Use of thermal imaging and the photochemical reflectance index (PRI) to detect wheat response to elevated CO2 and drought

The spectral-based photochemical reflectance index (PRI) and leaf surface temperature (T_leaf) derived from thermal imaging are two indicative metrics of plant functioning. The relationship of PRI with radiation-use efficiency (RUE) and T_leaf with leaf transpiration could be leveraged to monitor crop photosynthesis and water use from space. Yet, it is unclear how such relationships will change under future high carbon dioxide concentrations ([CO₂]) and drought. Here we established an [CO₂] enrichment experiment in which three wheat genotypes were grown at ambient (400 ppm) and elevated (550 ppm) [CO₂] and exposed to well-watered and drought conditions in two glasshouse rooms in two replicates. Leaf transpiration (T_r) and latent heat flux (LE) were derived to assess evaporative cooling, and RUE was calculated from assimilation and radiation measurements on several dates along the season. Simultaneous hyperspectral and thermal images were taken at $~$1.5 m from the plants to derive PRI and the temperature difference between the leaf and its surrounding air ($∆$T_leaf−air). We found significant PRI and RUE and $∆$T_leaf−air and T_r correlations, with no significant differences among the genotypes. A PRI–RUE decoupling was observed under drought at ambient [CO₂] but not at elevated [CO₂], likely due to changes in photorespiration. For a LE range of 350 W m^–2, the ΔT_leaf−air range was $~$10°C at ambient [CO₂] and only $~$4°C at elevated [CO₂]. Thicker leaves in plants grown at elevated [CO₂] suggest higher leaf water content and consequently more efficient thermoregulation at high [CO₂] conditions. In general, T_leaf was maintained closer to the ambient temperature at elevated [CO₂], even under drought. PRI, RUE, ΔT_leaf−air, and T_r decreased linearly with canopy depth, displaying a single PRI-RUE and ΔT_leaf−air T_r model through the canopy layers. Our study shows the utility of these sensing metrics in detecting wheat responses to future environmental changes.     

Increased Cuticle Permeability Caused by a New Allele of ACETYL-COA CARBOXYLASE1 Enhances CO2 Uptake

Carbon dioxide (CO₂) is an essential substrate for photosynthesis in plants. CO₂ is absorbed mainly through the stomata in land plants because all other aerial surfaces are covered by a waxy layer called the cuticle. The cuticle is an important barrier that protects against extreme water loss; however, this anaerobic layer limits CO₂ uptake. Simply, in the process of adapting to a terrestrial environment, plants have acquired drought tolerance in exchange for reduced CO₂ uptake efficiency. To evaluate the extent to which increased cuticle permeability enhances CO₂ uptake efficiency, we investigated the CO₂ assimilation rate, carbon content, and dry weight of the Arabidopsis (Arabidopsis thaliana) mutant excessive transpiration1 (extra1), whose cuticle is remarkably permeable to water vapor. We isolated the mutant as a new allele of ACETYL-COA CARBOXYLASE1, encoding a critical enzyme for fatty acid synthesis, thereby affecting cuticle wax synthesis. Under saturated water vapor conditions, the extra1 mutant demonstrated a higher CO₂ assimilation rate, carbon content, and greater dry weight than did the wild-type plant. On the other hand, the stomatal mutant slow-type anion channel-associated1, whose stomata are continuously open, also exhibited a higher CO₂ assimilation rate than the wild-type plant; however, the increase was only half of the amount exhibited by extra1. These results indicate that the efficiency of CO₂ uptake via a permeable cuticle is greater than the efficiency via stomata and confirm that land plants suffer a greater loss of CO₂ uptake efficiency by developing a cuticle barrier.

