Beata J. Wysocki

Endosomal Trafficking of Nanoformulated Antiretroviral Therapy Facilitates Drug Particle Carriage and HIV Clearance

ABSTRACT Limitations of antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance, and failure to penetrate viral reservoirs. Recent developments in nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at or adjacent to the sites of the viral replication cycle. Poloxamer 407-coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were ∼1,000 times higher in cells than those that could be achieved by the native drug. ATV particles in late and recycling endosome compartments were seen following pulldown by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of the compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1 p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000-fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The colocalization of nanoATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long-acting nanoART can serve as a drug carrier in both cells and subcellular compartments and, as such, can facilitate viral clearance. IMPORTANCE The need for long-acting ART is significant and highlighted by limitations in drug access, toxicity, adherence, and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells, and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly, and increasing ART concentrations in such sites enhances viral clearance. The current work uncovers a new mechanism by which nanoART can enhance viral clearance over native drug formulations.

The mixed lineage kinase-3 inhibitor URMC-099 improves therapeutic outcomes for long-acting antiretroviral therapy

During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the mixed lineage kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2Rγc-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance. From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the mixed lineage kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future.

Integrating mitosis, toxicity, and transgene expression in a telecommunications packet‐switched network model of lipoplex‐mediated gene delivery

Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms, including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet‐switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP‐induced toxicity can improve transfection efficiency by three‐fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels. Biotechnol. Bioeng. 2014;111: 1659–1671.

Modeling nonviral gene delivery as a macro-to-nano communication system

Abstract The principal role of any communication system is to deliver information from a source to a sink. Since gene delivery systems transport genetic information encoded as DNA to living cells, such systems can be considered as communication systems. Therefore, techniques developed for modeling conventional communication systems should be applicable to model gene delivery systems. The paper describes an approach to model nonviral gene delivery as a macro-to-nano communication system. To facilitate modeling, the gene delivery process is first described in terms of an abstractive layered communication protocol and then processing at each layer is implemented as M/M/ ∞ queues. To validate this approach, the model has been implemented in MATLAB/SIMULINK environment and the simulation results have been compared to experimental data from literature.

Layered Communication Protocol for Macro to Nano-Scale Communication Systems

Nanoparticle-based drug delivery systems provide a feasible method of efficiently delivering drugs in-vivo. These drug delivery systems could greatly benefit from a robust method of nano-scale communication. Additionally, there are numerous naturally occurring nano-scale communication systems and the opportunity to develop a system based on these principles is highly promising. A layered communication protocol provides a simple, succinct method of understanding macro to nano-scale communication systems. This protocol may be used to expand and eventually apply commonly used telecommunications techniques to nanoparticle signalling. Such a protocol may begin to pave the way for future communication systems at a nano-scale.

On an Orthogonal Space-Time-Polarization Block Code

Over the past several years, diversity methods such as space, time, and polarization diversity have been successfully implemented in wireless communications systems. Orthogonal space-time block codes efficiently combine space and time diversity, and they have been studied in detail. Polarization diversity has also been studied, however it is usually considered in a simple concatenation with other coding methods. In this paper, an efficient method for incorporating polarization diversity with space and time diversity is studied. The simple yet highly efficient technique is based on extending orthogonal space-time block codes into the quaternion domain and utilizing a description of the dual-polarized signal by means of quaternions. The resulting orthogonal space-timepolarization block codes have given promising results in simulations. In the example described in this paper, the achievable performance gain for two transmit and one receive antennas is approximately 6 dB at a bit error rate of 10-4 when compared with the Alamouti code.

A novel method for simulating insulin mediated GLUT4 translocation

Glucose transport in humans is a vital process which is tightly regulated by the endocrine system. Specifically, the insulin hormone triggers a cascade of intracellular signals in target cells mediating the uptake of glucose. Insulin signaling triggers cellular relocalization of the glucose transporter protein GLUT4 to the cell surface, which is primarily responsible for regulated glucose import. Pathology associated with the disruption of this pathway can lead to metabolic disorders, such as type II diabetes mellitus, characterized by the failure of cells to appropriately uptake glucose from the blood. We describe a novel simulation tool of the insulin intracellular response, incorporating the latest findings regarding As160 and GEF interactions. The simulation tool differs from previous computational approaches which employ algebraic or differential equations; instead, the tool incorporates statistical variations of kinetic constants and initial molecular concentrations which more accurately mimic the intracellular environment. Using this approach, we successfully recapitulate observed in vitro insulin responses, plus the effects of Wortmannin‐like inhibition of the pathway. The developed tool provides insight into transient changes in molecule concentrations throughout the insulin signaling pathway, and may be employed to identify or evaluate potentially critical components of this pathway, including those associated with insulin resistance. In the future, this highly tractable platform may be useful for simulating other complex cell signaling pathways. Biotechnol. Bioeng. 2014;111: 2454–2465.

Implementation and performance evaluation of QoS scheduling algorithms in Mobile WiMAX ns-2 simulator

Mobile WiMAXs scheduling service is a key factor in Radio Resource Management (RRM) in realizing Quality of Service (QoS). The choice in scheduling algorithm for Mobile WiMAX base stations, albeit outside of the scope of the standard, critically impacts the performance and robustness of Mobile WiMAX networks. This paper provides an in-depth study of four major scheduling algorithms: Round Robin (RR), Max CINR (MC), Fair Throughput (FT) and Proportional Fair (PF). Within the framework of Mobile WiMAX, we also discuss the implementation methodology and tradeoffs associated with these scheduling algorithms. Various Mobile WiMAX scenarios are simulated and analyzed using our Mobile WiMAX ns-2 model for performance throughput efficiency and resource allocation fairness.

Optimal receiver antenna location in indoor environment using dynamic differential evolution and genetic algorithm

Using the impulse responses of these multipath channels, the bit error rate (BER) performance for binary pulse amplitude modulation impulse radio ultra-wideband communication system is calculated. The optimization location of receiving antenna is investigated by dynamic differential evolution (DDE) and genetic algorithm (GA) to minimize the outage probability. Numerical results show that the performance for reducing BER and outage probability by DDE algorithm is better than that by GA.

A novel telecommunications-based approach to HIV modeling and simulation

Abstract It is well known that biological systems utilize communication in some form; one prolific example of this is the propagation of HIV (Human Immunodeficiency Virus) in the human body. By modeling HIV infection as a communication system, we hope to gain a unique insight into HIV and biological communication systems in general. Such a model would provide researchers a platform for experimenting and simulating various biological communication systems. We have previously developed a layered communication protocol for interpreting biological communication systems using telecommunications paradigms and will apply said model to HIV proliferation. We will also demonstrate the effectiveness of the model by implementing a communication-based simulation of HIV infection based on direct interpretation of this layered protocol.