyu (50.0 g) was smashed and homogenized for 0.5 h with 200 ml hydrochloric acid (0.01 mol/L). After centrifugation at 8,000 r/min and 4°C for 20 min, the supernatant was added with triploid ethanol for 20 min at 4°C and was then centrifuged to remove protein. The supernatant was lyophilized and stored at −80°C after desalination.Peptide extract was obtained according to the previous study . In brieThe assay of ACE inhibitory activity was performed using the ACE inhibitory assay kit  . In brieAblank1 was the absorbance without sample, Ablank2 was the absorbance without enzyme working solution, and Asample was the absorbance with samples or positive control.where +) at the range of 350–1,500 (m/z). The peptide sequences were identified using Proteome DiscovererTM version 2.4  according to the protein sequences of Siniperca chuatsi. False discovery rate <0.01, unique peptide ≥1, and Sequest HT >0 were used as the criteria to confirm the credible peptides. The peptides with ACE inhibitory activity from Chouguiyu were further predicted using AHTpin (http://crdd.osdd.net/raghava/ahtpin/).The peptidomics was conducted using the ultra-high performance liquid chromatography tandem mass as described previously  with somChouguiyu were obtained after centrifugation of fermented liquid in each sample. Shot-gun metagenomics was adopted to analyze the microorganisms as in the previous study (The microorganisms in us study . In brieus study .https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php). The isoelectric point of peptides was obtained from Expasy (https://web.expasy.org/compute_pi/). The hydrophobicity was predicted from PepDraw (http://www.tulane.edu/~biochem/WW/PepDraw/). The instability of ACE inhibitory peptides was calculated from ProtParam (https://web.expasy.org/protparam/). PepBank was used to determine whether the peptide was reported on http://pepbank.mgh.harvard.edu/search/basic, containing the searches of Pubmed, PDF, ASPD, and UniProt. The water solubility was searched using http://www.innovagen.com/proteomics-tools. The predicted enzymes of core ACE inhibitory peptides were obtained from BIOPEP-UWM (https://biochemia.uwm.edu.pl/) (https://www.brenda-enzymes.org/).The toxicity of peptides was obtained from ToxinPred (edu.pl/) , and thehttps://www.rcsb.org/) and prepared by Discovery Studio 2019 Client   was downloaded from RCSB PDB  . The strhttp://bioware.ucd.ie/peptigram) (https://www.R-project.org/). The interaction network was constructed using Gephi  and abundance heatmaps of peptides were generated using Origin 9.5C . The peptide profile was drawn using Peptigram (ptigram) . The corng Gephi .Chouguiyu at different fermentation times are shown in Chouguiyu exhibited good ACE inhibition activity as same as the positive drug captopril. The maximum ACE inhibition rate was observed in the D8 group, reaching 80.46%, significantly higher than that in the D0, D4, and positive control groups. To confirm which peptides contributed to the ACE inhibition activity, peptidomic analysis was used to identify the ACE inhibitory peptides. A previous study found that small-molecule peptides  with various hydrophobic amino acids in the structure possessed good ACE inhibitory activity  indicates better affinity between the peptides and receptor (Ebinding) of peptides was all lower than −145 kcal/mol, suggesting their stable binding with ACE. The electrostatic energy (ΔEele) of the peptides was much smaller than the van der Waals energy (ΔEvdw).Molecular docking technology has been widely used to study the ACE inhibition mechanism of peptides , 8. In treceptor , 40. In receptor . The binThe inhibition mechanisms of ACE inhibitory peptides were explored by intermolecular interactions at the atomic level . More kiChouguiyu is produced under the metabolism of the complex microbial community. In this study, the ACE inhibitory peptides with high activity might be formed by the hydrolysis of proteases from the microbial community. The relative abundance of the microbial community in Chouguiyu during the fermentation process was first studied , Acinetobacter (11.09%), Psychrobacter (1.71%), and Bacillus (1.06%), while the relative abundance of other genera was all less than 1%. With the increasing fermentation time, the abundance of Escherichia and Bacillus significantly decreased, and more microbial