To absorb carbon dioxide (CO₂) for photosynthesis, land plants expose their wet surfaces to a dry atmosphere and suffer evaporative water loss as a consequence (Hall et al., 1993). As too much water loss would result in dehydration, plants cover most of their aerial surfaces with a relatively impermeable layer, called the cuticle, and take in CO₂ mainly through stomatal pores, which make up only about 2% per a leaf area (Willmer and Fricker, 1996). In other words, the cuticle provides drought tolerance to plants in exchange for reduced efficiency in CO₂ uptake.The cuticle is a continuous membrane consisting of a polymer matrix (cutin), polysaccharides, and organic solvent‐soluble lipids (cuticular waxes; Holloway, 1982; Jeffree, 1996; Riederer and Schreiber, 2001). The cuticle is an important structure to protect plants against excess drought, high temperature, strong UV radiation, pathogens, and harmful insects (Kerstiens, 1996a, 1996b; Burghardt and Riederer, 2006; Riederer and Müller, 2006; Domínguez et al., 2011; Yeats and Rose, 2013). The cuticle limits the transpiration through plant surfaces other than through the stomatal pores to <10% of the total (Mohr and Schopfer, 1995). On the other hand, this impermeable layer also strongly restricts CO₂ influx. Boyer et al. (1997) and Boyer (2015a, 2015b) reported a lower conductance for CO₂ than for water vapor in cuticles of intact leaves of grape (Vitis vinifera) and sunflower (Helianthus annuus) due to the differences in molecular size and diffusion paths between the two gases. However, although many studies have explored the water permeability of cuticles in various conditions and species (Kerstiens, 1996a; Riederer and Müller, 2006; Kosma et al., 2009; Schreiber and Schönherr, 2009), much less attention has been directed to CO₂, despite its substantial role in photosynthesis.In this study, we verified the hypothesis that plants could absorb CO₂ more efficiently under non-drought stress conditions if their cuticles are more permeable. In addition, we also investigated the extent to which a permeable cuticle can enhance CO₂ uptake efficiency. To verify the hypothesis, we investigated whether the CO₂ uptake efficiency is increased in a mutant with a high cuticle permeability. For this research, we isolated an Arabidopsis (Arabidopsis thaliana) mutant named excessive transpiration1 (extra1), which exhibited marked evaporative water loss due to an increased cuticle permeability caused by a new allele of ACETYL-COA CARBOXYLASE1 (ACC1). ACC1 encodes a critical enzyme for the synthesis of malonyl-CoA, an essential substrate for fatty acid synthesis (Baud et al., 2003). To evaluate CO₂ uptake efficiency, we investigated CO₂ assimilation rate, carbon content, and dry weight of the extra1 mutant and compared them to that of wild-type plants as well as that of another mutant, slow-type anion channel-associated1 (slac1) with continuously open stomata (Negi et al., 2008; Vahisalu et al., 2008). Our results reveal that the increased cuticle permeability strongly and constantly enhances CO₂ uptake efficiency under non-drought stress conditions.     

Artifact quantification of venous stents in the MRI environment: Differences between braided and laser-cut designs

PurposeTo quantify B₀- and B₁-induced imaging artifacts of braided venous stents and to compare the artifacts to a set of laser-cut stents used in venous interventions.MethodsThree prototypes of braided venous stents with different geometries were tested in vitro. B₀ field distortion maps were measured via the frequency shift $\mathrm{\Delta }f$ using multi-echo imaging. B₁ distortions were quantified using the double angle method. The relative amplitudes $B_1^{\mathit{rel}}$ were calculated to compare the intraluminal alteration of B₁. Measurements were repeated with the stents in three different orientations: parallel, diagonal and orthogonal to B₀.ResultsAt 1.5 T, the braided stents induced a maximum frequency shift of $\left|\mathrm{\Delta }f\left(\mathit{x}\right)\right|<100\mathrm{H}\mathrm{z}$. Signal voids were limited to a distance of 2 mm to the stent walls at an echo time of 3 ms. No substantial difference in the B₀ field distortions was seen between laser-cut and braided venous stents. $B_1^{\mathit{rel}}$ maps showed strongly varying distortion patterns in the braided stents with the mean intraluminal $B_1^{\mathit{rel}}$ ranging from $\left(63\pm 18\right)\%$ in prototype 1 to $\left(98\pm 38\right)\%$ in prototype 2. Compared to laser-cut stents the braided stents showed a 5 to 9 times higher coefficient of variation of the intraluminal $B_1^{\mathit{rel}}$.ConclusionBraided venous stent prototypes allow for MR imaging of the intraluminal area without substantial signal voids due to B₀-induced artifacts. Whereas B₁ is attenuated homogeneously in laser-cut stents, the B₁ distortion in braided stents is more inhomogeneous and shows areas with enhanced amplitude. This could potentially be used in braided stent designs for intraluminal signal amplification.     

