An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.     

Depth-Independent Reproduction in the Reef Coral Porites astreoides from Shallow to Mesophotic Zones

Mesophotic coral ecosystems between 30–150 m may be important refugia habitat for coral reefs and associated benthic communities from climate change and coastal development. However, reduced light at mesophotic depths may present an energetic challenge to the successful reproduction of light-dependent coral organisms, and limit this refugia potential. Here, the relationship of depth and fecundity was investigated in a brooding depth-generalist scleractinian coral, Porites astreoides from 5–37 m in the U.S. Virgin Islands (USVI) using paraffin tissue histology. Despite a trend of increasing planulae production with depth, no significant differences were found in mean peak planulae density between shallow, mid-depth and mesophotic sites. Differential planulae production over depth is thus controlled by P. astreoides coral cover, which peaks at 10 m and ~35 m in the USVI. These results suggest that mesophotic ecosystems are reproductive refuge for P. astreoides in the USVI, and may behave as refugia for P. astreoides metapopulations providing that vertical larval exchanges are viable.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

Using simulations to evaluate Mantel‐based methods for assessing landscape resistance to gene flow

Mantel‐based tests have been the primary analytical methods for understanding how landscape features influence observed spatial genetic structure. Simulation studies examining Mantel‐based approaches have highlighted major challenges associated with the use of such tests and fueled debate on when the Mantel test is appropriate for landscape genetics studies. We aim to provide some clarity in this debate using spatially explicit, individual‐based, genetic simulations to examine the effects of the following on the performance of Mantel‐based methods: (1) landscape configuration, (2) spatial genetic nonequilibrium, (3) nonlinear relationships between genetic and cost distances, and (4) correlation among cost distances derived from competing resistance models. Under most conditions, Mantel‐based methods performed poorly. Causal modeling identified the true model only 22% of the time. Using relative support and simple Mantel r values boosted performance to approximately 50%. Across all methods, performance increased when landscapes were more fragmented, spatial genetic equilibrium was reached, and the relationship between cost distance and genetic distance was linearized. Performance depended on cost distance correlations among resistance models rather than cell‐wise resistance correlations. Given these results, we suggest that the use of Mantel tests with linearized relationships is appropriate for discriminating among resistance models that have cost distance correlations <0.85 with each other for causal modeling, or <0.95 for relative support or simple Mantel r. Because most alternative parameterizations of resistance for the same landscape variable will result in highly correlated cost distances, the use of Mantel test‐based methods to fine‐tune resistance values will often not be effective.     

The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow,Energy-Dependent Quenching,and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation

When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H⁺ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H⁺ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes.Oxygenic photosynthesis involves the conversion of light energy into chemical bond energy by plants, green algae, and cyanobacteria and the use of that energy to fix CO₂. The photosynthetic electron transport system, located in thylakoid membranes, involves several major protein complexes: PSII (water-plastoquinone oxidoreductase), cytochrome b₆f (cyt b6f; plastoquinone-plastocyanin oxidoreductase), PSI (plastocyanin-ferredoxin oxidoreductase), and the ATP synthase (CF_oCF₁). Light energy absorbed by the photosynthetic apparatus is used to establish both linear electron flow (LEF) and cyclic electron flow (CEF), which drive the production of ATP and NADPH, the chemical products of the light reactions needed for CO₂ fixation in the Calvin-Benson-Bassham (CBB) cycle.With the absorption of light energy by pigment-protein complexes associated with PSII, energy is funneled into unique chlorophyll (Chl) molecules located in the PSII reaction center (RC), where it can elicit a charge separation that generates a large enough oxidizing potential to extract electrons from water. In LEF, electrons from PSII RCs are transferred sequentially along a set of electron carriers, initially reducing the plastoquinone (PQ) pool, then the cyt b₆f complex, and subsequently the lumenal electron carrier plastocyanin (PC). Light energy absorbed by PSI excites a special pair of Chl molecules (P700), causing a charge separation that generates the most negative redox potential in nature (Nelson and Yocum, 2006). The energized electron, which is replaced by electrons from PC, is sequentially transferred to ferredoxin and ferredoxin NADP⁺ reductase, generating reductant in the form of NADPH.Electron transport from water to NADPH in LEF is accompanied by the transport of H⁺ into the thylakoid lumen. For each water molecule oxidized, two H⁺ are released in the thylakoid lumen. In addition, H⁺ are moved into the lumen by the transfer of electrons through cyt b₆f (Q cycle). H⁺ accumulation in the thylakoid lumen dramatically alters the lumenal pH, and the transmembrane H⁺ gradient (ΔpH) together with the transmembrane ion gradient constitute the proton motive force (pmf), which drives ATP formation by ATP synthase (Mitchell, 1961, 1966, 2011). This pmf also promotes other cellular processes, including the dissipation of excess absorbed excitation energy as heat in a photoprotective process (see below; Li et al., 2009; Erickson et al., 2015). The NADPH and ATP molecules generated by LEF and CEF fuel the synthesis of reduced carbon backbones (in the CBB cycle) used in the production of many cellular metabolites and fixed carbon storage polymers.A basic role for CEF is to increase the ATP-NADPH ratio, which can satisfy the energy requirements of the cell and augment the synthesis of ATP by LEF, which is required to sustain CO₂ fixation by the CBB cycle (Allen, 2003; Kramer et al., 2004; Iwai et al., 2010; Alric, 2014). There are two distinct CEF pathways identified in plants and algae. In both pathways, electrons flow from the PQ pool through cyt b₆f to reduce the oxidized form of P700 (P700⁺). In one CEF pathway, electrons are transferred back to the PQ pool prior to the formation of NADPH. This route involves the proteins PGR5 and PGRL1 (DalCorso et al., 2008; Tolleter et al., 2011; Hertle et al., 2013) and is termed PGR5/L1-dependent CEF. A second route for CEF includes an NADPH dehydrogenase that oxidizes NADPH (product of LEF) to NADP⁺, simultaneously reducing the PQ (Allen, 2003; Kramer et al., 2004; Rumeau et al., 2007). The reduced PQ pool is then oxidized by cyt b₆f, causing H⁺ translocation into the thylakoid lumen, followed by the transfer of electrons to P700⁺ via PC. In the green alga Chlamydomonas reinhardtii, this second route for CEF involves a type II NADPH dehydrogenase (NDA2; Jans et al., 2008; Desplats et al., 2009).Oxygenic photosynthetic organisms have inhabited the planet for approximately 3 billion years and have developed numerous strategies to acclimate to environmental fluctuations. These acclimation processes confer flexibility to the photosynthetic machinery, allowing it to adjust to changes in conditions that impact the metabolic/energetic state of the organism and, most importantly, the formation of reactive oxygen species that may damage the photosynthetic apparatus and other cellular components (Li et al., 2009). Several ways in which the photosynthetic apparatus adjusts to environmental fluctuations have been established. A well-studied acclimation process, nonphotochemical quenching (NPQ), reduces the excitation pressure on PSII when oxidized downstream electron acceptors are not available (Eberhard et al., 2008; Li et al., 2009; Erickson et al., 2015). Several processes constitute NPQ, as follows. (1) qT, which involves the physical movement of light-harvesting complexes (LHCs) from one photosystem to another (this is also designated state transitions; Rochaix, 2014). (2) qE, which involves thermal dissipation of the excitation energy. This energy-dependent process requires an elevated ΔpH and involves an LHC-like protein, LHCSR3 (in C. reinhardtii) or PSBS (in plants), as