Annexin Research Articles

Ameliorative potential of Astaxanthin on glyphosate-induced neurocognitive functional changes in adult zebrafish via acid phosphatase and annexin A2 dysfunction

Glyphosate (GYP) is a phosphonate type of herbicide and potentially causes neurodegenerations. Astaxanthin (AST) is a red pigment and it is a group of carotenoids. It possesses the potential antioxidants and neuroprotective actions. However, the ameliorative role of AST in GYP- induced neurocognitive dysfunction has not been studied yet. Hence, the present study is designed to investigate the ameliorative potential of AST in GYP-induced cognitive dysfunction via regulations of acid phosphatase (AP) and annexin A2 (AXA2) actions in the zebrafish model. The cognitive dysfunction was induced by exposure to GYP (0.5 mg/L for 30 minutes/day) for 14 consecutive days. The AST (50, 75, and 100 mg/L for 30 minutes/day) and reference drug i.e., donepezil (DP; 1 µg/mL for 30 minutes/day) were exposed for 14 consecutive days before GYP exposure. On the 14th day, cognitive changes i.e., three horizontal compartment test,optokinetic motor response (OMR), startle response (SR), and T-maze tests were assessed. The changes of biomarkers i.e., acetylcholinesterase (AChE) activity, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), AP, and AXA2 levels were estimated in brain tissue. The administration of AST ameliorates the GYP-induced neurocognitive dysfunctions via the antioxidant, regulation of AP and AXA2 actions. Hence, it can be used for neurocognitive disorders against the phosphonate type of herbicide (GYP) toxicity.

Changes in milk fat globule membrane proteins along lactation stage of Laoshan dairy goat

The milk fat globule membrane (MFGM) is a complex structure with numerous functions, and its composition is affected by many factors. There have been few systematic investigations on goat MFGM proteome profiling during lactation. Individual milk samples from 15 healthy dairy goats were obtained at six lactation time points for investigation of the MFGM proteome using both data-independent acquisition (DIA) and data-dependent acquisition (DDA) proteomics techniques combined with multivariate statistical analysis. Using the DIA method, 890 variably abundant MFGM proteins were discovered throughout the lactation cycle. From 1 to 240 d, butyrophilin subfamily 1 member A1, lipoprotein lipase, perilipin-2, and adipose triglyceride lipase were upregulated, while APOE, complement C3, clusterin, and IgG were downregulated. Furthermore, from 1 to 90 d, annexin A1, annexin A2, and antithrombin-III were downregulated, then upregulated by d 240. Albumin had a high degree of connectedness, indicating that it was a key protein, according to protein–protein interaction research. Overall, our findings gave new insights into the biological features of MFGM protein in goat milk throughout lactation, which may aid in the creation of specialized MFGM products and infant formula.

Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

MiR-374a/b-5p Suppresses Cell Growth in Papillary Thyroid Carcinoma Through Blocking Exosomal ANXA1-Induced Macrophage M2 Polarization.

Papillary thyroid carcinoma (PTC), comprising 85% of all thyroid cancers, is an epithelial malignancy. The potential for malignant transformation in normal cells by thyroid cancer cells via exosomal Annexin A1 (ANXA1) delivery is investigated in this study. Our aim is to determine the impact of PTC cells on macrophage polarization through exosomal ANXA1 secretion and its implications for tumor progression.Exosomes in PTC cells were examined using transmission electron microscopy, exosome labeling, and nanoparticle tracking analysis. Real-time quantitative polymerase chain reaction was employed to quantify gene expression levels. Protein levels were determined through Western blot analysis. The interplay between genes was assessed using luciferase reporter and RNA pull-down assays. Functional experiments were conducted to investigate PTC cell proliferation and apoptosis.Our findings reveal that ANXA1 promotes PTC cell proliferation and inhibits apoptosis. Exosomes derived from PTC cells were found to promote macrophage M2 polarization. ANXA1 stimulates M2 polarization through the activation of the PI3K/AKT pathway. MicroRNA-374a-5p (miR-374a-5p) and microRNA-374b-5p (miR-374b-5p) were identified as inhibitors of ANXA1 expression and PI3K/AKT pathway activity, thereby inhibiting macrophage M2 polarization. Furthermore, miR-374a-5p and miR-374b-5p were observed to suppress PTC cell proliferation through their regulatory action on ANXA1.Our study suggests that miR-374a/b-5p inhibits PTC cell growth by blocking the macrophage M2 polarization induced by exosomal ANXA1.