genera with high abundance occurred. In the D4 group, there were 16 genera with an abundance of more than 1%, mainly including Vagococcus (18.79%), Acinetobacter (17.23%), Enterococcus (5.06%), Peptostreptococcus (5.06%), and Psychrobacter (4.63%). After fermentation for 8 days, the abundance of Vagococcus, Psychrobacter, and Enterococcus reached the maximum, respectively, reaching 33.77, 10.40, and 8.91%. Besides, the genera with an abundance of more than 1% in the D8 group also included Acinetobacter (8.63%), Peptostreptococcus (7.48%), Streptococcus (4.66%), Lactococcus (2.76%), and Carnobacterium (2.23%). Similar dominant genera such as Vagococcus, Psychrobacter, Lactococcus, Fusobacterium, Vibrio, and Psychrilyobacter were found in the late fermentation period in Chouguiyu , microbial cluster 2 , and microbial cluster 3 . Interestingly, most ACE inhibitory peptides in Chouguiyu showed negative relations with microbial cluster 1 but positive correlations with the genera in microbial cluster 2 and microbial cluster 3. The correlation network map was then built using the Pearson's correlation coefficient between the ACE inhibitory peptides and microbial genera (|r|>0.6 and p < 0.05)  . Meanwhi < 0.05) . Among t < 0.05) . InteresChouguiyu for preparing 7 core ACE inhibitory peptides were further analyzed (Aeromonas (r = 0.69), as well as by serine endopeptidases  and metalloendopeptidases  from Bacillus (r = 0.62). P2 could be prepared by serine endopeptidases , aspartic endopeptidases (COG0616), and metalloendopeptidases  from Escherichia (r = 0.84) and by serine endopeptidases  and metalloendopeptidases  from Bacillus (r = 0.82). P4 could be obtained by serine endopeptidases  and aspartic endopeptidases (COG0616) from Escherichia (r = 0.63), as well as by serine endopeptidases  from Bacillus (r = 0.60). Many microbial genera could secrete various proteases for P7, P8, and P9 production. These proteases were concentrated on serine endopeptidases from Clostridium, Lactococcus, Peptostreptococcus, Citrobacter, Streptococcus, Vagococcus, Enterococcus, Fusobacterium, Morganella, Hafnia, Carnobacterium, and Psychrobacter and aspartic endopeptidases from Lactococcus, Citrobacter, Vagococcus, Enterococcus, Fusobacterium, Morganella, Hafnia, Carnobacterium, and Psychrobacter. In addition, fewer microbial genera could produce cysteine endopeptidases for P7, P8, and P9 production, mainly including Citrobacter (COG3672) and Psychrobacter (ENOG4111IPD). For P10, only Psychrobacter (r = 0.64) contributed most to the generation of P10 by serine endopeptidases , cysteine endopeptidases (ENOG4111IPD), and aspartic endopeptidases (COG0616).After prediction at BIOPEP-UWM, the cleavage proteases from the microbial genera in analyzed . P1 coulChouguiyu exhibited good ACE inhibition, and the inhibition rate was significantly enhanced after fermentation for 8 days , P2 (FAVMVKG), P4 (EITWSDDKK), P7 (DFDDIQK), P8 (IGDDPKF), P9 (INDDPKIL), and P10 (GVDNPGHPFI). Molecular docking showed that the inhibition of ACE mainly resulted from unsaturated oxygen atoms in the seven peptides forming a salt bridge and conventional hydrogen bond with ACE as H-acceptors, followed by active carbon atoms forming alkyl and pi-alkyl interactions. The N-terminal and C-terminal amino acid residues of these peptides played an important role in their ACE inhibitory activity. P7, P8, and P9 were mainly produced by the proteases from LAB including Lactococcus, Enterococcus, Vagococcus, Peptostreptococcus, and Streptococcus, while P1, P2, P4, and P10 were mainly produced by Aeromonas, Bacillus, Escherichia, and Psychrobacter after analysis by hydrolysis prediction and correlation network. This study provides a theoretical basis for understanding the formation and inhibition mechanism of ACE inhibitory peptides in Chouguiyu and important information for the related microbial strains and proteases to produce the responding ACE inhibitory peptides.Peptide extract of PXD033251.The data presented in the study are deposited in ProteomeXchange, accession number Ethical review and approval was not required for the animal study because Frozen mandarin fish were used in this study.DY: methodology and writing – original draft. LL: conceptualization. CL: writing – review and editing and conceptualization. SC and JD: visualization. SY: data curation. All authors contributed to the article and approved the submitted version.This research was financially supported by the National Key R&D Program of China (2019YFD0901903), the China Agriculture Research System of MOF and MARA (CARS-46), the Guangdong Basic and Applied Basic Research Foundation (2021A1515010872), the Young S&T Talent Training Program of Guangdong Provincial Association for S&T, China (SKXRC202210), and the Central Public-interest Scientific Institution Basal Research Fund, CAFS (2020TD69).The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study.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.


