Collective cell movement is critical to the emergent properties of many multicellular systems, including microbial self-organization in biofilms, embryogenesis, wound healing, and cancer metastasis. However, even the best-studied systems lack a complete picture of how diverse physical and chemical cues act upon individual cells to ensure coordinated multicellular behavior. Known for its social developmental cycle, the bacterium Myxococcus xanthus uses coordinated movement to generate three-dimensional aggregates called fruiting bodies. Despite extensive progress in identifying genes controlling fruiting body development, cell behaviors and cell-cell communication mechanisms that mediate aggregation are largely unknown. We developed an approach to examine emergent behaviors that couples fluorescent cell tracking with datadriven models. A unique feature of this approach is the ability to identify cell behaviors affecting the observed aggregation dynamics without full knowledge of the underlying biological mechanisms. The fluorescent cell tracking revealed large deviations in the behavior of individual cells. Our modeling method indicated that decreased cell motility inside the aggregates, a biased walk toward aggregate centroids, and alignment among neighboring cells in a radial direction to the nearest aggregate are behaviors that enhance aggregation dynamics. Our modeling method also revealed that aggregation is generally robust to perturbations in these behaviors and identified possible compensatory mechanisms. The resulting approach of directly combining behavior quantification with data-driven simulations can be applied to more complex systems of collective cell movement without prior knowledge of the cellular machinery and behavioral cues.agent-based simulation | image processing | emergent behavior | fluorescent imaging | cell communication C ollective cell migration is essential for many developmental processes, including fruiting body development of myxobacteria (1) and Dictyostelium (2), embryonic gastrulation (3, 4), and neural crest development (5). Conversely, cancer cell metastases represent detrimental migratory events that disseminate dysfunctional cells (6). In all these processes, a population of cells leaves its current location and migrates in a coordinated manner to new locations where motility becomes reduced. Remarkable progress has been made in studying the intracellular machinery of these organisms (7). Much less is known about the systemlevel coordination of cell migration. Cell movement in these systems is a 3D, dynamic process coordinated by a combination of diverse physical and chemical cues acting on the cells (3,5,8). Recent developments in tracking individual cell movement in vivo have provided unprecedented detail and revealed surprising levels of heterogeneity (5, 7). Reverse engineering of how these individual cell movements lead to collective migration patterns has proved difficult. Whereas computational models are able to test whether a given set of ad hoc assumptions lead to e...

The 2009 H1N1 pandemic (H1N1pdm) viruses have evolved to contain an E47K substitution in the HA2 subunit of the stalk region of the hemagglutinin (HA) protein. The biological significance of this single amino acid change was investigated by comparing A/California/7/2009 (HA2-E47) with a later strain, A/Brisbane/10/2010 (HA2-K47). The E47K change was found to reduce the threshold pH for membrane fusion from 5.4 to 5.0. An inter-monomer salt bridge between K47 in HA2 and E21 in HA1, a neighboring highly conserved residue, which stabilized the trimer structure, was found to be responsible for the reduced threshold pH for fusion. The higher structural and acid stability of the HA trimer caused by the E47K change also conferred higher viral thermal stability and infectivity in ferrets, suggesting a fitness advantage for the E47K evolutionary change in humans. Our study indicated that the pH of HA fusion activation is an important factor for influenza virus replication and host adaptation. The identification of this genetic signature in the HA stalk region that influences vaccine virus thermal stability also has significant implications for influenza vaccine production.

Defective viral genomes (DVGs) are generated during virus replication. DVGs bearing complementary ends are strong inducers of dendritic cell (DC) maturation and of the expression of antiviral and pro-inflammatory cytokines by triggering signaling of the RIG-I family of intracellular pattern recognition receptors. Our data show that DCs stimulated with virus containing DVGs have an enhanced ability to activate human T cells and can induce adaptive immunity in mice. In addition, we describe the generation of a short Sendai virus (SeV)-derived DVG RNA (DVG-324) that maintains strong immunostimulatory activity in vitro and in vivo. DVG-324 induced high levels of IFN-β expression when transfected into cells and triggered fast expression of pro-inflammatory cytokines and mobilization of dendritic cells when injected into the footpad of mice. Importantly, DVG-324 enhanced the production of antibodies to a prototypic vaccine after a single intramuscular immunization in mice. Notably, the proinflammatory cytokine profile induced by DVG-324 was different from that induced by poly I:C, the only viral RNA analogue currently used as an immunostimulant in vivo, suggesting a distinct mechanism of action. SeV-derived oligonucleotides represent novel alternatives to be harnessed as potent adjuvants for vaccination.

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Immune evasion is a defining feature of the virus-host relationship. During infection, herpes simplex virus type 1 (HSV-1) utilizes multiple proteins to manipulate the host immune response. In the present study, we investigated the mechanism by which the virion host shutoff (vhs) protein blocks the activation of dendritic cells (DCs). Previously, we found that coinfection of wild-type HSV-1 with a panel of RNA viruses resulted in a block to DC activation that was attributable to vhs. These observations led us to hypothesize that the vhs-mediated inhibition was dependent on signaling through the RIG-I-like receptor (RLR) signaling pathway. By examining DCs generated from MAVS (IPS-1) knockout (KO) mice, we determined that RLR/MAVS signaling is not essential for the DC response to HSV-1. We also evaluated the requirement for the type I interferon ( Herpes simplex virus type 1 (HSV-1) is a highly successful human pathogen belonging to the Alphaherpesviridae subfamily of herpesviruses. Initial exposure to virus results in lifelong infection, and it is estimated that between 60 and 80% of humans are seropositive for the virus (52). Normal HSV infections are characterized by cycling between lytic infection at epithelial surfaces and stages of latency in neuronal cells (reviewed in reference 47). The pathology of HSV infection is greatly influenced by the immune status of the host, which impacts both disease severity and the frequency of reactivation (21,35,42,48,69).Early during primary infection of the epithelium, HSV encounters a specialized type of immune cell, the dendritic cells (DCs). DCs function as a crucial link between innate and adaptive immune responses (reviewed in reference 4). These cells survey peripheral tissues in an immature state and undergo a process referred to as maturation (or activation) upon encounter with virus-associated molecules (5, 32). DC maturation is initially characterized by the secretion of type I and III interferons (IFNs) and proinflammatory cytokines (e.g., interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-␣], and IL-12) and regulation of molecules necessary for migration to peripheral lymph nodes (32). En route to these secondary lymphoid organs, the DCs upregulate several costimulatory markers (CD86 and CD80) and load viral antigen onto major histocompatibility complex (MHC) molecules, which in concert serve to stimulate naïve B and T cells (4).Numerous viral proteins are utilized by HSV to evade the host immune response at all stages of the virus life cycle (7,9,31,33,39,63). Immunomodulatory proteins are either produced during the virus replication cycle or prepackaged in viral particles in the tegument and deposited into the cell immediately following virus envelope-host cell membrane fusion. The virion-host shutoff (vhs) protein is one such tegument-localized viral protein synthesized with late kinetics and packaged into mature virion particles (14,25,51,59). Functionally, vhs is a viral RNase that is known to preferentially degrade both host and viral mRNA species (1...