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface.

Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

Abstract 3866: Azvudine: An emerging immunomodulator in the treatment of cancer and viral infections

Abstract Azvudine (FNC) is a nucleoside analog that has secured approval from the CDE in China for the treatment of both AIDS and COVID-19. In these contexts, it has showed remarkable efficacy and safety when taken orally. Typically, nucleoside analog drugs function by integrating into DNA and impeding DNA synthesis, evident in drugs like 5-FU, Capecitabine, Ara-C, and Gemcitabine. Furthermore, FNC has been reported to exhibit robust anti-tumor properties, curbing cell proliferation, initiating G1 and S phase arrests, and advancing apoptosis across various human cancer cell lines. Grounded in this mode of action, we have ascertained that the anti-tumor effects of FNC intricately align with the expression of deoxycytidine kinase (dCK) both in vitro and in vivo. Remarkably, our studies delved into the effects and underlying mechanisms of FNC in enhancing the tumor microenvironment within solid tumors. Our findings, stemming from cell-based assays and in vivo animal experiments, elucidate the following: 1. FNC can trigger immunogenic cell death (ICD) in tumor cells. This is evidenced by the surface presentation and liberation of damage-associated molecular patterns (DAMPs) such as Calreticulin, HSP70, HSP90, Annexin A1, and HMGB1, which in turn stimulate both innate and adaptive immune responses. 2. FNC substantially reduces the population of monocytic-myeloid derived suppressor cells (M-MDSC) and Polymorphonuclear-myeloid derived suppressor cells (PMN-MDSC) in the tumor microenvironment. Concurrently, it boosts the infiltration and proliferation of CD8+T, CD4+T and NK cells. This instigates the transformation of the tumor microenvironment from a 'cold' state to a 'hot' one, enhancing the synergistic effects of PD-1 when used in tandem with FNC. This mechanism was substantiated through a high proportion of MDSCs observed in H22 and CT-26 syngeneic models, and contrastingly, a low proportion in MC38 and LL/2 in vivo syngeneic models. Notably, every animal subjected to combined FNC and PD-1 treatment in the H22 and CT-26 in vivo models witnessed complete tumor regression. Given these revelations, there's a compelling argument that FNC monotherapy possesses immense potential as an anti-tumor agent. The potent anticancer effects of FNC in vitro and in vivo warrants further investigation of its anticancer potential as immunomodulator. Citation Format: Pan Li, Limin Jia, Zhaoyang Wang, Jinfa Du. Azvudine: An emerging immunomodulator in the treatment of cancer and viral infections [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3866.

Effect of Annexin A2 on prognosis and sensitivity to immune checkpoint plus tyrosine kinase inhibition in metastatic renal cell carcinoma

