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Background In endothelial cells, phospholipase C (PLC) β1-activated Ca 2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation. Methods Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. Results The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with K d of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs. Conclusions Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca 2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.

Pioglitazone (PIO) attenuates cisplatin nephrotoxicity whereas the underlying mechanism remains unknown. Apoptosis is associated with mitochondrial dysfunction and SIRT1 activation can decrease cell apoptosis in cisplatin nephrotoxicity. Therefore, we explored whether the protective effect of PIO in cisplatin nephrotoxicity is achieved by suppressing mitochondria‐mediated apoptosis through SIRT1/p53 signalling regulation. Cell viability, apoptosis, survival rate, renal pathology and function were examined. Moreover, we also analysed the expression of SIRT1, Acetyl‐p53, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP) opening, adenosine triphosphate (ATP) and apoptosis‐related protein in vivo and in vitro. Pioglitazone treatment significantly increased cell viability, promoted SIRT1‐p53 interaction, upregulated Bcl‐2 expression, activated SIRT1 and elevated mitochondrial ATP synthesis after cisplatin treatment. However, PIO decreased the generation of ROS, opening of mPTP, dissipation of MMP and translocation of cytochrome c after cisplatin treatment. Pioglitazone also reduced the activation of caspase‐3 and caspase‐9, lowered the ratio of Bax/Bcl‐2, attenuated kidney pathological damage and dysfunction, down‐regulated the expression of Acetyl‐p53, PUMA‐α and Bax and abated cell apoptosis after cisplatin treatment. The SIRT1 inhibitor, EX527, clearly reversed the protective effects of PIO. These results implied PIO attenuated cisplatin nephrotoxicity by suppressing mitochondria‐mediated apoptosis through regulating SIRT1/p53 signalling.

A sugar-lipid molecule called OAcGD2 is a novel marker for breast cancer stem cells. Treatment with anti-OAcGD2 mAb8B6 may have superior anticancer efficacy by targeting cancer stem cells, thereby reducing metastasis and recurrence of cancer. Cancer stem cells (CSCs) that drive tumor progression and disease recurrence are rare subsets of tumor cells. CSCs are relatively resistant to conventional chemotherapy and radiotherapy. Eradication of CSCs is thus essential to achieve durable responses. GD2 was reported to be a CSC marker in human triple-negative breast cancer, and anti-GD2 immunotherapy showed reduced tumor growth in cell lines. Using a specific anti-OAcGD2 antibody, mAb8D6, we set out to determine whether OAcGD2 cells exhibit stem cell properties and mAb8D6 can inhibit tumor growth by targeting OAcGD2 CSCs. OAcGD2 expression in patient-derived xenografts (PDXs) of breast cancer was determined by flow cytometric analyses using mAb8D6. The stemness of OAcGD2 cells isolated by sorting and the effects of mAb8B6 were assessed by CSC growth and mammosphere formation and tumor growth using PDX models. We found that the OAcGD2 expression levels in six PDXs of various molecular subtypes of breast cancer highly correlated with their previously defined CSC markers in these PDXs. The sorted OAcGD2 cells displayed a greater capacity for mammosphere formation and tumor initiation than OAcGD2 cells. In addition, the majority of OAcGD2 cells were aldehyde dehydrogenase (ALDH ) or CD44 CD24 , the known CSC markers in breast cancer. Treatment of PDXs-bearing mice with mAb8B6, but not doxorubicin, suppressed the tumor growth, along with reduced CSCs as assessed by CSC markers and tumorigenicity. , mAb8B6 suppressed proliferation and mammosphere formation and induced apoptosis of OAcGD2 breast cancer cells harvested from PDXs, in a dose-dependent manner. Finally, administration of mAb8B6 dramatically suppressed tumor growth of OAcGD2 breast CSCs (BCSCs) with complete tumor abrogation in 3/6 mice. OAcGD2 is a novel marker for CSC in various subtypes of breast cancer. Anti-OAcGD2 mAb8B6 directly eradicated OAcGD2 cells and reduced tumor growth in PDX model. Our data demonstrate the potential of mAb8B6 as a promising immunotherapeutic agent to target BCSCs.

