IDH-mutant oligodendrogliomas and astrocytomas are still indistinguishable. We also detected altered performance in the previous diagnostic transcriptomic molecular biomarkers  and prognostic gene signature  after reclassification. Thus, we conclude that further efforts are needed to reconsider glioma molecular biomarkers.In 2021, the fifth edition of the WHO classification of tumors of the central nervous system (WHO CNS5) was published. Molecular features of tumors were directly incorporated into the diagnostic decision tree, thus affecting both the typing and staging of the tumor. It has changed the traditional approach, based solely on histopathological classification. The Cancer Genome Atlas project (TCGA) is one of the main sources of molecular information about gliomas, including clinically annotated transcriptomic and genomic profiles. Although TCGA itself has played a pivotal role in developing the WHO CNS5 classification, its proprietary databases still retain outdated diagnoses which frequently appear incorrect and misleading according to the WHO CNS5 standards. We aimed to define the up-to-date annotations for gliomas from TCGA’s database that other scientists can use in their research. Based on WHO CNS5 guidelines, we developed an algorithm for the reclassification of TCGA glioma samples by molecular features. We updated tumor type and diagnosis for 828 out of a total of 1122 TCGA glioma cases, after which available transcriptomic and methylation data showed clustering features more consistent with the updated grouping. We also observed better stratification by overall survival for the updated diagnoses, yet WHO grade 3  Diffusely infiltrating gliomas are the most common primary malignant brain tumors in adults . They inhttps://portal.gdc.cancer.gov/, accessed on 21 December 2022). Integrated TCGA data collections include clinical information, whole exome sequencing, copy number variation, DNA methylation, gene expression, and protein expression profiles for thousands of individual tumors. This source of multi-omics data is widely used for studying the molecular mechanisms of carcinogenesis and for the discovery and validation  of cancer biomarkers.The Cancer Genome Atlas (TCGA) is an integrative multicenter comprehensive project aiming to collect and publish clinically annotated omics profiles for 33 human cancer types; its data are publicly available through the NCI Genomic Data Commons portal  [https://portal.gdc.cancer.gov/projects/TCGA-LGG, accessed on 21 December 2022) [In TCGA, the highest number of samples were collected for the gliomas. Due to considerable molecular and clinical differences between pediatric and adult gliomas , pediatrer 2022) ,4. In TCer 2022) .TCGA-GBM includes glioblastoma samples collected between 1989 and 2011; 414 patients 76%) were diagnosed in or after 2002 ,4. TCGA-% were diThe WHO CNS5 principles of how molecular features affect the typing and staging of adult-type diffuse gliomas are illustrated in As an obvious consequence of molecular biomarker implementation in the WHO CNS classification, the diagnosis must change for many previously diagnosed glioma cases, including those from the TCGA-LGG and TCGA-GBM databases. For molecular biology investigations and for the search and validation of new diagnostic, prognostic, and predictive biomarkers, the correct classification of TCGA tumor samples may be necessary. Owing to versatile molecular information about the TCGA samples, such a reclassification is possible in most cases, as is demonstrated by the present study.Unfortunately, this was not the case for the Chinese Glioma Genome Atlas (CGGA), another large-scale databank of human glioma molecular profiles . In thisIn the current study, for the first time we re-classified available TCGA glioma cases according to the WHO CNS5 guidelines. We also found better correlation between the updated diagnoses and the clinical, epigenomic, and transcriptomic features of the TCGA gliomas.TCGA-LGG and TCGA-GBM glioma cases (n = 1122) were initially diagnosed according to the 2000 and 2007 WHO criteria ,7 based In this study, we extracted the available molecular information to reclassify TCGA glioma cases according to the WHO CNS5 criteria. The algorithm for reclassification of TCGA glioma cohorts according to the WHO CNS5 guidelines is visualized in It is worth noting that in the fifth edition of WHO Blue Books the grade designation has changed from the traditionally used Roman to Arabic numerals. Therefore, throughout the text, Roman and Arabic numerals are used to distinguish the old and the revised WHO CNS tumor nomenclature (WHO CNS5).IDH1/2 mutation is the major molecular diagnostic criterion for the classification of gliomas. In the TCGA annotation, the IDH1/2 mutation status was unknown for 119 glioblastomas, one oligodendroglioma, and one astrocytoma case. For such samples, we analyzed Mutect2 MAF data from the GDC Data Portal to establish the status for this type of mutation. IDH1 missense mutation in codon 132 was confirmed for the astrocytoma and oligodendroglioma cases, and IDH-wildtype status was confirmed for 13 glioblastomas (IDH-wildtype» (WHO grade 4), or «astrocytoma, IDH-mutant» (WHO grade 4) types.The presence of lastomas . No genoIDH-wildtype” as a diffuse, astrocytic glioma that is IDH-wildtype and H3-wildtype, all IDH-wildtype gliomas (n = 551) were checked for H3 K27 and G34 mutations. Most IDH-wildtype gliomas (n = 463) were also H3-wildtype, for 85 gliomas there was no genomic data, and in three cases mutation of H3 was detected. Two patients (age of diagnosis 21 and 30 years) had H3 G34R substitution and one patient (age of diagnosis 32 years) had H3 K27M mutation. Younger age and H3 mutations favor pediatric-type high-grade H3-mutant gliomas, namely “H3 G34–mutant diffuse hemispheric glioma” and “H3 K27–altered diffuse midline glioma”.According to the WHO CNS5 definition of “glioblastoma, IDH1/2 mutation status (n = 106); and (2) IDH-wildtype gliomas with unknown H3 mutation status (n = 82).As a result, 828 cases were reclassified out of 1047 TCGA gliomas analyzed . A totalWe performed unsupervised k-means consensus clustering of TCGA glioma transcriptomic (n = 634) and DNA methylation data (n = 601) in order to check how molecular clusters match with the tumor types according to the “old”  and “new” (WHO CNS5) classifications. Cases with unknown histology were excluded from the analysis.IDH-wildtype or IDH-mutant tumors  have two or three transcriptome profiles in the database for different tissue aliquots  and that grade 4 “astrocytomas, IDH-mutant” were not clustered together with “glioblastomas, IDH-wildtype”  , but it IDH-wildtype” according to the WHO CNS5 criteria were analyzed. The Mesenchymal cluster (n = 66) remained almost unchanged, while Proneural and Classical have changed drastically  were “molecular glioblastomas” , which were previously classified as grade II-III astrocytomas, oligoastrocytomas, and oligodendrogliomas. Newly introduced grade 4 “astrocytomas, IDH-mutant” were localized only in the Proneural cluster. As in the case of whole transcriptome unsupervised analysis . The goal was to check how “old” and “new” glioma types reflect patient survival records. Only samples with survival data were used in the analysis (n = 1043).Our next step was to analyze overall survival (OS) by Kaplan–Meier and Cox regression both for the basic TCGA glioma cohorts  and for the revised cohorts formed in accordance with the WHO CNS5 classification  B and IDH-mutant gliomas was the same for both classifications. Despite perturbations in the group of glioblastomas .As the median OS for glioblastomas did not change after reclassification  vs 14.0  months), this cohort was selected as a reference group for Cox proportional hazards regression . In the lastomas , the surrade IV” . This isIDH-mutant, grade 3” and “oligodendroglioma, IDH-mutant, and 1p/19q-codeleted, grade 3” were indistinguishable in terms of survival prognosis. The difference between the OS of “astrocytoma, IDH-mutant, grade 2” and that of “oligodendroglioma, IDH-mutant, and 1p/19q-codeleted, grade 2” was also not statistically significant. In contrast, the differences in OS were more pronounced for grade 3 vs grade 2 oligodendrogliomas   .As an indicator of prognosis value, we estimated the proportion of statistically significant differences in pairwise comparisons made for the “old” and “new” glioma classes, which was 57% (12/21) for the basic TCGA groups and 73% (11/15) for the WHO CNS5 types .Finally, we conclude that, on the available TCGA sampling, the integrated WHO CNS5 classification, which considers molecular features of gliomas, generally better serves to distinguish between the survival prognosis for the different groups, as compared to the outdated basic classification.FREM2 (FRAS1 Related Extracellular Matrix 2) and SPRY1  is specific to glioblastomas [FREM2 expression and overexpression of CRNDE  non-coding RNA are associated with worse prognosis for glioblastoma patients [FREM2 gene expression level itself to discriminate glioblastomas and lower grade gliomas and to predict survival within different glioma subgroups [FN1, ITGA5, OSMR, and NGFR) was proposed.Glioma management still lacks suitable diagnostic, predictive, and prognostic biomarkers. Nonetheless, a number of diagnostic and prognostic biomarkers were proposed . In partlastomas ,30,31. Lpatients . We had ubgroups . In addiubgroups . Many prubgroups , a risk All the above-mentioned biomarkers were developed on the basis of the outdated WHO classification of CNS tumors. In this study we aimed to interrogate their efficacy for the WHO CNS5-classified gliomas and to answer the question of whether reconsidering the previous biomarkers may be necessary for the new, WHO CNS5-based glioma diagnoses.CRNDE, FREM2, and SPRY1 genes were connected with the “old” and “new” tumor types. In addition, three variants of the FREM2 pathway with sequentially interacting nodes for one, two, or three levels of interactions were reconstructed as previously described [To this end, we tested the performance of the above biomarkers for the revised groups of TCGA gliomas. We investigated how the expression levels of escribed . The firescribed . Activatescribed . The metescribed ,37. The CRNDE, FREM2, and SPRY genes allowed the diagnosis of “glioblastoma, grade IV” to be differentiated from the lower grade gliomas . However, the use of the WHO CNS5 classification has transformed CRNDE into the diagnostic biomarker of IDH-mutant lower grade gliomas. FREM2 appeared a relatively weak diagnostic biomarker as it showed low or statistically unreliable AUC values for almost all pairwise comparisons except “glioblastoma, IDH-wildtype” vs “oligodendroglioma, IDH-mutant, and 1p/19q-codeleted”. Much better results were obtained for the activation of FREM2 pathway, as PALs returned relatively higher AUC values and allowed all major types of gliomas to be distinguished from each other, with the only exception being low- and high-grade “astrocytomas, IDH-mutant”. A similar figure was observed for another biomarker under analysis, expression of SPRY1 gene. At the same time, discussed biomarkers are not equivalent to IDH1/2 mutation status, since they allow glioma type-specific differential diagnostics within the group of IDH-mutant tumors with AUC > 0.7.We found a controversial situation regarding the diagnostic biomarkers under investigation . When thIDH-wildtype” group.To evaluate the performance of prognostic 4-gene signature risk scores were calculated for each glioblastoma patient  accordinOverall, it can be concluded that the changes introduced by the WHO CNS5 classification of gliomas have dramatically influenced the