Recognition of cyclic dinucleotides and folates by human SLC19A1.

Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life1,2. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase after detecting cytosolic DNA signals3-7. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses8-21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A122,23. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.

Interrogating glioma-M2 macrophage interactions identifies Gal-9/Tim-3 as a viable target against PTEN-null glioblastoma.

Genomic alteration can reshape tumor microenvironment to drive tumor malignancy. However, how PTEN deficiency influences microenvironment-mediated cell-cell interactions in glioblastoma (GBM) remains unclear. Here, we show that PTEN deficiency induces a symbiotic glioma-M2 macrophage interaction to support glioma progression. Mechanistically, PTEN-deficient GBM cells secrete high levels of galectin-9 (Gal-9) via the AKT-GSK3ß-IRF1 pathway. The secreted Gal-9 drives macrophage M2 polarization by activating its receptor Tim-3 and downstream pathways in macrophages. These macrophages, in turn, secrete VEGFA to stimulate angiogenesis and support glioma growth. Furthermore, enhanced Gal-9/Tim-3 expression predicts poor outcome in glioma patients. In GBM models, blockade of Gal-9/Tim-3 signaling inhibits macrophage M2 polarization and suppresses tumor growth. Moreover, α-lactose attenuates glioma angiogenesis by down-regulating macrophage-derived VEGFA, providing a novel antivascularization strategy. Therefore, our study suggests that blockade of Gal-9/Tim-3 signaling is effective to impair glioma progression by inhibiting macrophage M2 polarization, specifically for PTEN-null GBM.

Combination of levetiracetam and IFN-α increased temozolomide efficacy in MGMT-positive glioma.

PURPOSE: Glioma, especially glioblastoma (GBM), is the most aggressive malignant brain tumor and its standard therapy is often ineffective because of temozolomide (TMZ) resistance. Reversal of the TMZ resistance might improve the prognosis of glioma patients. We previously found that interferon-α (IFN-α) and anti-epileptic drug levetiracetam (LEV) could sensitize glioma to TMZ, respectively. In this study, we further investigated the efficiency of combining of LEV and IFN-α for improving the efficacy of TMZ. METHODS: We evaluated whether LEV and IFN-α could increase TMZ efficacy using colony formation assay and cell viability assay with MGMT-positive and MGMT-negative glioma cell lines in vitro. Subcutaneous xenografts and orthotopic xenografts mice models were used in vivo to observe the tumor growth and mice survival upon treatments with TMZ, TMZ + IFN-α, TMZ + LEV, or TMZ + LEV + IFN-α. The expression levels of MGMT, markers of pro-apoptotic and anti-apoptotic in tumor samples were analyzed by Western blotting. RESULTS: The combinational use of IFN-α, LEV, and TMZ showed the best anti-tumor activity in MGMT-positive cell lines (U138, GSC-1, U118, and T98 G). TMZ + LEV + IFN-α further obviously increased TMZ + LEV or TMZ + IFN-α efficiency in MGMT-positive cell lines, while not in negative cell lines (SKMG-4, U87, U373, and U251) in vitro, which were also observed in subcutaneous mice models (U138, GSC-1 compared to SKMG-4, U87) and orthotopic models (GSC-1) in vivo. Strikingly, the combination of LEV and IFN-α together with TMZ significantly prolonged the survival of mice with orthotopic GSC-1 glioma. Furthermore, we confirmed that the combination of LEV and IFN-α enhanced the inhibition of MGMT and the activation of apoptosis in U138 tumor on the basis of TMZ treatment. CONCLUSIONS: The combination use of LEV and IFN-α could be an optimal method to overcome TMZ resistance through obvious MGMT inhibition in MGMT-positive glioma.

Transcriptome profile analysis reveals cardiotoxicity of maduramicin in primary chicken myocardial cells.

Maduramicin, an excellent ionophore antibiotic, is extensively used to control coccidiosis in poultry. Numerous maduramicin intoxications have been reported in farm animal and human due to its relatively narrow safety range, with necrosis or degeneration of cardiac and skeletal muscles as hallmark. To date, the mechanisms of maduramicin-induced cardiotoxicity remain unclear in chicken and other animals. Maduramicin (5 µg/mL)-treated primary chicken myocardial cells were used for RNA sequencing (RNA-Seq) and bioinformatics analysis in this study. A total of 1442 differential expressed genes were identified. 810 genes were up-regulated and the rest 632 genes were down-regulated. Transcriptome analysis revealed that the cytokine-cytokine receptor interaction, apoptosis, calcium signal and cytoplasmic vacuolization pathways were significantly affected. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that gene expression patterns were consistent with RNA-Seq analysis. Pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8), apoptosis ratios, cleaved caspase-3, intracellular calcium level and Ca2+-ATPase activity were elevated after maduramicin (0.05, 0.5 and 5 µg/mL) treatment. Massive vacuole formation was found in the cytoplasm by morphology and transmission electron microscopy observation. Taken together, the results suggested that maduramicin exerted its cardiotoxicity by multiple molecular mechanisms in primary chicken myocardial cells.