well as the accumulation of specific xanthophylls (mainly lutein in C. reinhardtii and zeaxanthin in plants; Niyogi et al., 1997b; Li et al., 2000, 2004; Peers et al., 2009). (3) qZ, which is energy independent and involves the accumulation of zeaxanthin (Dall’Osto et al., 2005; Nilkens et al., 2010). (4) qI, which promotes quenching following physical damage to PSII core subunits (Aro et al., 1993). Additional mechanisms that can impact LEF and CEF are the synthesis and degradation of pigment molecules, changes in levels of RC and antenna complexes, and the control of electron distribution between LEF and CEF as the energetic demands of the cell change (Allen, 2003; Kramer et al., 2004). In addition, electrons can be consumed by mitochondrial and chlororespiratory activities (Bennoun, 1982; Peltier and Cournac, 2002; Johnson et al., 2014; Bailleul et al., 2015). The latter mainly involves the plastid terminal oxidase PTOX2, which catalyzes the oxidation of the PQ pool and the reduction of oxygen and H⁺ to form water molecules (Houille-Vernes et al., 2011; Nawrocki et al., 2015).Photosynthetic processes also must be modulated as organisms experience changes in the levels of available nutrients (Grossman and Takahashi, 2001). The macronutrient nitrogen (N), which represents 3% to 5% of the dry weight of photosynthetic organisms, is required to synthesize many biological molecules (e.g. amino acids, nucleic acids, and various metabolites) and also participates in posttranslational modifications of proteins (e.g. S-nitrosylation; Romero-Puertas et al., 2013). Importantly, N is highly abundant in chloroplasts in the form of DNA, ribosomes, Chl, and polypeptides (e.g. Rubisco and LHCs; Evans, 1989; Raven, 2013). Furthermore, there is a strong integration between N and carbon assimilation. During N limitation under photoautotrophic conditions, the inability of the organism to synthesize amino acids and other N-containing molecules necessary for cell growth and division can feed back to inhibit both carbon fixation by the CBB cycle and electron transport processes and also can negatively impact the expression of genes encoding key CBB cycle enzymes (Terashima and Evans, 1988; Huppe and Turpin, 1994; Nunes-Nesi et al., 2010).C. reinhardtii is a well-established model organism in which to study photosynthesis and acclimation processes, including acclimation to nutrient limitation (Wykoff et al., 1998; Grossman and Takahashi, 2001; Moseley et al., 2006; Grossman et al., 2009; Terauchi et al., 2010; Aksoy et al., 2013). This unicellular alga grows rapidly as a photoheterotroph (on fixed carbon in the light) or as a heterotroph (on fixed carbon in the dark), has completely sequenced nuclear, chloroplast, and mitochondrial genomes, can be used for classical genetic analyses, and is haploid, which makes some aspects of molecular manipulation (e.g. the generation of knockout mutants) easier (Merchant et al., 2007; Blaby et al., 2014). In the past few years, there have been many studies on the ways in which C. reinhardtii responds to N deprivation (Bulté and Wollman, 1992; Blaby et al., 2013; Goodenough et al., 2014; Schmollinger et al., 2014; Wei et al., 2015; Juergens et al., 2015). Cells deprived of N under photoheterotrophic conditions (i.e. acetate as an external carbon source) minimize the use of N (referred to as N sparing) and induce mechanisms associated with scavenging N from both external and internal pools, all of which eventually lead to proteome modifications and an elevated carbon-N ratio (Schmollinger et al., 2014). Acclimation under photoheterotrophic conditions also causes dramatic modifications of cellular metabolism and energetics: photosynthesis is down-regulated at multiple levels, with a portion of its N content recycled (mainly Chl and polypeptides of the photosynthetic apparatus), while there is enhanced accumulation of mitochondrial complexes leading to increased respiratory activity (Schmollinger et al., 2014; Juergens et al., 2015). Additionally, while fixed carbon cannot be used for growth in the absence of N, it may be stored as starch and triacylglycerol (Work et al., 2010; Siaut et al., 2011; Davey et al., 2014; Goodenough et al., 2014).In contrast to the acclimation of photoheterotrophically grown C. reinhardtii to N deprivation, little is known about how the photosynthetic machinery in this alga adjusts in response to N deprivation under photoautotrophic conditions, when the cells absolutely require photosynthetic energy generation for maintenance. Specifically, we sought to understand how photosynthesis adjusts to metabolic restrictions that slow down the CBB cycle, which in turn could cause the accumulation of photoreductant, particularly NADPH, as the demand for electrons declines (Peltier and Schmidt, 1991; Rumeau et al., 2007). Based on analyses of mutants and the use of spectroscopic and fluorescence measurements, we established a critical role for NDA2 in the acclimation of C. reinhardtii to N deprivation under photoautotrophic conditions, including (1) an augmented capacity for alternative routes of electron utilization (which decrease the NADPH-NADP⁺ ratio) based on increased NDA2-dependent CEF and chlororespiration, and (2) elevated qE, which relies on the H⁺ gradient generated by NDA2-dependent CEF.     