Conditions moderated after deposition of the silica‐rich, CaCO3‐poor rocks as reflected by the microfossil changes indicating a relatively stable water column although episodes of enhanced eutrophy did continue into the lower Turonian at Site U1516.Oceanic Anoxic Event 2, spanning the Cenomanian/Turonian boundary (93.9 Ma), was an episode of major perturbations in the global carbon cycle. To investigate the response of biota and the paleoceanographic conditions across this event, we present data from International Ocean Discovery Program sites U1513 and U1516 in the Mentelle Basin  that register the first complete records of OAE 2 at southern high latitudes. Calcareous nannofossils provide a reliable bio‐chronostratigraphic framework. The distribution and abundance patterns of planktonic and benthic foraminifera, radiolaria, and calcispheres permit interpretation of the dynamics of the water mass stratification and provide support for the paleobathymetric reconstruction of the two sites, with Site U1513 located northwest of the Mentelle Basin depocenter and at a deeper depth than Site U1516. The lower OAE 2 interval is characterized by reduced water mass stratification with alternating episodes of enhanced surface water productivity and variations of the thickness of the mixed layer as indicated by the fluctuations in abundance of the intermediate dwelling planktonic foraminifera. The middle OAE 2 interval contains lithologies composed almost entirely of radiolaria reflecting extremely high marine productivity; the low CaCO Documentation of first complete record of the Late Cretaceous Oceanic Anoxic Event 2 (OAE 2) at southern high latitudes (60°S) in the Indian OceanDynamics of the water mass stratification inferred from distribution patterns of foraminifera, radiolaria, calcispheresOAE 2 is characterized by alternating episodes of enhanced surface water productivity and variations of the thickness of the mixed layer This event, known as Heterohelix shift  Expedition 369 at Site U1516 in the Mentelle Basin Figure , allowinThe Mentelle Basin, located off southwest Australia on the eastern flank of the Naturaliste Plateau Figure , is a ri13C excursion and highlight similarities and differences between the sites.This study is focused on planktonic foraminiferal population dynamics, the benthic foraminifera, radiolaria and calcispheres distributions and the carbon isotope record across the Cenomanian‐Turonian boundary interval and OAE 2 at Site U1513 and their comparison of these observations with results from Site U1516 previously published in Petrizzo et al. . Conside2IODP Sites U1513  and U1516  lie at 2,800 and 2,676.6 m water depth, respectively, on the western margin of the Mentelle Basin and off the southwestern margin of Australia in the southeast Indian Ocean , U1513D (core 15R to core 22R) and from holes U1516C (core 28R to core 35R) and U1516D (core 2R to core 5R) previously studied by Petrizzo et al. . The ovehttp://web.iodp.tamu.edu/LORE/). The lithologies in Hole U1513A (core 39X to core 45X‐2) and in Hole U1513D (core 15R to core 19R‐2) are assigned to lithostratigraphic Unit II  revised by S. Batenburg (pers. comm. 2020) from the shipboard splice  and U1513D (core 15R –22R) spanning approximately 46.7 m of the composite section were prepared at the University of Nebraska and studied for biostratigraphic determination. At least 400 fields of view at 1,000x were observed using light microscopy for each sample to calculate species richness and identify the distributions of biostratigraphically‐significant taxa. The biozonation for calcareous nannofossils is according to Perch‐Nielsen . Ages we3 content. Values were chemically detected using a Dietrich–Frühling