Mycobacterium tuberculosis (Mtb) infection. S100A4, a calcium-binding protein that plays an important role in promoting cancer progression as well as the pathophysiological development of various non-tumor diseases, has not been explored in Mtb-infected hosts. In this study, transcriptome analysis of the peripheral blood of patients with pulmonary tuberculosis (PTB) revealed that S100A4 and GSDMD were significantly up-regulated in PTB patients’ peripheral blood. Furthermore, there was a positive correlation between the expression of GSDMD and S100A4. KEGG pathway enrichment analysis showed that differentially expressed genes between PTB patients and healthy controls were significantly related to inflammation, such as the NOD-like receptor signaling pathway and NF-κB signaling pathway. To investigate the regulatory effects of S100A4 on macrophage pyroptosis, THP-1 macrophages infected with Bacillus Calmette-Guérin (BCG) were pre-treated with exogenous S100A4, S100A4 inhibitor or si-S100A4. This research study has shown that S100A4 promotes the pyroptosis of THP-1 macrophages caused by BCG infection and activates NLRP3 inflammasome and NF-κB signaling pathways, which can be inhibited by knockdown or inhibition of S100A4. In addition, inhibition of NF-κB or NLRP3 blocks the promotion effect of S100A4 on BCG-induced pyroptosis of THP-1 macrophages. In conclusion, S100A4 activates the NF-κB/NLRP3 inflammasome signaling pathway to promote macrophage pyroptosis induced by Mtb infection. These data provide new insights into how S100A4 affects Mtb-induced macrophage pyroptosis.Pyroptosis is a host immune strategy to defend against  Mycobacterium tuberculosis (Mtb) [Tuberculosis (TB) is a respiratory infectious disease caused by is (Mtb) . Accordiis (Mtb) ,3. Despiis (Mtb) .Alveolar macrophages are the main immune cells involved in Mtb infection and TB progression . When MtPyroptosis is characterized by cellular swelling, plasma membrane rupture, and the release of the cell’s cytoplasmic contents, which promotes an inflammatory response . This pr2+ homeostasis, inflammation, and cell death [S100A4 is an important member of the S100 protein family, expressed in various cell types, including lymphoid and myeloid cells such as macrophages, neutrophils, mast cells, and memory T cells. It plays a crucial role in promoting cancer occurrence and metastasis [S100A4 plays different roles in the pathogenesis of infectious viruses. Yang’s studies show that S100A4+ macrophages promote the infiltration of ZIKV and the continuous presence of interferon serotonin secreted by the macrophages in the seminiferous tubule, which can increase the permeability of the blood–testis barrier [S100A4 is involved in the process of epithelial–mesenchymal transition (EMT) in human immunodeficiency virus (HIV)-associated nephropathy (HIVAN), and renal cell EMT plays a significant role in the development of proliferative HIVAN phenotype [S100A4 can lead to the development of liver cancer, which suggests that it could be a potential therapeutic target inducing liver cancer in patients with HBV [S100A4 is involved in the pathological and physiological processes of viral infectious diseases.The S100 protein family is a group of calcium-binding proteins, which now has 25 members with high sequence and structure similarity . Within ll death . S100A4 tastasis ,22,23,24henotype . Zhu repwith HBV . These sS100A4 in the host response to Mtb infection remains unexplored. In this study, THP-1 macrophages were infected with Bacillus Calmette-Guérin (BCG) to explore the regulatory effect of S100A4 on pyroptosis based on transcriptome analysis of peripheral blood of PTB patients. Our experiments indicate that S100A4 is a positive regulator of pyroptosis through si-S100A4, exogenous S100A4, or inhibition of S100A4 pre-treatment. Moreover, the addition of NF-κB inhibitor Triptolide (TPL) or NLRP3 inhibitor MCC950 prevented the up-regulation of pyroptosis by exogenous S100A4. These results indicated that S100A4 promoted pyroptosis by activating the NF-κB/NLRP3 signaling pathway.However, the role of S100A4 and GSDMD were significantly up-regulated in PTB patients compared with healthy controls (p < 0.001)  from healthy individuals, patients with clinically active pulmonary tuberculosis (PTB), and patients with extrapulmonary tuberculosis (EPTB) provided by the UK National Institute for Medical Research  , the inf< 0.001) A,B. Spea = 0.460 C. The AUas 0.940 D,E, indip < 0.05 A,B. KEGGp < 0.05 C,D.S100A4 on macrophages pyroptosis induced by Mtb infection, a Western blot was performed to evaluate the expression of S100A4 and GSDMD in THP-1 macrophages at different time points  after BCG infection. The results showed that BCG infection up-regulated the expression of S100A4, which peaked at 24 h  had higher expression levels of S100A4 and GSDMD compared to the control group  were added prior to treatment. Western blot revealed that the expression of S100A4 was significantly increased in THP-1 macrophages at a concentration of 1.5 ug/mL , has been used to treat inflammatory, autoimmune, and malignant diseases for centuries [Triptolide (TPL), one of the major bioactive ingredients in the Chinese traditional Herb enturies . It is aenturies ,35; afteS100A4 could down-regulate the protein expression of GSDMD-N, NLRP3, and NF-κB in BCG-infected THP-1 macrophages. This suggests that S100A4 promotes BCG-induced pyroptosis of THP-1 macrophages by activating NF-κB/NLRP3 inflammasome signaling pathway. THP-1 macrophages were pr-treated with TPL or MCC950 and infected with BCG. The results of Western blot showed that the addition of TPL and MCC950 inhibited the promotion effect of S100A4 on pyroptosis during BCG infection  and NLRP3 inflammasome related proteins  in BCG-infected THP-1 macrophages. Previous studies have demonstrated the involvement of the NF-κB/NLRP3 inflammasome signaling pathway in regulating pyroptosis in various diseases, such as spinal cord injury , diabetieratitis . In thisS100A4 expression in macrophages promotes inflammasome activation and pyroptosis, leading to the formation of a beneficial inflammatory microenvironment for host defense against Mtb infection. However, the persistence of Mtb infection leads to the development of an increasingly excessive or uncontrollable inflammatory microenvironment. Pyroptosis ensues with the release of intracellular live Mtb and their diffusion to surrounding cells, thereby facilitating Mtb spread. This dynamic inflammatory response reflects the delicate balance between protective immunity and immunopathology, thereby enhancing our comprehension of the intricate regulatory mechanisms governing cellular inflammation and pyroptosis signaling pathways during pathogen infection.During Mtb infection, the up-regulation of Mycobacterium bovis and did not fully replicate the process of Mtb infection in humans. However, certain experiments have validated that the expression patterns of IL-1β and other pro-inflammatory factors, as well as immunomodulatory genes induced by H37RV, BCG, and H37RA infections in THP-1 macrophages, are consistent, along with the activation patterns of Caspase-1 and Cleaved-Caspase-1 signal transduction pathways [S100A4−/− mice.Our results were obtained from macrophages infected with an attenuated strain of pathways . PerhapsS100A4 expression in macrophages promotes pyroptosis by NF-κB/NLRP3 inflammasome signaling pathway. This study enhances our comprehension of the intricate regulatory mechanisms governing cellular inflammation and pyroptosis signaling pathways during pathogen infection.In summary, during Mtb infection, the up-regulation of 2 tissue culture incubator until it reached the early logarithmic phase of growth.BCG was purchased from the Centers for Disease Control and Prevention  and cultured in Middlebrooks 7H9 broth medium  containing 10% oleic acid albumin dextrose catalase  and 0.05% Tween-80 at 37 °C in a 5% CO2 incubator until 80–90% confluent. These cells were then inoculated into plates containing 6 wells (1 × 106 cells/well) with media containing 50 ng/mL Phorbol myristate acetate (PMA) to induce adherence. After being cultured for 48 h, the media were replaced with fresh culture media, and cells were used for infection after an additional 24 h culture period. For bacterial infection, adherent cells were infected with BCG at an MOI of 10.Human monocyte-macrophage THP-1 cells were acquired from the cell bank of the Chinese Academy of Sciences and cultivated in RPMI-1640 containing 10% FBS at 37 °C in a 5% CORNA and small interfering RNA sequences against S100A4 (si-S100A4), as listed in 6 cells per well in a 6-well plate the day before transfection. The siRNAs were transfected into the cells using LipofectamineTM RNAi MAX reagent according to the manufacturer’s instructions. After incubating for an additional 24 h at 37 °C in a CO2 incubator, the cells were harvested to analyze the expression of genes of interest using Western blot assays and quantitative reverse-transcription PCR (qRT-PCR).Nontarget control siS100A4 was purchased from Sino Biological . The S100A4 inhibitor Niclosamise (Nic) was purchased from MedChemExpress . THP-1 macrophages were pr-treated with Exogenous S100A4 at concentrations of 0, 0.25, 0.50, 1.00, and 1.50 μg/mL, Nic at concentrations of 0, 0.5, 1.0, 3.0, and 5.0 μM for 2 h before being infected with BCG for 24 h, respectively. Cellular protein samples were extracted and stored at −20 °C for Western blot analysis.NF-κB inhibitor Triptolide (TPL) was purchased from MedChemExpress , and NLRP3 inhibitor MCC950 was purchased from MedChemExpress . THP-1 macrophages were pr-treated for 2 h using TPL (5 nM) and MCC950 (10 μM), respectively. Followed by infection with BCG for 24 h. Exogenous S100A4 ; β-actin ; anti-GSDMD-N ; anti-Caspase1 p48 ; anti-ASC ; anti-NLRP3 ; anti-IL-1β p17 ; anti-IL-18 .Key antibodies used included anti-S100A4 (1:1000), GSDMD (1:1000), NLRP3 (1:1000), Caspase-1 p48 (1:1000), ASC (1:500), IL-1β (1:1000), IL-18 (1:1000), NF-κB/p-NF-κB (1:1000) at 4 °C. Blots were probed with an HRP-conjugated secondary antibody (1:3000) for 1 h at room temperature. Protein bands were detected using a chemiluminescence kit  and an Amersham Imager 6000. A total protein extraction kit was used to extract protein from cells. The protein levels were then quantified and separated using SDS-PAGE before being transferred onto an appropriate membrane . The blots were blocked for 1 h at room temperature, followed by overnight probing with antibodies specific for β-actin (1:3000), −ΔΔCt method in triplicates in three different independent experiments. The primers used for qRT-PCR are listed in Total cellular RNA was extracted using Trizol and then reverse transcribed into cDNA following the instructions of the reverse transcription kit. qRT-PCR was performed using cDNA as a template. The results were analyzed using the 25 cells/well) for appropriate periods. After that, the cells were fixed with 4% paraformaldehyde for 30 min, rinsed thrice with PBS, permeabilized using 0.5% Triton-X 100 for 20 min, washed thrice with PBS, and incubated overnight with anti-S100A4, anti-GSDMD, and anti-NLRP3. Following three additional washes with PBS, the samples were probed for 1 h using FITC-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) antibody , rinsed thrice with PBS, and nuclei were counterstained using DAPI. After three additional