Genomic regions associate with major axes of variation driven by gas exchange and leaf construction traits in cultivated sunflower (Helianthus annuus L.).

Stomata and leaf veins play an essential role in transpiration and the movement of water throughout leaves. These traits are thus thought to play a key role in the adaptation of plants to drought and a better understanding of the genetic basis of their variation and coordination could inform efforts to improve drought tolerance. Here, we explore patterns of variation and covariation in leaf anatomical traits and analyze their genetic architecture via genome-wide association (GWA) analyses in cultivated sunflower (Helianthus annuus L.). Traits related to stomatal density and morphology as well as lower-order veins were manually measured from digital images while the density of minor veins was estimated using a novel deep learning approach. Leaf, stomatal, and vein traits exhibited numerous significant correlations that generally followed expectations based on functional relationships. Correlated suites of traits could further be separated along three major principal component (PC) axes that were heavily influenced by variation in traits related to gas exchange, leaf hydraulics, and leaf construction. While there was limited evidence of colocalization when individual traits were subjected to GWA analyses, major multivariate PC axes that were most strongly influenced by several traits related to gas exchange or leaf construction did exhibit significant genomic associations. These results provide insight into the genetic basis of leaf trait covariation and showcase potential targets for future efforts aimed at modifying leaf anatomical traits in sunflower.

Data-driven modeling reveals cell behaviors controlling self-organization during Myxococcus xanthus development.

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 data-driven 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 Modeling Reveals Possible Mechanisms for Observed Aggregation Cell Behaviors.

Myxococcus xanthus is a soil bacterium that serves as a model system for biological self-organization. Cells form distinct, dynamic patterns depending on environmental conditions. An agent-based model was used to understand how M. xanthus cells aggregate into multicellular mounds in response to starvation. In this model, each cell is modeled as an agent represented by a point particle and characterized by its position and moving direction. At low agent density, the model recapitulates the dynamic patterns observed by experiments and a previous biophysical model. To study aggregation at high cell density, we extended the model based on the recent experimental observation that cells exhibit biased movement toward aggregates. We tested two possible mechanisms for this biased movement and demonstrate that a chemotaxis model with adaptation can reproduce the observed experimental results leading to the formation of stable aggregates. Furthermore, our model reproduces the experimentally observed patterns of cell alignment around aggregates.

CD55 is a key complement regulatory protein that counteracts complement-mediated inactivation of Newcastle Disease Virus.

Newcastle disease virus (NDV) is being developed as an oncolytic virus for virotherapy. In this study we analysed the regulation of complement-mediated inactivation of a recombinant NDV in different host cells. NDV grown in human cells was less sensitive to complement-mediated virus inactivation than NDV grown in embryonated chicken eggs. Additionally, NDV produced from HeLa-S3 cells is more resistant to complement than NDV from 293F cells, which correlated with higher expression and incorporation of complement regulatory proteins (CD46, CD55 and CD59) into virions from HeLa-S3 cells. Further analysis of the recombinant NDVs individually expressing the three CD molecules showed that CD55 is the most potent in counteracting complement-mediated virus inactivation. The results provide important information on selecting NDV manufacture substrate to mitigate complement-mediated virus inactivation.

A single amino acid in the stalk region of the H1N1pdm influenza virus HA protein affects viral fusion, stability and infectivity.

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.

Identification of critical residues in the hemagglutinin and neuraminidase of influenza virus H1N1pdm for vaccine virus replication in embryonated chicken eggs.