calcimeter at the University of Milan that measures the volume of CO2 developed by hydrochloric acid reacting with the bulk sample, which is proportional to the carbonate concentration. CaCO3 results are reported in weight percentage (%) and included in Petrizzo, Amaglio, et al.  connected to a Delta V Advantage (Thermo Fisher Scientific Inc.) isotopic ratio mass spectrometer. Carbonate powders (about 200 μg each) were reacted with >99% orthophosphoric acid at 70°C for 1 hr. The carbon and oxygen isotope compositions are expressed in the conventional delta notation calibrated to the Vienna Pee‐Dee Belemnite scale by the international standards NBS18 and IAEA‐603 as well as internal standards (Carrara marble) during each run of samples. Analytical reproducibility for these analyses was better than ±0.1‰ for both δr et al. . Analyti, et al. .2 was detected with TCD. For calibration, 2.5‐bis (5‐tert‐butylbenzoxazol‐2‐yl) thiophene (BBOT) was used as a standard. A K‐factor method was employed to obtain a regression line for quantitative analyses. Each TOC value was expressed as a percentage of dry total sample weight, and the value was calculated as the mean of three analyses on a sample. The TOC data are included in Petrizzo, Amaglio, et al.  content was measured in 40 samples at Kanazawa University. About 40 mg of each sample was powdered using a mortar and pestle and reacted with 1 M HCl for 8 hours to remove carbonate, then washed with deionized water to neutralize, and dried. Approximately 2 mg of the powder was placed in a tin film cup and weighed. The samples were combusted at about 1,000°C in a Thermoquest NA2500 elemental analyzer, and the produced CO, et al. .33.13 values ranging from 0.2% to 0.6%  are also observed at the base of the stratigraphic section from 265.52 to 271.31 m rCCSF which coincides with a lithologic shift to massive black nannofossil claystone. In the overlying interval the CaCO3 content increases to about 30% before the abrupt drop to 0.4% at 247.52 m rCCSF. The interval above 244.21 m rCCSF is composed of an alternating sequence of gray and black nannofossil‐rich claystone. In this upper interval CaCO3 content increases with values fluctuating from 15% to 71%  of Gartnerago obliquum and Gartnerago segmentatum, which is placed at 98.3 Ma  of G. nanum which occurs at 258.49 m rCCSF  above the barren interval , Eprolithus moratus (at 236.65 m rCCSF), and Quadrum gartneri (at 227.80 m rCCSF).The overlying succession from 224.50 to 241.78 m rCCSF Figure . The latQ. gartneri that marks the base of Zone CC 11 and the disappearance of H. chiastia that defines the base of Subzone CC 10b are both reliable datums for approximating the base of the Turonian Stage in the Global Stratotype Sections and Points (GSSP) type section at Pueblo, Colorado, whose stage criterion is the lowest occurrence of the ammonite Watinoceras devonense  useful to constrain the Cenomanian/Turonian boundary have been found similar to the record at Site U1516  specimens whereas large‐sized specimens (>125 μm) are always a minor component. In general, planktonic foraminifera dominate assemblages throughout except for the intervals where radiolaria comprise nearly the total microfossil assemblages Figure .The stratigraphic interval from 265.52 to 271.31 m rCCSF Figure  is mainlThe overlying interval from 247.60 to 264.47 m rCCSF Figure . CalcareThe interval of low carbonate content , Gyroidinoides, and Clavulinoides. Praebulimina is common only from above the interval of low CaCO3 content.Calcareous taxa are more abundant whereas agglutinated taxa represent only a minor component and never exceed 10% of the total assemblages. The most abundant taxa throughout the studied interval are gavelinellids  display a scattered distribution and low number of specimens ranging from 1 to 12 specimens per gram of dry sediment whereas the keeled Dicarinella and Marginotruncana are absent.The overlying interval from 247.60 to 264.47 m rCCSF Figure . The assMicrohedbergella is still the dominant genus. It ranges from about 20 to 900 specimens per gram of dry sediments with a peak value close to 3,600 specimens per gram of dry sediments at 225.01 m rCCSF. Muricohedbergella is the next most common genus varying from 5 to 1,653 specimens per gram of dry sediment. Whiteinella ranges from 2 to 70 specimens per