Macrophages form functional vascular mimicry channels in vivo.

Macrophages, key cells of the innate immune system, are known to support angiogenesis but are not believed to directly form vessel walls. Here we show that macrophages structurally form primitive, NON-ENDOTHELIAL "vessels" or vascular mimicry (VM) channels in both tumor and angiogenesis in vivo models. These channels are functionally connected to the systemic vasculature as they are perfused by intravenously injected dye. Since both models share hypoxic micro-environments, we hypothesized that hypoxia may be an important mediator of VM formation. Indeed, conditional genetic depletion of myeloid-specific HIF-1α results in decreased VM network formation, dye perfusion and tumor size. Although the macrophage VM network shares some features with an endothelial vasculature, it is ultrastructurally different. Cancer stem cells have been shown to form vascular mimicry channels. Our data demonstrates that tumor-associated macrophages also form them. The identification of this novel type of vascular mimicry may help in the development of targeted cancer therapeutics.

Cyclophilin Inhibitors Remodel the Endoplasmic Reticulum of HCV-Infected Cells in a Unique Pattern Rendering Cells Impervious to a Reinfection.

The mechanisms of action by which cyclophilin inhibitors (CypI) interfere with the HCV life cycle remain poorly understood. We reported that CypI and NS5A inhibitors (NS5Ai), but not other classes of anti-HCV agents, prevent assembly of double membrane vesicles (DMVs), which protect replication complexes. We demonstrated that both NS5A and the isomerase cyclophilin A (CypA) are required for DMV formation. Here, we examined whether CypI mediate an additional antiviral effect that could further explain the high efficacy of CypI. We identified a unique action of CypI. CypI remodel the organization of the endoplasmic reticulum (ER) of HCV-infected cells, but not of uninfected cells. This effect is specific since it was not observed for other classes of anti-HCV agents including NS5Ai, and has no effect on the viability of CypI-treated cells. Since ER serves as platform for the establishment of HCV replication complexes, we asked whether the ER reorganization by CypI would prevent cells from being newly infected. Remarkably, CypI-treated HCV-pre-infected cells remain totally impervious to a reinfection, suggesting that the CypI-mediated ER reorganization prevents a reinfection. This block is not due to residual CypI since CypI-resistant HCV variants also fail to infect these cells. The ER reorganization by CypI is rapid and reversible. This study provides the first evidence that CypI trigger a unique ER reorganization of infected cells, rendering cells transiently impervious to a reinfection. This study further suggests that the HCV-induced ER rearrangement represents a key target for the development of new therapies.

Approaching rational epitope vaccine design for hepatitis C virus with meta-server and multivalent scaffolding.

Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314-324) and E2 (residues 412-423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a "scaffolding meta-server" to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a "multivalent scaffolding" approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.

Assembly of the Ebola Virus Nucleoprotein from a Chaperoned VP35 Complex.

Ebolavirus NP oligomerizes into helical filaments found at the core of the virion, encapsidates the viral RNA genome, and serves as a scaffold for additional viral proteins within the viral nucleocapsid. We identified a portion of the phosphoprotein homolog VP35 that binds with high affinity to nascent NP and regulates NP assembly and viral genome binding. Removal of the VP35 peptide leads to NP self-assembly via its N-terminal oligomerization arm. NP oligomerization likely causes a conformational change between the NP N- and C-terminal domains, facilitating RNA binding. These functional data are complemented by crystal structures of the NP°-VP35 complex at 2.4 Å resolution. The interactions between NP and VP35 illuminated by these structures are conserved among filoviruses and provide key targets for therapeutic intervention.

Coxsackievirus can exploit LC3 in both autophagy-dependent and -independent manners in vivo.

RNA viruses modify intracellular membranes to produce replication scaffolds. In pancreatic cells, coxsackievirus B3 (CVB3) hijacks membranes from the autophagy pathway, and in vivo disruption of acinar cell autophagy dramatically delays CVB3 replication. This is reversed by expression of GFP-LC3, indicating that CVB3 may acquire membranes from an alternative, autophagy-independent, source(s). Herein, using 3 recombinant CVB3s (rCVB3s) encoding different proteins (proLC3, proLC3(G120A), or ATG4B(C74A)), we show that CVB3 is, indeed, flexible in its utilization of cellular membranes. When compared with a control rCVB3, all 3 viruses replicated to high titers in vivo, and caused severe pancreatitis. Most importantly, each virus appeared to subvert membranes in a unique manner. The proLC3 virus produced a large quantity of LC3-I which binds to phosphatidylethanolamine (PE), affording access to the autophagy pathway. The proLC3(G120A) protein cannot attach to PE, and instead binds to the ER-resident protein SEL1L, potentially providing an autophagy-independent source of membranes. Finally, the ATG4B(C74A) protein sequestered host cell LC3-I, causing accumulation of immature phagophores, and massive membrane rearrangement. Taken together, our data indicate that some RNA viruses can exploit a variety of different intracellular membranes, potentially maximizing their replication in each of the diverse cell types that they infect in vivo.

