Interferon-b Modulates Inflammatory Response in Cerebral Ischemia

BACKGROUND: Stroke is a leading cause of death in the world. In >80% of strokes, the initial acute phase of ischemic injury is due to the occlusion of a blood vessel resulting in severe focal hypoperfusion, excitotoxicity, and oxidative damage. Interferon-β (IFNβ), a cytokine with immunomodulatory properties, was approved by the US Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis for more than a decade. Its anti-inflammatory properties and well-characterized safety profile suggest that IFNβ has therapeutic potential for the treatment of ischemic stroke. METHODS AND RESULTS: We investigated the therapeutic effect of IFNβ in the mouse model of transient middle cerebral artery occlusion/reperfusion. We found that IFNβ not only reduced infarct size in ischemic brains but also lessened neurological deficits in ischemic stroke animals. Further, multiple molecular mechanisms by which IFNβ modulates ischemic brain inflammation were identified. IFNβ reduced central nervous system infiltration of monocytes/macrophages, neutrophils, CD4(+) T cells, and γδ T cells; inhibited the production of inflammatory mediators; suppressed the expression of adhesion molecules on brain endothelial cells; and repressed microglia activation in the ischemic brain. CONCLUSIONS: Our results demonstrate that IFNβ exerts a protective effect against ischemic stroke through its anti-inflammatory properties and suggest that IFNβ is a potential therapeutic agent, targeting the reperfusion damage subsequent to the treatment with tissue plasminogen activator

Determining exciton bandwidth and film microstructure in polythiophene films using linear absorption spectroscopy

We analyze the linear absorption spectrum of regioregular poly(3-hexylthiophene) films spun from a variety of solvents to probe directly the film microstructure and how it depends on processing conditions. We estimate the exciton bandwidth and the percentage of the film composed of aggregates quantitatively using a weakly interacting H-aggregate model. This provides a description of the degree and quality of crystallites within the film and is in turn correlated with thin-film field-effect transistor characteristics.Comment: Applied Physics Letters (in press); 9 pages, three figure

Isolation of Mouse Cerebral Microvasculature for Molecular and Single-Cell Analysis

Brain microvasculature forms a specialized structure, the blood-brain barrier (BBB), to maintain homeostasis and integrity of the central nervous system (CNS). The BBB dysfunction is emerging as a critical contributor to multiple neurological disorders, including stroke, traumatic brain injury, autoimmune multiple sclerosis, and neurodegenerative diseases. The brain microvasculature exhibits highly cellular and regional heterogeneity to accommodate dynamic changes of microenvironment during homeostasis and diseases. Thus, investigating the underlying mechanisms that contribute to molecular or cellular changes of the BBB is a significant challenge. Here, we describe an optimized protocol to purify microvessels from the mouse cerebral cortex using mechanical homogenization and density-gradient centrifugation, while maintaining the structural integrity and functional activity of the BBB. We show that the isolated microvessel fragments consist of BBB cell populations, including endothelial cells, astrocyte end-feet, pericytes, as well as tight junction proteins that seal endothelial cells. Furthermore, we describe the procedures to generate single-cell suspensions from isolated microvessel fragments. We demonstrate that cells in the single-cell suspensions are highly viable and suitable for single-cell RNA-sequencing analysis. This protocol does not require transgenic mice and cell sorting equipment to isolate fluorescence-labeled endothelial cells. The optimized procedures can be applied to different disease models to generate viable cells for single-cell analysis to uncover transcriptional or epigenetic landscapes of BBB component cells

Dimethyl fumarate attenuates reactive microglia and long-term memory deficits following systemic immune challenge

BACKGROUND: Systemic inflammation is associated with increased cognitive decline and risk for Alzheimer's disease. Microglia (MG) activated during systemic inflammation can cause exaggerated neuroinflammatory responses and trigger progressive neurodegeneration. Dimethyl fumarate (DMF) is a FDA-approved therapy for multiple sclerosis. The immunomodulatory and anti-oxidant properties of DMF prompted us to investigate whether DMF has translational potential for the treatment of cognitive impairment associated with systemic inflammation. METHODS: Primary murine MG cultures were stimulated with lipopolysaccharide (LPS) in the absence or presence of DMF. MG cultured from nuclear factor (erythroid-derived 2)-like 2-deficient (Nrf2 -/- ) mice were used to examine mechanisms of DMF actions. Conditioned media generated from LPS-primed MG were used to treat hippocampal neuron cultures. Adult C57BL/6 and Nrf2 -/- mice were subjected to peripheral LPS challenge. Acute neuroinflammation, long-term memory function, and reactive astrogliosis were examined to assess therapeutic effects of DMF. RESULTS: DMF suppressed inflammatory activation of MG induced by LPS. DMF suppressed NF-κB activity through Nrf2-depedent and Nrf2-independent mechanisms in MG. DMF treatment reduced MG-mediated toxicity towards neurons. DMF suppressed brain-derived inflammatory cytokines in mice following peripheral LPS challenge. The suppressive effect of DMF on neuroinflammation was blunted in Nrf2 -/- mice. Importantly, DMF treatment alleviated long-term memory deficits and sustained reactive astrogliosis induced by peripheral LPS challenge. DMF might mitigate neurotoxic astrocytes associated with neuroinflammation. CONCLUSIONS: DMF treatment might protect neurons against toxic microenvironments produced by reactive MG and astrocytes associated with systemic inflammation