washes with PBS, the samples were sealed and imaged via an inverted fluorescence microscope .Cells were cultivated in plates containing 12 wells  for 2 h, washed three times with 0.1 M phosphate buffer (pH 7.2), and fixed in 1% osmic acid at 4 °C for another 2 h. These samples were subsequently dehydrated using a graded ethanol series before being embedded in Epon–Araldite resin and placed into a model for polymerization. Ultrathin slices (70–80 nm) are treated with the anti-stain uranium acetate and imaged using a Hitachi H-7650 Electron Microscope .An enzyme-linked immunosorbent assay (ELISA) kit for human IL-1β  and IL-18  was purchased from Boster. Supernatants (500 μL) were collected from each group, and ELISAs were performed according to the instructions provided with the ELISA kit. Cytokine concentrations in individual samples were quantified by measuring absorbance at 450 nm with a microplate reader.4·mL−1, then inoculated into 96 well culture plates. After culturing cells for 48 h and infecting them with BCG for 24 h, 10 μL of CCK-8 solution was added to each well. The absorbance (OD) of each well was measured at a wavelength of 450 nm using a fluorescence microplate reader, and data were recorded after 4 h. Cell viability (%) = /OD value of control group × 100%.Cell Counting Kit-8 was purchased from Absin . THP-1 macrophages at the logarithmic growth stage were taken and treated with a cell density of 1 × 10GSDMD and S100A4 in the healthy group and PTB group were compared, and the t-test was used to determine the significance level of difference. The ggpubr package of the R programming language was used to draw box plots and correlation scatter plots, and the pROC package of the R programming language was utilized for conducting ROC analysis and drawing ROC curves. The limma algorithm was employed to analyze differences in all genes between samples from the healthy and PTB groups. FC ≥ 1.5, p < 0.05 were the criterion of analysis for DEGs. Volcano plots were plotted using the R package ggplot2. Histograms of gene numbers were plotted using GraphPad Prism 8.0. For the differential expressed genes obtained, the R programming language ClusterProfiler was used for KEGG pathway enrichment analysis, and the top 30 most significantly enriched pathways were screened. Bubble maps were then drawn using the ggpubr package.The GSE83456 dataset is from the Gene Expression Omnibus (GEO) database, which includes peripheral blood transcriptome sequencing information from 61 healthy controls (controls), 47 patients with extrapulmonary tuberculosis (EPTB), and 45 patients with pulmonary tuberculosis (PTB). Healthy people (N = 61) and PTB patients (N = 45) in these datasets were selected as research objects. These datasets were centralized and normalized using the R language scale function. Then, the expression levels of t-tests or one-way ANOVAs. These data were expressed as mean ± standard deviation (SD), and * p < 0.05; ** p < 0.01; *** p < 0.001.GraphPad Prism 8.0 was used for all statistical analyses, and results from triplicate experiments were compared using S100A4 is a positive regulator of pyroptosis in BCG-infected macrophages and promotes pyroptosis by activating the NF-κB/NLRP3 inflammasome signaling pathway (Our results indicate that  pathway .


KRAS variant alleles may have differential biological properties which impact prognosis and therapeutic options in pancreatic ductal adenocarcinomas (PDA).KRAS mutation variants with and without co-occurring PI3K or other genomic alterations and evaluated the association of these mutations with clinicopathological characteristics and survival using a Cox proportional hazards model.We retrospectively identified patients with advanced PDA who received first-line therapy and underwent blood and/or tumor genomic sequencing at the University of Washington between 2013 and 2020. We examined the incidence of KRAS mutations were identified in 111 PDA and included the following variants: G12D (43)/G12V (35)/G12R (23)/other (10). PI3K pathway mutations (26% vs. 8%) and homologous recombination DNA repair (HRR) defects (35% vs. 12.5%) were more common among KRAS G12R vs. non-G12R mutated cancers. Patients with KRAS G12R vs. non-G12R cancers had significantly longer overall survival (OS) (HR 0.55) and progression-free survival (PFS) (HR 0.58), adjusted for HRR pathway co-mutations among other covariates. Within the KRAS G12R group, co-occurring PI3K pathway mutations were associated with numerically shorter OS (HR 1.58), while no effect was observed on PFS.One hundred twenty-six patients had genomic sequencing data; KRAS/PI3K genomic profile could inform therapeutic vulnerabilities in patients with PDA.Patients with PDA harboring KRAS G12R vs. non-G12R mutations have longer survival, but this advantage was offset by co-occurring PI3K alterations. The  This retrospective analysis of patients with metastatic and locally advanced pancreatic ductal adenocarcinoma characterizes prognostic or predictive genomic markers for this patient population, with a focus on KRAS mutational variants and their interaction with concurrent molecular alterations. KRAS and co-occurring genetic mutations and assessed their impact on survival outcomes for patients with advanced pancreatic cancer. KRAS mutation variants have distinct biological interactions with downstream pathways. Preclinical data suggest that unlike KRAS G12D and G12V, the G12R variant cannot interact with a key protein, PI3K p110α (PIK3CA) and cannot activate its growth-promoting pathway. The data demonstrate biologically and potentially therapeutically relevant differences among cancers with coexisting KRAS G12R and PI3K pathway alterations and support further investigation of a rational combination of targeted therapies aimed at cancers with specific KRAS variants.In this study, the authors examined the incidence of 1 Exome sequencing studies demonstrated alterations in several prevalent core signaling pathways, among which KRAS is almost ubiquitous, affecting more than 90% of pancreatic cancers.3 Emerging evidence in the last decade suggests that not all KRAS mutation variants are equally pathogenic even when they affect the same codon.4 Most KRAS mutations in pancreatic cancers occur in exon 2 at codon G12, with a lower incidence at codons G13 and Q61. Within codon G12, mutational substitutions occur more frequently at G12D (41-44%), followed by G12V (29-34%), G12R (16-20%), and G12C (1-3%).5Pancreatic cancer is one of the most aggressive malignancies, with more than 60 000 new cases and, despite several multiagent chemotherapies in use since 2011, 47 000 deaths in 2021.KRAS variants are conflicting, with some suggesting that the G12R6-8 or Q619 variants may be associated with better survival compared to G12D variants, and others showing no effect or an inverse relationship.10-14 The molecular underpinnings of these differences are being elucidated, and the disparate ability of KRAS variants to activate the PI3K pathway appears to play a central role.16Data on the prognostic impact of various 17 Similarly, the downstream effectors PI3K/AKT/mTOR and RAF/MEK/ERK have not been successfully targeted in unselected patients.19 Despite preclinical evidence suggesting antitumor efficacy from MEK inhibitors in KRAS G12R mutated cancers due to the potential lack of activation of the downstream PI3K targets,16 single agent selumetinib showed no benefit among patients with KRAS G12R mutated pancreatic cancers.20 A better understanding of the molecular drivers and the consequent dysregulated signaling pathways in pancreatic cancer is critical to the development of targeted therapies and identifying patient subsets most likely to benefit.KRAS has not been targetable to date, with the exception of the very rare KRAS G12C variant.KRAS mutational variants and their interaction with concurrent molecular alterations.We conducted a retrospective analysis of all patients with metastatic and locally advanced pancreatic ductal adenocarcinoma (PDA) who received at least first-line systemic therapy and underwent next-generation sequencing from tumor or blood samples at any point during their follow-up. Our objective was to characterize additional prognostic or predictive genomic markers for this patient population, with a focus on All patients with locally advanced or metastatic PDA who were seen at the University of Washington/Fred Hutchinson Cancer Center  between August 2013 and July 2020, received at least first-line therapy for advanced disease, had somatic genomic sequencing information available, and had any follow up information were included in this retrospective study. Clinical and pathologic information was abstracted from a chart review from the UW/FHCC electronic medical record. The study was approved by the UW Institutional Review Board and conformed to the Declaration of Helsinki.Data were collected on patient demographics, Eastern Cooperative Oncology Group (ECOG) performance status (PS) at the time of initiating first-line systemic therapy for the advanced disease, genomic sequencing results, treatment history , date of disease progression, and date of death or last follow-up.TM  (11.1%), Tempus xT/xF  (3.2%), Guardant 360  (2.4%), CellNetix  (0.8%), and Biotheranostics  (0.8%).Genomic sequencing results included only genetic alterations that were adjudicated to be pathogenic by the reporting sequencing platform. No variants of unknown significance were included. The next-generation sequencing (NGS) platforms included FoundationOne  (80.1%), UW-OncoPlexOverall survival (OS) was defined as the duration from the start of first-line systemic therapy for advanced disease until the date of death from any cause. OS for patients last known to be alive was censored at the date of the last follow-up. Progression-free survival (PFS) was measured from the start of first-line systemic therapy for an advanced disease until the date of progression on first-line systemic therapy or death, whichever came first. PFS for patients last known to be alive and progression-free was censored at the date of the last follow-up.t-tests were used to test for associations between continuous variables and mutation status. The distribution of time-to-event outcomes (PFS and OS) was estimated using the method of Kaplan–Meier. Comparisons of distributions were done using a log-rank test. For multivariable modeling, potential confounders, to be included as variables in the model, were identified primarily based on whether they were statistically significant or near significantly (P < .15) associated with the variable of interest (KRAS G12R or PI3K pathway mutation status) in bivariate analysis and were thought to be clinically relevant to the outcome being modeled . If two variables showed significant collinearity, only one was used in the model. For example, given the co-occurrence of PI3K and SMAD4 mutations within the KRAS G12R mutated group, SMAD4 mutations were not considered as a separate covariate in the multivariable analysis model. For multivariable models of survival data, hazard ratios were calculated using the Cox proportional hazards model with P-values based on the likelihood ratio test. An a priori value of P < .05 was considered statistically significant. All statistical analyses were performed using JMP software .The associations between binary categorical variables and mutation status were evaluated using