Disarming Pseudomonas aeruginosa virulence factor LasB by leveraging a Caenorhabditis elegans infection model.

The emergence of antibiotic resistance places a sense of urgency on the development of alternative antibacterial strategies, of which targeting virulence factors has been regarded as a "second generation" antibiotic approach. In the case of Pseudomonas aeruginosa infections, a proteolytic virulence factor, LasB, is one such target. Unfortunately, we and others have not been successful in translating in vitro potency of LasB inhibitors to in vivo efficacy in an animal model. To overcome this obstacle, we now integrate in silico and in vitro identification of the mercaptoacetamide motif as an effective class of LasB inhibitors with full in vivo characterization of mercaptoacetamide prodrugs using Caenorhabditis elegans. We show that one of our mercaptoacetamide prodrugs has a good selectivity profile and high in vivo efficacy, and confirm that LasB is a promising target for the treatment of bacterial infections. In addition, our work highlights that the C. elegans infection model is a user-friendly and cost-effective translational tool for the development of anti-virulence compounds.

Cyclophilin and NS5A inhibitors, but not other anti-hepatitis C virus (HCV) agents, preclude HCV-mediated formation of double-membrane-vesicle viral factories.

Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.

Comprehensive bioimaging with fluorinated nanoparticles using breathable liquids.

Fluorocarbons are lipophobic and non-polar molecules that exhibit remarkable biocompatibility, with applications in liquid ventilation and synthetic blood. The unique properties of these compounds have also enabled mass spectrometry imaging of tissues where the fluorocarbons act as a Teflon-like coating for nanostructured surfaces to assist in desorption/ionization. Here we report fluorinated gold nanoparticles (f-AuNPs) designed to facilitate nanostructure imaging mass spectrometry. Irradiation of f-AuNPs results in the release of the fluorocarbon ligands providing a driving force for analyte desorption. The f-AuNPs allow for the mass spectrometry analysis of both lipophilic and polar (central carbon) metabolites. An important property of AuNPs is that they also act as contrast agents for X-ray microtomography and electron microscopy, a feature we have exploited by infusing f-AuNPs into tissue via fluorocarbon liquids to facilitate multimodal (molecular and anatomical) imaging.

The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase.

Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2(-/-) mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2(-/-) mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2(-/-) brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.

Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.

Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.

ROS and Sympathetically Mediated Mitochondria Activation in Brown Adipose Tissue Contribute to Methamphetamine-Induced Hyperthermia.

Methamphetamine (Meth) abuse has been shown to induce alterations in mitochondrial function in the brain as well as to induce hyperthermia, which contributes to neurotoxicity and Meth-associated mortality. Brown adipose tissue (BAT), a thermogenic site known to be important in neonates, has recently regained importance since being identified in significant amounts and in correlation with metabolic balance in human adults. Given the high mitochondrial content of BAT and its role in thermogenesis, we aimed to investigate whether BAT plays any role in the development of Meth-induced hyperthermia. By ablating or denervating BAT, we identified a partial contribution of this organ to Meth-induced hyperthermia. BAT ablation decreased temperature by 0.5°C and reduced the length of hyperthermia by 1 h, compared to sham-operated controls. BAT denervation also affected the development of hyperthermia in correlation with decreased the expression of electron transport chain molecules, and increase on PCG1a levels, but without affecting Meth-induced uncoupling protein 1 upregulation. Furthermore, in isolated BAT cells in culture, Meth, but not Norepinephrine, induced H2O2 upregulation. In addition, we found that in vivo Reactive Oxygen Species (ROS) play a role in Meth hyperthermia. Thus, sympathetically mediated mitochondrial activation in the BAT and Meth-induced ROS are key components to the development of hyperthermia in Meth abuse.

Pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo.