Time-resolved FTIR difference spectroscopy for the study of quinones in the A1 binding site in photosystem I: Identification of neutral state quinone bands

Infrared absorption bands associated with the neutral state of quinones in the A₁ binding site in photosystem I (PSI) have been difficult to identify in the past. This problem is addressed here, where time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study PSI with six different quinones incorporated into the A₁ binding site. (P700⁺A₁⁻ – P700A₁) and (A₁⁻ – A₁) FTIR difference spectra (DS) were obtained for PSI with the different quinones incorporated, and several double-difference spectra (DDS) were constructed from the DS. From analysis of the DS and DDS, in combination with density functional theory based vibrational frequency calculations of the quinones, the neutral state bands of the incorporated quinones are identified and assigned. For neutral PhQ in the A₁ binding site, infrared absorption bands were identified near 1665 and 1635 cm⁻¹, that are due to the C₁O and C₄O stretching vibrations of the incorporated PhQ, respectively. These assignments indicate a 30 cm⁻¹ separation between the C₁O and C₄O modes, considerably less than the ~80 cm⁻¹ found for similar modes of PhQ⁻. The C₄O mode downshifts due to hydrogen bonding, so the suggestion is that hydrogen bonding is weaker for the neutral state compared to the anion state, indicating radical-induced proton dynamics associated with the quinone in the A₁ binding site in PSI.     

Allosteric kidney-type glutaminase (GLS) inhibitors with a mercaptoethyl linker

A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a mercaptoethyl (SCH₂CH₂) linker were synthesized in an effort to further expand the structural diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl linker between the two thiadiazole rings was found to potently inhibit GLS with an IC₅₀ value of 50 nM. Interestingly, the corresponding derivative with an n-propyl (CH₂CH₂CH₂) linker showed substantially lower inhibitory potency (IC₅₀ = 2.3 μM) while the derivative with a dimethylsulfide (CH₂SCH₂) linker showed no inhibitory activity at concentrations up to 100 μM, underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and tested as GLS inhibitors to further explore SAR within this scaffold including derivatives possessing a pyridazine as a replacement for one of the two thiadiazole moiety.     

Phospholipid headgroups govern area per lipid and emergent elastic properties of bilayers

Phospholipid bilayers are liquid-crystalline materials whose intermolecular interactions at mesoscopic length scales have key roles in the emergence of membrane physical properties. Here we investigated the combined effects of phospholipid polar headgroups and acyl chains on biophysical functions of membranes with solid-state ²H NMR spectroscopy. We compared the structural and dynamic properties of phosphatidylethanolamine and phosphatidylcholine with perdeuterated acyl chains in the solid-ordered (s_o) and liquid-disordered (l_d) phases. Our analysis of spectral lineshapes of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d₆₂) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d₆₂) in the s_o (gel) phase indicated an all-trans rotating chain structure for both lipids. Greater segmental order parameters ($S_{\text{CD}}$) were observed in the l_d (liquid-crystalline) phase for DPPE-d₆₂ than for DPPC-d₆₂ membranes, while their mixtures had intermediate values irrespective of the deuterated lipid type. Our results suggest the $S_{\text{CD}}$ profiles of the acyl chains are governed by methylation of the headgroups and are averaged over the entire system. Variations in the acyl chain molecular dynamics were further investigated by spin-lattice ($R_{1\text{Z}}$) and quadrupolar-order relaxation ($R_{1\text{Q}}$) measurements. The two acyl-perdeuterated lipids showed distinct differences in relaxation behavior as a function of the order parameter. The R_1Z rates had a square-law dependence on $S_{\text{CD}}$, implying collective mesoscopic dynamics, with a higher bending rigidity for DPPE-d₆₂ than for DPPC-d₆₂ lipids. Remodeling of lipid average and dynamic properties by methylation of the headgroups thus provides a mechanism to control the actions of peptides and proteins in biomembranes.     