the two-tailed Fisher’s exact test. The associations between non-binary categorical variables and mutation status were evaluated using the likelihood-ratio chi-squared test. Two-sided ERBB2 amplification status, BRCA1/BRCA2/PALB2, KRAS G12R, and TP53 mutation status. Variables included in the multivariable model of OS and PFS for the KRAS-mutated cohort included KRAS G12R status (variable of interest), stage and ECOG performance status at the start of first-line systemic therapy for advanced disease, type of first-line chemotherapy, ERBB2 amplification status, homologous recombination DNA repair (HRR), and PI3K pathway mutation status. Variables included in the multivariable model of OS and PFS for the KRAS G12R mutated cohort included PI3K pathway mutation status (the variable of interest), stage at diagnosis and the receipt of both FOLFIRINOX and Gemcitabine/nab-Paclitaxel within the first two lines of therapy. Variables included in the multivariable model of OS and PFS for the non-G12R KRAS mutated cohort included the PI3K pathway (the variable of interest) and BRCA1/BRCA2/PALB2 mutation status.Variables included in the multivariable model of OS and PFS for the whole cohort included PI3K pathway mutation status (the variable of interest), sex, stage at diagnosis, We identified 126 patients who met the selection criteria. As of the data cut-off on July 1, 2020, 96 patients died, 25 patients were alive, and 5 patients were censored at the last follow-up. Among patients alive at the last follow-up, the median follow-up duration was 8.1 months (range 2.4-42.2). Next-generation sequencing was performed on tumor tissue for 121 patients (96%) and on circulating tumor deoxyribonucleic acid (ctDNA) for 5 patients (4%).n = 109, 86.5%), and received first-line treatment with FOLFIRINOX (51.6%) or with gemcitabine/nab-paclitaxel (31.7%). Demographic and clinicopathologic characteristics for patients with cancers with PI3K pathway mutations vs. wildtype in the entire cohort and in the KRAS G12R and non-G12R mutated subgroups are shown in Demographic and clinicopathologic characteristics of the patient population for the KRAS wild-type, G12R and non-G12R mutated subgroups are shown in KRAS genetic defects were noted in 111 (88.0%) PDA: KRAS G12D , G12V , G12R , G12C , Q61 , and KRAS amplification . Fifteen (11.9%) cancers harbored 16 PI3K pathway mutations: PIK3CA (n = 7), AKT2 and RICTOR (n = 3 each), PTEN, PIK3C2B, and PIK3R1 (n = 1 each). Core , and any HRR mutations  occurred in 13 (10.3%) and 22 (17.5%) cancers, respectively. The most common genomic alterations in addition to KRAS occurred in TP53 (70.6%), CDKN2A (38.1%), and SMAD4 (25.4%), consistent with data from The Cancer Genome Atlas.21BRAF fusions (n = 2), and one each with RET fusion plus BRCA2 loss of function mutation, IDH1 mutation, RAF1 fusion, and EML4-NTRK3 fusion.P = .027), and more HRR gene defects . KRAS G12R mutated cancers with co-occurring PI3K pathway mutations vs. those without PI3K pathway genetic alterations harbored significantly more SMAD4 gene defects   . No signP > .05 for all). There were numerically greater proportions of locally advanced vs. metastatic, ECOG PS 0 vs. 1, and first-line treatment with FOLFIRINOX vs. gemcitabine/nab-paclitaxel in the KRAS G12R compared to non-G12R mutated groups, but these differences did not reach statistical significance . However, as these associations had P < .15, these factors were incorporated into multivariable analyses of survival outcomes.There were no statistically significant differences in demographic and clinical characteristics between patients with KRAS G12R vs. non-G12R cancers (n = 23), patients with cancers harboring PI3K pathway alterations (n = 6) vs. none (n = 17) were more likely to have metastatic disease at diagnosis (P = .098), and to have received both FOLFIRINOX and gemcitabine/nab-paclitaxel during their disease course (P = .045) (Within the KRAS G12R mutated group ( = .045) .P = .91), and median PFS 8.6 vs. 9.0 months (P = .30) with first-line systemic therapies. Patients with KRAS G12R vs. non-G12R mutated cancers had significantly longer median OS  and PFS  (P = .17), but had similar PFS  with first-line systemic therapies (P = .62) or PFS  for KRAS non-G12R mutated cancers  , 2B. No  = .009) , 3D. Witherapies , 3E. Co- cancers , 3F.P = .60) or any HRR defects , although statistical significance was not observed  and PFS with first-line systemic therapies  in patients with cancers harboring KRAS G12R vs. non-G12R mutations (P = .44) or PFS (P = .49). Within the KRAS G12R mutated group, co-occurring PI3K mutations were associated with a trend towards shorter OS .In the adjusted analysis, multivariate Cox proportional hazards models confirmed significantly longer OS  progressed and died within 2 months.32 Nevertheless, selumetinib monotherapy had modest activity in refractory KRAS G12R mutated PDA, with median PFS and OS of 3 and 9 months, respectively.20 Several larger studies evaluating MEK inhibitors for unselected patients with advanced PDA showed no significant efficacy.33-36 Nevertheless, combination strategies with MEK or ERK inhibitors should be studied for pancreatic cancers with KRAS G12R mutations.KRAS G12R mutated PDA may be differentially targeted with MEK/ERK inhibitors, particularly in the absence of co-occurring PI3K pathway alterations. Preclinically, KRAS G12R mutated cells and organoids have increased sensitivity to MEK/ERK and PI3K inhibitors, with synergistic inhibition from combination MEK/ERK with PI3Kγ or mTOR inhibitors.37-40PI3K inhibitors were similarly ineffective.42 There is currently little clinical trial-level biomarker data to determine if there are specific PDA subsets that benefit from PI3K pathway inhibition. Preclinical data suggest that PDA harboring KRAS G12R or PI3K mutations may be more susceptible to PI3K inhibitors.43 Whether patients with KRAS G12R mutated cancers benefit from PI3K pathway inhibitors is yet unknown.Clinical trials with the mTOR inhibitor everolimus alone and in combination with chemotherapy or with molecularly targeted therapies, including EGFR and MEK inhibitors have mostly shown increased toxicity and limited activity.45 but clinical trials of unselected patients noted limited efficacy and poor tolerability.46-50 Whether KRAS G12R mutations with and without co-occurring PI3K mutations could confer specific therapeutic vulnerabilities to combined PI3K/MEK inhibition at lower, more tolerable doses is unknown and deserves further study.Dual PI3K/MEK pathway blockade in preclinical models has shown synergism in some studies,Limitations of our analysis include the retrospective nature of our data collection, the use of a relatively small patient population limited to a single institution, and the heterogeneity of the next-generation sequencing platform used to identify genomic alterations.KRAS G12R variant alleles. However, we report a greater incidence of co-occurring PI3K pathway alterations within this subgroup, which appears to offset some of their survival advantages. These exploratory results need to be further validated and support the development of clinical trials in pancreatic cancer to uniquely target KRAS mutation variants within context-specific signaling networks.Our analysis of patients with advanced PDA treated with contemporary multiagent chemotherapy supports improved prognosis associated with oyac179_suppl_Supplementary_Table_S1Click here for additional data file.oyac179_suppl_Supplementary_Table_S2Click here for additional data file.oyac179_suppl_Supplementary_Table_S3Click here for additional data file.oyac179_suppl_Supplementary_Table_S4Click here for additional data file.


Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f. C-repeat-binding factor 1 (AmCBF1) into the upland cotton variety R15. We found that Gossypium hirsutum Tubulin beta-1 (GhTUBB1) was downregulated in L28 and L30, which suggested that this gene may have contributed to the dwarf phenotype of L28 and L30. Here, we tested this hypothesis by silencing GhTUBB1 expression in R15 and found that decreased expression resulted in a dwarf phenotype. Interestingly, we found that repressing AmCBF1 expression in L28 and L30 partly recovered the expression of GhTUBB1. Thus, AmCBF1 expression presented a negative relationship with GhTUBB1 expression in L28 and L30. Moreover, yeast one-hybrid and dual-luciferase assays suggest that AmCBF1 negatively regulates GhTUBB1 expression by directly binding to C-repeat/dehydration-responsive (CRT/DRE) elements in the GhTUBB1 promoter, potentially explaining the dwarf phenotypes of L28 and L30. This study elucidates the regulation of GhTUBB1 expression by AmCBF1 and suggests that GhTUBB1 may be a new target gene for breeding dwarf and compact cultivars.The original ‘Green Revolution’ genes are associated with gibberellin deficiency. However, in some species, mutations in these genes cause pleiotropic phenotypes, preventing their application in dwarf breeding. The development of novel genotypes with reduced plant height will resolve this problem. In a previous study, we obtained two dwarf lines, L28 and L30, by introducing the  Vigorous vegetative growth during plant development may eventually lead to a serious yield reduction . BalanciREDUCED HEIGHT-B1b (Rht-B1b) and REDUCED HEIGHT-D1b (Rht-D1b) encode truncated DELLA proteins that cannot be degraded in response to GA signaling, resulting in improved yield traits [Rht24b, a dwarfing allele of Rht24 which encodes the GA metabolic enzyme gibberellin 2-oxidase (TaGA2ox-A9) [TaGA2ox-A9, which decreases endogenous bioactive GA content in the stems and reduces plant height without causing a yield penalty [Rht24b does not change the plant’s resistance to FHB [sd1 alleles produce inactive GA20 oxidase 2 (GA20ox-2), which disrupts the conversion of GA53 to GA20 in the stem and also generates a semi-dwarf phenotype [BRI1, which is involved in brassinosteroid (BR) signaling, also cause a dwarf phenotype [Arabidopsis), was found to phosphorylate and stabilize Rht-B1b. A gain-of-function allele of GSK3 was found to enhance the stability of Rht-B1b and reduce plant height in wheat [CBF overexpression also reduces plant height by inactivating GA [The ‘Green Revolution’ genes contribute to yield increases by affecting gibberellin (GA) biosynthesis or signaling pathways ,2. Mutatd traits  and a red traits ,9. Howevd traits ,8,9. RhtA2ox-A9) , has an  penalty . Moreovee to FHB . In ricehenotype ,14,15,16henotype ,18,19,20henotype ,22. Intein wheat . In addiating GA ,26. TherAS98, in which the upregulation of GhDREB1 confers a dwarf phenotype [pag1 mutant is due to overexpression of the BR catabolism gene PAGODA1 (PAG1) and the resulting deficiency in endogenous bioactive BRs [asd mutant, abscisic acid (ABA) biosynthesis or signaling may reduce plant height [The identification of target dwarfing genes is of particular interest in cotton, one of the most economically important textile crops. A dwarf stature can improve planting density and enable the replacement of manual with mechanical picking, both of which directly affect the yield and cost of cotton production. Traditionally, the dwarfing of cotton has been induced by chemical spraying, artificial topping and pruning ,28,29. Ahenotype ,31. The tive BRs . In the t height .Ammopiptanthus mongolicus Cheng f. transcription factor CBF1 (AmCBF1) into the upland cotton variety