usability of the previously discovered and validated biomarkers, sometimes making them ineffective. Thus, there is a need to refine the existing pool or to search for the new diagnostic biomarkers of human gliomas that would be based on an updated molecular diagnosis, such as the update to the TCGA glioma database communicated in this report.Due to the large-scale versatile clinical and multi-omics molecular data for human cancer cases including gliomas, TCGA is now a premium open access database for molecular biomedical studies. However, as it is currently on the project web site, “TCGA-LGG” and “TCGA-GBM” databases dedicated to human gliomas contain obsolete diagnosis annotations based solely on histological laboratory assessments. Since the correct categorization of published glioma cases is crucial for the correct molecular investigations, including the search for new biomarkers, we reclassified the available TCGA glioma cases to make the diagnosis consistent with the current WHO classification of central nervous system tumors (WHO CNS5), which also includes the interrogation of molecular data such as diagnostic mutations and chromosomal rearrangements. This was not possible for the CGGA database, another large collection of molecular data on human gliomas, because of insufficient annotation of the profiles in respect to the diagnostic mutations and rearrangements.We also tested the way these changes in the groups of gliomas were related to molecular clusters (based on transcriptomic or DNA methylation profiles) and found an overall better agreement with the WHO CNS5 tumor types than with the basic TCGA classification. The evaluation of glioblastoma case distribution among distinct molecular subtypes  showed that “molecular glioblastomas”  belong distinctly to the Classical glioblastoma-intrinsic transcriptional subtype.Overall, our results showed that molecular marker-based diagnosis provides a more objective determination of glioma type. However, several “bottlenecks” are still present, e.g., that profiles for different tissue aliquots may fall into different molecular clusters, which is related to the well-known intratumoral heterogeneity in CNS tumors, especially in the glioblastomas ,26. FurtOur next goal was to check the survival prognosis performance of the new glioma classification using TCGA data. We found that the “new” (WHO CNS5) tumor types generally more accurately reflect survival estimates compared with the “old” ones which were based on histological diagnosis alone. Better survival stratification increases the likelihood of discovering new effective prognostic biomarkers.In addition, we tested several previously established and published diagnostic and prognostic biomarkers to investigate whether reclassification according to the WHO CNS5 has affected their clinical relevance. All examined single genes, gene signatures, and molecular pathway-based expression biomarkers behaved differently for the older and newer glioma classes. Some of the previously established gene expression-based molecular markers that performed well for the “old” glioma classes could be of little value for the updated groups of the gliomas. This suggests that the previous studies including high-throughput biomarker investigations based on TCGA, CGGA , and othAnother important conclusion is that previous secondary molecular databanks that aggregate published molecular and clinical data including patient diagnosis and response on treatment must be reconsidered as well . FinallyThus, we claim that there is a need to search for and to validate new diagnostic biomarkers for diffuse gliomas that would be consistent with the latest WHO CNS5 classification.Although our findings don’t have a direct clinical impact, it can be considered of high importance for many applied studies aimed at developing molecular biomarkers for gliomas, because molecular criteria of previous (WHO CNS3-4) and updated (WHO CNS5) glioma classes differ dramatically. Our results also show that the changes occurred in the glioma classification cannot be ignored when comparing and interpreting the results of clinical trials conducted before and after the introduction of the WHO CNS5, since the clinical features of gliomas, such as survival prognosis, have also changed.IDH1/2, ATRX, and TERTp mutation statuses, EGFR amplification, gain of chromosome 7 and loss of chromosome 10, and 1p/19q-codeletion were taken from [IDH1/2 mutation status was unknown for 121 gliomas. For such samples, we analyzed Mutect2 MAF data from the GDC Data Portal to establish the status for this type of mutation. Additionally, mutation in histone H3 subgroup has been determined: specifically, mutations p.Lys28Met (K27M) in genes H3-3A (H3F3A), H3C2 (HIST1H3B), and H3C3 (HIST1H3C) and p.Gly35Arg (G34R) in gene H3-3A. EGFR amplification, CDKN2A/B deletion were extracted from corresponding CNV files from the GDC Data Portal  [https://portal.gdc.cancer.gov/, accessed on 1 August 2022) [There are a total of 1122 glioma cases in TCGA-LGG and TCGA-GBM datasets. Cases with unknown histology (n = 75) were excluded from the analysis. ken from . In the er 2022) . RNA-seqer 2022) ) were dost 2022) . RNA-seqst 2022) . Overallst 2022) .The clustering of transcriptional profiles based on tumor-intrinsic gene signatures from Verhaak et al.  was condpPAL is PAL for pathway p; nCNR is case-to-normal ratio, the ratio of gene n expression in a sample under study to an average level in the control group; and ARR (activator/repressor role) is a value that depends on the function of this gene product in pathway p. ARRs are values defined as follows: −1 when product of gene n inhibits p; 1 when product of n activates p; 0 when product of n has an ambiguous or unclear role in the pathway; and 0.5 or −0.5, when the product of n is an activator or an inhibitor of p, respectively. As the reference gene expression profile we used the artificial gene expression profile obtained by gene-by-gene averaging of all gene expression data in the cohort under investigation.Pathway activation level (PAL) is an integral quantitative and qualitative characteristic of changes in the expression levels of genes participating in a certain molecular pathway . PALs weFREM2 pathway was reconstructed as described by Zolotovskaia M. et al. .p-value. Hazard ratios (HR) were calculated in the Cox model to assess differences in survival among the groups under comparison. Cox survival analysis for OS and PFS was performed between those two groups using R packages “survival” v3.2-11 [ROC AUC value was used as the measure of biomarker quality. Overall survival was assessed by Kaplan–Meier analysis; the statistical significance of differences was measured by log-rank test  v3.2-11  and “sur v3.2-11 . Kaplan– v3.2-11 . Tables  v3.2-11 . Hierarc v3.2-11 .


Diagnostic and Statistical Manual of Mental Disorders, is a person-centered instrument for systematically appraising the impact of cultural factors in psychiatric assessment. A number of key areas in the future development of the CFI have been identified in order to ensure further clinical uptake. In this paper, we suggest that applying a Therapeutic Assessment (TA) approach in using the CFI—i.e., framing the interview in a way that gives primacy to its self-transformative potential by explicitly focusing on those issues that are seen as the most urgent, relevant, and meaningful by the patient—could prove helpful in alleviating patients’ suffering beyond what is achieved by merely collecting relevant cultural information that may inform diagnosis and subsequent treatment interventions. The TA methodology has been designed as a collaborative approach to psychological assessment in which the assessment procedure itself is meant to induce therapeutic change. This is achieved by explicitly focusing on the particular questions and queries that patients have about themselves with respect to their mental health problems or psychosocial well-being; these questions are then allowed to guide the assessment process and the interpretation of the findings. We suggest a number of potential modifications to the related Outline for Cultural Formulation and to the CFI content that could strengthen a TA-inspired focus. With this paper, we do not claim to offer a definitive integration of the TA approach in using the CFI but hope to further the discussion of a therapeutic potential of the instrument.The Cultural Formulation Interview (CFI), included in the fifth edition of the  Cultural differences in how patients and clinicians conceptualize health, illness, and treatment can create barriers in terms of help seeking, patient–clinician communication and assessment, making diagnoses, engaging patients in treatment, and evaluating outcomes. Differences in gender, age, ethnicity, socioeconomic background, educational level, language, sexual orientation, religious beliefs, occupation, and/or disability, etc. may contribute to misunderstandings, lack of insight by clinicians into the patients’ contexts, and failure to establish rapport and trust. Failing to recognize and address such barriers can ultimately result in poor treatment adherence, negative outcomes, and prolonged illness.Diagnostic and Statistical Manual of Mental Disorders (DSM-IV)   was introduced in the fourth edition of the In the DSM-5 field trials, the CFI was found to be a feasible, acceptable, and clinically useful tool for the assessment of relevant cultural factors —an approach where the assessment procedure itself is designed to induce change in the patient—and compare it to conceptual features of the CFI. Our objective is to demonstrate that the use of an explorative instrument such as the CFI, when applied in a way that gives primacy to its self-transformative potential by explicitly focusing on those issues that are seen as the most urgent, relevant, and meaningful by the patient, could prove helpful in alleviating patients’ suffering beyond what may be achieved by mainly collecting relevant cultural information and allowing it to inform subsequent treatment interventions. Thus, we argue that in addition to informing clinical decision making related to diagnosis and treatment, the CFI could hold a therapeutic potential in and by itself.While we fully agree with these suggestions, we would like to suggest a fourth potential avenue for the CFI, namely that it could be useful not only as an assessment tool but also additionally as a therapeutic tool in its own right. Surely, it is often emphasized that the CFI can be useful in improving treatment outcomes; however, this usefulness generally hinges on aiding the clinician in gathering information on the cultural context of the patient, making a correct culturally informed diagnosis, exploring patients’ help-seeking habits, and improving trust and treatment adherence. As a complement to this perspective, we want to discuss the literature on so-called In the history of mental healthcare, psychological assessment has mostly been thought of as a tool in diagnosing psychiatric disorders, facilitating communication between clinicians, and planning treatment interventions  intended to support culturally informed psychiatric assessment, including formal nosological categorization that is not necessarily an integral part of psychological assessment, psychotherapy, or counseling. In contrast, TA constitutes an overall attitude towards the assessment procedure. Even so, the CFI and TA share many common grounds. Certainly, a focus on patients’ subjective concerns and queries is not new to the field of cultural consultation—for example, the clinicians behind the Cultural Consultation Service in Montreal, Canada, stress the importance of discussing the patient’s own framing of vital questions before the assessment, since it may often differ substantially from the issues described in the referral , the guide to the clinician performing the CFI , and the OCF that could help incorporate a TA approach in the assessment of an individual’s social and cultural context and history. These suggestions are to be seen as illustrations of how the CFI could be modified, not as definitive edits—we readily realize that there are numerous considerations involved in updating a guideline such as the DSM section on cultural formulation. For example, as an alternative to the adaptations to the core CFI suggested below, a TA perspective could be introduced by the creation of a complementary TA-focused version of the CFI, analogous to the current supplementary modules. In this way, a TA road map would be made available without compromising the practical necessity of keeping the CFI concise In the introductory phrase directed to the individual undergoing the interview, it is clear that although the clinician wants to know about the patient’s experience and ideas, they are first and foremost vehicles for the clinician’s own understanding: “I would like to understand the problems that bring you here so that I can help you more effectively. I want to know about your experience and ideas.” . This perspective could be further emphasized throughout the interview, such as in question 14: “What kinds of help do you think would be most useful to you at this time for your [PROBLEM]? What would be useful for you to find out about your [PROBLEM] in order to better deal with it?”