Generation and preclinical characterization of an antibody specific for SEMA4D

Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.     

An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells

Oscillations in cytosolic-free Ca²⁺ concentration ([Ca2+]_i) have been proposed to encode information that controls stomatal closure. [Ca2+]_i oscillations with a period near 10 min were previously shown to be optimal for stomatal closure in Arabidopsis (Arabidopsis thaliana), but the studies offered no insight into their origins or mechanisms of encoding to validate a role in signaling. We have used a proven systems modeling platform to investigate these [Ca2+]_i oscillations and analyze their origins in guard cell homeostasis and membrane transport. The model faithfully reproduced differences in stomatal closure as a function of oscillation frequency with an optimum period near 10 min under standard conditions. Analysis showed that this optimum was one of a range of frequencies that accelerated closure, each arising from a balance of transport and the prevailing ion gradients across the plasma membrane and tonoplast. These interactions emerge from the experimentally derived kinetics encoded in the model for each of the relevant transporters, without the need of any additional signaling component. The resulting frequencies are of sufficient duration to permit substantial changes in [Ca2+]_i and, with the accompanying oscillations in voltage, drive the K⁺ and anion efflux for stomatal closure. Thus, the frequency optima arise from emergent interactions of transport across the membrane system of the guard cell. Rather than encoding information for ion flux, these oscillations are a by-product of the transport activities that determine stomatal aperture.Stomata in the leaf epidermis are the main pathway both for CO₂ entry for photosynthesis and for foliar water loss by transpiration. Guard cells surround the stomatal pore and regulate the aperture, balancing the often conflicting demands for CO₂ and water conservation. Guard cells open and close the pore by expanding and contracting through the uptake and loss, respectively, of osmotic solutes, notably of K⁺, Cl⁻, and malate²⁻ (Mal²⁻; Pandey et al., 2007; Kim et al., 2010; Roelfsema and Hedrich, 2010; Lawson and Blatt, 2014). These transport processes comprise the final effectors of a regulatory network that coordinates transport across the plasma membrane and tonoplast, and maintains the homeostasis of the guard cell. A number of well-defined signals—including light, CO₂, drought and the water stress hormone abscisic acid (ABA)—act on this network, altering transport, solute content, turgor and cell volume, and ultimately stomatal aperture.Much research has focused on stomatal closure, underscoring both Ca²⁺-independent and Ca²⁺-dependent signaling. Of the latter, elevated cytosolic-free Ca²⁺ concentration ([Ca2+]_i) inactivates inward-rectifying K⁺ channels (I_K,in) to prevent K⁺ uptake and activates Cl⁻ (anion) channels (I_Cl) at the plasma membrane to depolarize the membrane and engage K⁺ efflux through outward-rectifying K⁺ channels (I_K,out; Keller et al., 1989; Blatt et al., 1990; Thiel et al., 1992; Lemtiri-Chlieh and MacRobbie, 1994). ABA, and most likely CO₂ (Kim et al., 2010), elevate [Ca2+]_i by facilitating Ca²⁺ entry at the plasma membrane to trigger Ca²⁺ release from endomembrane stores, a process often described as Ca²⁺-induced Ca²⁺ release (Grabov and Blatt, 1998, 1999). The hormone promotes Ca²⁺ influx by activating Ca²⁺ channels (I_Ca) at the plasma membrane, even in isolated membrane patches (Hamilton et al., 2000, 2001), which is linked to reactive oxygen species (Kwak et al., 2003; Wang et al., 2013). In parallel, cADP-ribose and nitric oxide promote endomembrane Ca²⁺ release and [Ca2+]_i elevation (Leckie et al., 1998; Neill et al., 2002; Garcia-Mata et al., 2003; Blatt et al., 2007). Best estimates indicate that endomembrane release accounts for more than 95% of the Ca²⁺ entering the cytosol to raise [Ca2+]_i (Chen et al., 2012; Wang et al., 2012).One feature of stomatal response to ABA, and indeed to a range of stimuli both hormonal as well as external, is its capacity for oscillations both in membrane voltage and [Ca2+]_i. Guard cell [Ca2+]_i at rest is typically around 100 to 200 nm, as it is in virtually all living cells. In response to ABA, [Ca2+]_i can rise above 1 μm—and locally, most likely above 10 μm—often in cyclic transients of tens of seconds to several minutes’ duration in association with oscillations in voltage and stomatal closure (Gradmann et al., 1993; McAinsh et al., 1995; Webb et al., 1996; Grabov and Blatt, 1998, 1999; Staxen et al., 1999; Allen et al., 2001). In principle, cycling in voltage and [Ca2+]_i arises as closure is accelerated with a controlled release of K⁺, Cl⁻, and Mal²⁻ from the guard cell and is subject to extracellular ion concentrations (Gradmann et al., 1993; Chen et al., 2012). However, it has been proposed that these, and similar oscillations in a variety of plant cell models, serve as physiological signals in their own right (McAinsh et al., 1995; Ehrhardt et al., 1996; Taylor et al., 1996). In support of such a signaling role, experiments designed to impose [Ca2+]_i (and voltage) oscillations in guard cells have yielded an optimal frequency for closure with a period near 10 min (Allen et al., 2001). Nonetheless, the studies offer no mechanistic explanation for this optimum that could validate a causal role in signaling, and none has been forthcoming since. Here we address questions of how such optimal frequencies in [Ca2+]_i oscillation arise and their relevance for stomatal closure, using quantitative systems analysis of guard cell transport and homeostasis. Our findings indicate that oscillations in voltage and [Ca2+]_i, and their optima associated with stomatal closure, are most simply explained as emerging from the interactions between ion transporters that drive stomatal closure. Thus, we conclude that these oscillations do not control, but are a by-product of the transport that determines stomatal aperture.     

