Cell Surface Expression of the Vaccinia Virus Complement Control Protein is Mediated by Interaction with the Viral A56 Protein and Protects Infected Cells from Complement Attack

The vaccinia virus (VACV) complement control protein (VCP) is the major protein secreted from VACV-infected cells. It has been reported that VCP binds to the surfaces of uninfected cells by interacting with heparan sulfate proteoglycans (HSPGs). In this study, we show that VCP is also expressed on the surfaces of infected cells and demonstrate that surface localization occurs independently of HSPGs. Since VCP does not contain a transmembrane domain, we hypothesized that VCP interacts with a membrane protein that localizes to the infected-cell surface. We show that the VACV A56 membrane protein is necessary for the cell surface expression of VCP and demonstrate that VCP and A56 interact in VACV-infected cells. Since the surface expression of VCP was abrogated by reducing agents, we examined the contribution of an unpaired cysteine residue on VCP to VCP surface expression and VCP\u27s interaction with A56. To do this, we mutated the unpaired cysteine in VCP and generated a recombinant virus expressing the altered form of VCP. Following the infection of cells with the mutant virus, VCP was neither expressed on the cell surface nor able to interact with A56. Importantly, the cell surface expression of VCP was found to protect infected cells from complement-mediated lysis. Our findings suggest a new function for VCP that may be important for poxvirus pathogenesis and impact immune responses to VACV-based vaccines

The Vaccinia Virus Complement Control Protein Modulates Adaptive Immune Responses during Infection

Complement activation is an important component of the innate immune response against viral infection and also shapes adaptive immune responses. Despite compelling evidence that complement activation enhances T cell and antibody (Ab) responses during viral infection, it is unknown whether inhibition of complement by pathogens alters these responses. Vaccinia virus (VACV) modulates complement activation by encoding a complement regulatory protein called the vaccinia virus complement control protein (VCP). Although VCP has been described as a virulence factor, the mechanisms by which VCP enhances VACV pathogenesis have not been fully defined. Since complement is necessary for optimal adaptive immune responses to several viruses, we hypothesized that VCP contributes to pathogenesis by modulating anti-VACV T cell and Ab responses. In this study, we used an intradermal model of VACV infection to compare pathogenesis of wild-type virus (vv-VCPwt) and a virus lacking VCP (vv-VCPko). vv-VCPko formed smaller lesions in wild-type mice but not in complement-deficient mice. Attenuation of vv-VCPko correlated with increased accumulation of T cells at the site of infection, enhanced neutralizing antibody responses, and reduced viral titers. Importantly, depleting CD8(+) T cells together with CD4(+) T cells, which also eliminated T helper cell-dependent Ab responses, restored vv-VCPko to wild-type levels of virulence. These results suggest that VCP contributes to virulence by dampening both antibody and T cell responses. This work provides insight into how modulation of complement by poxviruses contributes to virulence and demonstrates that a pathogen-encoded complement regulatory protein can modulate adaptive immunity

Organization of GC/MS and LC/MS metabolomics data into chemical libraries

<p>Abstract</p> <p>Background</p> <p>Metabolomics experiments involve generating and comparing small molecule (metabolite) profiles from complex mixture samples to identify those metabolites that are modulated in altered states (e.g., disease, drug treatment, toxin exposure). One non-targeted metabolomics approach attempts to identify and interrogate all small molecules in a sample using GC or LC separation followed by MS or MSⁿdetection. Analysis of the resulting large, multifaceted data sets to rapidly and accurately identify the metabolites is a challenging task that relies on the availability of chemical libraries of metabolite spectral signatures. A method for analyzing spectrometry data to identify and <b>Qu</b>antify <b>I</b>ndividual <b>C</b>omponents in a <b>S</b>ample, (QUICS), enables generation of chemical library entries from known standards and, importantly, from unknown metabolites present in experimental samples but without a corresponding library entry. This method accounts for all ions in a sample spectrum, performs library matches, and allows review of the data to quality check library entries. The QUICS method identifies ions related to any given metabolite by correlating ion data across the complete set of experimental samples, thus revealing subtle spectral trends that may not be evident when viewing individual samples and are likely to be indicative of the presence of one or more otherwise obscured metabolites.</p> <p>Results</p> <p>LC-MS/MS or GC-MS data from 33 liver samples were analyzed simultaneously which exploited the inherent biological diversity of the samples and the largely non-covariant chemical nature of the metabolites when viewed over multiple samples. Ions were partitioned by both retention time (RT) and covariance which grouped ions from a single common underlying metabolite. This approach benefitted from using mass, time and intensity data in aggregate over the entire sample set to reject outliers and noise thereby producing higher quality chemical identities. The aggregated data was matched to reference chemical libraries to aid in identifying the ion set as a known metabolite or as a new unknown biochemical to be added to the library.</p> <p>Conclusion</p> <p>The QUICS methodology enabled rapid, in-depth evaluation of all possible metabolites (known and unknown) within a set of samples to identify the metabolites and, for those that did not have an entry in the reference library, to create a library entry to identify that metabolite in future studies.</p