.Another way of integrating a TA approach to the CFI is to adapt specific interview items so as to highlight the direct importance of how the patient can make use of the findings. Perhaps the most obvious starting point is question 3, “What troubles you most about your problem?,” which could easily be adjusted so as to shift the focus to the patients’ own queries , this instruction could strengthen the emphasis on making the assessment meaningful for the patient. A similar perspective could be promoted throughout the CFI instructions, such as in the section regarding cultural factors affecting current help seeking where it is suggested that the clinician “[c]larify individual’s current perceived needs and expectations of help, broadly defined”. Here too, an added sentence  could help underscore that the patient’s queries are given full priority.The guidance offered in the left-hand column of the CFI could also be adapted according to a TA framework. For instance, the instruction associated with question 3, “Focus on the aspects of the problem that matter most to the individual,” may already seem to introduce a perspective similar to that of the TA approach. In its current context, however, this is probably most often taken as a recommendation to use the most urgent or relevant aspect as an example when asking about cultural perceptions and meanings; this is also reflected in the recurrent reference to this as the “[PROBLEM]” to be inserted in the succeeding CFI questions. If slightly modified  or projective personality tests such as the Rorschach test. This, however, seems to be mostly tied to tradition—there is nothing that formally prevents the application of the central TA principles of enlisting patients’ curiosity, motivation, and willingness to explore their problems in the use of other instruments or techniques. Indeed, assessor flexibility in modifying test practices when it is seen as helpful for the patient and using their creativity in collaboratively applying psychological assessment instruments are explicitly mentioned as strengths of the TA model (Finn et al., Given the numerous conceptual similarities between TA and the CFI outlined above, a straightforward strategy for strengthening a culturally responsive use of TA could be to promote the uptake of the CFI as a routine instrument within the TA community. Based on published TA case reports that focus on issues related to cultural identity, it is our belief that the CFI can prove highly useful in this context. For example, Martin and Jacklin describe working with a 27-year-old man who had migrated to the United States with his family from a Middle Eastern country as a child and who had now been referred for a learning disability assessment (Martin and Jacklin, In sum, we have argued that the CFI could benefit from the application of a TA perspective by engaging patients in making the assessment meaningful for them, inducing change in how they conceptualize and cope with the issues that are subject to assessment, and avoiding treatment disruption. While there are certainly conceptual differences between the CFI and the TA approach, the models also share a number of important similarities and intersections that call for further development. Various modifications of the CFI content may be necessary in order to ensure a strengthened TA-inspired focus on what the patients wish to find out about themselves and on how to render the findings meaningful in their everyday lives. Alternatively, it may suffice to simply use the existing CFI within an explicit TA framework, much as a TA clinician would do with any assessment instrument. Moreover, we would like to highlight the CFI as a key assessment tool and promote its use among clinicians, therapists, and counselors already involved in TA. Embracing the CFI within a TA practitioner context could lead to further cross-fertilization between fields and potentially give rise to new insights into how the CFI can be optimally adapted to incorporate a therapeutic intention. We do not attempt to present a finished, ready-to-use solution for how a TA perspective could be integrated into the CFI and vice versa—instead, we would like to encourage a discussion regarding the direct therapeutic potential of the CFI and how a greater emphasis on what the patient sees as meaningful can be achieved in the use of this important assessment instrument.


ORF 73 (LANA), ORF 72 (vCyclin), ORF 71 or ORFK13 (vFLIP), ORF 74 (vGPCR), ORF 16 (vBcl-2), ORF K2 (vIL-6), ORF K9 (vIRF 1)/ORF K10.5, ORF K10.6 (vIRF 3), ORF K1 (K1), ORF K15 (K15), and ORF 36 (vPK)] that have the potential to induce malignant phenotypic characteristics of Kaposi’s sarcoma. These oncogenes can be explored in prospective studies as future therapeutic targets of Kaposi’s sarcoma.Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human gammaherpesvirus 8 (HHV-8), contains oncogenes and proteins that modulate various cellular functions, including proliferation, differentiation, survival, and apoptosis, and is integral to KSHV infection and oncogenicity. In this review, we describe the most important KSHV genes [ Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human gammaherpesvirus 8 (HHV-8), is the causative agent of several malignancies, including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD) . KS is aKSHV can establish either latent or lytic infections. The latent phase aims to enhance KSHV persistence in the host by transcription of a limited number of latent transcripts required for episomal maintenance of KSHV in dividing cells and limiting host immune responses . During KSHV has a double-stranded linear DNA genome with a length of approximately 165–170 kb. The genome comprises a unique central coding region of approximately 145 kb, and both sides of the region comprise highly GC-rich terminal repeats (TRs) of approximately 30 kb ,9. The KSeveral of these genes are viral oncogenes and are host homologs or unique to KSHV. These oncogenes and proteins modulate a variety of cellular functions, including proliferation, differentiation, survival, and apoptosis, and are integral in KSHV infection and oncogenicity . In thisKaposi sarcoma (KS) is an angioproliferative neoplasm, firstly described by Moritz Kaposi, an Austro-Hungarian dermatologist in 1872 . KS is tKS varies from an indolent to an aggressive presentation. Classic and Iatrogenic KS usually present progressive, multicentric, or even ulcerated violaceous, reddish or brownish macules, patches, plaques or nodules on the skin, but possible mucosa involvement following its development stages. Nodular lesions can bleed or become hyperkeratotic as well as polychromatic color change, collarette signs, white lines, among other dermoscopic findings that suggest KS. Usually, these lesions are not invasive and are localized on the lower extremities, with head and trunk, oral or upper limbs possibilities. Iatrogenic KS aggressivity depends on immunosuppression levels and period, being potentially fatal if not regressed ,13.Endemic and AIDS-associated KS are more aggressive. Endemic form typically has a lymph node involvement with more diffuse presentation on the body surface, such as the trunk, besides the lower limbs. This form can be divided into four subgroups: nodular, florid, infiltrative, and lymphadenopathic. Nodular African KS is limited to cutaneous lesions. Florid and Infiltrative African KS are aggressive infiltrative local lesions, being deep and extensive cutaneous lesions with bone involvement. Lymphadenopathic African KS is a particular fatal form, affecting children and has a rapid development with viscera involvement ,15,16. AIDS-associated KS has a predilection for viscera involvement, especially gastrointestinal tract, and pulmonary systems. Usually, oral mucosa is affected, as well as upper limbs, neck and head, besides lower limbs. Due to immunodeficiency in these HIV-patients, AIDS-associated KS has high morbidity and mortality with organ commitment. Patients treated with highly active antiretroviral therapy (HAART) tend to improve immunodeficiency, leading to regression of size and reduction of aggressivity of cancer, resembling a classic KS form ,15,16.In men who have sex with men (MSM), the epidemiological and clinical features are quite different from the AIDS-associated KS, resembling more the Classic KS clinic, with particular epidemiology and increasing incidence over the last years, despite the popularization of HIV therapies. There is a proposition of a fifth KS form specifically to this group, called nonepidemic KS ,17,18.Staging is not common for Kaposi Sarcoma. However, there is the validated staging classification for only AIDS-associated KS, created in 1980’s pre-HAART era, including the measurement of disease as localized or disseminated (T), immunodeficiency levels by measuring CD4 count as high or low (I) and the presence of systemic symptoms (S), just to classify a poor or good diagnosis. For the other KS forms, there is no universally accepted staging classification [Thus, KS negatively influences the quality of life of patients causing many physical and psychosocial problems. Some gastrointestinal KS lesions, for example, may cause pain, bleeding, difficulty with feeding, diarrhea, intestinal obstruction, malabsorption, and weight loss. Pain, severe edema, and cellulitis can also accompany ulcerated skin lesions, and edema can also be present in the absence of skin lesions. Edema of the external genitalia may also obstruct urination. Pulmonary lesions and effusions may be associated with dyspnea, cough, hemoptysis, and restricted activity. Facial and periorbital edema is disfiguring and, in extreme cases, may obstruct vision. Skin lesions, particularly those on the face that are hard to camouflage but also those on the torso and extremities, may lead to self-imposed social isolation, exclusion by others and psychological distress. Many of these problems have most commonly been associated with AIDS-related KS, but they may also occur with all epidemiological forms of the disease .Oncogenes are genes that have the ability to cause cancer and the activation of these is a major driver of cancer ,22. OncoViral oncogenes play a central role in KSHV infection and oncogenicity cancer ,22. DuriORF73 measures approximately 4437 kb . This onLANA was found to bind and interact with multiple cellular proteins, including tumor suppressors p53, pRb, and von Hippel Lindau (VHL), transcription factors- ATF4/CREB2 and STAT3, chromatin-binding proteins HP1, H2A/H2B, MeCP2, and Brd4, as well as signal transducer GSK-3β, in order to inhibit apoptosis and stimulate spindle cell proliferation. LANA has also been proposed to bind to several viral promoters and suppress viral lytic gene transcription, thereby influencing latency maintenance. Therefore, LANA is a highly versatile oncogenic protein that plays a central role in the pathogenesis of KSHV [In 1996, Chang et al. identified that KSHV ORF 72 encodes a protein, similar to the human cellular D-type cyclin, called KSHV-Cyclin (vCyclin). In 1997, the length and size of the protein (257 amino acids in length and 29 to 30 kDa in size) and a 53% similarity with human D2 cyclins was discovered ,28. ORF In human cyclins, vCyclin interacts with cyclin-dependent kinase (CDK)2, CDK4, and CDK6, as well as phosphorylates the cyclin/CDK inhibitory proteins p21 and p27, mediating and inducing the acceleration of the G1/S phase transition. However, unlike D-type human cyclins, whose expression is cell cycle-dependent, the level of vCyclin remains stable throughout the cell cycle and the constitutive activation of vCyclin/CDK complexes in KSHV infected cells appears to result from the extended half-life of vCyclin, which may explain its role in the deregulated proliferation of Kaposi’s sarcoma ,31.On the other hand, the ectopic expression of vCyclin in cells with elevated levels of CDK6 has demonstrably proven to lead to apoptotic cell death after the cells enter the S phase, called oncogene-induced senescence . HoweverORF 71  measures approximately 566 bp . This onThe best-characterized function of KSHV vFLIP is the utilization of the NF-κB pathway by directly binding to the IκB kinase γ (IKKγ) complex to initiate an extensive range of cellular processes that promote survival, proliferation, differentiation, cytokine secretion, and oncogenic transformation. vFLIP protects cells against growth factor withdrawal-induced apoptosis and plays a very important role in KS infection . vFLIPs ORF 74 measures approximately 1025 bp . This onAlthough ORF 74 is constitutively active, it is also activated by several human CXC chemokines. Hence, both constitutive and chemokine-induced vGPCR mediated functions may be used by KSHV to promote survival and replication of the virus, while the host immune system also uses the same functions to keep viral survival and replication under control. The transgenic expression of ORF 74 in mice was sufficient for the development of vascular KS-like lesions, indicating that vGPCR plays a critical role in the initiation of KS . The vGPORF 16 measures 525 bp  Table 1Table 1. L, and Bcl-w) and pro-apoptotic  proteins, which cooperate by forming homodimers or heterodimers to regulate the commitment of cells to apoptosis. Besides Bcl-2, which has the ability to interact with and inhibit pro-apoptotic family members such as Bax and BH3-only proteins, the hydrophobic pocket of Bcl-2 also binds Beclin 1 , which is part of class III PI3 kinase complex, required for the initiation of autophagosome membrane. In addition, the dual roles of Bcl-2 in apoptosis and autophagy suggest that a coordinated regulation may exist for Bcl-2 to conduct these two activities [Cellular Bcl-2 was originally discovered as an oncogenic protein in B-cell lymphomas. Since then, a number of proteins belonging to the Bcl-2 family have been identified, each possessing the signature Bcl-2 homology (BH) domain. The Bcl-2 family consists of both anti-apoptotic  to prevent the premature death of infected cells from sustained viral replication and associated diseases . vBcl-2 ORF K2 measures approximately 676 bp  Table 1Table 1. vIL-6 is an oncoprotein responsible for the development of Kaposi’s sarcoma  and induThe KSHV genome encodes two viral interferon regulatory factor oncoproteins (vIRF1 and vIRF3) and is a viral homolog of the cellular IRF transcription factor family . ORF K9 The ORFs K10.5 and K10.6 measure around 2040 bp . The vIRvIRFs are known to interfere with cellular responses to viral infections ,61. By dORF K1 measures approximately 1080 bp  Table 1Table 1. The K1 protein has multiple roles in cellular signal transduction, viral reactivation, endothelial cell immortalization, and host immune recognition . K1 has ORF K15 measures approximately 6245 bp  Table 1Table 1. K15 is a transmembrane viral protein with structural similarity of LMP2A of the virus Epstein Barr, and like LMP2A, K15 can block BCR signaling ,74. K15 ORF 36 measures approximately 2900 bp  Table 1Table 1 aStandard Kaposi’s sarcoma therapy has not changed in the last years and as KS manifests in many forms, therapies should also be divided into multiple application scenarios . Thus, t2-laser and superficial cryotherapy can be temporarily efficient in superficial lesions [Several therapies have been developed for localized lesions but without randomized trial comparisons. Radiotherapy is one of the most efficient treatments for this KS type. However, higher doses per fractions and concomitant administration of systemic therapies should be avoided to reduce the risk of sequelae. The possible risks are out-of-field recurrence, and radiotherapy-induced skin toxicity  . Surgica lesions ,86,87. I lesions  and vinc lesions  are exam lesions . Electro lesions  or topic lesions  can also lesions .Systemic treatments are performed for locally aggressive extensive and disseminated KS. The most recommended therapies are pegylated liposomal doxorubicin (PLD) and paclitaxel (PCT) chemotherapies . The PLDIn classic KS, PLD or low-dose interferon-alfa is the recommended first-line agents in younger patients. In AIDS KS, combination antiretroviral therapy is the first treatment option, but specific systemic treatment is recommended in case of extensive disease. Systemic treatment is also used in the prevention and treatment of immune reconstitution inflammatory syndrome in these patients . PLD is The use of KSHV oncoproteins as therapeutic targets has been gradually becoming more in focus as a novel strategy to treat and prevent the KS lesions growth. However, these studies are preliminary ,105,106.LANA binds in the terminal repeat region of the KSHV genome and, after this, docks onto the host chromosome, which ensures the viral genome replication and segregation during cell mitosis ,26 Figu. Kirsch Another potential target is the vFLIP, a potent activator of the NF-kB pathway and neutralizer apoptosis  Figure . SilencivGPCR develops angioproliferative tumors in multiple organs, such as KS. This tumorigenesis is mediated through numerous pathways, the most important of which appears to be the phosphatidylinositol 3-kinase (PI3K) pathway ,123,124),124123,1Other therapies and their respective therapeutic targets in signaling pathways modulated by KSHV oncoproteins, such as vIL-6 , have beKSHV encodes many viral oncogenes. The information on KSHV oncogenes associated with Kaposi’s sarcoma reported here has provided a basis for molecular studies of KS tumorigenesis. The development of inhibitors of proteins associated with these oncogenes would be a novel possibility for anticancer therapy of Kaposi’s sarcoma.


This is a review of the current state of molecular profiling in gastrointestinal (GI) cancers and what to expect from this evolving field in the future. Individualized medicine is moving from broad panel testing of numerous genes or gene products in tumor biopsy samples, identifying biomarkers of prognosis and treatment response, to relatively noninvasive liquid biopsy assays, building on what we have learned in our tumor analysis and growing into its own evolving predictive and prognostic subspecialty. Hence, the field of GI precision oncology is exploding, and this review endeavors to summarize where we are now in preparation for the journey ahead. Comprehensive molecular profiling has evolved over the last decade. The evolution of molecular profiling changed the face of oncology from standard chemotherapy based on histology to personalized therapy. Using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and whole-genome sequencing, oncologists are able to recognize the genomic drivers of tumorigenesis and provide patients with prognostic biomarkers and targeted therapy options Fig. ; Table 1Molecular profiling using tissue next-generation sequencing (NGS) has become a standard of care practice, and recently, circulating tumor DNA (ctDNA) has emerged as a tool for molecular profiling, a predictor of response to systemic treatment, and a powerful way to measure minimal residual disease (MRD) using less invasive approaches –3.KRAS mutant CRC do not respond to epidermal growth factor rector (EGFR)-targeted agents such as the monoclonal antibodies cetuximab and panitumumab  of the Year . An incr


Vibrio vulnificus necrotizing soft tissue infection (NSTI) is associated with higher mortality compared with non-Vibrio NSTI. This study’s objective was to compare outcomes including in-hospital mortality and prognosis between patients with V. vulnificus NSTI and those with non-Vibrio NSTI.It remains unclear whether V. vulnificus infection. We performed multiple imputation using chained equations followed by multivariable regression analyses fitted with generalized estimating equations to account for clustering within matched pairs. All-cause in-hospital mortality and length of stay during hospitalization were compared for NSTI patients with and without V. vulnificus.A retrospective 1:2 matched-pair cohort study of hospitalized patients with NSTI diagnosed by surgical finding was conducted in two tertiary hospitals in southern Taiwan between January 2015 and January 2020. In-hospital outcomes  were compared between patients with and without V. vulnificus NSTI group and 90 in non-Vibrio group. The V. vulnificus NSTI patients had higher mortality and longer hospital stays. Multivariable logistic regression analysis revealed that V. vulnificus NSTI was significantly associated with higher in-hospital mortality compared with non-Vibrio NSTI .A total of 135 patients were included, 45 in Vibrio vulnificus NSTI was associated with higher in-hospital mortality and longer hospital stay which may increase health care costs, suggesting that preventing V. vulnificus infection is essential. Vibrio vulnificus is a life-threatening condition with rapid progression and high mortality. It usually occurs through the injuries sustained when handling seafood, wound exposure to seawater, and ingestion of contaminated under-cooked seafood. Immunocompromised patients are also at higher risk of developing this disease  = 12.7), and 66.4% of the patients were male. Table V. vulnificus NSTI and non-Vibrio NSTI after 1:2 matching. The V. vulnificus NSTI group tended to have hypotension, bacteremia, history of seawater or seafood contact, comorbidity with liver disease, and higher serum lactate. All-cause in-hospital mortality was 13.3% in the V. vulnificus NSTI, whereas it was 7.8% in the non-Vibrio NSTI . Higher mortality was significantly associated with higher serum lactate, higher LRINEC score, and with liver disease.A total of 135 patients were included, 45 in Vibrio vulnificus is a gram-negative marine bacterium that is usually present in warm coastal waters. The main clinical manifestations of V. vulnificus infections in humans are gastrointestinal illnesses, primary septicemia, and wound infections. The