Macromolecular Crowding and Size Effects on Probe Microviscosity

Development of biologically relevant crowding solutions necessitates improved understanding of how the relative size and density of mobile obstacles affect probe diffusion. Both the crowding density and relative size of each co-solute in a mixture will contribute to the measured microviscosity as assessed by altered translational mobility. Using multiphoton fluorescent correlation spectroscopy, this study addresses how excluded volume of dextran polymers from 10 to 500 kDa affect microviscosity quantified by measurements of calmodulin labeled with green fluorescent protein as the diffusing probe. Autocorrelation functions were fit using both a multiple-component model with maximum entropy method (MEMFCS) and an anomalous model. Anomalous diffusion was not detected, but fits of the data with the multiple-component model revealed separable modes of diffusion. When the dominant mode of diffusion from the MEMFCS analysis was used, we observed that increased excluded volume slows probe mobility as a simple exponential with crowder concentration. This behavior can be modeled with a single parameter, β, which depends on the dextran size composition. Two additional modes of diffusion were observed using MEMFCS and were interpreted as unique microviscosities. The fast mode corresponded to unhindered free diffusion as in buffer, whereas the slower agreed well with the bulk viscosity. At 10% crowder concentration, one finds a microviscosity approximately three times that of water, which mimics that reported for intracellular viscosity

Design and Development of a Low-Cost, High Resolution, High Mobility Acceleration Data Logger

To understand the forces exerted on structures during tsunamis, model cities are subjected to human-generated tsunamis. The study of vertical evacuation, wherein individuals could take refuge in the upper levels of strong buildings, has resulted in a growing need for a low cost wireless acceleration data logger. Such a device could be placed inside the walls of scale buildings to measure the forces encountered. This document describes the development and implementation of a suitable module. The major implication of this project is that researchers can more accurately record the forces that are exerted on structures and better understand the types of construction that best withstand tsunamis

An exploration of function analysis and function allocation in the commercial flight domain

The applicability is explored of functional analysis methods to support cockpit design. Specifically, alternative techniques are studied for ensuring an effective division of responsibility between the flight crew and automation. A functional decomposition is performed of the commercial flight domain to provide the information necessary to support allocation decisions and demonstrate methodology for allocating functions to flight crew or to automation. The function analysis employed 'bottom up' and 'top down' analyses and demonstrated the comparability of identified functions, using the 'lift off' segment of the 'take off' phase as a test case. The normal flight mission and selected contingencies were addressed. Two alternative methods for using the functional description in the allocation of functions between man and machine were investigated. The two methods were compared in order to ascertain their relative strengths and weaknesses. Finally, conclusions were drawn regarding the practical utility of function analysis methods

Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes

Prior exposure to an attenuated Listeria vaccine does not reduce immunogenicity: pre-clinical assessment of the efficacy of a Listeria vaccine in the induction of immune responses against HIV

Abstract Background We have evaluated an attenuated Listeria monocytogenes (Lm) candidate vaccine vector in nonhuman primates using a delivery regimen relying solely on oral vaccination. We sought to determine the impact of prior Lm vector exposure on the development of new immune responses against HIV antigens. Findings Two groups of rhesus macaques one Lm naive, the other having documented prior Lm vector exposures, were evaluated in response to oral inoculations of the same vector expressing recombinant HIV-1 Gag protein. The efficacy of the Lm vector was determined by ELISA to assess the generation of anti-Listerial antibodies; cellular responses were measured by HIV-Gag specific ELISpot assay. Our results show that prior Lm exposures did not diminish the generation of de novo cellular responses against HIV, as compared to Listeria-naïve monkeys. Moreover, empty vector exposures did not elicit potent antibody responses, consistent with the intracellular nature of Lm. Conclusions The present study demonstrates in a pre-clinical vaccine model, that prior oral immunization with an empty Lm vector does not diminish immunogenicity to Lm-expressed HIV genes. This work underscores the need for the continued development of attenuated Lm as an orally deliverable vaccine

Toxicology and Biodistribution Studies for MGH2.1, an Oncolytic Virus that Expresses Two Prodrug-activating Genes, in Combination with Prodrugs

MGH2.1 is a herpes simplex virus type 1 (HSV1) oncolytic virus that expresses two prodrug-activating transgenes: the cyclophosphamide (CPA)-activating cytochrome P4502B1 (CYP2B1) and the CPT11-activating secreted human intestinal carboxylesterase (shiCE). Toxicology and biodistribution of MGH2.1 in the presence/absence of prodrugs was evaluated in mice. MGH2.1 ± prodrugs was cytotoxic to human glioma cells, but not to normal cells. Pharmacokinetically, intracranial MGH2.1 did not significantly alter the metabolism of intraperitoneally (i.p.) administered prodrugs in mouse plasma, brain, or liver. MGH2.1 did not induce an acute inflammatory reaction. MGH2.1 DNA was detected in brains of mice inoculated with 108 pfus for up to 60 days. However, only one animal showed evidence of viral gene expression at this time. Expression of virally encoded genes was restricted to brain. Intracranial inoculation of MGH2.1 did not induce lethality at 108 pfus in the absence of prodrugs and at 106 pfus in the presence of prodrugs. This study provides safety and toxicology data justifying a possible clinical trial of intratumoral injection of MGH2.1 with peripheral administration of CPA and/or CPT11 prodrugs in humans with malignant gliomas

350 mV, 5 GHz Class-D Enhanced Swing Differential and Quadrature VCOs in 65 nm CMOS

A new enhanced swing class-D VCO which operates from a supply voltage as low as 300 mV is presented. The architectural advantages are described along with an analysis for the oscillation frequency. Prototype differential and quadrature variants of the proposed VCO have been implemented in a 65 nm RF CMOS process with a 5 GHz VCO oscillation frequency. At a 350 mV supply, the measured phase noise performance for the quadrature VCO with a 5% tuning range is -137.1 dBc/Hz at 3 MHz offset with a power dissipation of 2.1 mW from a 0.35 V supply. The highest resulting figure-of-merit (FoM) is 198.3 dBc/Hz.©2015 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other users, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works for resale or redistribution to servers or lists, or reuse of any copyrighted components of this work in other works

Community as a source of health in three racial/ethnic communities in Oregon: a qualitative study

BACKGROUND: A 2011 report by the Oregon Health Authority and the Department of Human Services documented disparities in its Latino and American Indian populations on multiple individual-level health indicators. However, research is lacking on the social contexts in which Latinos and American Indians in Oregon live and how these environments influence the health of communities as a whole. To help fill this gap, this study sought to contextualize the social environments that influence the health of Latinos and American Indian residents in three Oregon communities. METHODS: Guided by an ecological framework, we conducted one-time semi-structured qualitative interviews with 26 study participants to identify the prominent health-related issues in the communities and to examine the factors that study participants perceived as enabling or inhibiting healthy lifestyles of community residents. We used a grounded theory approach to perform content and thematic analyses of the data. RESULTS: Study participants identified preventable chronic conditions, such as diabetes, obesity, and hypertension, as the most pressing health concerns in their communities. Results showed that traditional and cultural activities and strong family and community cohesion were viewed as facilitators of good community health. Poverty, safety concerns, insufficient community resources, and discrimination were perceived as barriers to community health. Three themes emerged from the thematic analyses: social connectedness is integral to health; trauma has an ongoing negative impact on health; and invisibility of residents in the community underlies poor health. CONCLUSIONS: This study’s findings provide insight to the social contexts which operate in the lives of some Latinos and American Indians in Oregon. While participants identified community-level factors as important to health, they focused more on the social connections of individuals to each other and the relationships that residents have with their communities at-large. Our findings may also help to explain how the intra- and inter-personal levels, the community/institutional level, and the macro level/public policy contexts can serve to influence health in these communities. For example, trauma and invisibility are not routinely examined in community health assessment and improvement planning activities; nonetheless, these factors appear to be at play affecting the health of residents.Keywords: American Indian, Latino, Migrant and seasonal farmworkers, Trauma, Community healt

Oncolytic HSV Vectors and Anti-Tumor Immunity

The therapeutic promise of oncolytic viruses (OVs) rests on their ability to both selectively kill tumor cells and induce anti-tumor immunity. The potential of tumors to be recognized and eliminated by an effective anti-tumor immune response has been spurred on by the discovery that immune checkpoint inhibition can overcome tumor-specific cytotoxic T cell (CTL) exhaustion and provide durable responses in multiple tumor indications. OV-mediated tumor destruction is now recognized as a powerful means to assist in the development of anti-tumor immunity for two important reasons: (i) OVs, through the elicitation of an anti-viral response and the production of type I interferon, are potent stimulators of inflammation and can be armed with transgenes to further enhance anti-tumor immune responses; and (ii) lytic activity can promote the release of tumor-associated antigens (TAAs) and tumor neoantigens that function as in situ tumor-specific vaccines to elicit adaptive immunity. Oncolytic herpes simplex viruses (oHSVs) are among the most widely studied OVs for the treatment of solid malignancies, and Amgen's oHSV Imlygic® for the treatment of melanoma is the only OV approved in major markets. Here we describe important biological features of HSV that make it an attractive OV, clinical experience with HSV-based vectors, and strategies to increase applicability to cancer treatment