Autophagy protects against many infections by inducing the lysosomal-mediated degradation of invading pathogens. However, previous in vitro studies suggest that some enteroviruses not only evade these protective effects but also exploit autophagy to facilitate their replication. We generated Atg5(f/f)/Cre(+) mice, in which the essential autophagy gene Atg5 is specifically deleted in pancreatic acinar cells, and show that coxsackievirus B3 (CVB3) requires autophagy for optimal infection and pathogenesis. Compared to Cre(-) littermates, Atg5(f/f)/Cre(+) mice had an ∼2,000-fold lower CVB3 titer in the pancreas, and pancreatic pathology was greatly diminished. Both in vivo and in vitro, Atg5(f/f)/Cre(+) acinar cells had reduced intracellular viral RNA and proteins. Furthermore, intracellular structural elements induced upon CVB3 infection, such as compound membrane vesicles and highly geometric paracrystalline arrays, which may represent viral replication platforms, were infrequently observed in infected Atg5(f/f)/Cre(+) cells. Thus, CVB3-induced subversion of autophagy not only benefits the virus but also exacerbates pancreatic pathology.

Characterization of circulating endothelial cells in acute myocardial infarction.

Acute myocardial infarction (MI), which involves the rupture of existing atheromatous plaque, remains highly unpredictable despite recent advances in the diagnosis and treatment of coronary artery disease. Accordingly, a clinical measurement that can predict an impending MI is desperately needed. Here, we characterize circulating endothelial cells (CECs) using an automated and clinically feasible CEC three-channel fluorescence microscopy assay in 50 consecutive patients with ST-segment elevation MI and 44 consecutive healthy controls. CEC counts were significantly elevated in MI cases versus controls, with median numbers of 19 and 4 cells/ml, respectively (P = 1.1 × 10(-10)). A receiver-operating characteristic (ROC) curve analysis demonstrated an area under the ROC curve of 0.95, suggesting near-dichotomization of MI cases versus controls. We observed no correlation between CECs and typical markers of myocardial necrosis (ρ = 0.02, creatine kinase-myocardial band; ρ = -0.03, troponin). Morphological analysis of the microscopy images of CECs revealed a 2.5-fold increase (P < 0.0001) in cellular area and a twofold increase (P < 0.0001) in nuclear area of MI CECs versus healthy controls, age-matched CECs, as well as CECs obtained from patients with preexisting peripheral vascular disease. The distribution of CEC images that contained from 2 to 10 nuclei demonstrates that MI patients were the only subject group to contain more than 3 nuclei per image, indicating that multicellular and multinuclear clusters are specific for acute MI. These data indicate that CEC counts may serve as a promising clinical measure for the prediction of atherosclerotic plaque rupture events.

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet.

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2-3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.

Fine-tuning of Drp1/Fis1 availability by AKAP121/Siah2 regulates mitochondrial adaptation to hypoxia.

Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.

Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo.

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.

Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture.

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

Short-term fasting induces profound neuronal autophagy.

Disruption of autophagy--a key homeostatic process in which cytosolic components are degraded and recycled through lysosomes--can cause neurodegeneration in tissue culture and in vivo. Upregulation of this pathway may be neuroprotective, and much effort is being invested in developing drugs that cross the blood brain barrier and increase neuronal autophagy. One well-recognized way of inducing autophagy is by food restriction, which upregulates autophagy in many organs including the liver; but current dogma holds that the brain escapes this effect, perhaps because it is a metabolically privileged site. Here, we have re-evaluated this tenet using a novel approach that allows us to detect, enumerate and characterize autophagosomes in vivo. We first validate the approach by showing that it allows the identification and characterization of autophagosomes in the livers of food-restricted mice. We use the method to identify constitutive autophagosomes in cortical neurons and Purkinje cells, and we show that short-term fasting leads to a dramatic upregulation in neuronal autophagy. The increased neuronal autophagy is revealed by changes in autophagosome abundance and characteristics, and by diminished neuronal mTOR activity in vivo, demonstrated by a reduction in levels of phosphorylated S6 ribosomal protein in Purkinje cells. The increased abundance of autophagosomes in Purkinje cells was confirmed using transmission electron microscopy. Our data lead us to speculate that sporadic fasting might represent a simple, safe and inexpensive means to promote this potentially therapeutic neuronal response.

Structural insights into Nox4 and Nox2: motifs involved in function and cellular localization.

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22(phox), creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H(2)O(2) production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22(phox) Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O(2)(-) generation. In contrast, Nox4 released H(2)O(2) and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.

Talin 1 and 2 are required for myoblast fusion, sarcomere assembly and the maintenance of myotendinous junctions.

Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we show that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking beta1 integrins. Talin 1/2-deficient myoblasts express functionally active beta1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.