Der Einfluss der Temperatur Auf die Lage des CO2-Kompensationspunktes

Zusammenfassung Der Begriff CO₂-Kompensationspunkt wird im Vergleich zum Begriff Licht-Kompensationspunkt an Hand von Meßergebnissen erläutert. Die Lage des CO₂-Kompensationspunktes ist von der Temperatur abhängig derart, daß sich bei höherer Temperatur das Gleichgewicht zwischen CO₂-Aufnahme und-abgabe im belichteten Gewebe bei einem höheren CO₂-Partialdruck der Umgebung einstellt. Die Temperaturkoeffizienten für die Dunkelatmung, die apparente und die gesamte Photosynthese werden in Form vonQ ₁₀-Werten wiedergegeben. Da diese Werte bei der photosynthetischen CO₂ Verarbeitung wesentlich kleiner sind wie die bei photochemischen und chemischen Reaktionen beobachteten Temperaturkoeffizienten, wurde vermutet, daß der mit zunehmender Temperatur beobachtete steile Abfall der apparenten Photosynthese sowie die Temperaturabhängigkeit der Gesamtphotosynthese in Form einer Optimumkurve auf eine temperaturabhängige Veränderung der Reaktionsbedingungen zurückzuführen ist. Solche temperaturbedingten Veränderungen sind in bezug auf das CO₂-Diffusionsgefälle zwischen der die Pflanze umgebenden Luft und den Reaktionsorten für die Bindung und photosynthetische Verarbeitung des Kohlendioxyds im Gewebe gegeben, so daß anzunehmen ist, daß die beobachteten Temperaturwirkungen in erster Linie auf temperaturbedingte Veränderungen physikalisch-physiologischer Größen bei dem vor der eigentlichen photochemischen und chemischen CO₂-Verarbeitung stattfindenden Diffusionsvorgang zurückzuführen sind.Mit 8 Textabbildungen.Herrn Prof. Dr.O. Renner zum 70. Geburtstag gewidmet.     

A Gs-RhoGEF interaction: An old G protein finds a new job

The heterotrimeric G proteins are known to have a variety of downstream effectors, but G_s was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated G_s can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by G_s-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gα_s subfamily regulates activity of Rho GTPases extends our understanding of Gα_s activity and establishes RhoGEF coupling as a universal Gα function.