clinical course can progress rapidly by releasing hemolysins and proteases and result in hemorrhagic bullae and severe skin necrosis [V. vulnificus include wound infections while handling seafood, exposure of a preexisting wound to seawater, and ingestion of contaminated under-cooked seafood [necrosis , 12, 13. seafood .V. vulnificus NSTI than those in non-Vibrio group. In our study, in-hospital mortality among patients with V. vulnificus NSTI was 13.3%. Previous studies have reported vulnificus NSTI mortality around 10–13% [Aeromonas spp. revealed high mortality rates, even through aggressive surgical debridement and antibacterial therapies. They all raised concern for increasing risk of monomicrobial infection and gram-negative pathogen. As comparison, the non-Vibrio group are including gram-positive, negative, and culture no growth patients, and possibly monomicrobial or polymicrobial. Mainly pathogens included Aeromonas spp., Pseudomonas, Escherichia coli, Klebsiella pneumoniae, Staphylococcus, and Streptococcus spp. Using two tertiary hospital database in Southern Taiwan, our study showed that mortality was higher in patients with d 10–13% , 15, whid 10–13%  found thd 10–13%  revealedV. vulnificus NSTI, there are 17 patients with positive blood and wound culture (37.8%), 12 patients with positive blood culture (26.7%), and 16 patients with positive wound culture (35.5%). In non-Vibrio group, there are 19 patients with bacteremia (21.1%). V. vulnificus group had higher incidence of bacteremia than non-Vibrio group (64.4% vs 21.1%). Tsai et.al conducted a study about NSTI patients with V. vulnificus and methicillin-resistant Staphylococcus aureus (MRSA). The incidence of bacteremia was 65% vs 18.75%; V. vulnificus group was also higher than MRSA group [V. vulnificus are more likely to develop blood stream infections [V. vulnificus group. Vibrio vulnificus can produce Vibrio vulnificus hemolysin/cytolysin (VVH), multifunctional autoprocessing RTX toxin, Vibrio vulnificus serine protease (VvsA), and Vibrio vulnificus protease (VVP). Both VVH and RTX toxin contribute to bacterial invasion to the blood stream and play a role in the development of hypotensive septic shock. The RTX toxin may promote the colonization at the primary infection site and cause severe tissue damage by releasing degrading cytotoxins to induce the inflammatory responses, that Vibrio vulnificus can then spread to the blood stream to result in systemic manifestations. Both VVP and VvsA are produced in the interstitial tissues of limbs and cause serious collagenolytic, hemorrhagic, or edematous skin damage in the extremities [V. vulnificus may explain a relatively higher rate of blood steam infection than other pathogens. However, the comparison of Vibrio vulnificus bacteremia between NSTI and food-borne infection settings needs further studies.In culture 2.7%, and remities –23. The Aeromonas species than for those with V. vulnificus [V. vulnificus and Klebsiella pneumoniae [V. vulnificus and MRSA [V. vulnificus versus non-Vibrio NSTI. In the present study, we clearly demonstrated that mortality was higher in patients with V. vulnificus than in patients with non-Vibrio NSTI. Besides, longer hospital stay may lead to higher hospitalization cost in V. vulnificus NSTI.Studies have shown conflicting results on the difference in mortality between patients with NSTI in subgroups. One study had shown higher mortality for NSTI patients with lnificus . One stueumoniae . Anotherand MRSA . But fewV. vulnificus infections and mortality. There were studies considered liver cirrhosis as risk factor with increased mortality among NSTI patients [V. vulnificus infection. Because NSTI and its rapidly progressive infection remain associated with high mortality, the LRINEC score, developed by Wong et al. [V. vulnificus [V. vulnificus NSTI but display a significant role with prognosis, associated with higher mortality in V. vulnificus NSTI patients.Our study showed that patients with hepatic dysfunction had significant associations with patients , 26–29. patients , 31. Theg et al.  based onlnificus . HoweverThis study still has some limitations. First, this study lacks external validation. The validity of the result still requires other studies to confirm. Second, the retrospective design of this study had its inherent limitation in data collection, such as unverified comorbidities. Third, the sample size is relatively small due to the rarity of NSTI. Fourth, the degree of liver disease in each group is not clearly stated.V. vulnificus NSTI than for those with non-Vibrio NSTI in this matched-pair cohort study. Preventing V. vulnificus infection is essential. The validity of the result still needs to be confirmed by further studies.In summary, adjusted in-hospital mortality was significantly higher for patients with


The prognostic relevance of prostate cancer (PCa) molecular subtypes remains controversial, given the presence of multiple foci with the possibility of different subtypes in the same patient.SPOP, ERG(+), EZH2, NKX3.1, and SPINK‐1 subtypes were analyzed.To determine the clonal origin of heterogeneity in PCa and its association with disease progression, TMPRSS2‐ERG fusion and ERG, EZH2, NKX3.1, and SPINK‐1 expression levels, using RT‐PCR. Mutations in exons 6 and 7 of the SPOP gene were determined by sequencing. The presence of subtypes and molecular patterns were identified by combining all subtypes analyzed. To establish the clonal origin of multifocal PCa, molecular concordance between different foci of the same patient was determined. Association of these subtypes with histopathological groups and time to biochemical recurrence (BCR) was assessed.A total of 103 samples from 20 PCa patients were analyzed; foci of adjacent non‐tumor prostate tissue, HGPIN, GL3, GL4, GL5, and LN were examined to determine the presence of the SPOP in any sample. The ERG(+) subtype was the most frequent. The molecular pattern containing all four PCa subtypes was only detected in 3 samples (4%), all LN, but it was the most frequent (40%) in patients. Molecular discordance was the predominant status (55%) when all analyzed molecular characteristics were considered. It was possible to find all subtypes, starting as a preneoplastic lesion, and all but one LN molecular subtype were ERG(+) and NKX3.1 subtypes. Only the expression of the NKX3.1 gene was significantly different among the histopathological groups. No association was found between BCR time in patients and molecular subtypes or molecular concordance or between clinicopathological characteristics and molecular subtypes of ERG, EZH2, and SPINK‐1.No mutation was found in NKX3.1 gene expression is the only molecular characteristic that shows a progression pattern by sample.The predominance of molecular discordance in prostatic foci per patient, which reflects the multifocal origin of PCa foci, highlights the importance of analyzing multiple samples to establish the prognostic and therapeutic relevance of molecular subtypes in a patient. All the subtypes analyzed here are of early onset, starting from preneoplastic lesions.  NKX3.1 also binds to TMPRSS2 upstream sequences, and in TMPRSS2‐ERG cases, it negatively regulates ERG expression and is a negative feedback control since ERG directly represses NKX3.1.EZH2 and NKX3.1 had also been described as PCa subtypes. Another subtype reported in PCa is characterized by SPINK‐1 overexpression, which specifically happens in a subset of ETS(−) and is associated with another subtype, the SPOP subtype, characterized by a mutation in this gene.SPINK‐1 protein is a trypsin inhibitor, which can function as an autocrine growth factor,SPOP is an E3 ubiquitin ligase substrate‐binding subunit of the proteasome complex that mediates the ubiquitination of target proteins, leading more frequently to their proteasomal degradation.Numerous studies have proposed different classifications of PCa molecular subtypes that can contribute to determining prognosis; among them, ETS(+) subtypes characterized by chromosomal fusions that cause the overexpression of ETS transcription factors. The most prevalent ETS fusion in PCa is The prognostic relevance of PCa molecular subtypes remains controversial.ERG(+), SPOP, EZH2, SPINK‐1, and NKX3.1 subtypes were analyzed in 103 samples from prostatic foci and regional metastasis to lymph nodes (LN) from 20 PCa patients with poor prognosis. Molecular patterns are also established by sample and patient, combining the presence of all subtypes analyzed.To elucidate the origin of heterogeneity in PCa and its association with disease progression, 22.1Formalin‐fixed paraffin‐embedded (FFPE) tissue samples obtained from radical prostatectomy (RP) of 20 PCa patients with poor prognosis  and 5‐year follow‐up were included. Cases were chosen from the project “Exploration of potential predictor biomarkers in patients diagnosed with prostate cancer,” approved by the Ethics Committee of the Instituto Nacional de Cancerología  to develop this study. Since tissues were obtained as part of routine care and not specifically for this study, and the data used in this study were previously anonymized, the Ethics Committee of the Instituto Nacional de Cancerología  waived the need for informed consent for this study.n = 17), PCa foci with different Gleason scores , and LN (n = 13). A sample of adjacent non‐tumor prostate tissue was used from each patient as a reference (n = 20). Two frozen PCa tissue samples were also used as a positive control of the TMPRSS2‐ERG fusion, as well as the PC3 cell line was assessed as a negative control for gene fusions. All methods complied with relevant guidelines and regulations at the national and international levels.Four to five FFPE samples per patient were selected to identify foci with different degrees of differentiation; a total of 83 samples were analyzed. These samples corresponded to HGPIN , supplemented with 10% fetal bovine serum and a cocktail of penicillin, streptomycin, and 1% amphotericin B.The PC3 cell line was derived from androgen‐independent PCa. This line was kindly provided by Dr. Fabio Aristizabal, from the Universidad Nacional de Colombia. The culture conditions were as follows: 5% CO2.3DNA/RNA was isolated from FFPE tissue by automated extraction in the QIAcube using a modified protocol of the commercial kit AllPrep DNA/RNA FFPE . The modification consisted of incubating the sample before starting the automated protocol with proteinase K at 56°C for 3 h, instead of 15 min. The RNA was quantified by using the Qubit® 2.0 fluorometer RNA HS assay . The RNA was stored at −70°C until its use. Good quality DNA was obtained with this same kit, which was also quantified using the Qubit® 2.0 fluorometer DNA HS assay and stored at −20°C.2.4TMPRSS2‐ERG fusion (exon 1 of TMPRSS2: exon 4 of ERG), using the KAPA SYBR FAST One‐Step kit to carry out qRT‐PCR , following the recommendations of the manufacturer. The primers used are presented in Table TMPRSS2‐ERG fusion was determined by visualizing the corresponding amplimers in 2% agarose gels with SYBR Safe.RT‐PCR was performed to determine the presence of the 2.5SPOP mutations were identified by sequencing exons 6 and 7, which were amplified through PCR with the Platinum High Fidelity PCR kit , using the SPOP‐6 and SPOP‐7 primers2.6ERG, Upper Bavaria, Germany) was used for RT‐qPCR. To obtain the melting curves, the PCR products were heated at 95°C for 5 s, at 65°C for 1 min, at 97°C continuously with 5–10 adq/°C, and then cooled at 40°C for 10 s. UBC was used as a reference expression gene. All tests were carried out in duplicate. All 103 study samples showed similar values of UBC gene expression: cross points (CPs) median = 29.61, mean = 29.68.RT‐PCR was carried