Body Fat Percentage and Hormonal Intrauterine Device Use Are Independently Associated with Self-Reported Menstrual Regularity in Young Adult Females

Menstrual regularity is a key indicator of energy availability, long-term bone density, and other important health information in females. The occurrence of a regular menstrual cycle indicates that an individual’s level of estrogen is supportive of strong bones and that they are achieving the caloric intake required to support their activity level. In contrast, an irregular menstrual cycle can be indicative of insufficient energy availability which may, over time, result in low bone mineral density and thus a higher risk of bone stress injuries. However, hormonal contraceptive use, including the rising use of intrauterine devices (IUDs), may mask these changes in menstrual regularity. PURPOSE: The purpose of this study was to examine factors related to self-reported menstrual regularity among a population of young, generally healthy females. METHODS: Participants were included if they were no more than 50 years of age at the time of enrollment and had less than 50% body fat as assessed via dual-energy x-ray absorptiometry (DXA). Participants were asked via questionnaire if they reported having a regular menstrual cycle, defined as menstrual periods occurring at predictable intervals and no missed periods in the past six months. Additionally, participants were asked if they were currently using any form of hormonal contraception, and if so, what type. A logistic regression was run with menstrual regularity (1 = regular; 0 = irregular) as the dependent variable and body fat percentage (BFP) and contraceptive type as the predictors. RESULTS: Out of the 76 participants (mean±SD age: 23.2±5.1 years; height: 164.5±6.5 cm; weight: 65.2±13.6 kg; BFP: 32.3±8.5%), 54 (71%) reported having a regular menstrual cycle. Of the 45 (59%) participants using hormonal contraception, 27 (60%) used a combined oral contraceptive pill, six (13%) used a progestin-only pill, nine (20%) used an IUD, two (4%) used a hormonal implant, and one (2%) used a vaginal ring. Overall, a higher BFP was associated with a greater likelihood of menstrual regularity (coefficient±SE: 0.08 ± 0.04; p = 0.04) while IUD use was associated with a lower likelihood (coefficient±SE: -1.8 ± 0.9; p = 0.04). No other hormonal contraception type was independently associated with self-reported menstrual regularity. CONCLUSION: These results collectively suggest, within a population of generally healthy, young adult females, that lower BFP and hormonal IUD use are both independently associated with a lower likelihood of having a regular menstrual cycle. When assessing the lack of a regular menstrual cycle, practitioners may consider hormonal IUD use as one potential factor in addition to a general assessment of body composition and energy availability. However, this analysis was limited by a relatively small sample size, which may have reduced the ability to detect the relationship between menstrual regularity and less commonly used contraceptive types. Future research is required to determine the relationship between these contraceptive types and menstrual regularity in generally healthy adult females

Thermal Damage on LX-04 Mock Material and Gas Permeability Assessment

RM-04-BR, a mock material for the plastic-bonded HMX-based explosive LX-04, is characterized after being thermally damaged at 140 C and 190 C. We measured the following material properties before and after the thermal experiments: sample volume, density, sound speed, and gas permeability in the material. Thermal treatment of the mock material leads to de-coloring and insignificant weight loss. Sample expanded, resulting in density reductions of 1.0% to 2.5% at 140 C and 190 C, respectively. Permeability in the mock samples was found to increase from 10{sup -15} to 10{sup -16} m{sup 2}, as the porosity increased. The permeability measurements are well represented by the Blake-Kozeny equation for laminar flow through porous media. The results are similar to the gas permeability in PBX-9501 obtained by other researchers

The Effects of Severity of Losses of Well Clear on Minimum Operations Performance Standards End-To-End Verification and Validation Simulation Study for Integrating Unmanned Aircraft Systems into the National Airspace System Using Detect and Avoid Systems

As Unmanned Aircraft Systems (UAS) make their way to mainstream aviation operations within the National Airspace System (NAS), research efforts are underway to develop a safe and effective environment for their integration into the NAS. Detect and Avoid (DAA) systems are required to account for the lack of eyes in the sky due to having no human on-board the aircraft. The technique, results, and lessons learned from a detailed End-to-End Verification and Validation (E2-V2) simulation study of a DAA system representative of RTCA Special Committee(SC)-228s proposed Phase I DAA Minimum Operational Performance Standards (MOPS), based on specific test vectors and encounter cases, will be presented in this paper

ALE3D Simulation and Measurement of Violence in a Fast Cookoff Experiment for LX-10

Fast cookoff is of interest in the areas of fire hazard reduction and the development of directed energy systems for defense. During a fast cookoff (thermal explosion), high heat fluxes cause rapid temperature increases and ignition in thin boundary layers. We are developing ALE3D models to describe the thermal, chemical, and mechanical behavior during the heating, ignition, and explosive phases. The candidate models and numerical strategies are being evaluated using benchmark cookoff experiments. Fast cookoff measurements were made in a Scaled-Thermal-Explosion-eXperiment (STEX) for LX-10 (94.7% HMX, 5.3% Viton A) confined in a 4130 steel tube with reinforced end caps. Gaps were present at the side and top of the explosive charge to allow for thermal expansion. The explosive was heated until explosion using radiant heaters. Temperatures were measured using thermocouples positioned on the tube wall and in the explosive. During the explosion, the tube expansion and fragment velocities were measured with strain gauges, Photonic-Doppler-Velocimeters (PDVs), and micropower radar units. A fragment size distribution was constructed from fragments captured in Lexan panels. ALE3D models for chemical, thermal, and mechanical behavior were developed for the heating and explosive processes. A multi-step chemical kinetics model is employed for the HMX while a one-step model is used for the Viton. A pressure-dependent deflagration model is employed during the expansion. A Steinberg-Guinan model represents the mechanical behavior of the solid constituents while polynomial and gamma-law expressions are used for the equation of state of the solid and gas species, respectively. Parameters for the kinetics model were specified using measurements of the One-Dimensional-Time-to-Explosion (ODTX), while measurements for burn rate were employed to determine parameters in the burn front model. The simulations include radiative and conductive transport across the dynamic gaps between the explosive charge and metal case. Model results are compared to measurements for the temperature fields, time to explosion, and wall expansion rates

LX-17 Deflagration at High Pressures and Temperatures

We measure the laminar deflagration rate of LX-17 (92.5 wt% TATB, 7.5 wt% Kel-F 800) at high pressure and temperature in a strand burner, thereby obtaining reaction rate data for prediction of thermal explosion violence. Simultaneous measurements of flame front time-of-arrival and temporal pressure history allow for the direct calculation of deflagration rate as a function of pressure. Additionally, deflagrating surface areas are calculated in order to provide quantitative insight into the dynamic surface structure during deflagration and its relationship to explosion violence. Deflagration rate data show that LX-17 burns in a smooth fashion at ambient temperature and is represented by the burn rate equation B = 0.2P{sup 0.9}. At 225 C, deflagration is more rapid and erratic. Dynamic deflagrating surface area calculations show that ambient temperature LX-17 deflagrating surface areas remain near unity over the pressure range studied

CHARACTERIZATION OF DAMAGED MATERIALS

Thermal damage experiments were conducted on LX-04, LX-10, and LX-17 at high temperatures. Both pristine and damaged samples were characterized for their material properties. A pycnometer was used to determine sample true density and porosity. Gas permeability was measured in a newly procured system (diffusion permeameter). Burn rate was measured in the LLNL strand burner. Weight losses upon thermal exposure were insignificant. Damaged pressed parts expanded, resulting in a reduction of bulk density by up to 10%. Both gas permeabilities and burn rates of the damaged samples increased by several orders of magnitude due to higher porosity and lower density. Moduli of the damaged materials decreased significantly, an indication that the materials became weaker mechanically. Damaged materials were more sensitive to shock initiation at high temperatures. No significant sensitization was observed when the damaged samples were tested at room temperature