BackgroundImmunotherapy (IO) plus tyrosine kinase inhibitor (TKI) therapy is the first-line recommendation for advanced renal cell carcinoma (RCC), but no biomarker has been approved for it. Annexin A2 (ANXA2) can induce immune escape in tumors.MethodsTwo independent cohorts of advanced RCC treated by IO + TKI were utilized for survival analysis (ZS-MRCC, n = 45; Javelin-101, n = 726). ANXA2 expression was determined by RNA-sequencing. The impact of ANXA2 on the tumor microenvironment was assessed by RNA-sequencing, flow cytometry and immunohistochemistry in two localized RCC datasets (ZS-HRRCC, n = 40; TCGA-KIRC, n = 530).ResultsANXA2 was upregulated in non-responders of IO + TKI therapy (p = 0.027). High-ANXA2 group showed poor progression-free survival (PFS) in both the ZS-MRCC cohort (HR, 2.348; 95% CI 1.084–5.085; P = 0.025) and the Javelin-101 cohort (HR, 1.472; 95% CI 1.043–2.077; P = 0.027). Multivariate Cox regression determined ANXA2 as an independent prognostic factor (HR, 2.619; 95% CI 1.194–5.746; P = 0.016). High-ANXA2 was correlated with decreased proportion of granzyme B+ CD8+ T cells (Spearman’s ρ = − 0.40, P = 0.01), and increased TIM-3+ (Spearman’s ρ = 0.43, P < 0.001) and CTLA4+ (Spearman’s ρ = 0.49, P < 0.001) tumor-infiltrating lymphocytes. A random forest (RF) score was further build by integrating ANXA2 and immune genes, which stratified patients who would benefit from IO + TKI therapy (low-RF score, IO + TKI vs TKI, HR = 0.453, 95% CI 0.328–0.626; high-RF score, IO + TKI vs TKI, HR = 0.877, 95% CI 0.661–1.165; interaction P = 0.003).ConclusionsUpregulated ANXA2 was associated with poor PFS and therapeutic resistance in RCC treated by IO + TKI therapy, and related with T cell exhaustion. The integrated RF score could stratify patients who would benefit from IO + TKI therapy.

Abstract 3288: Identification of Annexin A1 as a novel driver of lenvatinib resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Lenvatinib has recently been approved by the FDA for front-line treatment of HCC patients at advanced stages. However, the survival benefit of lenvatinib is modest due to the development of acquired drug resistance. In this study, we aimed to investigate the mechanisms underlying lenvatinib resistance in HCC and to identify potential therapeutic targets to combat lenvatinib resistance. For this purpose, we established a lenvatinib-resistant (LenR) HCC patient-derived tumor xenograft (PDTX) by administering four rounds of lenvatinib treatment, and their genetic profiles were analyzed using single-cell RNA sequencing (scRNA-seq). Based on single-cell regulatory network inference and clustering (SCENIC) analysis, Annexin A1 (ANXA1), the top gene that was enriched in the HCC cell-like cluster of LenR tumors, was identified and was consistently observed in LenR HCC cell lines and LenR tumors derived from Tp53KO/C-MycOE and RIL175 HCC mouse models. Using an in vitro differentiation model, limiting dilution assay, apoptosis assay and correlation analysis, we demonstrated the role of ANXA1 in the regulation of cancer stemness-driven lenvatinib resistance. Mechanistically, ANXA1 regulates cancer stemness and drug resistance by driving the STAT3/S100A6 signaling cascade, and ANXA1 expression is regulated by SOX2 via promoter activation, as demonstrated by a chromatin immunoprecipitation (ChIP) assay. Using the same set of scRNA-seq data, we showed that LenR HCC tumors reshaped the tumor microenvironment (TME) by inducing M2 macrophage polarization, as evidenced by a higher M2/M1 ratio of tumor-associated macrophages (TAM) in LenR HCC tumors than in their mock counterparts. Based on this observation, we further demonstrated the in vitro role of secretory ANXA1 in LenR HCC cells in inducing macrophage polarization towards the M2 phenotype. In conclusion, ANXA1 may regulate lenvatinib resistance intrinsically via the promotion of cancer stemness and extrinsically via the induction of an immunosuppressive TME. Citation Format: Catherine Yujia GU, Carmen Oi Ning LEUNG, Terence Kin Wah LEE. Identification of Annexin A1 as a novel driver of lenvatinib resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3288.

Abstract 3003: Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling

Abstract Background: Glioblastoma (GBM) is one of the most lethal forms of cancer. The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which upon activation, are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling resulting in hyperactive RTK signaling. We demonstrated that the alternative splicing of the tumor suppressor Annexin A7 (ANXA7) supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. Exon six of ANXA7 can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). Methods: We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates first in U251 malignant glioma followed by stem-like brain tumor initiating cells (BTICs) originating from patient-derived xenografts (PDXs). U251s and primary BTICs (JX14) were transduced with lentivirus to overexpress ANXA7 and GFP for enrichment by flow cytometry. JX14 empty vector (EV; I2 only) and I1 (I2 and I1 expressing) cells were plated onto Geltrex and ligand starved overnight in serum-free media to maintain BTICs. We assessed EGFR protein levels following EGF stimulation in a time dependent manner. Immunofluorescence experiments were conducted to measure EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway, which included the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Results: Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P&amp;gt;0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (Rab11: R2=0.9267, P&amp;lt;0.0001). Immunoblotting revealed a significant reduction in EGFR protein in I1 cells only. Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P&amp;lt;0.0001). ANXA7 siRNA in EV cells did not alter EGFR trafficking, which implicates I2 as nonfunctional. On the other hand, ANXA7 KD in I1 cells impaired EGFR sorting via a 40% increase (P&amp;lt;0.0010) in total EGFR protein and a 40% reduction (P&amp;lt;0.0001) in colocalization with EEA1. Loss of I1 also shifted the degradation of EGFR back towards recycling. Conclusion: GBMs express I2, which supports EGFR hyperactivity by favoring receptor recycling. I1 overexpression significantly reduced EGFR signaling by promoting degradation. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for BTICs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition. Citation Format: Aran Merati, Sindhu Nair, Rajani Rajbhandari, Nicholas Johnson, Markus Bredel. Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3003.

Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1.

Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium. We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA. LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin - 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10. ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.

Bending of a lipid membrane edge by annexin A5 trimers

Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a “neck”-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

The structural properties of full-length annexin A11.

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38-59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.

Banxia Xiexin Decoction inhibiting colitis-associated colorectal cancer infected with Fusobacterium nucleatum by regulating Wnt/β-catenin pathway

This paper investigates the intervention effect and mechanism of Banxia Xiexin Decoction(BXD) on colitis-associated colorectal cancer(CAC) infected with Fusobacterium nucleatum(Fn). C57BL/6 mice were randomly divided into a control group, Fn group, CAC group [azoxymethane(AOM)/dextran sulfate sodium salt(DSS)](AOM/DSS), model group, and BXD group. Except for the control and AOM/DSS groups, the mice in the other groups were orally administered with Fn suspension twice a week. The AOM/DSS group, model group, and BXD group were also injected with a single dose of 10 mg·kg~(-1) AOM combined with three cycles of 2.5% DSS taken intragastrically. The BXD group received oral administration of BXD starting from the second cycle until the end of the experiment. The general condition and weight changes of the mice were monitored during the experiment, and the disease activity index(DAI) was calculated. At the end of the experiment, the colon length and weight of the mice in each group were compared. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-2, IL-4, and IL-6 inflammatory factors in the serum. Immunohistochemistry(IHC) was used to detect the expression of Ki67, E-cadherin, and β-catenin in the colon tissue. Western blot was used to detect the protein content of Wnt3a, β-catenin, E-cadherin, annexin A1, cyclin D1, and glycogen synthase kinase-3β(GSK-3β) in the colon tissue. The results showed that compared with the control group, the Fn group had no significant lesions. The mice in the AOM/DSS group and model group had decreased body weight, increased DAI scores, significantly increased colon weight, and significantly shortened colon length, with more significant lesions in the model group. At the same time, the colon histology of the model group showed more severe adenomas, inflammatory infiltration, and cellular dysplasia. The levels of IL-4 and IL-6 in the serum were significantly increased, while the IL-2 content was significantly decreased. The IHC results showed low expression of E-cadherin and high expression of Ki67 and β-catenin in the model group, with a decreased protein content of E-cadherin and GSK-3β and an increased protein content of Wnt3a, β-catenin, annexin A1, and cyclin D1. After intervention with BXD, the body weight of the mice increased; the DAI score decreased; the colon length increased, and the tumor decreased. The histopathology showed reduced tumor proliferation and reduced inflammatory infiltration. The levels of IL-6 and IL-4 in the serum were significantly decreased, while the IL-2 content was increased. Meanwhile, the expression of E-cadherin was upregulated, and that of Ki67 and β-catenin was downregulated. The protein content of E-cadherin and GSK-3β increased, while that of Wnt3a, β-catenin, annexin A1, and cyclin D1 decreased. In conclusion, BXD can inhibit CAC infected with Fn, and its potential mechanism may be related to the inhibition of Fn binding to E-cadherin, the decrease in annexin A1 protein level, and the regulation of the Wnt/β-catenin pathway.