The Genetics of Leaf Flecking in Maize and Its Relationship to Plant Defense and Disease Resistance

Physiological leaf spotting, or flecking, is a mild-lesion phenotype observed on the leaves of several commonly used maize (Zea mays) inbred lines and has been anecdotally linked to enhanced broad-spectrum disease resistance. Flecking was assessed in the maize nested association mapping (NAM) population, comprising 4,998 recombinant inbred lines from 25 biparental families, and in an association population, comprising 279 diverse maize inbreds. Joint family linkage analysis was conducted with 7,386 markers in the NAM population. Genome-wide association tests were performed with 26.5 million single-nucleotide polymorphisms (SNPs) in the NAM population and with 246,497 SNPs in the association population, resulting in the identification of 18 and three loci associated with variation in flecking, respectively. Many of the candidate genes colocalizing with associated SNPs are similar to genes that function in plant defense response via cell wall modification, salicylic acid- and jasmonic acid-dependent pathways, redox homeostasis, stress response, and vesicle trafficking/remodeling. Significant positive correlations were found between increased flecking, stronger defense response, increased disease resistance, and increased pest resistance. A nonlinear relationship with total kernel weight also was observed whereby lines with relatively high levels of flecking had, on average, lower total kernel weight. We present evidence suggesting that mild flecking could be used as a selection criterion for breeding programs trying to incorporate broad-spectrum disease resistance.The plant hypersensitive response (HR) is a form of programmed cell death (PCD) characterized by rapid, localized cell death at the point of attempted pathogen penetration, usually resulting in disease resistance (Coll et al., 2011). It is often associated with other responses, including ion fluxes, an oxidative burst, lipid peroxidation, and cell wall fortification (Hammond-Kosack and Jones, 1996). van Doorn et al. (2011) suggested that HR is a type of PCD sharing features with, but distinct from, both vacuolar cell death and necrosis.HR has been associated with resistance to almost every class of pathogen and pest, including bacteria, viruses, fungi, nematodes, insects, and parasitic plants (Wu and Baldwin, 2010), and generally is most effective against biotrophic pathogens, since biotrophs require a long-term feeding relationship with living host cells. It is generally mediated by dominant resistance (R) genes whose activation is triggered by the direct or indirect detection of specific pathogen-derived effector proteins (Bent and Mackey, 2007). R proteins are maintained in their inactive state if their corresponding effector is not present. Mutants in which HR is constitutively active have been identified in many plant species, including maize/corn (Zea mays; Walbot et al., 1983; Johal, 2007), Arabidopsis (Arabidopsis thaliana; Lorrain et al., 2003), barley (Hordeum vulgare; Wolter et al., 1993), and rice (Oryza sativa; Yin et al., 2000).One well-known class of plant mutants spontaneously form lesions (patches of dead or chlorotic cells) in the absence of any obvious injury, stress, or infection to the plant. Since these lesions in some cases resemble HR, they have been termed disease-lesion mimics (Neuffer and Calvert, 1975). These mutants, which we will here collectively term Les mutants, have been studied extensively, especially in maize (Walbot et al., 1983; Johal et al., 1995; Johal, 2007) and Arabidopsis (Coll et al., 2011). While some of these lesion phenotypes are indeed caused by perturbations in the plant defense response (Hu et al., 1996; Rustérucci et al., 2001), some of the genes underlying this mutant class affect various other pathways that cause cell death if their function is perturbed (Johal, 2007). For instance, the Arabidopsis gene acd2 and the maize gene lls1 are defective in chlorophyll degradation (Gray et al., 1997; Mach et al., 2001).We have defined leaf flecking as the mild, genetically determined spotting observed on many maize inbred cultivars (Vontimitta et al., 2015; Fig. 1). The trait is qualitatively and visually similar to, but quantitatively less severe than, Les mutant phenotypes. The distinction between what constitutes a flecking versus a mild Les trait is necessarily somewhat arbitrary, but for our purposes, we have defined any nonproliferating and distinct leaf-spotting phenotype as flecking.Open in a separate windowFigure 1.A, Examples of variation in the flecking phenotype among inbred lines, with severity increasing from left to right (flecking scores in parentheses, from 0 to 4, scored on a scale of 1–10). B, Leaves of the lines nearly isogenic to inbred Mo20W, into which specific indicated dominant Les mutant genes have been introgressed (Rp1-D21 mutation in an H95 inbred background). Photographs were taken in Clayton, North Carolina, 12 weeks after planting. This figure is adapted from Figure 1 of Vontimitta et al. (2015).Leaf flecking is familiar to most corn breeders, appearing in such well-known and widely used lines such as Mo17 (Zehr et al., 1994) and in several other species such as barley (Makepeace et al., 2007), wheat (Triticum aestivum; Nair and Tomar, 2001), and oat (Avena sativa; Ferdinandsen and Winge, 1930). Flecking tends to be more noticeable in inbreds compared with their derived hybrids (M. Goodman and W. Dolezal, personal communication). Anecdotally, it is often thought to be indicative of a constitutive low-level defense response and as a marker for increased disease resistance.In previous work, we and others have defined the genetic architectures associated with resistance to several maize diseases, including southern leaf blight (SLB; causal agent, Cochliobolus heterostrophus), northern leaf blight (NLB; causal agent, Exserohilum turcicum), and gray leaf spot (GLS; causal agent, Cercospora zeae-maydis; Kump et al., 2011; Poland et al., 2011; Wisser et al., 2011; Benson et al., 2015), and with the control of the maize HR (Chintamanani et al., 2010; Chaikam et al., 2011; Olukolu et al., 2013). For much of this work, we used two powerful mapping populations: the maize association population (Flint-Garcia et al., 2005), a collection of 302 diverse inbred lines with low linkage disequilibrium, and the 5,000-line nested association mapping (NAM) population (McMullen et al., 2009), which is made up of 25 200-line recombinant inbred line (RIL) subpopulations derived from crosses between the common parent B73 and 25 diverse inbreds. Using these populations, it is possible to both sample a diverse array of germplasm and map quantitative trait loci (QTLs) precisely, in some cases to the gene level (Tian et al., 2011; Cook et al., 2012; Hung et al., 2012; Larsson et al., 2013; Olukolu et al., 2013; Wang and Balint-Kurti, 2016).A recent study using 300 lines from the maize intermated B73 × Mo17 population advanced intercross line mapping population identified low but moderately significant positive correlations between increased flecking and increased disease resistance and defense response (Vontimitta et al., 2015). Loci associated with variation in flecking were mapped, although these loci did not colocalize with QTLs identified previously for disease resistance and defense response traits (Balint-Kurti et al., 2007, 2008, 2010; Olukolu et al., 2013). In this study, we have extended this work to examine the genetic basis of leaf flecking over a much more diverse set of maize germplasm using a substantially larger population. We mapped loci associated with variation in leaf flecking and identified candidate genes and pathways that may be involved in this phenotype. Additionally, we have examined the correlations between leaf flecking and disease resistance, the hypersensitive defense response, and total kernel weight.     