R15 and generated two dwarf lines, L28 and L30 [GhTUBB1 (Gh_D06G2276.1), was downregulated in L28 and L30, suggesting that it may be involved in height regulation in upland cotton. To explore the role of GhTUBB1 expression in plant height determination, we obtained plants with knockdown of GhTUBB1 using virus-induced gene silencing (VIGS) technology. In addition, to explore the evolutionary divergence of TUB proteins between several dicot species, we identified the members of the cotton TUB family and examined their phylogenetic relationships. Furthermore, we demonstrated that AmCBF1 can directly bind to the upstream promoter of GhTUBB1 to repress its transcription. This study provides new insight into the AmCBF1-mediated regulation of cotton plant height and suggests GhTUBB1 as a novel locus for breeding dwarf cotton varieties.Dwarf varieties of upland cotton are more suitable for mechanical picking , which w and L30 . The comArabidopsis thaliana (L.) Heynh., Vitis vinifera L. and Theobroma cacao. According to the BLASTP results, 35 Gossypium hirsutum L., 31 Gossypium barbadense L., 17 Gossypium raimondii L., 19 Gossypium arboreum L., 9 Arabidopsis thaliana, 11 Vitis vinifera and 11 Theobroma cacao proteins were identified as highly reliable hits with a threshold of E-value = 0 or percentage identity greater than 90%  contains more orthologous clusters than other groups (Gossypium (Using the full-length GhTUBB1 (Gh_D06G2276.1) protein as a query, we performed BLASTP searches to identify TUB family members in four species of cotton, than 90% . To detethan 90% . The TUBthan 90% . Ortholor groups . GhTUBB1ossypium .G. hirsutum TUBs. All of the GhTUB genes contained three exons and two introns, except Gh_A07G1077.1, Gh_D09G1534.1 and Gh_Sca005103G03.1  exhibits several interesting characteristics, such as a lack of synteny with the G. raimondii genome and a large first intron, which indicate that the expression of GhTUBB1 may be regulated by the intron and that it may play an important role in growth and development specifically in G. hirsutum. A previous analysis revealed that GhTUBB1 expression was lower in L28 and L30 than in R15  assay. The relative locations of three CRT/DRE cis-elements in the GhTUBB1 promoter and the structures of relevant vectors are shown in pGADT7-AD and pAbAi-GhTUBB1 pro were inviable on SD/− Leu/−Ura medium supplemented with AbA (500 ng/mL), whereas those expressing pGADT7-AmCBF1 and pAbAi-GhTUBB1 survived well under the same conditions  activity (approximately 30% lower) compared with the control  is approximately equal to the sum of GrTUBs (17 genes) and GaTUBs (19 genes). However, there are fewer than 31 GbTUBs (31 genes)  , indicat species .ZmRPH1 in maize reduces plant height by repressing the internode cell elongation without significantly reducing yield [In the past decades, studies of the regulation of microtubule assembly and depolymerization have mainly focused on microtubule-associated proteins (MAPs), which can regulate microtubule dynamics by directly binding to microtubule lattices . Previoung yield , suggestGhTUBB1 in the R15 variety reduced plant height. GhTUBB1 is directly downstream of AmCBF1 and its expression is positively associated with plant height in L28 and L30. This indicates that GhTUBB1 is a new practical potential target for breeding dwarf and compact cultivars.Several MAPs and microtubule proteins have been reported to be involved in cotton fiber development, such as GhMAP20L5, GhTUB1 and GhTUA9 ,44,45. HGhTUBB1 by a CBF-like transcription factor, which results in dwarfing in upland cotton  superfamily of transcription factor with a conserved AP2 domain. CBFs bind to DRE/CRT elements to regulate plant growth and development . This std cotton . In a pr and L30 . PP2C fat growth . The GhTelements , suggestA. thaliana, V. vinifera and T. cacao. The hits with an E-value = 0 or a percentage identity greater than 90% were kept. The Arabidopsis genome was downloaded from The Arabidopsis Information Resource . The V. vinifera genome (Genome assembly: PN40024.v4) was downloaded from Ensemble plant  [T. cacao genome (Cocoa Criollo B97-61/B2 version 2) was downloaded from Cocoa Genome Hub  [G. hirsutum  [G. barbadense  [G. arboreum  [G. raimondii  [https://cottonfgd.net, accessed on 28 May 2023) [The full-length GhTUBB1 (Gh_D06G2276.1) protein was used as a query in BLASTP searches of the databases of four cotton species, ay 2023) . The T. ay 2023) . The genssembly) , G. barbssembly) , G. arbossembly) , and G. ssembly)  were doway 2023) . A. thaliana, V. vinifera and T. cacao were used for phylogenetic analysis using MEGA 11 [p-distance was 0.07; identity = 0.93), which indicated the small evolutionary distances of TUBs and were appropriate for using the Neighbor-Joining method. Therefore, a phylogenetic tree was constructed using the Neighbor-Joining method with 2000 bootstrap replicates [https://itol.embl.de/, accessed on 7 June 2023) [The amino acid sequences of the TUBs were aligned using Clustal W in MEGA 11 . The ful MEGA 11 . The TUBplicates . The phyne 2023) .GhTUB genes were extracted from the genome GFF annotation file and depicted using TBtools [https://www.ncbi.nlm.nih.gov/, accessed on 31 May 2023). Finally, the gene structure, conserved domains and phylogenetic tree of GhTUBs were visualized using Gene Structure View (Advanced) in TBtools [GhTUBs were submitted to the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify the potential cis-elements [cis-elements in the GhTUB promoters was visualized using TBtools [The exon-intron structures of  TBtools . The con TBtools . The upselements . The dis TBtools .TUB genes were determined using TBtools [https://cottonfgd.net, accessed on 28 May 2023) [TUB genes were analyzed using One-Step MCScanX-SuperFast software in TBtools [https://cottonfgd.net, accessed on 28 May 2023) [The chromosomal locations of the cotton  TBtools  accordinay 2023) . The syn TBtools  accordinay 2023) .AmCBF1 transgenic lines of L28 and L30 were generated in our previous study by introducing the full-length coding sequence of AmCBF1 of Ammopiptanthus mongolicus into the cotton variety R15 [AmCBF1 transgenic lines of L28 and L30, and tobacco (Nicotiana benthamiana) were grown in a greenhouse under the following conditions: 25 ± 3°C, 50% relative humidity, and a 16 h light/8 h dark cycle.The iety R15 . The pla−ΔΔCt method. For all qPCR assays, the cotton UBQ gene (Gh_D13G1489) was used as an internal control. The primers used for qPCR are listed in Total RNA was extracted from cotton leaves and reverse transcribed into first-strand cDNA for quantitative PCR (qPCR) using HiScript III RT SuperMix . The qPCR reaction mixture was run on an ABI Prism 7500 Fast Real-Time PCR System  in a 20 μL reaction containing 10 μL ChamQ Universal SYBR qPCR Master Mix , 0.4 μL 10 μM primers, 200 ng cDNA and nuclease-free water. The PCR program was as follows: 95 °C (30 s), followed by 40 cycles at 95 °C (10 s), 60 °C (30 s) and 72 °C (15 s). The relative expression levels were calculated using the 2GhTUBB1 and AmCBF1 genes by following a previously reported protocol. In brief, the specific gene fragment was inserted into the pTRV2 vector via the Xba I and Kpn I restriction sites. Then, the constructed plasmids were transferred into the Agrobacterium strain GV3101. The positive-transformed strains were incubated overnight in liquid Luria-Burtani (LB) medium at 28 °C and 200 rpm. Cells were resuspended to a density of OD600 = 1.2 with permeate solution  and incubated for 3 h at room temperature in a dark chamber. Then, the suspension containing the pTRV1 plasmid was mixed with the suspension containing one of the above pTRV2 plasmids at a volume ratio of 1:1. The cell suspensions containing pTRV2 and pTRV2-CLA1 plasmids mixed with the pTRV1 suspension were regarded as the negative and positive controls, respectively. The mixed suspensions were injected into cotton seedlings on the abaxial surface of the expanded cotyledons. The infected plants were cultured for 24 h in a dark chamber, and then grown for 4 weeks in a 23 °C chamber with a photoperiod of 16 h light/8 h dark and 50% relative humidity. The primers used for VIGS vector construction and qRT-PCR detection are listed in VIGS has been widely used to silence target genes in various plant species . We sileGhTUBB1 gene. A Y1H analysis was conducted to verify the binding of AmCBF1 to the GhTUBB1 promoter. The full-length AmCBF1 coding sequence was inserted into the pGADT7 vector via the EcoR I and BamH I restriction sites to construct the prey plasmid pGADT7-AmCBF1-AD. The GhTUBB1 promoter containing all CRT/DRE elements was inserted into the pAbAi vector via the Kpn I and Sal I restriction sites to generate the bait plasmid pAbAi-GhTUBB1 pro. Then, the bait plasmid was linearized by Bstb I digestion and introduced into the Y1H Gold yeast strain to generate a decoy reporter yeast strain. An aureobasidin A (AbA) inhibition assay was used to select the positive strains. The prey plasmid was then transformed into the bait reporter strains to test DNA–protein interactions. The yeast cells containing the prey and bait plasmids were cultured on SD/−Leu/−Ura medium for 3 days at 30 °C. Subsequently, these yeast cultures were diluted  and cultured on SD/−Leu/−Ura media supplemented with different concentrations of AbA. The pGADT7 vector was used as the negative control. The primers used in this experiment are listed in Three CRT/DRE elements were identified in the 3 kb promoter region upstream of the pCambia1302-35S::AmCBF was constructed by inserting the full-length coding sequence of AmCBF1 into the pCambia1302 vector via the Nco I restricted site. The GhTUBB1 promoter containing three CRT/ DRE elements was inserted into the pGreenII 0800-LUC vector to generate the reporter plasmid pGreenII 0800-GhTUBB1 pro::LUC. Then, the recombinant plasmids and the negative control plasmid were introduced into Agrobacterium strain EHA105. The positive strains were cultured to OD600 = 1.0 at 28 °C and 200 rpm. The strains were resuspended to OD600 = 1.2 with permeate solution . The suspensions were incubated for 3 h at room temperature in the dark. Then, the suspensions containing effector and reporter plasmids were mixed and injected into tobacco leaves. After 48–60 h of growth in a chamber at 23 °C with a photoperiod of 16 h light/8 h dark and 50% relative humidity, the leaves were sprayed with 10 μM D-Luciferin potassium salt and then imaged using a LB985 Night SHADE fluorescence imaging system . The dual LUC activity was measured using a GloMax 20/20 luminometer , and relative activity was calculated according to the Dual-Luciferase Reporter Assay System instructions (Promega). The primers used in this experiment are listed in The effector plasmid GhTUBB1 was identified as being differentially expressed in L28 and L30 vs. R15 according to previous RNA-sequencing data. The silencing of GhTUBB1 expression caused a dwarf phenotype in cotton plants. GhTUBB1 expression was negatively associated with AmCBF1 expression in L28 and L30. Moreover, AmCBF1 directly bound to CRT/DRE elements and negatively regulated the expression of GhTUBB1, which may explain the dwarf morphology of L28 and L30 (GhTUBB1 expression by AmCBF1 and suggested GhTUBB1 as a new potential target gene for breeding dwarf and compact cultivars. and L30 F. This s