gram of dry sediments and increase to 276 specimens at 225.01 m rCCSF. Planoheterohelix ranges from 3 to 2,603 specimens per gram of dry sediment whereas Planohedbergella ranges from 1 to 813 specimens per gram of dry sediment. The keeled Dicarinella and Marginotruncana show an almost continue stratigraphic distribution but never exceed 50 specimens per gram of dry sediments  and become common in the overlying interval from 219.97 to 243.21 m rCCSF Figure  where sp44.13 content supported by the biostratigraphic events  are characterized by sparse (<5% of sediment volume) and poorly preserved assemblages with generally low species richness (in average 16–19 species at both sites). Preservation improves and abundance increases at both sites starting from the base of Subzone CC 10a, and the interval from the FAD of L. acutus to the base of the interval with low CaCO3 content  and exhibit the same sequence of bioevents as in coeval tropical to temperate assemblages.Although the sites are 69 km apart and are not separated by any obvious physiographic barriers, there are substantial differences in the composition of the assemblages in the two sites for this interval. For example, L. bentonensis precedes the LAD of A. biramiculatus, whereas the opposite is observed at Site U1513. Similarly, the lowest occurrence of D. hagni is recorded above the FAD of E. octopetalus at Site U1516, whereas at Site U1513 that sequence of events is inverted  the correlation coefficient of 0.688 for the combined calcareous plankton groups indicates a reasonable consistency  to the first positive shift in δ13C values at the top (460.13 m rCCSF) just above an interval of lower δ13C values based on correlation with the low latitude Eastbourne section in England and according to the OAE 2 definition by Jarvis et al.  in Figures Carbon isotope data and microfossil distributions and abundances are plotted on a common age scale to highlight similarities and discrepancies between sites U1513 and U1516 Figure . Age est3 content, which might result from erosion of sediments by seafloor currents and winnowing. This observation is consistent with the sharp contact between the gray and black clay‐rich claystone observed in core U1513A‐45X‐2, 137 cm  seems remarkably different between sites both in the carbon isotope record and in the composition of the microfossil assemblages  is characterized by the dominance of radiolaria with the exception of three samples in the middle part of the interval that contain calcispheres  at Site U1513 and foraminifera  at Site U1516  and above the interval of low CaCOt Figure  from thet Figure  or by nut Figure  likely t3 content, but they are significantly different in the interval from 93.5 to 93.9 Ma , and the co‐occurring benthic foraminifera, radiolaria and calcispheres.The paleoceanographic conditions of the water column across the OAE 2 interval from 93.2 to 96.0 Ma at sites U1513 and U1516 Figure  are inte18O and δ13C stable isotope ratios at Site U1516, and the information on the planktonic foraminiferal paleoecology derived from the literature  inhabited the thick mixed layer and occupied ecological niches in the surface mixed layer and the thermocline.Small‐sized (38–125 μm) Radiolaria, especially Nassellaria, and calcispheres are interpreted to be shallow mixed layer dwellers and are generally associated with conditions of very high fertility in outer shelf to upper slope environments including upwelling regions  and agglutinated taxa tolerated sub‐ to anoxic conditions  and the consistent occurrences of the opportunistic and eutrophic calcispheres.Changes in the composition and abundance of the microfossil assemblages at the two sites are pretty similar and indicate a paleoceanographic setting characterized by reduced water mass stratification resulting from enhanced surface water productivity or vertical mixing. This interpretation is based on the abundance of the opportunistic vel Haq,  or alterAll together these observations suggest fluctuations in surface water marine productivity and variations in the thickness of the mixed layer likely driven by changes of the nutrient provenance, including either delivery from terrigenous influx from southwest Australia via fluvial input and consequent enhanced delivery of continentally derived nutrients that could have stimulated the marine