Adenosine signaling is a crucial immunosuppressive pathway within the tumor microenvironment, making it a promising target for cancer therapy. In this study, it is demonstrated that Globo H ceramide (GHCer), the most prevalent tumor‐associated glycosphingolipid, influences the tumor microenvironment by activating adenosine signaling, which results in dual immunosuppressive effects on T cells. It is demonstrated that GHCer interacts with the adenosine receptor 2A (A2AR), triggering cyclic AMP (cAMP) and protein kinase A (PKA) signaling. This interaction leads to a reduction in the proliferation of CD4+ T cells while simultaneously promoting the differentiation of regulatory T cells (Tregs). Furthermore, GHCer enhances the suppressive capacity of Treg cells by upregulating inhibitory molecules such as Lymphocyte‐activation gene 3 (LAG3), cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4), Programmed cell death 1 ligand 1 (PD‐L1), and it stimulates the secretion of the immunosuppressive cytokine Interleukin 35 (IL‐35). Additionally, GHCer‐induced Tregs express CD39 and CD73, which further enhances adenosine production and creates a positive feedback loop in the adenosinergic pathway and A2AR signaling. Mechanistically, it is found that GHCer forms a complex with TRAX (translin‐associated factor‐X) and the C‐terminus of A2AR, which facilitates the activation of A2AR and promotes an immunosuppressive tumor microenvironment. This study identifies Globo H ceramide (GHCer) as a novel immune checkpoint molecule that suppresses the activation of conventional T lymphocytes while promoting the differentiation and function of regulatory T cells. The immunosuppressive effects of GHCer are mediated through the activation of the A2AR/cAMP/PKA pathway, which involves its interactions with both TRAX and A2AR.

Background Fat grafting has been extensively used in plastic surgery practice, yet unstable retention in the recipient site remains a significant clinical challenge. The limited tolerance of injected adipose tissue to ischemia has prompted strategies aiming at timely enhancing the vascularity of the grafted fat. Various modified fat graft preparations have been used, and the mechanically processed tissue stromal vascular fraction (tSVF) derived from fat tissue has garnered considerable interest for enhancing rate of fat graft retention. Further enhancement of the graft retention and quality through supplements to tSVF is worthy of investigation. Methods The arteriovenous (AV) shunt in rats has been used to evaluate tSVF in vivo. We employed this animal model to investigate the regenerative potential of glycolipid Globo H Ceramide (GHCer) added to tSVF isolated from male Lewis rats. Sixty-two rats divided into four groups were studied. Study parameters included gene expression of vascular endothelial growth factor A (VEGFA) and fatty acid binding protein 4 (FABP4), percentages of the CD45 − CD31 + endothelial cell, fat tissue retention and fibrotic changes. In vitro studies on adipose-derived mesenchymal stromal cells (AD-MSCs) included angiogenesis by tube formation assay and adipogenesis. Results The addition of GHCer resulted in superior retention of the tSVF grafts at one-, two-, and eight-week post-grafting ( p  < 0.05). Elevated expression VEGFA was observed from one week ( p  < 0.05), followed by FABP4 at two weeks post-grafting in the tSVF + GHCer grafts ( p  < 0.01). After eight weeks, the numbers of CD45 − CD31 + endothelial cells and adipocytes were significantly increased in the tSVF + GHCer grafts ( p  < 0.01), while collagen deposition was reduced ( p  < 0.05). Given that GHCer potentially exerted its effects on tSVF through AD-MSCs within, we performed in vitro studies and demonstrated that GHCer promoted AD-MSC differentiation into neovessels ( p  < 0.05) and adipocytes ( p  < 0.001). Conclusions Supplementing GHCer to tSVF effectively reduced fat reabsorption and fibrotic changes of the grafts, while enhancing angiogenesis and adipogenesis, potentially through facilitating AD-MSC differentiation within tSVF. These findings support the potential clinical application of GHCer to enhance the stability and long-term outcomes of fat grafting procedures. Trial registration Not applicable. Clinical trial number Not applicable.

Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment. A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals. The average TRACP level of the high-UA individuals (1.47 ± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p < 0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24). High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.

Invariant natural killer T cell (NKT) cells (iNKT cells) produce both T-helper 1 (Th1) and T-helper 2 cytokines in response to α-Galactosylceramide (α-GalCer) stimulation and are thought to be the important effectors in the regulation of both innate and adaptive immunity involved in autoimmune disorders, microbial infections, and cancers. However, the anticancer effects of α-GalCer were limited in early clinical trial. In this study, several analogs of α-GalCer, containing phenyl groups in the lipid tails were found to stimulate murine and human iNKT cells to secrete Th1-skewed cytokines and exhibit greater anticancer efficacy in mice than α-GalCer. We explored the possibility of different Vβ usages of murine Vα14 iNKT or human Vα24 iNKT cells, accounting for differential cytokine responses. However, T-cell receptor Vβ analysis revealed no significant differences in Vβ usages by α-GalCer and these phenyl glycolipid analogs. On the other hand, these phenyl glycolipids showed greater binding avidity and stability for iNKT T-cell receptor when complexed with CD1d. These findings suggest that CD1d–phenyl glycolipid complexes may interact with the same population of iNKT cells but with higher avidity and stability to drive Th1 polarization. Thus, this study provides a key to the rational design of Th1 biased CD1d reactive glycolipids in the future.

In order to enhance surface property of brake materials, compacted graphite cast iron (CGI) with non-smooth surface was processed by laser local processing. This study focuses on the influence of the work media on wear resistance and thermal fatigue resistance of specimens. The results showed that when the work media were air and water respectively, laser local processing could enhance surface property of CGI. Changing work media could not change phase compositions of the laser processing area, but refined their microstructures, which enhanced their micro-hardness. By this way, the specimens’ wear resistance and resistance to thermal crack initiation were further improved, while their resistance to thermal crack propagation was influenced by cracks on bionic units.

Gallbladder cancer (GBC) has poor prognosis, is primarily treated with gemcitabine-based chemotherapy, which is limited by gemcitabine correlates with GBC progression. This study investigate the role of Globo H ceramide (GHCer) in GR and explore whether targeting Glob H could overcome resistance Globo H expression was assessed by immunohistochemistry in 81 GBC samples. GHCer-induced GR and ABCG2 upregulation were assessed in GBC cell lines, patient-derived xenograft (PDX), and a thioacetamide (TAA)-induced rat cholangiocarcinoma model. A2AR/cAMP/PKA signaling involvement was examined using inhibitors and siRNA. The efficacy of anti-Globo H antibody (mAb VK9) or vaccine (OBI-833/OBI-821) combined with gemcitabine was evaluated and . Immune responses were assessed by ELISA and multiplex immunohistochemistry. High Globo H expression correlated with shorter survival in GBC patients receiving gemcitabine. GHCer promoted GR via A2AR/cAMP/PKA-mediated ABCG2 upregulation, which was reversed by mAb VK9 or pathway inhibition. mAb VK9 or OBI-833/821 enhanced gemcitabine efficacy in GBC PDX and TAA cholangiocarcinoma models. OBI-833/821 induced anti-Globo H IgG/IgM, reduced Foxp3⁺ Tregs, and increased CD161⁺ NK cells in TAA model. A compassionate clinical use of 833/821 led to stable disease. GHCer promotes GR by upregulating ABCG2 via A2AR/cAMP/PKA signaling. Targeting Globo H may improve chemotherapy response in GBC.

Background In endothelial cells, phospholipase C (PLC) [beta]1-activated Ca.sup.2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLC[beta]1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLC[beta]1 activation. Methods Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLC[beta]1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. Results The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with Kd of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 [+ or -] 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 [+ or -] 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLC[beta]1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLC[beta]1 in cells by GHCer derived from EVs. Conclusions Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLC[beta]1, leading to Ca.sup.2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments. Keywords: Globo H ceramide, TRAX, Phospholipase C[beta]1, Angiogenesis, Glycosphingolipids, Extracellular vesicles