Phosphoinositide 3-kinases (PI3Ks) are lipid enzymes that regulate a wide range of intracellular functions. In contrast to Class I and Class III PI3K, which have more detailed descriptions, Class II PI3K has only recently become the focus of functional research. PIK3C2A is a classical member of the PI3Ks class II. However, the role of PIK3C2A in cancer prognosis and progression remains unknown.in vitro experiments. Furthermore, the correlation of PIK3C2A expression with tumor immunity was investigated in KIRC. The TCGA database was employed to investigate PIK3C2A functional networks.The expression pattern and prognostic significance of PIK3C2A in human malignancies were investigated using multiple datasets and scRNA-seq data. The PIK3C2A expression in renal clear cell carcinoma (KIRC) was then validated utilizing Western blot. The functional role of PIK3C2A in KIRC was assessed using combined function loss experiments with Significant decrease in PIK3C2A expression in KIRC, demonstrated that it potentially influences the prognosis of diverse tumors, particularly KIRC. In addition, PIK3C2A was significantly correlated with the T stage, M stage, pathologic stage, and histologic grade of KIRC. Nomogram models were constructed and used to predict patient survival based on the results of multivariate Cox regression analysis. PIK3C2A knockdown resulted in significantly increased KIRC cell proliferation. Of note, PIK3C2A expression demonstrated a significant correlation with the infiltrating levels of primary immune cells in KIRC.These findings support the hypothesis that PIK3C2A is a novel biomarker for tumor progression and indicates dynamic shifts in immune infiltration in KIRC. Furthermore, aberrant PIK3C2A expression can influence the biological activity of cancer cells. Renal cell carcinoma (RCC), which arises from the renal epithelium, accounts for approximately 90% of kidney tumors and 3% of all cancers , and theThe immune system protects the body’s homeostasis against exogenous and endogenous, infectious and non-infectious threats, as well as eliminating defective cells before they develop into malignant cells. A subset of malignant cells may evade immune cell surveillance, resulting in tumorigenesis and metastatic spread. Interaction of infiltrating immune cells, one of the major components of the tumor microenvironment, with cancer cells has been shown to influence tumor onset and progression , 6. CompPhosphoinositide 3-kinases (PI3Ks) are a type of enzyme that catalyzes the formation of second messengers for a variety of signaling pathways . This fain vitro. Last but not least, the underlying mechanism of PIK3C2A was evaluated using the TCGA database. The findings of this research shed light on the effects of PIK3C2A on KIRC prognosis.The prognostic landscape of PIK3C2A was investigated using multiple databases. The correlation of PIK3C2A expression with infiltrating immune cells in various types of cancer was analyzed using the TIMER, TCGA, and GEPIA databases. Utilizing loss-of-function assays, the effect of PIK3C2A on the proliferation of KIRC cells was evaluated https://portal.gdc.cancer.gov/) and analyzed using R v4.0.3. Screening distinct mRNAs was performed with the R package ‘DESeq2’  data portal website  with the Seurat package (version 3.1.1). Filtering was performed on sample KIRC cells with < 200 and > 2500 genes and a mitochondrial gene percentage of >10%. The top 2000 most variable genes in the scRNA-seq data were used for principal components analysis (PCA), and the first 20 principal components were used for UMAP analysis. The FindClusters function divided all cells into 14 clusters, with resolution = 0.5.via multivariate Cox regression to determine its independence as a predictor of patient survival. Nomogram-based models for predicting patient survival were developed using the R package ‘rms’. The area under the time-dependent ROC curve (AUC) was generated using the ‘timeROC’ (version: 0.4) package to examine the sensitivity and specificity of PIK3C2A in survival prediction.Univariate Cox regression analysis was used to assess the relationship between PIK3C2A and the prognosis of malignancies. Based on the median expression value of PIK3C3A, KIRC survival curves were generated using the ‘survival’ R package. PIK3C2A was evaluated The human KIRC cell lines 769P, CAKI-1, 786-O, and ACHN cells were obtained from the cancer institute of Zhejiang Caner Hospital. KIRC cell lines were grown in RPMI-1640 medium  containing 10% fetal bovine serum (FBS), 100 units of penicillin, and 50 units of streptomycin per ml.PIK3C2A siRNA was purchased from Zorinbio Company (China); the PIK3C2A siRNA1 sequence was 5’-CCACUUAUGCUUUACCUUCUATT-3’, the PIK3C2A siRNA2 sequence was 5’-GCUAGUGUGAAGGUCUCCAUUTT-3’, and the PIK3C2A siRNA3 sequence was 5’-GAUGACAGUUUCGAGACUAAATT-3’. Lipofectamine 2000 was used to transfect KIRC cells with siRNA. Control cells were transfected with non-targeting siRNA.Real-time PCR assayThe TRIzol Reagent  was used to isolate total RNA, and the PrimeScript RT Reagent Kit was used to reverse-transcribe this RNA into cDNA, per the manufacturer’s instructions . The mRNA expressions of PIK3C2A and GAPDH were measured using an RT-PCR quantitative PCR kit .Cells were lysed in ice-cold RIPA buffer containing protease and phosphatase inhibitors. Proteins from each group were separated using SDS-PAGE and transferred in equal amounts to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk for one hour and then treated overnight at 4°C with PIK3C2A Mouse mAb . The membranes were then washed thrice in Tris-buffered saline with Tween-20 (TBST) and incubated at room temperature for 1 hour with a secondary antibody conjugated to HRP. Blots were examined with an ECL detection kit  and processed with Quantity One 1-D Analysis Software .Colony formation tests were performed as described previously . Briefly4 cells/well) were seeded in the glass bottom dish and cultured overnight. Afterward, the cells were incubated with 10 μM EdU buffer at 37°C for 2 hours, fixed with 4% formaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 25 minutes, and nuclear-stained with Hoechst. The cells were then examined using a fluorescence microscope. The ratio of EdU-positive cells (red cells) to all Hoechst-positive cells (blue cells) was defined as the EdU incorporation rate.For cell proliferation assessment, a 5-ethynyl-20-deoxyuridine (EdU) test kit was utilized . KIRC cells  has confirmed that this protocol quantifies a terrific amount of specific cell types . ThereupThe association of PIK3C2A with protein-coding genes (PCGs) was evaluated using Spearman correlation coefficients, and genes with a Spearman correlation coefficient greater than 0.50 were identified as PIK3C2A-associated PCGs. GO and KEGG pathway enrichment studies on the PIK3C2A-associated PCGs were then performed using the clusterProfiler program . P-valuep-value 0.05), based on the expression patterns between the high-expression and low-expression groups. This analysis was conducted with the clusterProfile application , breast invasive carcinoma (BRCA), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), thyroid carcinoma (THCA), and (UCEC)  and 3575 cells from the left KIRC tissue (KIRC2). The data were subjected to UMAP analysis following quality control. Cells in KIRC were classified into 14 distinct categories, including CD8+ cell, T helper cell, Treg cell, NKT cell, effector CD8+ memory T cell, tumor cell, macrophage, vascular endothelial cell, endothelial cell, neutrophil, mesangial cell, B cell, mast cell and collecting duct principal cell, according to data on previously reported markers , M stage , pathologic stage , and histologic grade   Table 1.The independence of PIK3C2A from other clinicopathological characteristics of KIRC was determined using univariate and multivariate Cox regression analysis. Results from the univariate Cox regression analysis of KIRC patients revealed that PIK3C2A expression, T stage, N stage, M stage, histologic grade, and pathologic stage were all significant predictors of OS, DFS, and PFS Table 2.P<0.001; in vitro.Having found that PIK3C2A expression decreased in KIRC tissues, we analyzed the influence of PIK3C2A on the cellular biology of KIRC cells. Firstly, we assessed the relationship between PIK3C2A and tumor-related signal pathways using ssGSEA. According to the results, PIK3C2A expression was negatively correlated with tumor proliferation signature and DNA replication pathways  < 0.05. The results revealed that 1039 DEGs were up-regulated in the high-expression group, while 2815 PCGs were down-regulated in the low-expression group  >1 and an adjusted P = 8.92E–05), CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and dendritic cells  in KIRC , B cell plasma , T cell CD4+ memory resting , NK cell resting , Monocyte , Macrophage M1 , Macrophage M2 , Myeloid dendritic cell resting , Mast cell activated  and Neutrophil  in the PIK3C2A high-expression group than in the PIK3C2A low-expression group , T cell CD8+ , T cell follicular helper , T cell regulatory , T cell gamma delta , NK cell activated , Macrophage M0  and Mast cell resting  was lower in the PIK3C2A high-expression group than in the PIK3C2A low-expression group , T regulatory cells (Tregs), T helper cells (Th cells), and T cell exhaustion in KIRC or normal tissues. The results demonstrated that the markers of B cells, including CD19 and CD79A ; the top 50 positively correlated and negatively correlated PCGs are shown in p-value<0.001; p-value<0.001; The correlation of PIK3C2A with PCGs was evaluated using Spearman correlation analysis to determine the precise mechanism of PIK3C2A in KIRC carcinogenesis. The results revealed that 2345 PCGs and 466 PCGs were positively and negatively correlated with PIK3C2A, respectively  in KIRC.In addition, an inverse correlation was found between the level of PIK3C2A and the gene markers of T-cell exhaustion. Cancer cells rely heavily on exhausted T cells evade the immune system because these cells have low cytokine levels and effector function . In addiFurther analysis revealed strong relationships between PIK3C2A levels and Treg genetic markers. Foxp3 is the most prominent transcription factor in Treg, and its expression is inversely related to that of PIK3C2A. Although it plays an important in escape mechanisms against autoimmunity, FOXP3+ Treg dampens tumor immunity . MoreoveMoreover, according to the function-rich study, the genes co-expressed with PIK3C2A were primarily associated with ubiquitin, which is the essential mechanism through which its regulates the translation of innate immune response signals . MoreoveIn conclusion, PIK3C2A may be an independent prognostic marker and it regulates the infiltration of immune cells in many types of malignancies. Specifically, in KIRC, PIK3C2A overexpression is associated with a better prognosis and increased immunocyte infiltration. Moreover, KIRC’s proliferative capacities are altered by aberrant PIK3C2A expression. Further research is needed to develop anti-tumor agents based on these results.The original contributions presented in the study are included in the article/DM and YY contributed to the conception and design of the study. CQ, SL, and QW drafted the manuscript. CQ, SL, SZ, XX, JH, XY, and ZW collected and analyzed the data. DM and YY revised the manuscript. All authors contributed to the manuscript revision and read and approved the submitted version.


