Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.National Institutes of Health (U.S.)Massachusetts Life Sciences Center (New Investigator Award)National Institute of General Medical Sciences (U.S.) (Pre-Doctoral Grant in the Biological Sciences (5-T32-GM007287-33))Studienstiftung des deutschen VolkesCancer Research Institute (New York, N.Y.)Cleo and Paul Schimmel FoundationBayer HealthcareHuman Frontier Science Program (Strasbourg, France

Monitoring the Morphology of M87* in 2009-2017 with the Event Horizon Telescope

The Event Horizon Telescope (EHT) has recently delivered the first resolved images of M87*, the supermassive black hole in the center of the M87 galaxy. These images were produced using 230 GHz observations performed in 2017 April. Additional observations are required to investigate the persistence of the primary image feature—a ring with azimuthal brightness asymmetry—and to quantify the image variability on event horizon scales. To address this need, we analyze M87* data collected with prototype EHT arrays in 2009, 2011, 2012, and 2013. While these observations do not contain enough information to produce images, they are sufficient to constrain simple geometric models. We develop a modeling approach based on the framework utilized for the 2017 EHT data analysis and validate our procedures using synthetic data. Applying the same approach to the observational data sets, we find the M87* morphology in 2009-2017 to be consistent with a persistent asymmetric ring of ∼40 μas diameter. The position angle of the peak intensity varies in time. In particular, we find a significant difference between the position angle measured in 2013 and 2017. These variations are in broad agreement with predictions of a subset of general relativistic magnetohydrodynamic simulations. We show that quantifying the variability across multiple observational epochs has the potential to constrain the physical properties of the source, such as the accretion state or the black hole spin

Intracellular Trafficking of Guanylate-Binding Proteins Is Regulated by Heterodimerization in a Hierarchical Manner

Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins

N-glycosylation regulates intrinsic IFN-γ resistance in colorectal cancer: implications for immunotherapy

Background & Aims: Advanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored. Methods: Spontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines. Results: The conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N-glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N-acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ–resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N-glycosylation, as well as IFNγRα protein stability and signaling. Conclusions: Together, our results demonstrated that tumor-associated changes in N-glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC

Roflumilast inhibits tumor growth and migration in STK11/LKB1 deficient pancreatic cancer

Pancreatic cancer is a malignant tumor of the digestive system. It is highly aggressive, easily metastasizes, and extremely difficult to treat. This study aimed to analyze the genes that might regulate pancreatic cancer migration to provide an essential basis for the prognostic assessment of pancreatic cancer and individualized treatment. A CRISPR knockout library directed against 915 murine genes was transfected into TB 32047 cell line to screen which gene loss promoted cell migration. Next-generation sequencing and PinAPL.py- analysis was performed to identify candidate genes. We then assessed the effect of serine/threonine kinase 11 (STK11) knockout on pancreatic cancer by wound-healing assay, chick agnosia (CAM) assay, and orthotopic mouse pancreatic cancer model. We performed RNA sequence and Western blotting for mechanistic studies to identify and verify the pathways. After accelerated Transwell migration screening, STK11 was identified as one of the top candidate genes. Further experiments showed that targeted knockout of STK11 promoted the cell migration and increased liver metastasis in mice. Mechanistic analyses revealed that STK11 knockout influences blood vessel morphogenesis and is closely associated with the enhanced expression of phosphodiesterases (PDEs), especially PDE4D, PDE4B, and PDE10A. PDE4 inhibitor Roflumilast inhibited STK11-KO cell migration and tumor size, further demonstrating that PDEs are essential for STK11-deficient cell migration. Our findings support the adoption of therapeutic strategies, including Roflumilast, for patients with STK11-mutated pancreatic cancer in order to improve treatment efficacy and ultimately prolong survival

First M87 Event Horizon Telescope Results. IV. Imaging the Central Supermassive Black Hole