productivity (Chen et al., 5.23 zone (Figure 3 content interval (Figure The lithology in the subinterval below the low CaCOl Figure  is reprel Figure . The mic3 undersaturation of ambient waters due to prolonged ocean acidification during the thermal maximum and the δ13C excursion, which have been interpreted to be forced by the high atmospheric pCO2 released by volcanic activity associated with the Large Igneous Provinces eruptions (Barclay et al., 13C record measured at the base of the low CaCO3 interval at Site U1516 is instead not registered at Site U1513 as it falls within the time of deposition of the low carbonate sediments (Figure At Site U1513 a hiatus of about 200 ky is inferred near the top of the subinterval based on the age model adopted in this study. The hiatus at Site U1513, which is interpreted as resulting from seafloor erosion, might also represent an interval coinciding with unfavorable conditions for the preservation of pelagic carbonate on the seafloor with waters that were not anoxic enough to allow accumulation of organic matter Jenkyns,  or to shs Figure .3 content (Figure The subinterval with low CaCOt Figure  is markeThe high abundance of radiolaria and the rarity of calcareous microfossils has been previously interpreted (Petrizzo et al., 3 content (Figure Gavelinella and Stensioeina and the opportunist calcareous Praebulimina show a significant increase in abundance that suggests the presence of oxic‐dysoxic bottom waters. The planktonic foraminiferal record at sites U1513 and U1516 slightly differs, pointing to differences in water column stratification. At Site U1513 the dominance of Microhedbergella over the other planktonic foraminiferal genera, the presence of lower mixed layer planispiral and scattered keeled taxa, and the radiolaria distributions and abundances might suggest the presence of a thick mixed layer with significant thermal differences between surface and thermocline waters. On the contrary at Site U1516 Microhedbergella is progressively replaced in abundance by Muricohedbergella to indicate a relatively stable water column with a thick mixed layer and a thin thermocline and frequent episodes of eutrophy toward the top.The subinterval above the low CaCOt Figure  is marke13C and δ18O data from foraminiferal tests (Petrizzo et al., The increase in marine productivity seems more pronounced at Site U1516 and likely resulted from the delivery of continentally derived nutrients to the ocean based on the δThe OAE 2 interval can be subdivided into three subintervals: 1. below, 2. within and above the low CaCOe Figure .The lithRelatively high terrestrial sediment influx is consistent with the proximity of the two sites to the southwestern Australian margin, with Site U1516 being the more proximal and thus more affected by episodic eutrophy as also corroborated by geochemical data and clay mineralogy proxies (Chen et al., Planoheterohelix, whose abundance indicates high productivity and low oxygen conditions, is almost absent in the lower intervals, it becomes relatively common in this interval, but it does not show the abrupt peak in abundance (Heterohelix shift: Leckie, Planoheterohelix was able to adapt to different water masses in a wide range of depth habitats and thus was not strictly indicative of oxygen depleted conditions.Although the genus 5.313C curve reflects the global carbon cycle with minimal effects on regional conditions. That is, the paleoceanographic scenario for the interval above OAE 2 is similar to that inferred in upper subinterval of OAE 2 with a relatively stable and thermally stratified water column that was interrupted by episodic phases of enhanced eutrophy, particularly at Site U1516.The termination of the OAE 2 interval Figure  is ident6High resolution carbon isotope and microfossil assemblage data from Site U1513 are compared with previously published records from Site U1516. Together, these sites yield the most complete OAE 2 and Cenomanian/Turonian boundary sedimentary sections in the Southern Hemisphere and reveal a detailed history of the microfossil assemblage changes. The two sites are located 69 km apart in the Mentelle Basin in the SE Indian Ocean and were positioned at a paleolatitude of 59°–60°S during the mid‐Cretaceous.13C values following previous definitions and no remarkable changes are registered by the