The canonical G protein pathway consists of a cell surface receptor, a heterotrimeric G protein, and an effector protein that controls signaling within the cells. This fundamental paradigm, familiar to every biologist, is rooted in discoveries by the laboratories of Sutherland, Rodbell, and Gilman, which in the 1970s and 1980s dissected biochemical mechanisms of adenylyl cyclase activation by hormones. Their breakthrough came after experiments showing that the G protein G_s is essential to transfer agonist stimulation from the receptor to adenylyl cyclase (1). This G protein consists of the ∼42-kDa α subunit, which binds and hydrolyzes GTP, and the permanently associated dimer of 35-kDa β and ∼10-kDa γ subunits (Gβγ). Their findings helped establish a canonical model in which the agonist-bound receptor causes the G protein to release GDP, and the heterotrimer dissociates into Gα-GTP and free Gβγ; in this state, the G protein can activate its effector (i.e. Gα_s will activate adenylyl cyclase until GTP is hydrolyzed). Although the rod photoreceptor G protein, transducin, was discovered by that time (2), the ubiquitously expressed G_s can be considered the founding member of the G protein family.The subsequent cloning and identification of the other three families (G_i, G_q, and G₁₂) completed the rough map of G protein–mediated transduction. These initial studies suggested that the α subunits were responsible for activation of one type of effector (e.g. Gα_s for adenylyl cyclase and cAMP; Gα_q for phospholipase C, phosphoinositides, and Ca²⁺; and Gα_i for ion channels and inhibition of adenylyl cyclase), whereas the free Gβγ complexes interact with a remarkably large number of binding partners, including some effector enzymes and ion channels (3). Later, Gα₁₂ and Gα₁₃ were found to regulate a distinct type of effectors, the RhoGEFs (4, 5). These multidomain proteins contain pleckstrin homology (PH) domains, which facilitate their membrane localization, and Dbl homology (DH) domains, which catalyze GDP-for-GTP exchange (guanine nucleotide exchange factor; GEF) in the Rho family of small (∼20-kDa) G proteins. At the time, the G₁₂-RhoGEF pathway seemed odd as it contained two G proteins: the receptor-activated “large” G₁₂ class protein and the “small” Rho G protein, which is activated by RhoGEF. However, it was then discovered that Gα_q could activate a RhoGEF called Trio (6), and that Gβγ complexes activate other RhoGEFs, indicating that this pathway, if unusual, is at least popular. Gα_s, however, mostly appeared to be faithful to its originally determined role—to stimulate adenylyl cyclase(s)—possibly contributing to the enduring perception that regulation of a second messenger–generating enzyme is the “real” function of a heterotrimeric G protein.In the current issue of JBC, Castillo-Kauil et al. (7) force a reexamination of the existing canon, presenting data that show Gα_s can also interact with a specific RhoGEF, in this case PDZ-RhoGEF (PRG). The authors made this discovery as part of an examination of the regulation of cell shape by the Rho family. They began by expressing a series of short constructs of three RhoGEF proteins, p115RhoGEF, PRG, and LARG, all of which activated RhoA as expected, promoting cell contraction. However, they noticed that the DH/PH domain of PRG also activated Cdc42 and induced filopodia-like cell protrusions. To investigate which G protein is responsible for activation of this Cdc42-mediated pathway, they overexpressed constitutively active mutants of different Gα subunits. These mutants are stabilized in the active GTP-bound state due to substitution of the glutamine residue crucial for GTP hydrolysis. Surprisingly, the PRG-Cdc42 pathway was stimulated by Gα_sQ227L, the one Gα subtype not known for interaction with RhoGEFs. Furthermore, they showed that binding of PRG to Cdc42 was promoted only by G_s-coupled receptors, and not by G_q- or G_i-coupled GPCRs. The authors then investigated the PRG site responsible for the interaction with Gα_s, narrowing it down to the isolated PRG DH and PH domains and their linker region. A construct encompassing these domains was able to inhibit (i) GPCR-mediated activation of Cdc42, (ii) the Gα_sQ227L-promoted interaction of PRG with Cdc42, and (iii) some protein phosphorylation events downstream of the canonical cAMP pathway. Taken together, their work identifies PRG as a novel effector for G_s; the Gα_s-PRG interaction mediates activation of Rho family protein Cdc42, leading to cytoskeletal remodeling.The unexpected results of Castillo-Kauil et al. open up new opportunities to explore this mechanism at different levels of biology. The experiments described in the paper were performed in vitro using cultured cells, imaging, and pulldown of protein complexes containing the overexpressed Gα_s Q227L mutant. Considering the multitude of G_s-coupled receptors and RhoGEFs in the body (8, 9), it will be important to understand the physiological context where the new G_s-mediated pathway plays a significant role. This will require experimentation in vivo and possibly reevaluation of the phenotypes associated with known pathogenic mutations in Gα_s (GNAS) and other relevant genes. At the molecular level, it would be important to delineate the biochemical mechanisms of Gα_s interaction with PRG. For example, at what stage of the GTP/GDP cycle does Gα_s bind to PRG: in the GTP-bound state, which also activates adenylate cyclase, or in the transition state (i.e. just before the terminal phosphate of GTP is removed)? Indeed, there is precedent for proteins that bind preferentially with the transition state—specifically RGS proteins, which accelerate the GTPase reaction. Another possibility is that, by analogy with p115RhoGEF, which stimulates GTPase activity of Gα₁₂ and Gα₁₃, PRG (and other RhoGEFs with similar DH-PH sequences) can influence interaction of Gα_s with nucleotides, Gβγ, and other partners.Since defining the receptor, G protein, and effector as the three essential members of the G protein pathway, researchers have discovered many additional proteins that regulate the amplitude and duration of the stimulus and/or participate in cross-talk with other signaling circuits. These “new” proteins include arrestins, receptor kinases, nonreceptor exchange factors, GTPase-activating proteins, special chaperones, etc. Thus, in a way, discovering a novel binding partner for a signaling molecule is not as surprising as it would have been 20 years ago. However, the new partner identified by Castillo-Kauil et al. makes the result of extra significance; until now, we knew that three of four G protein subfamilies could regulate Rho GTPases by activating RhoGEFs: G₁₂ and G_q via their α subunits and G_i via the Gβγ subunits (10). The demonstration that the G_s subfamily is no exception shows that activation of RhoGEFs by heterotrimeric G proteins may be a truly universal mechanism (Fig. 1). The significance of this insight is that the multitude of biological processes regulated by Rho-GTPase networks can potentially respond to the entire repertoire of GPCR-mediated stimuli.Open in a separate windowFigure 1.Activation of the Rho family by heterotrimeric G proteins. The Rho family of small GTPases is activated by RhoGEF proteins, some of which can be stimulated by heterotrimeric G proteins. Of four families of heterotrimeric G proteins, three (G₁₂, G_q, and G_i, shown in shades of gray) were known to activate certain RhoGEFs. The new results (highlighted in orange) (7) show that G_s, the G protein known to stimulate production of cAMP, can also stimulate a particular RhoGEF; this suggests that the Rho GTPases can potentially be stimulated by the multitude of signals from the entire class of GPCRs, including those coupled to G_s. IP₃, inositol 1,4,5-trisphosphate.