Mosaicism in a new Eocene pufferfish highlights rapid morphological innovation near the origin of crown tetraodontiforms

Tetraodontiformes (pufferfishes and kin) is a taxonomically and structurally diverse, widely‐distributed clade of acanthomorphs, whose members often serve as models for genomics and, increasingly, macroevolutionary studies. Morphologically disparate Palaeogene fossils suggest considerable early experimentation, but these flattened specimens often preserve limited information. We present a three‐dimensionally preserved beaked tetraodontiform from the early Eocene (c. 53 Ma) London Clay Formation, UK. Approximately coeval with the oldest crown tetraodontiforms, ?Ctenoplectus williamsi gen. et sp. nov. presents an unprecedented combination of characters, pairing a fused beak‐like dentition with prominent dorsal‐fin spines that insert atop transversely‐expanded pterygiophores roofing the skull. Bayesian total‐evidence tip‐dating analysis indicates that ?Ctenoplectus represents the sister lineage of Triodontidae and highlights considerable levels of homoplasy in early tetraodontiform evolution. According to our dataset, rates of morphological character evolution were elevated at the origin of crown Tetraodontiformes, especially within gymnodonts, but declined after the principal body plans were established. Such ‘early burst’ patterns are regarded as a hallmark of adaptive radiations, but are typically associated with diversification at smaller spatiotemporal scales. However, denser sampling of Neogene and Recent taxa is needed to confirm this pattern.     

Drivers of <Emphasis Type="Italic">Bromus tectorum</Emphasis> Abundance in the Western North American Sagebrush Steppe

Bromus tectorum can transform ecosystems causing negative impacts on the ecological and economic values of sagebrush steppe of the western USA. Although our knowledge of the drivers of the regional distribution of B. tectorum has improved, we have yet to determine the relative importance of climate and local factors causing B. tectorum abundance and impact. To address this, we sampled 555 sites distributed geographically and ecologically throughout the sagebrush steppe. We recorded the canopy cover of B. tectorum, as well as local substrate and vegetation characteristics. Boosted regression tree modeling revealed that climate strongly limits the transformative ability of B. tectorum to a portion of the sagebrush steppe with dry summers (that is, July precipitation <10 mm and the driest annual quarter associated with a mean temperature >15°C) and low native grass canopy cover. This portion includes the Bonneville, Columbia, Lahontan, and lower Snake River basins. These areas are likely to require extreme efforts to reverse B. tectorum transformation. Our predictions, using future climate conditions, suggest that the transformative ability of B. tectorum may not expand geographically and could remain within the same climatically suitable basins. We found B. tectorum in locally disturbed areas within or adjacent to all of our sample sites, but not necessarily within sagebrush steppe vegetation. Conversion of the sagebrush steppe by B. tectorum, therefore, is more likely to occur outside the confines of its current climatically optimal region because of site-specific disturbances, including invasive species control efforts and sagebrush steppe mismanagement, rather than climate change.     

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology,mycelial growth and plant infection

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4′,6‐diamidino‐2‐phenylindole (DAPI) staining and PcSDA1‐green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non‐filamentous yeasts and human cells.