microfossils assemblages that strictly correlate with the onset and the termination of the event.The microfossil and carbon isotope records are compared to highlight similarities and differences between the sites and to verify the response of biota to the paleoenvironmental perturbations associated with OAE 2. The comparison between sites is based on bioevents that allow direct correlation and identification of the OAE 2 interval whose duration is estimated to be 950 ky. The onset of OAE 2 is identified according to the δ3 content that coincides with the dominance of radiolaria and a rise in the CCD presumably resulting from ocean acidification during the thermal maximum and the δ13C excursion. These changes interpreted to have been forced by the high atmospheric pCO2 released by volcanic activity associated with the Central Kerguelen Plateau and other LIP eruptions. The interval of low CaCO3 content is more extended at Site U1513 and spans the entire interval where peak values of the δ13C positive excursion are registered at Site U1516 and elsewhere. Just below the interval of low CaCO3 content, a short hiatus of about 200 ky at Site U1513 is inferred from the age‐depth model and may result from seafloor erosion and/or winnowing and redistribution of sediments by currents.The sedimentary record corresponding to OAE 2 is marked by an interval of low CaCOChanges in abundance of the planktonic foraminiferal genera and the co‐occurring benthic foraminifera, radiolaria and calcispheres across OAE 2 depict a paleoceanographic scenario dominated by eutrophy resulting from a strong influence of continentally derived nutrients leading to enhanced marine productivity. The terrestrial sediment influx is consistent with the proximity of the two sites to the southwestern Australian margin and is corroborated by published geochemical and mineralogical data from Site U1516.3 undersaturation. This extreme paleoenvironment lasted about 100 ky before returning to a relatively stable paleoceanographic regime as revealed by more diverse planktonic foraminiferal assemblages in the upper OAE 2 interval, including the occurrence of lower mixed layer to thermocline dweller taxa thriving in stratified waters. However, enhanced eutrophic episodes still occurred either because of upwelling of nutrient‐rich and δ13C‐depleted intermediate water masses or delivery of continental nutrients.The distribution and abundance of the planktonic foraminiferal genera, each of them with specific paleoecological preferences, indicates that sites U1513 and U1516 were above the CCD before and during the onset of OAE 2 in a water column characterized by a predominantly eutrophic regime with variations of marine productivity and thickness of the mixed layer, as indicated by the fluctuations in abundance of the intermediate dwelling planktonic foraminifera. This phase is followed by shoaling of the CCD, as indicated by the dominance of radiolaria that reflect extremely high marine productivity and ocean acidification because of CaCOThe sedimentation rate was higher at Site U1513 than at Site U1516 from within OAE 2 through the lowermost Turonian, indicating an increase in carbonate production and dilution by terrigenous sediments. This conclusion is corroborated by the occurrence of diverse and abundant pelagic calcifiers reflecting a more stable and stratified water column. Variations in the composition of the microfossil assemblages coupled with the calculated sedimentation rates provide support for the paleobathymetric reconstruction of the two sites, with Site U1513 located northwest of the Mentelle Basin depocenter and at a deeper depth than Site U1516. Thus, Site U1513 was probably more influenced by water circulation in the nearby Perth Abyssal Basin.pCO2 at high latitudes in the Southern Hemisphere. The results presented here provide valuable paleontological and geochemical insights that improve our knowledge of the causes and consequences of paleoenvironmental changes associated with OAE 2 at high latitudes in comparison with concomitant records from lower latitudes.Finally, analysis of the microfossils and carbon isotope records at sites U1516 and U1513 provide a robust chronostratigraphic framework and a comprehensive understanding of the response of biota to the environmental perturbations associated with warming and high