out in a single step using the qScript 1‐Step SYBR Green qRT‐PCR kit , following the manufacturer's instructions. The protocol begins with reverse transcription at 50°C for 10 min for 1 cycle; activation of reverse transcriptase at 95°C for 5 min for 1 cycle; denaturation of samples at 95°C for 10 s, annealing for 20 s, and extension at 72°C for 1 min for 45 cycles. The primers used for each gene and respective amplicon length, concentration, and annealing temperature are described in Table 2.7http://cels.uri.edu/gsc/cndna.html). Seven serial dilutions were made in duplicate, between 1 × 101 and 1 × 107 copy number. The efficiency of the amplification (E) was calculated as: E = 10[−1/slope]. E values were: 1.903 for UBC, 1.873 for EZH2, 2.003 for NKX3.1, and 1.904 for SPINK‐1; SDs were <0.2 with respect to the duplicates. The validity of the points, efficiency calculations, and errors in the equipment were verified by the method of the second derivative.The standard curves were prepared with PCR products obtained from a FFPE sample of adjacent non‐tumor prostate tissue, purified from agarose gel using the Illustra GFX PCR DNA and Gel Band Purification commercial kit , and quantified using the Qubit® 2.0 fluorometer DNA HS assay . The number of copies of the cDNA was calculated using the URI Genomics & Sequencing Center's online calculator (2004) ; CPtarget is the cross point of the target gene; CPref is the CP of the reference gene (UBC). Calibrator is the adjacent non‐tumor prostate tissue for each patient. To analyze the relative expression of ERG, EZH2, NKX3.1, and SPINK‐1, the values obtained were converted into three categories: highly expressed (>1.20), normally expressed (between 0.80 and 1.20), and lowly expressed (<0.80).With the E values from the standard curves, the relative expression of each sample was calculated using the method of Pfaffl,2.9ERG(+) subtype, when TMPRSS2‐ERG fusion is present and/or ERG mRNA is highly expressed; SPOP subtype, in the presence of mutations in the SPOP gene; EZH2 subtype, when EZH2 mRNA is highly expressed; NKX3.1 subtype, when NKX3.1 mRNA is lowly expressed; and SPINK‐1 subtype, when SPINK‐1 mRNA is highly expressed.PCa molecular subtypes were assigned as follows: These subtypes were assigned by sample and patient; to consider that a patient is positive for a subtype, at least one sample must have this subtype. A molecular pattern is the combination of the presence of all subtypes analyzed in samples and patients.2.10TMPRSS2‐ERG fusion, SPOP mutations, and ERG, EZH2, NKX3.1, and SPINK‐1 gene expression was assessed separately and together, aiming to establish the clonal origin of all prostatic foci (HGPIN and PCa) analyzed by patient. LN were not used for concordance analyses, given that they are extraprostatic lesions. Figure Concordance for the presence/absence of the 2.11TMPRSS2‐ERG fusion, SPOP mutations, and relative expression of ERG, EZH2, NKX3.1, and SPINK‐1. The values obtained were converted into three categories: highly expressed (>1.20), normally expressed (between 0.80 and 1.20), and lowly expressed (<0.80). The distribution of the relative expression of ERG, EZH2, NKX3.1, and SPINK‐1 was also analyzed.Clinicopathological characteristics were analyzed as continuous variables: age at diagnosis (years), prediagnosis PSA (ng/ml), tumor percentage in RP, index diameter (cm), and time to BCR (months), as well as categorical variables: Gleason score, Gleason grade group at RP, margin status in RP, perineural invasion, lymphovascular invasion, primary tumor size and/or amount of spread into nearby structures (pT), lymph node compromise (pN), and 5‐year BCR. Molecular characteristics were analyzed as categorical variables: detection of the SD) was used, while the median (interquartile range) was employed for those that did not. For comparison between groups, Student's t test for independent samples was applied in the case of variables with normal distribution, and the Wilcoxon signed‐rank test otherwise. In the case of categorical variables, absolute and relative frequencies were obtained as descriptive measures. Given the sample size, the Fisher exact test was applied to determine whether there was a statistically significant association between the categorical variables.The assumption of normality in the quantitative variables was validated using the Shapiro–Wilk test. For the variables that fulfilled the assumption of normality, the mean , HGPIN (n = 17), GL3 (n = 23), GL4 (n = 23), GL5 (n = 7), and LN (n = 13). The main clinicopathological characteristics of the 20 patients are summarized in Table A total of 103 samples from 20 PCa patients were analyzed: foci of adjacent non‐tumor prostate tissue (3.2ERG(+), EZH2, NKX3.1, and SPINK‐1. The ERG(+) subtype includes the presence of the TMPRSS2‐ERG fusion and/or highly expressed ERG mRNA. Since no mutations were found in the SPOP gene, this was not considered for analysis. A total of 83 samples (70 prostatic foci and 13 LN) obtained from 20 patients were analyzed. All the results by patient and sample are shown in Table The molecular subtypes analyzed in this study were: ERG(+) subtype, which is considered the most frequent and most widely studied finding in PCa. The presence of each subtype in the samples analyzed ranges from 41% to 60%; 50 samples (60%) were considered ERG(+), 45 (54%) were EZH2, 38 (46%) were NKX3.1, and 34 (41%) were SPINK‐1. A nonmolecular subtype was identified in 7 samples. The presence of each subtype in the samples was predominantly in combination with ERG(+), ranging from 60% to 74%: 27 (60%) of EZH2 samples, 27 (71%) of NKX3.1 samples, and 25 (74%) of SPINK‐1 samples. ERG(+) alone was found in only 1 sample , 17 (85%) with EZH2, 15 (75%) with NKX3.1, and 15 (75%) with SPINK‐1. The presence of each subtype in patients was predominantly in combination with ERG(+), ranging from 82 to 93%; ERG(+) alone was not found in any patient  with t Figure .ERG(+), and they were found in 50 samples (60%), while 7 were ERG(−) in 33 samples (40%) /EZH2/NKX3.1, which are related among them, was found in 11 samples (13%): 8 samples (9.6%) with this pattern and 3 samples (3.6%) also including SPINK‐1 (ERG(+)/EZH2/NKX3.1/SPINK‐1). The most frequent pattern was EZH2 alone (13%), followed by ERG(+)/EZH2/SPINK‐1 and ERG(+)/NKX3.1 (11% each).When considering the four subtypes simultaneously, 15 molecular patterns were identified in the samples, with the frequency of these patterns ranging from 1% to 13%; 8 molecular patterns were ) Figure . The simERG(+), and they were found in 17 patients (85%), while 3 patterns were ERG(−) in 3 patients (15%) /EZH2/NKX3.1, which are related among them, was found in 11 patients (55%): 3 samples (15%) with this pattern and 8 samples (40%) also including SPINK‐1 (ERG(+)/EZH2/NKX3.1/SPINK‐1). The latter was the most frequent pattern in patients. The 3 molecular patterns with ERG(−) are present in only 1 patient each (5%), but all have EZH2 subtype  Figure . The sime Figure .3.3TMPRSS2‐ERG fusion and the ERG, EZH2, NKX3.1, and SPINK‐1 gene expression in 70 prostatic foci (17 HGPIN and 53 PCa) , which is compatible with monoclonal origin. Discordance was the least common pattern (10%–30%), which would support a multiclonal origin , EZH2 (70%), and NKX3.1 (56%) (pattern A), as well as the absence of fusion (82%) and underexpression of SPINK‐1 (75%) (pattern B). The most common pattern for partially concordant cases was pattern C: acquisition of the fusion (100%) and progressive increase in gene expression of ERG (57%), EZH2 (75%), NKX3.1 (63%), and SPINK‐1 (67%)  Figure .3.4n = 70)  Table . A totalERG, NKX3.1, and SPINK‐1, which indicates intratumoral heterogeneity , the ERG expression is not decreased.3.6SPINK‐1 subtype had a low percentage (18%). In LN, the range was between 54% and 92%; the presence of each subtype increased compared to HGPIN, where the most frequent subtypes were ERG and NKX3.1 (92% each). It is clear that ERG overexpression increases in frequency from HGPIN to LN (53%–92%).Figure ERG(+)/EZH2/NKX3.1/SPINK‐1) was detected only in LN, in 23% of them. The constant in all but one LN molecular pattern was the NKX3.1 subtype.Figure ERG, EZH2, SPINK‐1, and NKX3.1 and histopathological group was also evaluated. The distribution of the relative gene expression of ERG, EZH2, and SPINK‐1 showed no significant differences between histopathological groups, but the relative gene expression of NKX3.1 showed a statistically significant difference between groups (p < 0.001). A lower expression in LN samples with respect to all the other groups , and between GL5 compared to GL4 samples (p = .0071), indicates that there is an inversely proportional association between PCa progression  and the expression levels of NKX3.1 , pT (p = 0.049), and pN (p = 0.029), but no association was found between BCR time and molecular subtypes or molecular concordance. A comparison of the clinicopathological characteristics by molecular subtype with ERG, EZH2, NKX3.1, and SPINK‐1 subtypes did not show any statistically significant association , EZH2, NKX3.1, SPINK‐1, and SPOP mutation, to establish their association with different lesion degrees and regional metastasis in LN in 20 PCa patients with poor prognosis, as well as the clonal origin of the foci in these patients. The ERG(+) subtype includes the TMPRSS2‐ERG fusion and ERG overexpression. Since no mutations were identified in the SPOP gene, which may be due to sample size, this was not considered for analysis.Although PCa is a multifocal disease, it is not clear to what extent molecular heterogeneity in these foci is a determining factor in the natural history of the disease or its role as a prognostic molecular biomarker for this disease.ERG(+) subtype was found in 60% of the samples and in 85% of the patients. The previously reported frequency of this subtype for PCa in Caucasic men was between 40% and 66%,Initially, when determining the presence of molecular subtypes, the EZH2 subtype was identified in 54% of the samples and 85% of the patients, which was more frequently associated with the ERG(+) subtype (60% in samples and 82% in patients). All patients with ERG(−)  had the EZH2 subtype. In the literature, the EZH2/ERG‐independent subtype has been reported in 30% of the cases by immunohistochemistry (IHC),ERG(+) focus.The reported NKX3.1 subtype, 46% of the foci and 75% of the patients had this subtype, and the presence of NKX3.1 subtype was more frequent in combination with ERG(+) (in 71% of the foci and 93% of the patients). Previous literature has not reported a frequency for this PCa subtype to compare our results. Given the relationship among the expressions of ERG, EZH2, and NKX3.1, these three subtypes (ERG(+)/EZH2/NKX3.1) were simultaneously present in 11 samples (13%) and in 11 patients (55%). It is important to highlight that these subtypes are also found separately, which indicates that the expression of these genes is more complex than the sole reciprocal relationship among them.With respect to the SPINK‐1 subtype was found in 41% of the samples and 75% of the patients. Although most studies reported this subtype in ERG(−) PCa in approximately 10%–15% of the cases,SPINK‐1 was present predominantly in combination with ERG(+) in our study (74% of SPINK‐1 samples and 87% of the patients). Although SPOP mutation has been linked to SPINK‐1 overexpression in various studies, mutations were not found in any foci.The ERG(−) pattern: EZH2/ERG‐independent, which was found in 11 samples (13%); although this number is not too high, there were 3 more patterns that ranged between 10% and 11%. In patients, the most common pattern (40%) was the simultaneous presence of the four subtypes (ERG(+)/EZH2/NKX3.1/SPINK‐1), which