Brassica allotetraploid species Brassica juncea (2n = AABB), Brassica carinata (2n = BBCC), and Brassica napus (2n = AACC) to generate AABC, BBAC, and CCAB interspecific hybrids and investigated chromosome inheritance and fertility in these hybrids and their self-pollinated progeny. Surprisingly, despite the presence of a complete diploid genome in all hybrids, hybrid fertility was very low. AABC and BBAC first generation (F1) hybrids both averaged ~16% pollen viability compared to 3.5% in CCAB hybrids: most CCAB hybrid flowers were male-sterile. AABC and CCAB F1 hybrid plants averaged 5.5 and 0.5 seeds per plant, respectively, and BBAC F1 hybrids ~56 seeds/plant. In the second generation (S1), all confirmed self-pollinated progeny resulting from CCAB hybrids were sterile, producing no self-pollinated seeds. Three AABC S1 hybrids putatively resulting from unreduced gametes produced 3, 14, and 182 seeds each, while other AABC S1 hybrids averaged 1.5 seeds/plant (0–8). BBAC S1 hybrids averaged 44 seeds/plant (range 0–403). We also observed strong bias towards retention rather than loss of the haploid genomes, suggesting that the subgenomes in the Brassica allotetraploids are already highly interdependent, such that loss of one subgenome is detrimental to fertility and viability. Our results suggest that relationships between subgenomes determine hybridization outcomes in these species.Interspecific hybridization is widespread in nature and can result in the formation of new hybrid species as well as the transfer of traits between species. However, the fate of newly formed hybrid lineages is relatively understudied. We undertook pairwise crossing between multiple genotypes of three The online version contains supplementary material available at 10.1007/s10577-023-09733-9. Brassica genus is the most prominent in the Brassicaceae family and includes 39 species. Many of the species in this genus are cultivated for their edible roots, stems, leaves, buds, flowers, and seeds (oil and mustard) (Rakow Brassica rapa (B. rapa) , Brassica nigra (B. nigra) , and Brassica oleracea (B. oleracea)  were determined to be the progenitors of the allopolyploid species Brassica juncea (B. juncea) , Brassica napus (B. napus) , and Brassica carinata (B. carinata)  (U N The d) Rakow . The six 17) U N .Brassica A, B, and C genomes has been well elucidated (Attia and Röbbelen B. nigra (B) lineage predicted to have diverged from the B. rapa and B. oleracea (A/C) lineage approximately 7.9 million years ago (Mya) followed by the separation of the B. rapa (A) and B. oleracea (C) lineages about 3.7 Mya  hybrid plants and genotypes used in this study is presented in Mason et al. (1 and second generation (S1) hybrid types (derived from self-pollination of the F1 hybrids), along with information on the later generations of this cross, have already been described in detail  from Canola Breeders Western Australia to obtain 133 AABC F1 hybrids. A total of 93 AABC S1 plants were produced by self-pollination of AABC F1 plants. One hundred and twenty four CCAB F1 plants from ten different cross combinations were obtained by crossing two genotypes of B. carinata (195923.3.2_01DH and 94024.2_02DH) hereafter referred to as “C1” and “C2”, with twelve genotypes of B. napus . The parental B. juncea genotype J1 was crossed with two different B. carinata genotypes “C1” and “C2”, respectively, to generate two separate BBAC F1 hybrid genotypes: “J1C1” and “J1C2.” A total of sixty-two BBAC F1 plants were produced. Two hundred and twenty-seven BBAC S1 seeds produced from self-pollination of the J1C1 and J1C2 BBAC F1 genotypes were sown directly into the field at Huazhong Agricultural University, Wuhan, China. An additional 44 seeds were grown under glasshouse conditions at The University of Queensland, while seeds from all other BBAC F1 hybrids were germinated in potting mix and grown in pots in a controlled environment room (CER) at 18 °C/13 °C day/night with a 16 h photoperiod and light intensity of approximately 500 μmol m-2s-1. The three most fertile plants from each BBAC F1 plant, as measured by total self-pollinated seed produced, were selected to produce the next generation .Total seed set was counted for all plants after encouraging self-pollination using micro-perforated plastic sleeves or paper bags to enclose racemes. Newly opened flowers were collected when plants started flowering and pollen stained with either fluorescein diacetate using the method detailed by Heslop-Harrison et al.  for theb-g. Plan1), plants were scored on one or multiple of the following: (1) plant morphology, (2) pollen morphology, (3) microsatellite marker inheritance of single alleles from both parents , only genome-wide SNP genotyping to determine if no unexpected alleles were present relative to the alleles predicted from the two parents was considered sufficient to distinguish between truly self-pollinated progeny and progeny resulting from foreign pollen contamination on to the maternal F1 plants.As all parent species and genotypes in the cross combinations were self-compatible, several measures were taken to establish true hybrid status for progeny resulting from interspecific hybridization .Leaf samples were collected in 2-ml micro-centrifuge tubes and stored at −20 °C until use. DNA was extracted using the “Microprep” method described in Fulton et al. , except bzh v8 reference sequence  of called SNPs or which had a “call” in > 10% of individuals within a haplotype block (r2 = 1) of “no-call” SNPs were removed from all hybrid types, in addition to SNPs showing patterns of segregation inconsistent with determined genomic locations. For the AABC S1 hybrids, the A genome was filtered to retain only SNPs which were polymorphic between the parent B. napus and B. juncea genotypes in the A genome, while in the CCAB S1 hybrids, the C genome was filtered to retain only SNPs which were polymorphic between the parent B. napus and B. carinata genotypes for each hybrid combination. No allelic segregation was expected for the B and C genomes in AABC hybrids, the A and C genomes in BBAC hybrids, or for the A and B genomes in CCAB hybrids, and hence SNPs which were heterozygous within these genomes (indicative of multi-locus amplification/aspecific probe binding) were filtered out with respect to parental genotype controls. As well, SNPs which mapped to the A genome but which amplified in B. carinata (2n = BBCC) and SNPs which mapped to the C genome but which amplified in B. juncea (2n = AABB) were filtered out. S1 generation individuals for each of the AABC, BBAC, and CCAB hybrids were determined to be the product of unintentional cross-pollination when these individuals showed presence of alleles  that were not present in either of the two parent genotypes.A total of 52 149 SNPs were exported for the A and C genomes  and the presence of non-homologous recombination events. Centromere locations used were initially mapped using the Darmor v.4.1 n et al.  for detaRoot tips were collected in 0.04% 8-hydroxyquinoline solution and incubated for 2 h at room temperature, followed by another 2 h at 4 °C. Root tips were then transferred to Carnoy’s I solution (3:1 parts ethanol: acetic acid) and incubated for 24 h before being transferred to 70% ethanol for storage at −20 °C. The procedure for mitosis slide preparation from root tips was as reported by Mason et al. , using t1 and S1 generation of AABC, CCAB, and BBAC hybrids. Boxplots and stripcharts were also produced in the base package of R v.4.0.2. Pearson’s χ2 test statistic values and barcharts were produced using the Microsoft Office Excel (2019), and chromosome images were combined and annotated using the Microsoft Office Powerpoint (2019).Genotypic effect of the trigenomic hybrids on total number of self-pollinated seeds and pollen viability was tested for using the one-way ANOVA in the base R version 4.0.2 , while two plants were derived from maternal self-pollination  parent and hence to have a genome complement of 2n = AAABCC; these showed morphology similar to the maternal parent  and was found to derive from an aneuploid (< n) B. carinata gamete  resulted from unintentional cross-pollination. The status of the 15 plants which were not SNP-genotyped could not be determined. A total of 191 BBAC S1 plants were produced. From these, SNP genotyping information was only available for 38 plants. All 38 plants were true S1 self-pollinated progeny of their F1 hybrid parent based on SNP information  × B. napus (Boomer) had the highest pollen viability while B. juncea (JN9-04) × B. napus (Monty_28DH) had the lowest. Bagged seed set was obtained for 91 out of 121 AABC F1 plants and ranged from 0 to 176 seeds per plant .Fertility was assessed using seed set and estimated pollen viability as fertility measures in all true AABC, CCAB, and BBAC F1 hybrid plants were produced. Pollen viability data was obtained for 73 true F1 plants and ranged from 0 to 29%, with an average of 3.5% .For the CCAB hybrids, a total of 116 F1 hybrid plants were produced. Pollen viability data was obtained for 31 of these plants, which ranged from 0 to 59%, with an average of 16% . There was no significant difference between the two hybrid combinations J1C1 and J1C2 in terms of pollen viability . Seed data in BBAC hybrids was obtained for 50 hybrid plants and ranged from 0 to 333 seeds per plant, with an average of 56 seeds per plant .For the BBAC hybrid population, a total of 62 true F1 hybrid types AABC, BBAC, and CCAB was compared for bagged seed set and pollen viability. BBAC hybrids produced the highest number of seeds per plant, followed by AABC hybrids and CCAB hybrids. Interestingly, similar proportions of AABC and BBAC F1 hybrids failed to produce any bagged seed, but fertile (at least one seed produced) BBAC F1 hybrids produced factorially more seeds per plant on average than fertile AABC F1 hybrids. CCAB F1 hybrids were more likely to be pollen-sterile, set no bagged seed, and to produce fewer seeds when fertile when compared to AABC and BBAC F1 hybrid types. Hybrid type was significantly associated with seed set and pollen viability . BBAC F1 hybrids were significantly different in the number of seed set compared to AABC and CCAB (Tukey’s HSD p < 0.05). BBAC hybrids were also significantly different in percentage pollen viability compared to CCAB but not AABC hybrids (Tukey’s HSD p < 0.05).The fertility of interspecific F1 plants were bagged to encourage self-pollination to produce S1 plants. The fertility of true AABC, CCAB, and BBAC S1 hybrid plants was assessed by pollen viability and bagged seed production , pollen viability ranged from 0 to 93% with an average of 47%  produced bagged seeds, indicating that these hybrids were completely sterile . A positive correlation was also observed between bagged seeds produced and pollen viability (r = +0.48).A total of 38 true BBAC S1 hybrid types was assessed using self-pollinated seed set and pollen viability in order to determine whether fertility is affected by AABC, CCAB, and BBAC S1 hybrid combinations or genotypes. Bagged seed set and pollen viability between AABC, BBAC, and CCAB S1 hybrids were not significantly different (ANOVA p > 0.05) .The fertility of the three different S05) Fig. , Fig. 4.1 generation . No chromosome information was obtained for true AABC S1 hybrids.The chromosome numbers of 29 hybrid plants were counted in the S1 hybrid plants had SNP genotyping information: five plants had A, B, and C genome data, and five plants had A and C genome data. For inheritance of the haploid B and C genome chromosomes across all ten individuals, we expected to see a 1:2:1 segregation ratio of 0 copies:1 copy:2 copies of each B- or C-genome chromosome (as a result of self-pollination of a parent AABC F1 hybrid with one copy of each B- and C-genome chromosome). However, 0 C-genome chromosome copies were observed only 13% of the time, with statistically significant bias towards retention of C-genome chromosomes relative to the expected distribution . Three individuals deviated significantly from expected chromosome segregation ratios , all showing higher heterozygosity than expected: 86%, 89%, and 87% heterozygosity in individuals A3-001, A-01, and A-02. Surprisingly, three individuals from the combination J1N2 with expected 50% heterozygosity (Pearson’s χ2 test p > 0.05) also demonstrated some bias towards retention of N2 alleles over J1 alleles , although this only reached significance (p = 0.02) for individual A-69.Of the ten AABC S1 individuals were genotyped using the AC array: 20 from the genotype combination J1C1 and 18 from the genotype combination J1C2. Partial chromosome inheritance (a product of non-homologous recombination) was extremely common for the A and C genomes: on average, 40% of chromosomes showed this pattern (ranging from 5% of chromosomes for chromosome A08 to 68% of chromosomes for chromosome C9). Presence of a whole A- or C-genome chromosome was observed 54% of the time on average (ranging from 29% for chromosome C9 to 92% for chromosome A08), while complete absence of an A- or C-genome chromosome was observed only 6% of the time on average .A total of 38 BBAC Sχ2 test, p <0.0001 and p = 0.003, respectively). Based on an expected 75% chance of each chromosome being present (following self-pollination of a parent F1 hybrid with one copy of each chromosome), chromosomes A04, A05, A06, and A08 were present more often than expected by chance , and chromosomes C4 and C5 were lost more often than expected by chance . Segregation of A- and C-chromosomes combined approximated the expected 25% inheritance of univalent chromosomes .Inheritance of centromeric regions was used to assess presence of A- and C-genome chromosomes independent of non-homologous recombination events  Fig. . On aver1 individuals were identified, and all were genotyped with the AC array. C1-019 showed no partial or complete loss of chromosomes, but at least one such event was observed for each other CCAB S1 individual. Complete loss of chromosome A06 was observed in two individuals; no other chromosomes were lost completely although chromosomes A01 and A04 had undergone recombination events where only a small telomeric chromosome fraction was retained . Large fractions of A-genome chromosomes were retained for events involving chromosomes A02 , A05, and A07.Only five true hybrid CCAB SB. napus and B. carinata parent genotypes across all genomic loci . One individual (C1-013) showed an excess of heterozygous loci , while another individual (C1-019) showed an excess of homozygous loci . Individual C1-017 showed significant bias towards inheritance of B. carinata alleles over B. napus alleles , while three other individuals  showed the opposite trend, inheriting 18% vs. 8%, 40% vs. 25%, and 27% vs. 14% B. napus alleles relative to B. carinata alleles at homozygous loci .Segregation of polymorphic loci in the C genome was surprisingly irregular: only one individual (C1-015) showed the expected 50% heterozygosity and approximately equal inheritance of alleles from the B. juncea × B. napus (AABC), B. juncea × B. carinata (BBAC), and B. napus × B. carinata (CCAB) F1 interspecific hybrids and S1 generation plants resulting from self-pollination of the F1 hybrids. Although pollen viability in AABC and BBAC F1 hybrids was similar and higher than that of CCAB F1 hybrids , only BBAC F1 hybrids produced substantial numbers of true S1 seeds. CCAB hybrids were all completely sterile by the S1 generation, while AABC S1hybrids were rescued from complete sterility by putative selection for unreduced gametes which transmitted complete sets of chromosomes from the F1 parent: three such individuals were identified and all produced at least a few seeds. Chromosome inheritance in BBAC S1 hybrids was biased towards retention of A-genome chromosomes and loss of C-genome chromosomes, and 40% of A- and C-genome chromosomes on average in these hybrids showed evidence of non-homologous recombination. Significant bias in allelic inheritance in the diploid A- and C-genomes of AABC S1 and CCAB S1 hybrids was also observed for several individuals, indicative of unreduced gamete involvement or other abnormal meiotic processes in both these hybrid types. As our experimental design mostly controlled for genotype-specific effects, our results suggest that fertility and viability of these hybrid lineages depend on genome structure, specifically on which genomes are present as haploid vs. diploid chromosome complements in the initial F1 hybrids.In this study, we analyzed fertility and chromosome inheritance in Brassica hybrid types. The poor fertility of hybrids can serve as a barrier preventing their maintenance as independent populations, thus hindering their long term speciation potentials  hybrid lineage completely failed to establish, with universally low fertility despite the generation of many F1 hybrids from different genotype combinations. Different molecular mechanisms causing hybrid infertility exist, including genetic incompatibilities (nuclear and cytonuclear) and changes in genome architecture (ploidy number and chromosome rearrangements) . Schelfhout et al.  were reported by Kumar et al.  also restored seed fertility. These results from our own and other studies suggest that a complete A or C genome resulting from the Brassica allotetraploid species B. juncea, B. napus, or B. carinata may no longer be sufficient for normal fertility in hybrids between these species. In support of this hypothesis, Pelé et al.   and one CCAB S1 hybrid (C1-013) in our study showed (1) inheritance of all or almost all chromosomes from the haploid subgenome/s and (2) significantly elevated heterozygosity in the diploid (A or C) genome, consistent with involvement of unreduced gametes produced via a first division restitution-like mechanism. Unreduced gametes are known to be involved more frequently than expected by chance in crosses between species and to be produced at higher levels in interspecific hybrids  and the other five AABC S1 hybrids were sterile. The CCAB S1 hybrid C1-013 did not produce any seed but also showed loss of most of chromosome A01, putatively due to a non-homologous recombination event. Another CCAB S1 hybrid (C1-019) may have been the result of an unreduced gamete produced via second division restitution (whereby both sister chromatids are inherited) or some other abnormal meiotic process: this individual inherited a copy of each A-genome chromosome and showed significant bias towards loss of heterozygosity. However, C1-013 and C1-019 had an estimated 43–44 chromosomes, which are low relative to the expectation (CCAB = 37 chromosomes for a 2n gamete). This is difficult to explain via a self-pollination event, but may suggest that unreduced ovules developed directly into embryos, which has previously been documented in Brassica  inheritance of chromosomes A04, A05, A06, and A08 in the A genome and preferential loss of chromosomes C4 and C5 in the C genome. These tend to be chromosomes which are less likely to be involved in A-C pairing, for which the most common pairs are A01-C1, A02-C2, A03-C3, and A07-C6  expression of kinetochore proteins controlling attachment of univalent chromosomes to microtubules, such that Festuca chromosomes were improperly attached and subsequently often lost as micronuclei following meiosis. Similar bias towards inheritance of Allium roylei over A. cepa chromosomes in A. cepa × A. roylei hybrids was attributed to female meiotic drive ESM 2(XLSX 23817 kb)


Corchorus olitorius seeds , followed by antimicrobial screening against various bacterial and fungal strains.Endophytic fungi are very rich sources of natural antibacterial and antifungal compounds. The main aim of this study is to isolate the fungal endophytes from the medicinal plant Penicillium, Fusarium, and Aspergillus. The seven isolated endophytic strains revealed selective noticeable activity against Escherichia coli (ATCC25922) with varied IC50s ranging from 1.19 to 10 µg /mL, in which Aspergillus sp. (Ar 6) exhibited the strongest potency against E. coli  and candida albicans  with IC50s 1.19 and 15 µg /mL, respectively. Therefore, the chemical profiling of Aspergillus sp. (Ar 6) crude extract was performed using LC-HR-ESI-MS and led to the dereplication of sixteen compounds of various classes (1–16). In-silico analysis of the dereplicated metabolites led to highlighting the compounds responsible for the antimicrobial activity of Aspergillus sp. extract. Moreover, molecular docking showed the potential targets of the metabolites; Astellatol (5), Aspergillipeptide A (10), and Emericellamide C (14) against E. coli and C. albicans.Seven endophytic fungal strains belonging to different three genera were isolated, including These results will expand the knowledge of endophytes and provide us with new approaches to face the global antibiotic resistance problem and the future production of undiscovered compounds different from the antibiotics classes.The online version contains supplementary material available at 10.1186/s12866-023-03092-5. The current health crisis that has arisen from the spread of antimicrobial resistance has become one of the major causes of death occurring worldwide, accounting for approximately more than 700,000 deaths annually . AntibioCorchorus olitorius L. , commonly known as Jute mallow, is an annual erect herb that grow on roadsides, fields, and home gardens +, for the predicted molecular formulas C11H20N2O4 gave hits of the amide type Aspergilliamide B (1), that were previously isolated from marine-derived Aspergillus westerdijkiae DFFSC 013. The mass ion peak at m/z 257.14, in consonance with the suggested molecular formula C12H22N2O4 [M-H]− was dereplicated as a peptide derivative Cyclo (isoleucylisoleucyl) (2) that were previously isolated from Aspergillus terreus. The mass ion peak at m/z 321.14, in conformity with the predicted molecular formula C21H22O3 [M-H]− was dereplicated as α-pyrone derivative, Asnipyrone A (3) previously isolated from Aspergillus niger DSM 2182 and MA-132 strains. While the mass ion peak m/z 344.14, corresponding to the suggested molecular formula C19H23NO5 [M-H]− fit an alkaloid compound aspyridone A (4) that were previously isolated from Aspergillus nidulans. Another mass ion peak at m/z 355.29 [M-H]−, corresponding to the molecular formulas C25H40O gave hits of the sesterterpenoid compound astellatol (5) that were previously isolated from Aspergillus variecolor. Likewise, the