In 2009, we successfully produced a high-yield live attenuated H1N1pdm A/California/7/2009 vaccine (CA/09 LAIV) by substitution of three residues (K119E, A186D, and D222G) in the hemagglutinin (HA) protein. Since then, we have generated and evaluated additional H1N1pdm vaccine candidates from viruses isolated in 2010 and 2011. The 2010 strains with the new HA substitutions near the HA receptor binding site (N125D and D127E or D127E and K209E) grew well in eggs and formed large plaques in Madin-Darby canine kidney (MDCK) cells. Introduction of these acidic amino acids into the HA of CA/09 also improved vaccine virus growth in eggs to a titer comparable to that of CA/09 LAIV. However, the high growth of A/Gilroy/231/2011 (Gil/11) vaccine virus required modification in both the HA and the NA segments. The residue at position 369 of the NA was found to be critical for virus replication in MDCK cells and eggs. These HA and NA residues had minimal impact on viral entry but greatly improved viral release from infected cells. Our data implied that the HA receptor binding and NA receptor cleaving function of the poor-growth H1N1pdm virus was not well balanced for virus replication in host cells. The high-growth vaccine candidates described in this study maintained vaccine virus antigenicity and induced high levels of neutralizing antibodies in immunized ferrets, making them suitable for vaccine production. The identification of the amino acids and their roles in viral replication should greatly help vaccine manufacturers to produce high-yield reassortant vaccine viruses against the future drifted H1N1pdm viruses.

The virion host shutoff protein of herpes simplex virus 1 blocks the replication-independent activation of NF-κB in dendritic cells in the absence of type I interferon signaling.

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 (IFN) signaling pathway in DC activation following infection with HSV-1 and found that stimulation of DCs with wild-type HSV-1 required intact type I IFN signaling for the production of cytokines, whereas the vhs deletion (vhs(-)) mutant virus activated DCs without the need for exogenous IFN signaling. Comparisons of transcription factor activation in DCs infected with wild-type HSV and the vhs(-) mutant virus revealed that NF-κB activation was inhibited by vhs in the early phase of the infection. In contrast, IRF3 activation was not influenced by vhs. In these studies, measurement of proinflammatory cytokines and type I IFN release from the infected DCs reflected the activation status of these transcription factors. Taken together, the work presented here (i) describes a novel role for the vhs protein as an inhibitor of the early activation of NF-κB during HSV-1 infection of DCs and (ii) offers a mechanistic explanation of how this protein interferes with DC activation.

Quantification of Myxococcus xanthus Aggregation and Rippling Behaviors: Deep-Learning Transformation of Phase-Contrast into Fluorescence Microscopy Images.

Myxococcus xanthus bacteria are a model system for understanding pattern formation and collective cell behaviors. When starving, cells aggregate into fruiting bodies to form metabolically inert spores. During predation, cells self-organize into traveling cell-density waves termed ripples. Both phase-contrast and fluorescence microscopy are used to observe these patterns but each has its limitations. Phase-contrast images have higher contrast, but the resulting image intensities lose their correlation with cell density. The intensities of fluorescence microscopy images, on the other hand, are well-correlated with cell density, enabling better segmentation of aggregates and better visualization of streaming patterns in between aggregates; however, fluorescence microscopy requires the engineering of cells to express fluorescent proteins and can be phototoxic to cells. To combine the advantages of both imaging methodologies, we develop a generative adversarial network that converts phase-contrast into synthesized fluorescent images. By including an additional histogram-equalized output to the state-of-the-art pix2pixHD algorithm, our model generates accurate images of aggregates and streams, enabling the estimation of aggregate positions and sizes, but with small shifts of their boundaries. Further training on ripple patterns enables accurate estimation of the rippling wavelength. Our methods are thus applicable for many other phenotypic behaviors and pattern formation studies.

Data-Driven Models Reveal Mutant Cell Behaviors Important for Myxobacterial Aggregation.

