Carnivore Territoriality: Simulating Economic Selection of Territories

We are developing theoretical models of territorial behavior of carnivores. This work will be useful for predicting the abundance of wolf (Canis lupus) territories in Montana and Idaho. Coupled with a patch occupancy model, it will provide more accurate estimates of abundance of wolves in each state. Ultimately, our work will also provide a better understanding of territorial behavior of a large carnivore. We are simulating the territory selection process for carnivores choosing patches on a landscape based on benefits of prey, where prey distribution ranges from overdispersed to highly clumped. Simulated carnivores will also consider hypothesized costs of patch ownership, including travel, competition, and mortality risk. In each simulation, carnivores will acquire patches for a territory as economically as possible based on these benefits and costs. Simulating various combinations of these hypothesized benefits and costs of patch ownership will provide predictions of territorial behavior. We can then compare these predictions to the territories of real wolves to determine which model is most predictive of actual wolf behavior. Starting with a model for benefits of prey and costs of travel, we found that prey distribution may influence mean size, quality, and fragmentation of simulated territories. Based on these preliminary results, we might expect differences in size or quality of territories in regions with different prey communities. Most importantly, this work provides a foundation from which we will build more complex models of territorial behavior of carnivores

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction

Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion

Ebola virus disease: In vivo protection provided by the PAMP restricted TLR3 agonist rintatolimod and its mechanism of action

Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks

AERCam Autonomy: Intelligent Software Architecture for Robotic Free Flying Nanosatellite Inspection Vehicles

The NASA Johnson Space Center has developed a nanosatellite-class Free Flyer intended for future external inspection and remote viewing of human spacecraft. The Miniature Autonomous Extravehicular Robotic Camera (Mini AERCam) technology demonstration unit has been integrated into the approximate form and function of a flight system. The spherical Mini AERCam Free Flyer is 7.5 inches in diameter and weighs approximately 10 pounds, yet it incorporates significant additional capabilities compared to the 35-pound, 14-inch diameter AERCam Sprint that flew as a Shuttle flight experiment in 1997. Mini AERCam hosts a full suite of miniaturized avionics, instrumentation, communications, navigation, power, propulsion, and imaging subsystems, including digital video cameras and a high resolution still image camera. The vehicle is designed for either remotely piloted operations or supervised autonomous operations, including automatic stationkeeping, point-to-point maneuvering, and waypoint tracking. The Mini AERCam Free Flyer is accompanied by a sophisticated control station for command and control, as well as a docking system for automated deployment, docking, and recharge at a parent spacecraft. Free Flyer functional testing has been conducted successfully on both an airbearing table and in a six-degree-of-freedom closed-loop orbital simulation with avionics hardware in the loop. Mini AERCam aims to provide beneficial on-orbit views that cannot be obtained from fixed cameras, cameras on robotic manipulators, or cameras carried by crewmembers during extravehicular activities (EVA s). On Shuttle or International Space Station (ISS), for example, Mini AERCam could support external robotic operations by supplying orthogonal views to the intravehicular activity (IVA) robotic operator, supply views of EVA operations to IVA and/or ground crews monitoring the EVA, and carry out independent visual inspections of areas of interest around the spacecraft. To enable these future benefits with minimal impact on IVA operators and ground controllers, the Mini AERCam system architecture incorporates intelligent systems attributes that support various autonomous capabilities. 1) A robust command sequencer enables task-level command scripting. Command scripting is employed for operations such as automatic inspection scans over a region of interest, and operator-hands-off automated docking. 2) A system manager built on the same expert-system software as the command sequencer provides detection and smart-response capability for potential system-level anomalies, like loss of communications between the Free Flyer and control station. 3) An AERCam dynamics manager provides nominal and off-nominal management of guidance, navigation, and control (GN&C) functions. It is employed for safe trajectory monitoring, contingency maneuvering, and related roles. This paper will describe these architectural components of Mini AERCam autonomy, as well as the interaction of these elements with a human operator during supervised autonomous control

Detection of Fastidious Vaginal Bacteria in Women with HIV Infection and Bacterial Vaginosis

Background. Fastidious bacteria have been associated with bacterial vaginosis (BV) using PCR methods. We assessed the prevalence of these bacteria in HIV-1 infected women and their relationship with vaginal pH and shedding of HIV-1 RNA. Methods. 64 cervicovaginal lavage (CVL) samples were collected from 51 women. Vaginal microbiota were characterized using 8 bacterium-specific quantitative PCR assays. Results. Women with the fastidious bacteria Bacterial Vaginosis Associated Bacterium (BVAB) 1, 2, and 3 showed a trend to increased HIV-1 shedding (OR 2.59–3.07, P = .14–.17). Absence of Lactobacillus crispatus (P < .005) and presence of BVAB2 (P < .001) were associated with elevated vaginal pH. BVAB1, 2, and 3 were highly specific indicators of BV in HIV-infected women, with specificities of 89%–93%. Conclusions. Fastidious bacteria (BVAB 1, 2, and 3) remain specific indicators of BV in HIV-infected women, and BVAB2 may contribute to the elevated vaginal pH that is a hallmark of this syndrome

A Multi-Compartment, Single and Multiple Dose Pharmacokinetic Study of the Vaginal Candidate Microbicide 1% Tenofovir Gel

Background: Tenofovir (TFV) gel is being evaluated as a microbicide with pericoital and daily regimens. To inhibit viral replication locally, an adequate concentration in the genital tract is critical. Methods and Findings: Forty-nine participants entered a two-phase study: single-dose (SD) and multi-dose (MD), were randomized to collection of genital tract samples (endocervical cells [ECC], cervicovaginal aspirate and vaginal biopsies) at one of seven time points [0.5, 1, 2, 4, 6, 8, or 24 hr(s)] post-dose following SD exposure of 4 mL 1% TFV gel and received a single dose. Forty-seven were randomized to once (QD) or twice daily (BID) dosing for 2 weeks and to collection of genital tract samples at 4, 8 or 24 hrs after the final dose, but two discontinued prior to gel application. Blood was collected during both phases at the seven times post-dose. TFV exposure was low in blood plasma for SD and MD; median C max was 4.0 and 3.4 ng/mL, respectively (C≤29 ng/mL). TFV concentrations were high in aspirates and tissue after SD and MD, ranging from 1.2×10 4 to 9.9×10 6 ng/mL and 2.1×10 2 to 1.4×10 6 ng/mL, respectively, and did not noticeably differ between proximal and distal tissue. TFV diphosphate (TFV-DP), the intracellular active metabolite, was high in ECC, ranging from 7.1×10 3 to 8.8×10 6 ng/mL. TFV-DP was detectable in approximately 40% of the tissue samples, ranging from 1.8×10 2 to 3.5×10 4 ng/mL. AUC for tissue TFV-DP was two logs higher after MD compared to SD, with no noticeable differences when comparing QD and BID. Conclusions: Single-dose and multiple-dose TFV gel exposure resulted in high genital tract concentrations for at least 24 hours post-dose with minimal systemic absorption. These results support further study of TFV gel for HIV prevention. Trial registration: ClinicalTrials.gov NCT00561496. © 2011 Schwartz et al

Promyelocytic Leukemia Protein Is a Cell-Intrinsic Factor Inhibiting Parvovirus DNA Replication

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses