Expression and characterization of the soluble form of recombinant mature HSV-2 glycoprotein G for use in anti-HSV-2 IgG serodiagnostic immunoassay.

Herpes simplex virus type-2 (HSV-2) specific glycoprotein G (gG-2) is widely used as the antigen of choice for serodiagnosis of HSV-2. In order to develop an ELISA for serodetection of HSV-2 IgG in patient sera, the soluble form of the mature gG-2 antigen (mgG-2), gG283-649, was expressed using a baculovirus expression system. gG283-649 contains the complete extracellular domain of mgG-2 including the C-terminal region, which despite homology to gG-1, does not cross-react with HSV-1 antibodies present in HSV-1 positive patient sera. gG283-649 had increased performance compared to a previously described gG-2 fragment and showed high sensitivity and specificity in a method comparison with HerpeSelect 1 & 2 Immunoblot IgG, a commercially available FDA-cleared assay for serodetection of HSV-1 and 2 antibodies. A total of 234 clinical samples consisting of 134 high risk samples, including 45 samples from pregnant subjects, and a panel of 100 mixed diagnosis samples, spanning the measurable range were tested in the method comparison. Clinical sensitivity and specificity were determined to be 94.2% and 100%, respectively. We conclude that this soluble form of mgG-2 is a novel antigen of choice for developing an ELISA for type-specific serodiagnosis of HSV-2.

Engineering of a miniaturized, robotic clinical laboratory.

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.

Endocytic pathway rapidly delivers internalized molecules to lysosomes: an analysis of vesicle trafficking, clustering and mass transfer.

Lysosomes play a critical role in intracellular drug delivery. For enzyme-based therapies, they represent a potential target site whereas for nucleic acid or many protein drugs, they represent the potential degradation site. Either way, understanding the mechanisms and processes involved in routing of materials to lysosomes after cellular entry is of high interest to the field of drug delivery. Most therapeutic cargoes other than small hydrophobic molecules enter the cells through endocytosis. Endocytosed cargoes are routed to lysosomes via microtubule-based transport and are ultimately shared by various lysosomes via tethering and clustering of endocytic vesicles followed by exchange of their contents. Using a combined experimental and numerical approach, here we studied the rates of mass transfer into and among the endocytic vesicles in a model cell line, 3T3 fibroblasts. In order to understand the relationship of mass transfer with microtubular transport and vesicle clustering, we varied both properties through various pharmacological agents. At the same time, microtubular transport and vesicle clustering were modeled through diffusion-advection equations and the Smoluchowski equations, respectively. Our analysis revealed that the rate of mass transfer is optimally related to microtubular transport and clustering properties of vesicles. Further, the rate of mass transfer is highest in the innate state of the cell. Any perturbation to either microtubular transport or vesicle aggregation led to reduced mass transfer to lysosome. These results suggest that in the absence of an external intervention the endocytic pathway appears to maximize molecular delivery to lysosomes. Strategies are discussed to reduce mass transfer to lysosomes so as to extend the residence time of molecules in endosomes or late endosomes, thus potentially increasing the likelihood of their escape before disposition in the lysosomes.

Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling.

Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs, progenitors, and, ultimately, neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, although a few endogenous factors are known to regulate neuronal differentiation. Astrocytes have been implicated, but their role in juxtacrine (that is, cell-cell contact dependent) signaling in NSC niches has not been investigated. We found that ephrin-B2 presented from rodent hippocampal astrocytes regulated neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies revealed a previously unknown role for ephrin-B2 in instructing neuronal differentiation. In addition, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activated ß-catenin in vitro and in vivo independently of Wnt signaling and upregulated proneural transcription factors. Ephrin-B2(+) astrocytes therefore promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.

Understanding intracellular transport processes pertinent to synthetic gene delivery via stochastic simulations and sensitivity analyses.

A major challenge in synthetic gene delivery is to quantitatively predict the optimal design of polymer-based gene carriers (polyplexes). Here, we report a consistent, integrated, and fundamentally grounded computational methodology to address this challenge. This is achieved by accurately representing the spatio-temporal dynamics of intracellular structures and by describing the interactions between gene carriers and cellular components at a discrete, nanoscale level. This enables the applications of systems tools such as optimization and sensitivity analysis to search for the best combination of systems parameters. We validate the approach using DNA delivery by polyethylenimine as an example. We show that the cell topology (e.g., size, circularity, and dimensionality) strongly influences the spatiotemporal distribution of gene carriers, and consequently, their optimal intracellular pathways. The model shows that there exists an upper limit on polyplexes' intracellular delivery efficiency due to their inability to protect DNA until nuclear entry. The model predicts that even for optimally designed polyethylenimine vectors, only approximately 1% of total DNA is delivered to the nucleus. Based on comparison with gene delivery by viruses, the model suggests possible strategies to significantly improve transfection efficiencies of synthetic gene vectors.

Theory of spatial patterns of intracellular organelles.

Here we report on a generalized theory of spatial patterns of intracellular organelles, which are controlled by cells using cytoskeleton-based movements powered by molecular motors. The theory reveals that organelles exhibit one of the four distinct, stable patterns, namely aggregation, hyperdispersion, radial dispersion, and areal dispersion. Existence of specific patterns is determined by the contributions from three transport mechanisms, characterized by two Peclet numbers. The predicted patterns compare well with experimental data. This study provides a firm theoretical ground for classification of spatial patterns of organelles and understanding their regulation by cells.

Dynamics and spatial organization of endosomes in mammalian cells.

We combine particle tracking and stochastic simulations to analyze the dynamics and organization of early endocytic vesicles in mammalian cells. At short time scales (<10(1) sec) vesicles exhibit 1D symmetric bidirectional motor-driven transport on microtubules such that the mean squared displacement (MSD) scales as t3/2, but the MSD shows a crossover to facilitated diffusion at longer times (>10(1) sec). Facilitated diffusion results in rapid equilibration of vesicles on microtubules. The asterlike organization of microtubules causes perinuclear accumulation of vesicles despite symmetric transport.