Single mutations frequently alter several aspects of cell behavior but rarely reveal whether a particular statistically significant change is biologically significant. To determine which behavioral changes are most important for multicellular self-organization, we devised a new methodology using Myxococcus xanthus as a model system. During development, myxobacteria coordinate their movement to aggregate into spore-filled fruiting bodies. We investigate how aggregation is restored in two mutants, csgA and pilC, that cannot aggregate unless mixed with wild-type (WT) cells. To this end, we use cell tracking to follow the movement of fluorescently labeled cells in combination with data-driven agent-based modeling. The results indicate that just like WT cells, both mutants bias their movement toward aggregates and reduce motility inside aggregates. However, several aspects of mutant behavior remain uncorrected by WT, demonstrating that perfect recreation of WT behavior is unnecessary. In fact, synergies between errant behaviors can make aggregation robust.IMPORTANCE Self-organization into spatial patterns is evident in many multicellular phenomena. Even for the best-studied systems, our ability to dissect the mechanisms driving coordinated cell movement is limited. While genetic approaches can identify mutations perturbing multicellular patterns, the diverse nature of the signaling cues coupled to significant heterogeneity of individual cell behavior impedes our ability to mechanistically connect genes with phenotype. Small differences in the behaviors of mutant strains could be irrelevant or could sometimes lead to large differences in the emergent patterns. Here, we investigate rescue of multicellular aggregation in two mutant strains of Myxococcus xanthus mixed with wild-type cells. The results demonstrate how careful quantification of cell behavior coupled to data-driven modeling can identify specific motility features responsible for cell aggregation and thereby reveal important synergies and compensatory mechanisms. Notably, mutant cells do not need to precisely recreate wild-type behaviors to achieve complete aggregation.

Detection of herpes simplex virus dependent apoptosis.

Subversion of the host response to virus infection is a universal theme of virology and viral immunology. Multiple mechanisms are in place to limit virus spread on behalf of the host, yet through evolution, viruses have adapted to either weaken or eliminate the effects of these host factors. Cell death or apoptosis is one such example of a host response to viral infection. As such, experimental techniques that enable analysis of viruses (and viral genes) involved in triggering, blocking, or perhaps augmenting this process represent important tools for virologists, immunologists, and cell biologists. Presented here are a series of techniques developed in our lab for the analysis of apoptosis that occurs as a consequence of herpes simplex virus type 1 infection.

Highly immunostimulatory RNA derived from a Sendai virus defective viral genome.

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 Ifnb 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 pro-inflammatory cytokine profile induced by DVG-324 was different from that induced by poly I:C, the only viral RNA analog 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.

A voluntary breath-hold treatment technique for the left breast with unfavorable cardiac anatomy using surface imaging.

PURPOSE: Breath-hold (BH) treatments can be used to reduce cardiac dose for patients with left-sided breast cancer and unfavorable cardiac anatomy. A surface imaging technique was developed for accurate patient setup and reproducible real-time BH positioning. METHODS AND MATERIALS: Three-dimensional surface images were obtained for 20 patients. Surface imaging was used to correct the daily setup for each patient. Initial setup data were recorded for 443 fractions and were analyzed to assess random and systematic errors. Real time monitoring was used to verify surface placement during BH. The radiation beam was not turned on if the BH position difference was greater than 5 mm. Real-time surface data were analyzed for 2398 BHs and 363 treatment fractions. The mean and maximum differences were calculated. The percentage of BHs greater than tolerance was calculated. RESULTS: The mean shifts for initial patient setup were 2.0 mm, 1.2 mm, and 0.3 mm in the vertical, longitudinal, and lateral directions, respectively. The mean 3-dimensional vector shift was 7.8 mm. Random and systematic errors were less than 4 mm. Real-time surface monitoring data indicated that 22% of the BHs were outside the 5-mm tolerance (range, 7%-41%), and there was a correlation with breast volume. The mean difference between the treated and reference BH positions was 2 mm in each direction. For out-of-tolerance BHs, the average difference in the BH position was 6.3 mm, and the average maximum difference was 8.8 mm. CONCLUSIONS: Daily real-time surface imaging ensures accurate and reproducible positioning for BH treatment of left-sided breast cancer patients with unfavorable cardiac anatomy.

The virion host shut-off (vhs) protein blocks a TLR-independent pathway of herpes simplex virus type 1 recognition in human and mouse dendritic cells.

Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.