Abnormal expression of oxylipins and related synthesizing/signaling pathways in inflammatory bowel diseases

We investigated selected oxylipins and related synthesizing/signaling pathways in 28 patients with Crohn's disease (CD), 19 patients with ulcerative colitis (UC), and 39 controls. Plasma and mucosal PUFA/oxylipin profiles were analyzed by LC-MS/MS. mRNA expression of 5, 12 and 15-lipooxygenases, FPR2/ALXR, FFAR4/GPR120, annexin A1, and interleukin-10 were analyzed by qRT-PCR. Oxylipin profile and related metabolic pathways were altered in both CD and UC patients. The patterns were characterized by increased prostaglandins, leukotrienes, and lipoxins and overexpression of 5-lipoxygenase, FPR2/ALXR, annexin A1, and interleukin-10 genes, but decreased n-3 PUFAs and 18-hydroxyeisapentaenoic acid. The gene of 15-lipoxygenase was under-expressed mainly in UC patients. CD and UC are associated with unbalanced n-6 ​​and n-3 derivatives and pro-inflammatory and anti-inflammatory/pro-resolving mediators favoring the former compounds. The findings suggest that oxylipins engage in the pathophysiology of the diseases. Targeting oxylipin's metabolic pathways would be a promising therapy for inflammatory bowel diseases.

The Relevance, Predictability, and Utility of Annexin A5 for Human Physiopathology.

As an important functional protein molecule in the human body, human annexin A5 (hAnxA5) is widely found in human cells and body fluids. hAnxA5, the smallest type of annexin, performs a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calcium-dependent manner and plays an important role in many human physiological and pathological processes. The free state hAnxA5 exists in the form of monomers and usually forms a polymer in a specific self-assembly manner when exerting biological activity. This review systematically discusses the current knowledge and understanding of hAnxA5 from three perspectives: physiopathological relevance, diagnostic value, and therapeutic utility. hAnxA5 affects the occurrence and development of many physiopathological processes. Moreover, hAnxA5 can be used independently or in combination as a biomarker of physiopathological phenomena for the diagnosis of certain diseases. Importantly, based on the properties of hAnxA5, many novel drug candidates have been designed and prepared for application in actual medical practice. However, there are also some gaps and shortcomings in hAnxA5 research. This in-depth study will not only expand the understanding of structural and functional relationships but also promote the application of hAnxA5 in the field of biomedicine.

Retraction: "Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2".