We present the first Event Horizon Telescope (EHT) images of M87, using observations from April 2017 at 1.3 mm wavelength. These images show a prominent ring with a diameter of similar to 40 mu as, consistent with the size and shape of the lensed photon orbit encircling the "shadow" of a supermassive black hole. The ring is persistent across four observing nights and shows enhanced brightness in the south. To assess the reliability of these results, we implemented a two-stage imaging procedure. In the first stage, four teams, each blind to the others' work, produced images of M87 using both an established method (CLEAN) and a newer technique (regularized maximum likelihood). This stage allowed us to avoid shared human bias and to assess common features among independent reconstructions. In the second stage, we reconstructed synthetic data from a large survey of imaging parameters and then compared the results with the corresponding ground truth images. This stage allowed us to select parameters objectively to use when reconstructing images of M87. Across all tests in both stages, the ring diameter and asymmetry remained stable, insensitive to the choice of imaging technique. We describe the EHT imaging procedures, the primary image features in M87, and the dependence of these features on imaging assumptions

Monitoring the Morphology of M87* in 2009–2017 with the Event Horizon Telescope

The Event Horizon Telescope (EHT) has recently delivered the first resolved images of M87*, the supermassive black hole in the center of the M87 galaxy. These images were produced using 230 GHz observations performed in 2017 April. Additional observations are required to investigate the persistence of the primary image feature—a ring with azimuthal brightness asymmetry—and to quantify the image variability on event horizon scales. To address this need, we analyze M87* data collected with prototype EHT arrays in 2009, 2011, 2012, and 2013. While these observations do not contain enough information to produce images, they are sufficient to constrain simple geometric models. We develop a modeling approach based on the framework utilized for the 2017 EHT data analysis and validate our procedures using synthetic data. Applying the same approach to the observational data sets, we find the M87* morphology in 2009–2017 to be consistent with a persistent asymmetric ring of ~40 μas diameter. The position angle of the peak intensity varies in time. In particular, we find a significant difference between the position angle measured in 2013 and 2017. These variations are in broad agreement with predictions of a subset of general relativistic magnetohydrodynamic simulations. We show that quantifying the variability across multiple observational epochs has the potential to constrain the physical properties of the source, such as the accretion state or the black hole spin

First M87 Event Horizon Telescope Results. VI. The Shadow and Mass of the Central Black Hole

We present measurements of the properties of the central radio source in M87 using Event Horizon Telescope data obtained during the 2017 campaign. We develop and fit geometric crescent models (asymmetric rings with interior brightness depressions) using two independent sampling algorithms that consider distinct representations of the visibility data. We show that the crescent family of models is statistically preferred over other comparably complex geometric models that we explore. We calibrate the geometric model parameters using general relativistic magnetohydrodynamic (GRMHD) models of the emission region and estimate physical properties of the source. We further fit images generated from GRMHD models directly to the data. We compare the derived emission region and black hole parameters from these analyses with those recovered from reconstructed images. There is a remarkable consistency among all methods and data sets. We find that >50% of the total flux at arcsecond scales comes from near the horizon, and that the emission is dramatically suppressed interior to this region by a factor >10, providing direct evidence of the predicted shadow of a black hole. Across all methods, we measure a crescent diameter of 42 +/- 3 mu as and constrain its fractional width to b

First M87 Event Horizon Telescope Results. VI. The Shadow and Mass of the Central Black Hole

We present measurements of the properties of the central radio source in M87 using Event Horizon Telescope data obtained during the 2017 campaign. We develop and fit geometric crescent models (asymmetric rings with interior brightness depressions) using two independent sampling algorithms that consider distinct representations of the visibility data. We show that the crescent family of models is statistically preferred over other comparably complex geometric models that we explore. We calibrate the geometric model parameters using general relativistic magnetohydrodynamic (GRMHD) models of the emission region and estimate physical properties of the source. We further fit images generated from GRMHD models directly to the data. We compare the derived emission region and black hole parameters from these analyses with those recovered from reconstructed images. There is a remarkable consistency among all methods and data sets. We find that >50% of the total flux at arcsecond scales comes from near the horizon, and that the emission is dramatically suppressed interior to this region by a factor >10, providing direct evidence of the predicted shadow of a black hole. Across all methods, we measure a crescent diameter of 42 +/- 3 mu as and constrain its fractional width to be <0.5. Associating the crescent feature with the emission surrounding the black hole shadow, we infer an angular gravitational radius of GM/Dc(2) = 3.8 +/- 0.4 mu as. Folding in a distance measurement of 16.8(-0.7)(+0.8) gives a black hole mass of M = 6.5. 0.2 vertical bar(stat) +/- 0.7 vertical bar(sys) x 10(9) M-circle dot. This measurement from lensed emission near the event horizon is consistent with the presence of a central Kerr black hole, as predicted by the general theory of relativity