is a relevant finding.Considering the simultaneous presence/absence of the four subtypes, more molecular patterns (15 patterns) were identified in the samples than in patients (8 patterns). It is noteworthy that the most frequent pattern in samples was an ERG, EZH2, NKX3.1, and SPINK‐1 subtypes, which did not show any statistically significant association.Similarly, a comparative analysis was carried out between clinicopathological characteristics, molecular subtypes, and BCR time in the 20 patients included in the study. No association was found between BCR time in patients, categorized as ≤3 and >3 months, and molecular subtypes or molecular concordance. Clinicopathological characteristics and the molecular subtype were compared with It is not surprising that the frequency of the analyzed subtypes increased when analyzed by patient (75%–85%) with respect to foci (41%–60%), while the presence of molecular patterns decreased. This is because most of the literature only analyzes one focus (index tumor)IDH1 and FOXA1 genes, described in the TCGA study.The presence of molecular alteration of a subtype in at least one sample is significant because a clone with a specific subtype can be the most aggressive one and can determine prognosis and response to therapies. Thus, considering more adaptative tumoral cells, the methodology employed in this study to establish the presence of a subtype in LN is unusual but very significant and increases the presence of subtypes found in patients. The analyzed nonmolecular subtype was identified in 7 prostatic foci (8%); nevertheless, there are other molecular subtypes not evaluated in this work, such as mutations in the TMPRSS2‐ERG fusion, expression of ERG, EZH2, NKX3.1, and SPINK‐1) was used to inquire about the clonal origin of PCa. When each molecular characteristic was evaluated separately, total concordance was the predominant state for most of them. Previous studies have found that 50%–60% of multifocal PCa tumors are concordant regarding the presence or absence of the TMPRSS2‐ERG fusion, similar to the 55% found in this study.ERG, NKX3.1, or EZH2.As for the concordance analysis of prostatic foci per patient, concordance among five molecular characteristics (ERG expression that characterizes the ERG(+) subtype of late appearance). A progressive decrease in gene expression related to PCa was less frequent, only in NKX3.1. These results are not comparable with previous literature since other studies of several foci only analyzed 2 or 3 tumor samples without considering HGPIN and did not present results regarding Gleason score per focus.The second most frequent pattern for these four alterations was partially concordant cases (20%–45%). Among these, the molecular characteristics vary in relation to disease progression, as observed in subclonal alterations acquired during tumor cell evolution. Of the 5 molecular characteristics, 4 were alterations considered PCa subtypes that appear with disease progression  for each alteration and supports a monoclonal origin with subclonal events; between 15% and 30% of the cases show discordance, which could be associated with a multiclonal origin. When analyzing all alterations simultaneously per patient, the degree of discordance (55%) was higher than when alterations were evaluated separately (10%–30%), as expected. Other studies have reported a decrease in molecular concordance as the number of analyzed molecular alterations increases; in particular, the whole genome or transcriptome shows that the foci vary widely in the same patient.It is noteworthy that there were intratumoral differences in foci with the same GL in 3 out of 6 patients who had samples with more than one focus with the same GL. Nevertheless, concordance in the other 3 patients supports the probability of a monoclonal origin. Wei et al.LN were not considered for concordance analysis, but it should be noted that none of the 13 LN studied was the same as the other foci analyzed, which underlines that malignant cells need other subclonal events to form metastatic foci in LN. This is in accordance with what was said above about subclonal alterations, which must provide additional advantages for such sophisticated processes as metastasis.ERG(+) and NKX3.1 subtype (92%). The molecular pattern with the four PCa subtypes (ERG[+]/EZH2/NKX3.1/SPINK‐1) was detected only in LN (23%). The 13 analyzed LN had more similarity among them than with the prostatic foci of the same patient. Findings in molecular patterns are in accordance with less heterogeneity in more advanced lesions (GL5 and LN). The result of less intermetastatic heterogeneity was expected when mutations are needed in the same genes to confer the same properties to a metastatic microenvironment at a distant site.Regarding the association between subtypes and histopathological groups, it was possible to observe all of them in HGPIN lesions, which leads to the conclusion that all the evaluated alterations are early events in the natural history of PCa. It is worth noting that the constant in all but one LN molecular subtype was the NKX3.1 gene was significantly different among the histopathological groups. Its expression decreased in more advanced lesions. Thus, the lowest levels were found in the LN group, indicating regional metastasis; this finding is consistent with other reports where NKX3.1 has been found downregulated in many PCa tissues. It has been reported lost or completely eliminated even in the most dysplastic and metastatic PCa tumors.Only the expression of the No association was found between BCR time and clinicopathological characteristics, molecular subtypes, or molecular concordance, which may be due to our sample size and highlights the importance of future studies with larger sample sizes that are more representative.5NKX3.1 expression level is the only molecular characteristic analyzed that shows a progression pattern.The results of this study support the notion that, given the molecular heterogeneity of PCa, the molecular evaluation of a single focus does not provide a complete molecular picture of the disease in each patient. Although it is possible to assign molecular subtypes in this way, this type of analysis hinders the association of molecular subtypes with prognosis and the choice of appropriate treatment. Therefore, it would have a significant clinical application, but it is necessary to have results from several studies that manage to demonstrate the impact of these molecular subtypes on the prognosis and progression of the disease. All the subtypes examined here are of early onset, starting in preneoplastic lesions. All authors have participated in the research that resulted in this work, as well as in the writing, reviewing, and final approval of this manuscript. Yenifer Yamile Segura‐Moreno and Martha Lucía Serrano wrote the main manuscript text and prepared the figures and tables.The authors declare no conflicts of interest.The present research was approved by the Ethics Committee of the Instituto Nacional de Cancerología, Bogotá, Colombia. Since tissues were obtained as part of routine care and not specifically for this study, and data used in this study were previously anonymized, the Ethics Committee of the Instituto Nacional de Cancerología  waived the need for informed consent for this study. All methods complied with relevant guidelines and regulations at the national and international levels.Appendix S1 Supplementary tables.Click here for additional data file.


These five peptides all behaved as mixed-type inhibitors and were predicted to form hydrogen bond interactions mainly with Asp333, a key residue located in the catalytic active site of sEH. Moreover, it was found that the corn gluten hydrolysates of Alcalase, Flavourzyme, pepsin and pancreatin all exhibited high sEH inhibitory activities, with IC50 values of 1.07 ± 0.08, 1.19 ± 0.24, and 1.46 ± 0.31 mg/mL, respectively. In addition, the sEH inhibitory peptides WYW, YFW, and YFY were successfully identified from the corn gluten hydrolysates by Alcalase using nano-LC-MS/MS. This study demonstrated the sEH inhibitory capacity of peptides for the first time and corn gluten might be a promising food protein source for discovering novel natural sEH inhibitory peptides.The objective of this study was to investigate the soluble epoxide hydrolase (sEH) inhibitory properties of corn gluten peptides. In total, 400 dipeptides and 8000 tripeptides were first virtually screened by molecular docking and 30 potential sEH inhibitory peptides were selected. Among them, WEY, WWY, WYW, YFW, and YFY showed the highest sEH inhibitory activities with IC Soluble epoxide hydrolase (sEH) is a member of the α/β-hydrolase family and is widely expressed in tissues, such as the liver, kidney, heart, lung, brain, and intestine . It is aAlisma orientale [Scutellaria baicalensis [Leonurus japonicus [Eucommia ulmoides [Discovering novel sEH inhibitors through chemical synthesis or from natural products has attracted a lot of attention. It has been found that most of the sEH inhibitors mainly include urea and amide groups . Comparerientale , Scutellcalensis , Leonuruaponicus , and Euculmoides . Howeverulmoides . All thiBioactive peptides prepared from food proteins by enzymatic hydrolysis or microbial fermentation have been considered promising nutraceutical treasures due to their excellent health-promoting benefits . Corn glMolecular docking has been widely used in the rapid screening of bioactive compounds by stimulating interactions between target proteins and candidate compounds. For example, Zhao et al. screened potential inhibitors against the main protease of SARS-CoV-2 from lactoferrin-derived peptides using molecular docking . Yu et aCorn gluten meal was purchased from Xi’an Guowei Starch Co., Ltd. . All peptides were synthesized by solid-phase procedures with a purity of over 95% by China Peptides Co., Ltd. . Bovine serum albumin (BSA), bis-Tris-HCl, dimethyl sulfoxide (DMSO), and methanol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . Human soluble epoxide hydrolase (sEH), 3-Phenyl-cyanomethylester-2-oxirane acetic acid (PHOME), and 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) were purchased from Cayman Chemical . Alcalase (1000 U/mg), papain (1000 U/mg), Flavourzyme (1000 U/mg), and Neutrase proteases (1000 U/mg) were purchased from Nanning Pangbo Bioengineering Co., Ltd. . Pepsin  and pancreatic  were purchased from Sigma-Aldrich . All other chemicals were of analytical grade.Corn gluten peptides were prepared through enzymatic hydrolysis according to the method by You et al. with minor modifications . The corThen, all the corn gluten hydrolysates were centrifuged at 4000 r/min for 10 min. The supernatant was fractionated using ultrafiltration with a molecular weight cut-off membrane of 1 kDa . The corn gluten hydrolysate fractions with a molecular weight less than 1 kDa were selected, lyophilized, and stored at −20 °C.Molecular docking was performed to screen potential sEH inhibitory peptides. The X-ray structure of human sEH (PDB code: 1ZD3) was used as the receptor . The strFor MOE, before docking, the force field of AMBER10: EHT and the implicit solvation model of the Reaction Field (R-field) were selected. The 2D structures of peptides were converted to 3D structures through an energy minimization module under the force field of AMBER10: EHT in MOE. The docking workflow followed the “induced fit” protocol, in which the side chains of the receptor pocket were allowed to move according to ligand conformations, with a constraint on their positions. The weight used for tethering side chain atoms to their original positions was 10. For each ligand, all docked poses of which were ranked