Retraction: "Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2" by Raffaella Belvedere, Elva Morretta, Emanuela Pessolano, Nunzia Novizio, Alessandra Tosco, Amalia Porta, James Whiteford, Mauro Perretti, Amelia Filippelli, Maria Chiara Monti, Antonello Petrella, J Cell Physiol. 2020, 236: 4926-4943. The above article, published online on 7 December 2020 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/full/10.1002/jcp.30207) has been retracted by agreement between the journal's Editor in Chief, Alexander Hutchison, and Wiley Periodicals LLC. The retraction has been agreed due to concerns raised by third parties. Some flaws and inconsistencies between results presented and experimental methods described were found. Specifically, manipulations have been detected affecting the Western Blot experiment presented in Figure 3a, and the microscopy images presented in Figure 5 and Figure S7. The corresponding author, Antonello Petrella, acknowledged not having noticed the anomalies at earlier stages and collaborated with the investigation. The original experimental data related to the concerns identified were no longer available; the authors have performed new Western Blot experiments. However, based on the editorial evaluation, the provided material failed to adequately address the concerns. For instance, the data intended to replicate the findings in Figure 3a were provided at a quality too low to ensure its accuracy. Furthermore, the concerns regarding the manipulation of microscopy images in Figure 5 and Figure S7 remained entirely unaddressed. Accordingly, the editors no longer have confidence in the integrity of the data presented in the article and consider the conclusions to be invalid. Antonello Petrella, on behalf of all authors, believes that the conclusions of the article are not affected by the identified issues, in part also corroborated by the new data; all authors disagree with the decision to retract the article.

Modeling of new markers for the diagnosis and prognosis of pancreatic cancer based on the transition from inflammation to cancer.

Pancreatic adenocarcinoma (PAAD) is a lethal disease with a poor prognosis. Genes involved in acute pancreatitis (AP) or chronic pancreatitis (CP) might be important for PAAD development. This study sought to identify potential PAAD diagnosis markers and to establish a PAAD prognosis prediction model based on AP- and CP-related genes. The significantly differentially expressed genes in both AP or CP and PAAD were obtained by a bioinformatics analysis. A risk-score model for predicting survival was constructed based on The Cancer Genome Atlas (TCGA) data and validated using an International Cancer Genome Consortium (ICGC) cohort. Protein expression and the effects of the genes in the risk models were validated by immunohistochemistry, or Cell Counting Kit-8 (CCK-8) and transwell assays. The study sample data included six AP tissue samples and five normal pancreatic tissue samples, six CP tissue samples and six normal pancreatic tissue samples from the Gene Expression Omnibus (GEO) expression profiling microarrays GSE109227 and GSE41418 data sets, respectively, and fragments per kilobase per million mapped fragments (FPKM) data from four normal controls and 150 PAAD cases from TCGA database, and 182 cancer patient samples with complete survival prognostic data from the ICGC database. In total, 508 significantly differentially expressed genes were found in both AP or CP and PAAD. Trefoil factor 2 (TFF2), tubulointerstitial nephritis antigen (TINAG), trefoil factor 1 (TFF1), aquaporin 5 (AQP5), SAM pointed domain containing ETS transcription factor (SPDEF), anterior gradient protein 2 (AGR2), apolipoprotein B messenger RNA editing enzyme catalytic subunit 1 (APOBEC1), kallikrein-related peptidase 6 (KLK6), dopa decarboxylase (DDC), mucin 13 (MUC13), claudin 18 (CLDN18), annexin A10 (ANXA10), and tetraspanin 1 (TSPAN1) were found to be present in PAAD and had the largest fold change. A risk-score model, comprising 19 genes, was constructed for prognostic prediction. A high-risk score indicated a poor prognosis. TINAG, DDC, SPDEF, and APOBEC1 proteins were increased in PAAD, while TINAG and DDC were correlated with the pathologic grade. Decreased TINAG, APOBEC1, transmembrane protein 94 (TMEM94), and kelch like family member 36 (KLHL36) expression inhibited PAAD cell proliferation, while decreased SPDEF, TMEM94, and KLHL36 expression significantly inhibited PAAD cell migration. The AP and CP co-related genes were significantly correlated with PAAD. TINAG, DDC, SPDEF, and APOBEC1 could serve as new PAAD predictors. The risk model developed in this study could be used to predict the prognosis of PAAD patients.

Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

FLIM-FRET-based analysis of S100A11/annexin interactions in living cells.

Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1-containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM-FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.