Myoglobin induces mitochondrial fusion, thereby inhibiting breast cancer cell proliferation.

Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Furthermore, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

Modeling Herpes Simplex Virus 1 Infections in Human Central Nervous System Neuronal Cells Using Two- and Three-Dimensional Cultures Derived from Induced Pluripotent Stem Cells.

Herpes simplex virus 1 (HSV-1) establishes latency in both peripheral nerve ganglia and the central nervous system (CNS). The outcomes of acute and latent infections in these different anatomic sites appear to be distinct. It is becoming clear that many of the existing culture models using animal primary neurons to investigate HSV-1 infection of the CNS are limited and not ideal, and most do not recapitulate features of CNS neurons. Human induced pluripotent stem cells (hiPSCs) and neurons derived from them are documented as tools to study aspects of neuropathogenesis, but few have focused on modeling infections of the CNS. Here, we characterize functional two-dimensional (2D) CNS-like neuron cultures and three-dimensional (3D) brain organoids made from hiPSCs to model HSV-1-human-CNS interactions. Our results show that (i) hiPSC-derived CNS neurons are permissive for HSV-1 infection; (ii) a quiescent state exhibiting key landmarks of HSV-1 latency described in animal models can be established in hiPSC-derived CNS neurons; (iii) the complex laminar structure of the organoids can be efficiently infected with HSV, with virus being transported from the periphery to the central layers of the organoid; and (iv) the organoids support reactivation of HSV-1, albeit less efficiently than 2D cultures. Collectively, our results indicate that hiPSC-derived neuronal platforms, especially 3D organoids, offer an extraordinary opportunity for modeling the interaction of HSV-1 with the complex cellular and architectural structure of the human CNS.IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model acute and latent HSV-1 infections in two-dimensional (2D) and three-dimensional (3D) CNS neuronal cultures. We successfully established acute HSV-1 infections and infections showing features of latency. HSV-1 infection of the 3D organoids was able to spread from the outer surface of the organoid and was transported to the interior lamina, providing a model to study HSV-1 trafficking through complex neuronal tissue structures. HSV-1 could be reactivated in both culture systems; though, in contrast to 2D cultures, it appeared to be more difficult to reactivate HSV-1 in 3D cultures, potentially paralleling the low efficiency of HSV-1 reactivation in the CNS of animal models. The reactivation events were accompanied by dramatic neuronal morphological changes and cell-cell fusion. Together, our results provide substantive evidence of the suitability of hiPSC-based neuronal platforms to model HSV-1-CNS interactions in a human context.

A new key player in VEGF-dependent angiogenesis in human hepatocellular carcinoma: dimethylarginine dimethylaminohydrolase 1.

BACKGROUND: Anti-angiogenic therapies, targeting VEGF, are a promising treatment for hepatocellular carcinoma (HCC). To enhance this potential therapy, identification of novel targets in this pathway is of major interest. Nitric oxide (NO) plays a crucial role in VEGF-dependent angiogenesis. NO production depends on arginine as substrate and asymmetric dimethylarginine (ADMA) as inhibitor. Dimethylarginine dimethylaminohydrolase 1 (DDAH-1) catabolizes ADMA and therefore regulates NO and VEGF expression. This study unravels additional mechanisms to improve VEGF targeting therapies. METHODS: The expression of DDAH-1 was examined in HCC specimen and non-tumorous background liver of 20 patients undergoing liver resection. Subsequently, arginine/ADMA balance, NO production, and VEGF expression were analyzed. The influence of hypoxia on DDAH-1 and angiogenesis promoting factors was evaluated in HepG2 cells and primary human hepatocytes. RESULTS: DDAH-1 expression was significantly induced in primary HCC tumors compared to non-tumorous background liver. This was associated with an increased arginine/ADMA ratio, higher NO formation, and higher VEGF expression in human HCC compared to non-tumorous liver. Hypoxia induced DDAH-1, iNOS, and VEGF expression in a time-dependent manner in HepG2 cells. CONCLUSIONS: Our results indicate that DDAH-1 expression is increased in human HCC, which is associated with an increase in the arginine/ADMA ratio and enhanced NO formation. Hypoxia may be an initiating factor for the increase in DDAH-1 expression. DDAH-1 expression is associated with promotion of angiogenesis stimulating factor VEGF. Together, our findings for the first time identified DDAH-1 as a key player in the regulation of angiogenesis in human HCC, and by understanding this mechanism, future therapeutic strategies targeting VEGF can be improved.

Basics of Digital Microscopy.

Modern digital microscopy combines the equipment of classical light microscopy with a computerized imaging system. The technique comprises image formation by optics, image registration by a camera, and saving of image data in a computer file. This chapter describes limitations that are particular to each of these processes, including optical resolution, efficiency of image registration, characteristics of image file formats, and data management. Further suggestions are given which serve, in turn, to help construct a set of guidelines aimed at optimization of digital microscopic imaging. © 2018 by John Wiley & Sons, Inc.

Sickle Cell Trait Increases Red Blood Cell Storage Hemolysis and Post-Transfusion Clearance in Mice.

BACKGROUND: Transfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. METHODS: We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). FINDINGS: Human and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. INTERPRETATION: The common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage.

Platelet-derived HMGB1 is a critical mediator of thrombosis.

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.