Home range size of adult Indo‐Pacific bottlenose dolphins (Tursiops aduncus) in a coastal and estuarine system is habitat and sex‐specific

This study examined sex‐specific differences in home range size of adult Indo‐Pacific bottlenose dolphins off Bunbury, Western Australia. We applied a new kernel density estimation approach that accounted for physical barriers to movements. A Bayesian mixture model was developed to estimate a sex effect in home range size with latent group partitioning constrained by association data. A post hoc analysis investigated group partitioning relating to the proportion of time spent in open vs. sheltered waters. From 2007 to 2013, photographic‐identification data were collected along boat‐based systematic transect lines (n = 586). Analyses focused on adult dolphins of known sex (sighted ≥ 30 times; n = 22 males and 34 females). The 95% utilization distributions of males varied between 27 and 187 km² (; 94.8 ± 48.15) and for females between 20 and 133 km² (65.6 ± 30.9). The mixture model indicated a 99% probability that males had larger home ranges than females. Dolphins mostly sighted in open waters had larger home ranges than those in sheltered waters. Home ranges of dolphins sighted in sheltered waters overlapped with areas of highest human activity. We suggest that sex differences in home ranges are driven by male mating strategies, and home range size differences between habitats may be influenced by prey availability and predation risk.     

Quantitative Analysis of the Whole-Body Metabolic Fate of Branched-Chain Amino Acids

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Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

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Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+?breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.     

Integrating life history traits into predictive phylogeography

Predictive phylogeography seeks to aggregate genetic, environmental and taxonomic data from multiple species in order to make predictions about unsampled taxa using machine‐learning techniques such as Random Forests. To date, organismal trait data have infrequently been incorporated into predictive frameworks due to difficulties inherent to the scoring of trait data across a taxonomically broad set of taxa. We refine predictive frameworks from two North American systems, the inland temperate rainforests of the Pacific Northwest and the Southwestern Arid Lands (SWAL), by incorporating a number of organismal trait variables. Our results indicate that incorporating life history traits as predictor variables improves the performance of the supervised machine‐learning approach to predictive phylogeography, especially for the SWAL system, in which predictions made from only taxonomic and climate variables meets only moderate success. In particular, traits related to reproduction (e.g., reproductive mode; clutch size) and trophic level appear to be particularly informative to the predictive framework. Predictive frameworks offer an important mechanism for integration of organismal trait, environmental data, and genetic data in phylogeographic studies.