mass ion peak at m/z 359.14, corresponding to the suggested molecular formula C21H18N4O2 [M + H]+ fit an alkaloid compound Fumiquinazoline F (6) that were previously isolated from marine-derived Aspergillus fumigatus. The mass ion peak at m/z 377.10, corresponding to the suggested molecular formula C18H22N2O3S2 [M-H]− fit an epidithiodioxopiperazines compound Dithiosilvatin (7) that was previously isolated from Aspergillus silvaticus. The mass ion peak at m/z 422.89, in conformity with the molecular formula C12H9ClN2O5S4 [M-H]− was identified as aspirochlorine, which is a tetrasulfide analogue compound (8) and previously isolated from Aspergillus flavus. The mass ion peak at m/z 463.20, corresponding to the suggested molecular formula C25H28N4O5 [M-H]−, in accordance with the peptide compound; Aspercolorin (9) that were previously isolated from Aspergillus versicolor. Another mass ion peak at m/z 515.25, corresponding to the molecular formula C26H36N4O7 [M-H]− in harmony with a peptide analogue compound aspergillipeptide A (10) that was previously isolated from marine-derived Aspergillus sp. SCSGAF 0076. The mass ion peak at m/z 525.25 [M + H]+ and mass ion peak at m/z 536.29 [M + H]+ corresponding to molecular formula C31H32N4O4 and C32H41NO6 were identified as Miyakamide A1(11) and Sulpinine C (12) that were previously isolated from Aspergillus flavus var. columnaris FKI-0739 and Aspergillus sulphureus, respectively. As well, the metabolite, namely aurasperone A (13), with the molecular formula C31H28O12, was dereplicated from the mass ion peak m/z 591.15 [M-H]− that was previously isolated from Aspergillus niger and Aspergillus awamori. In addition, the mass ion peak m/z 596.40 [M + H]+, in agreement with the molecular formula C30H53N5O7, was dereplicated as Emericellamide C (14) that was previously isolated from Aspergillus nidulans. The mass ion peak at m/z 793.34 [M-H]− for the predicted molecular formula C50H46N6O4, was dereplicated as Ditryptophenaline (15), that was previously isolated from Aspergillus flavus. The mass ion peak at m/z 907.40 [M + H]+ for the molecular formula C52H54N6O9, was identified as Stephacidin B (16) that was previously isolated from Aspergillus ochraceus as shown in Table The molecular ion mass peak at m/z 245.14 [MIn-silico studies have effectively integrated with experimental research to explain complicated chemical and biological processes. Combining computational and experimental techniques has proven highly beneficial in identifying the potential targets of biologically active compounds and their mechanism of action [Aspergillus sp. Ar6 crude extract that were responsible for exhibition its antimicrobial activity against E. coli and C. albicans, and to predict their mechanism of action. According to a literature survey, certain bioactive compounds of the extract were reported to have antibacterial or antifungal activities against E. coli and C. albicans, respectively as shown in  was predicted to be active against C. albicans while Asnipyrone A (3) was predicted to have no effect on C. albicans.After filtering the results, the selected compounds Aspergillipeptide A (10) and Emericellamide C (14) were applied for AntiBac-pred and AntiFun-pred tools to investigate which could exert antimicrobial activity against ectively . On the E. coli organism were collected for Aspergillipeptide A (10), while 27 bacterial targets were found for Emericellamide C (13)  and Kyoto Encyclopedia of Genes and Genomes (KEGG).In addition, PPI network was then exported to the software Cytoscape 3.9.1 which was utilized to analyze the network using CytoHubba plugin. CytoHubba plugin calculated, through 12 kinds of methods, the most significant and coinciding nodes which would be potential targets for the related compounds. The interactions and binding modes between selected compounds and filtered targets were examined through molecular docking.GO enrichment analysis is a method for interpreting sets of genes or genes products into their biological functions including biological processes (BB), cellular components (CC), and molecular functions (MF) . The colThe BB primarily involved two main processes Fig. , A. The In case of GO analysis for Emericellamide C (14), BP were also dominated by DNA biosynthetic process (GO:0071897), DNA replication (GO:0006260), and their other related process such as nucleobase-containing biosynthetic process GO:0034654), heterocycle biosynthetic process (GO:0018130), and organic cyclic biosynthetic process   and Emericellamide C (14) derived us to explore their structural similarity and flexible alignment.Flexible alignment is a computational procedure used for superposing small organic molecules for the assessment of their 3D similarity. The flexible alignment was constructed using Aspergillipeptide A and Emericellamide C to investigate the 3D structural similarity against each other through generation of the lowest energy each compound, and application of molecular overlay tool in Discovery Studio. Figure The CytoHubba plugin was utilized to calculate the 12 different metrics  of all nodes for each PPI network. After calculating the 12 centrality measures of all nodes  were probably showed pharmacophore matching with geranylgeranyltransferase-I (PDB ID: 3DRA), secreted aspartic proteinase (Sap) 5 (PDB ID: 2QZX), and N-acetylglucosamine-phosphate mutase (PDB ID: 2DKC). These targets are contributing in essential processes for fungal viability, virulence, and pathogenesis [For the investigation of antifungal targets against ogenesis –61. The E. coli DNA recombination protein (RecA) (PDB ID: 3CMT) and E. coli DNA polymerase III (Pol III) (PDB ID: 2NHN). Emericellamide C (14) was also investigated for interactions with bifunctional deaminase/reductase (RibD) of the riboflavin biosynthetic pathway (PDB ID: 2OBC). The docking analysis results are presented in Table The core targets were selected for molecular docking using AutoDock 4.2.6 to verify their binding affinity with Aspergillipeptide A (10) and Emericellamide C (14). Aspergillipeptide A (10) and Emericellamide C (14) were docked with E. coli DNA RecA with binding energy equals − 7.15 Kcal/mol. As shown in  exhibited the highest binding affinity for  in Fig. A and B,  in Fig. C and D.E. coli RecA monomer has three major structural domains: a small N-terminal domain (NTD), a central domain and a large C-terminal domain (CTD). The central domain contains the ATP binding site, which is essential for the catalytic activity of the protein [The  protein . AspergiE. coli, the replicative polymerase is DNA Pol III, which is composed of 10 different subunits that work together to coordinate the replication of both the leading and lagging strands of DNA. Recent research has identified six essential amino acid residues, specifically ARG362, ASP405, LYS553, TYR686, GLN688, and HIS760, for the activity of Pol III. In addition, three other important amino acid residues, TYR340, ARG390, and LYS758, also play crucial roles in the activity of Pol III [10) with DNA Pol III.In  Pol III . Molecul+) and substrate analogue in the binary complexes. The NADP+ nicotinamide ring makes a 3.5 Å pi-pi stacking interaction with Trp170. [RibD is one of crucial proteins that participate in riboflavin biosynthesis which is essential to bacterial growth. Hence, inhibition of ribD is considered as antibacterial drug target. RibD contains two separate domains for deamination and reduction. The reductase active site is lined by the loop GLY159–SER173, which makes important interactions with the cofactor (NADP Trp170.  EmericelN-acetylglucosamine-phosphate mutase (2DKC), Emericellamide C (14) has the lowest binding energy with N-acetylglucosamine-phosphate mutase, while Aspergillipeptide A (10) was found to be the highest binding amongst other molecules with − 8.15 Kcal/mol. The carbonyl groups of the compound 10 created H-bonds with LYS32 and ASP72  to be tested against the three fungal targets using AutoDock 4.2.6. The docking analysis results, presented in Table P72 Fig. , E and FAspergillus sp. Ar6 using LC-HR-ESI-MS revealed their richness in diverse metabolites and 16 compounds were annotated in positive and negative modes , belonging to different classes such as alkaloids, peptides and terpenoids based natural compounds, where peptide derivatives were found to be more predominate Fig. In silico screening of the dereplicated metabolites in the fungal extract revealed several compounds that could be potentially active entities. It was further proposed that these active compounds have antibacterial and antifungal activity. A combination of metabolomics and in-silico approaches have allowed a shorter route to search for antibacterial and antifungal natural products in a shorter time. The in-silico studies for target prediction of antibacterial compounds showed the ability of Aspergillipeptide A to interact with recA and the role of Emericellamide C in inhibiting the action of DNA Pol III and ribD targets. For target prediction of antifungal compounds, the result indicates that Astellatol could bind to two potential targets (geranylgeranyltransferase-I and Sap5), and Asperagillipeptide A could bind to N-acetylglucosamine-phosphate mutase. The accurate mechanism in view of molecular docking also demands further validation in biological experiments. In conclusion, the diversity of fungal endophytes isolated from Corchorus olitorius seeds has now been reported for the first time. According to these findings, C. olitorius is home to a diverse fungal endophytic community with antibacterial properties. A powerful antibacterial extract from Aspergillus sp. Ar6 was discovered to be a source of novel antimicrobial chemicals that may be used in the creation of pharmaceuticals.The growing spread of infectious diseases caused by microbes such as bacteria, viruses, and fungi has become one of the most significant challenges for the future of humanity. Plant Endophytes have become an important source of pharmacologically active metabolites. The chemical annotation of des Fig. , belongiAdditional file 1: Table S1. Reported antimicrobial activity of certain compounds of Aspergillus terreus extract against E. coli and C. albicans.  Table S2. PASS prediction scores for the unknown-activity compounds as antibacterial and antifungal agents. Table S3.AntiBac-pred and AntiFun-pred confidence scores for the selected compounds against E. coli and C. albicans from PASS online analysis. Table S4.Results of PharmMapper server forAspergillipeptide A (10) ranked by normalized fit score in descending order. Table S5. Results of PharmMapper server forEmericellamide C (14) ranked by normalized fit score in descending order. Table S6. E. coli protein targets for Aspergillipeptide A (10) and Emericellamide C (14) extracted from PharmMapper server and ranked by normalized fit score in descending order. Table S7. Results of top 10 nodes calculated by 12 different centrality measures for PPI networks of Aspergillipeptide A (10) and Emericellamide C (14). Figure S1. Number of occurrences for coinciding nodes from PPI networks. (A) Coinciding nodes of PPI network related to Aspergillipeptide A (10). (B) Coinciding nodes of PPI network related to Emericellamide C (14).