by London dG scoring first, and then a force field refinement was carried out on the top 30 poses followed by a rescoring of GBVI/WSA dG. The conformation with the lowest binding free energy was finally identified as the best probable binding mode.For AutoDock Vina, the X-ray structure of sEH was converted to a PDBQT file that contains a protein structure with hydrogens in all polar residues in AutoDock4. Additionally, the ligands were also converted to a PDBQT file and all the bonds were set to be rotatable in AutoDock4. All bonds of ligands were set to be rotatable. Lamarckian Genetic Algorithm (LGA) is used to calculate the flexible docking of protein-fixed ligands. AutoDock Vina Extended automatically calculates the grid maps and clusters the results in a way transparent to the user. The best conformation was chosen with the lowest binding energy after the docking search was completed.For CDOCKER, the water molecules and the native ligands were removed from the complex crystal structure and then prepared by Prepare Protein module in Discovery Studio (DS). The 2D structures of the ligands were prepared  by Prepare Ligands module in DS. The peptides were prepared based on their sequence, and then modification and energy minimization were performed by the Energy Minimize procedure. Finally, the Prepare Ligands module was carried out to make the peptides ready before docking. The CDOCKER protocol was conducted between the ligands and the receptor. The conformation with the lowest CDOCKER Energy was finally recognized as the most stable conformation.After the molecular docking, the interactions between the peptides and sEH including the hydrogen bonds and the bond lengths were analyzed using PyMol .t (RFU) and Ct (RFU) represent the fluorescence intensity of the sample and blank group at t min, respectively. The t (min) represents the reaction time.The effects of the concentrations of the enzyme, substrate, and the hydrolysis time of sEH on PHOME were determined according to the methods reported previously ,23. Brie40 (RFU) and C0 (RFU) represented the fluorescence intensity of the blank control group at 40 and 0 min, respectively. S40 (RFU) represented the fluorescence intensity of the sample group at 40 min.The sEH inhibitory activities of peptides were determined as above described. Briefly, 130 µL of 62.5 ng/mL human sEH solution  and 20 µL of peptides  were added to 96-well plate followed by adding 50 µL of 10 µM PHOME solution . After incubating at 37 °C for 5 to 40 min, the fluorescence intensity of the mixture at an emission wavelength of 465 nm and an excitation wavelength of 330 nm was determined. AUDA and bis-Tris-HCl instead of peptide samples were used as the positive and blank control, respectively. The sEH inhibitory activity was calculated as follows:50)—the concentration inhibiting 50% of sEH enzyme activity was calculated by through linear regression according to the percent inhibitory activity versus the sample concentration curve.The median inhibitory concentration , respectively.The peptide sequences of corn gluten hydrolysate were identified using nano-LC-MS/MS. Briefly, 5 μL of the sample was first loaded onto a nanocolumn  in an Easy-nLC 1200 system . The sample was eluted by a linear gradient with mobile phase B (20% 0.1% formic acid in water—80% acetonitrile) from 4% to 8% for 2 min, from 8% to 28% for 43 min, from 28% to 40% for 10 min, from 40% to 95% for 1 min, and from 95% to 95% for 10 min. The mobile phase A was 0.1% formic acid in water. The flow rate was set at 600 nL/min.Zea mays L. (corn) database using Byonic.Then, the sample was analyzed using Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific). The electrospray voltage was set at 2.2 kV and the capillary temperature was set at 270 °C. The m/z for the full scan was 300 to 1000. The activation type was selected as HCD and the normalized collision energy was set at 28. Up to the top 20, the most intense peptide ions from the preview scan in the Orbitrap were used for the data-dependent acquisition. The raw MS file was analyzed and searched against the UniProt http://www.innovagen.com/proteomics (accessed on 10 October 2022). The prediction of peptide toxicity using ToxinPred is available at https://webs.iiitd.edu.in/raghava/toxinpred/multisubmit.php (accessed on 10 October 2022).In addition, the sEH inhibitory peptides were analyzed for physicochemical properties and toxicity. The physicochemical properties of the identified peptides, including isoelectric point (pI), net charge at neutral pH, and water solubility, were analyzed by the peptide property calculator, which is available at n = 3). The difference in significance between the two groups was analyzed by Student’s t-test, and that between three and more groups was analyzed by one-way analysis of variance (ANOVA) followed by the Duncan multiple comparisons. Statistical analysis was performed by SPSS 19.0 with the significance level at p < 0.05.All data were presented as mean ± SD . In addition, YMW, YRW, RV, FRY, WF, WLR, and YHY also exhibited sEH inhibitory activities of more than 40% at 100 µM. All this suggests that the small peptides might have potential as sEH inhibitors.The 400 dipeptides and 8000 tripeptides were docked to sEH by MOE. After being docked, all peptides were ranked according to the score, namely, the binding free energy. The peptide–sEH complex with the lowest binding free energy was recognized as the most stable conformation and this peptide might have a strong binding ability to the active site of sEH. Finally, the top 20 tripeptides and the top 10 dipeptides were selected. It was noted that the tripeptides generally showed a lower binding free energy than dipeptides . These 350 values mainly range from 1 to 100 μM. For instance, quercetin and kaempferol showed sEH inhibitory activities with IC50 values of 22.5 ± 0.9 and 31.3 ± 2.6 μM, respectively [50 values of 39.3 ± 3.4, 84.5 ± 9.5, 51.3 ± 4.9, and 11.4 ± 2.7 μM, respectively [Recently, a number of natural phytochemicals have been demonstrated to display sEH inhibitory activities and their ICectively . In addiectively .Lepidium meyenii), papaya (Carica papaya), and watercress , have been isolated and shown to have sEH inhibitory activities [50 values of 5.6 ± 1.2 and 7.3 ± 0.7 μM, respectively [50 values of 93.68 ± 4.52, 6.06 ± 0.12, 7.83 ± 0.52, and 6.32 ± 0.31 μM, respectively [Many sEH inhibitors have urea and amide groups . Some nativities . The amiectively . In addip < 0.05), as shown in 50 value of 55.41 ± 1.55 μM, followed by WWY and WYW of 68.80 ± 7.72 and 70.66 ± 9.90 μM, and then YFW and YFY of 96.00 ± 7.5 and 94.06 ± 12.86 μM, respectively (p < 0.05)  ( < 0.05) . While tm) and the maximum rate (Vmax) were calculated. It was found that, as shown in The inhibition kinetics of peptides WYW, WWY, YFW, YFY, and WEY on sEH were determined. The reaction rates of the five peptides all increased and then remained at a high level with substrate PHOME ranging from 1 to 30 μM . To deteTo further understand the inhibition mechanisms of peptides on sEH, the intermolecular interactions between peptides and the sEH enzyme were analyzed. It is well-known that the hydrolase catalytic pocket of sEH is primarily composed of a catalytic triad Asp333-Asp495-His523 and two tyrosine residues, Tyr381 and Tyr465. When an epoxide enters the active site, the oxygen atom of the epoxide group will be recognized by Tyr381 and Tyr465, which in turn activate the epoxide ring-opening by Asp333 . Asp333 3+ of the N-terminal amino acid residue of WEY, WYW, and YFW were all predicted to behave as hydrogen bond donors to form hydrogen bonds with the oxygen atom of Asp333. Two hydrogen bonds between the oxygen atom of Asp333 and the hydrogen atoms of the carbon backbone of the N-terminal amino acid residue and the NH of the middle amino acid residue of YFY were also observed. In addition, the peptides were predicted to form hydrogen bonds with other residues that belonged to the active site of sEH. It was noted that hydrogen bonds between WWY and the residues of Ser413 and Met418 not Asp333 of sEH were predicted. The hydrogen bonds between peptides and sEH all had satisfactory bond distances [In the present study, it was found that the peptides WEY, WYW, YFW, WWY, and YFY were predicted to form multiple interactions mainly including hydrogen bonds and π stackings with the residues of the active site of sEH, as shown in istances . Moreove3+ not the NH of the amide group that formed hydrogen bond interactions with Asp333. In addition, different from the amide-based synthesized compounds, no hydrogen bond interactions between peptides Tyr381 and Tyr465 were observed. This might be associated with the relatively low sEH inhibitory activity of peptides than the amide-based synthetic inhibitors. Generally, the structure-activity relationships among these peptides can be used to further refine structural models of the sEH improving the power of future computational approaches.The amide group of chemically synthesized sEH inhibitors can form more hydrogen bond interactions with the key residues of the active site of sEH. For example, it was found that not only the NH of the amide group formed a hydrogen bond with the carboxyl of Asp333 (Asp335) but also the CO of the amide group formed hydrogen bonds with the hydroxy of Tyr381 (Tyr383) and Tyr465 (Tyr 466) of sEH ,32. In tThe physicochemical properties of the identified peptides were analyzed by the peptide property calculator . The pepp < 0.05) . However, the IC50 values of the corn gluten hydrolysates produced by Alcalase, Flavourzyme, and pepsin and pancreatin were all significantly lower than that of papain of 2.94 ± 0.26 mg/mL (p < 0.05). Different from Alcalase, papain, Flavourzyme, and pepsin and pancreatin, the corn gluten hydrolysates produced by Neutrase proteases had a low sEH inhibitory activity of less than 30% even at the concentration of 5 mg/mL (data not shown). Corn gluten hydrolysates by Alcalase, Flavourzyme, and pepsin and pancreatin were thought to be promising sources of natural sEH inhibitors.Different enzymes were used to determine the sEH inhibitory activities of corn gluten hydrolysates. The results showed that corn gluten hydrolysates by Alcalase, papain, Flavourzyme, and pepsin and pancreatin all displayed good sEH inhibitory activities with a significant dose effect ( < 0.05) . The corWhat should be noted is that although the sEH inhibitory activity of corn gluten hydrolysate was not as high as most of the traditional urea and amide-containing synthetic chemicals and some natural compounds , the corThe peptide sequences from the corn gluten hydrolysates by Alcalase were identified by nano-LC-MS/MS to further understand the sEH inhibitory activity of corn gluten hydrolysates. It was interesting to find that three of the above five peptides, WYW, YFW, and YFY, were successfully identified in the corn gluten hydrolysates by Alcalase . This maIn this study, 8400 dipeptides and tripeptides were screened for potential sEH inhibitors using molecular docking. Finally, 30 peptides were selected and tested for sEH inhibitory activities. Peptides WEY, WWY, WYW, YFW, and YFY showed the highest activities, and all five synthetic peptides were competitive and non-competitive mixed inhibitors. Furthermore, sEH inhibitory activities were found in corn gluten hydrolysates produced by Alcalase, Papain, Flavourzyme, pepsin and pancreatin. In addition, three sEH inhibitory peptides WYW, YFW, and YFY were successfully identified in the corn gluten hydrolysates by Alcalase. Our results suggest that corn gluten peptides have potential as natural sEH inhibitors and corn gluten may be a prominent protein source for sEH inhibitory peptides.