Efficient Prodrug Activator Gene Therapy by Retroviral Replicating Vectors Prolongs Survival in an Immune-Competent Intracerebral Glioma Model.

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials

Analysis of the functional conservation of ethylene receptors between maize and Arabidopsis

Ethylene, a regulator of plant growth and development, is perceived by specific receptors that act as negative regulators of the ethylene response. Five ethylene receptors, i.e., ETR1, ERS1, EIN4, ETR2, and ERS2, are present in Arabidopsis and dominant negative mutants of each that confer ethylene insensitivity have been reported. In contrast, maize contains just two types of ethylene receptors: ZmERS1, encoded by ZmERS1a and ZmERS1b, and ZmETR2, encoded by ZmETR2a and ZmETR2b. In this study, we introduced a Cys to Tyr mutation in the transmembrane domain of ZmERS1b and ZmETR2b that is present in the etr1-1 dominant negative mutant and expressed each protein in Arabidopsis. Mutant Zmers1b and Zmetr2b receptors conferred ethylene insensitivity and Arabidopsis expressing Zmers1b or Zmetr2b were larger and exhibited a delay in leaf senescence characteristic of ethylene insensitive Arabidopsis mutants. Zmers1b and Zmetr2b were dominant and functioned equally well in a hemizygous or homozygous state. Expression of the Zmers1b N-terminal transmembrane domain was sufficient to exert dominance over endogenous Arabidopsis ethylene receptors whereas the Zmetr2b N-terminal domain failed to do so. Neither Zmers1b nor Zmetr2b functioned in the absence of subfamily 1 ethylene receptors, i.e., ETR1 and ERS1. These results suggest that Cys65 in maize ZmERS1b and ZmETR2b plays the same role that it does in Arabidopsis receptors. Moreover, the results demonstrate that the mutant maize ethylene receptors are functionally dependent on subfamily 1 ethylene receptors in Arabidopsis, indicating substantial functional conservation between maize and Arabidopsis ethylene receptors despite their sequence divergence

Methodology for automatic fixture design in a computer-integrated environment

The objective of this research is to develop a general methodology for automatic fixture design (AFD) concerning the automation and integration issues in a computer environment. The research effort covers several major topics. These topics are studied as general methodology which can also be extended to solve other manufacturing problems. First, a set of proper workpiece orientations is determined. The orientation information is important for the planning of workholding, machining sequence, and production scheduling. To select the required workpiece orientations for all manufacturing activities, information about the workpiece geometry, the available manufacturing tools, and the job specification in general is considered in the analysis procedure. Second, a methodology for automatic workholding configuration is developed to generate the fixturing points for each workpiece orientation. The Projective Spatial Occupancy Enumeration (PSOE) approach is applied to develop the configuration algorithms which can automatically generate the fixturing points for any workpiece with general (prismatic or non-prismatic) shape. Third, an automatic workholding verification model is provided to verify the fixturing results for any workpiece. This model is developed under the domain of the general workholding; therefore, it can be used to verify other holding applications, such as the robot-hand (grasping) or mechanical manipulators. The model is developed by using non-linear programming to incorporate the fixturing conditions and constraints. These conditions include the force equilibrium, the frictional affects, the workpiece geometry constraints, and the fixturing force limits. Fixture design is one of numerous tasks in a complete manufacturing planning procedure. The AFD results are influenced by planning decisions other than fixturing, and are also used as inputs for these planning activities. Therefore, information integration between the AFD system and the other computer assisted design and planning modules are studied as the fourth research topic. In order to increase the degree of global automation and eliminate the human interaction for information transformation, an integration system shell, called MetaDesigner, is developed. The results of this research provide a global methodology to solve the automation and integration issues in fixture design

Surfaca and bulk melting of small metal clusters

We present an analytical solution to the two-parabola Landau model, applied to melting of metal particles with sizes in the nanoscale range. The results provide an analytical understanding of the recently observed pseudo-crystalline phase of nanoscale Sn particles. Liquid skin formation as a precursor of melting is found to occur only for particles with radii, greater than an explicitly given critical radius. The size effect of the melting temperature and the latent heat has been calculated and quantitative agreement with experiments on Sn particles was found