Funding and additional information—This work was supported in part by National Institutes of Health Grant R56DK119262 (to V. Z. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

PH

pleckstrin homology

DH

Dbl homology

GEF

guanine nucleotide exchange factor

PRG

PDZ-RhoGEF

GPCR

G protein–coupled receptor.

     

The energetic cost of NNT-dependent ROS removal

Under conditions of high nutrient availability and low ATP synthesis, mitochondria generate reactive oxygen species (ROS) that must be removed to avoid cell injury. Among the enzymes involved in this scavenging process, peroxidases play a crucial role, using NADPH provided mostly by nicotinamide nucleotide transhydrogenase (NNT). However, scarce information is available on how and to what extent ROS formation is linked to mitochondrial oxygen consumption. A new study by Smith et al. shows that NNT activity maintains low ROS levels by means of a fine modulation of mitochondrial oxygen utilization.

The rate of human energy expenditure fluctuates, increasing during periods of weight gain and decreasing during weight loss, to prevent large swings in body weight. Central to this ability are mitochondrial redox circuits responsible for nutrient oxidation and reactive oxygen species (ROS) generation. The redox circuits coupling the partial reduction of oxygen with ROS removal are linked with the major redox circuit represented by the complete reduction of oxygen into water at the level of the electron transport chain (ETC). This link enables cells to respond to changes in nutrient availability and energy demand. A better understanding of how these circuits are intertwined could lead to new therapeutic avenues for the treatment of metabolic disorders. New work by Smith et al. (1) reveals a mechanism by which mitochondria sense excess energy supply—particularly when energy demand is low—via ETC. This mechanism dependent on nicotinamide nucleotide transhydrogenase (NNT) couples the maintenance of low ROS levels with an increased oxygen consumption (i.e. energy expenditure).Within the inner mitochondrial membrane (IMM), the energetically favorable flow of electrons from NADH(H⁺) and FADH₂ to oxygen allows proton pumping from the matrix to the intermembrane space. The resulting proton gradient (Δp) is utilized for ATP synthesis, as well as for other mitochondrial processes, such as ion homeostasis, protein import, etc.A minor fraction of the electrons flowing through the ETC is diverted, causing the partial reduction of O₂ into superoxide, which is subsequently converted to H₂O₂ (2, 3). This electron detour is favored when flow is slowed down by a decrease in ATP demand. The carnitine-dependent β-oxidation of fatty acids exacerbates this situation, as it can increase NADH(H⁺) and FADH₂ availability, promoting mitochondrial ROS formation. This potentially deleterious process is counterbalanced by efficacious ROS removal systems. In particular, H₂O₂ reduction into water is catalyzed by peroxidases using reduced GSH and thioredoxin (Trx). The resulting oxidized forms of GSH and Trx are reduced by the reductases catalyzing NADPH(H⁺) oxidation. Various cytosolic and mitochondrial enzymes are involved in restoring the high NADPH(H⁺)/NADP⁺ ratio required not only for an optimal redox balance, but also for many anabolic processes. NNT, a ubiquitously expressed integral protein of the IMM (4), plays a major role among the enzymes contributing to NADP⁺ reduction.NNT functions as a redox-driven proton pump catalyzing the reversible reduction of NADP⁺ to NADPH(H⁺) at the expense of NADH(H⁺) oxidation into NAD⁺. Much of our current knowledge on the role of NNT derives from studies on the mouse strain C57BL/6J (B6J) displaying a markedly lower NNT protein expression as compared with the control B6N strain (5). The lack of NNT curtails NADPH(H⁺) availability and thus peroxidase activities leading to oxidative stress. A severe increase in mitochondrial ROS levels has been linked to various pathologies. On the other hand, a slight increase in mitochondrial ROS formation appears to contribute to endogenous defense mechanisms against cell injury (6). Because NNT is relevant for maintaining NADPH availability necessary for the peroxidase activities required for buffering H₂O₂, the following question arises: To what extent does NNT activity link mitochondrial ROS production resulting from excess substrate availability with mitochondrial oxygen consumption?Dr. Neufer''s group investigated whether an increase in H₂O₂ production due to high rates of substrate oxidation under resting conditions is counterbalanced by a corresponding increase in NNT-mediated oxygen consumption (1). Experiments in mitochondria isolated from hind limb skeletal muscle from B6N mice under conditions mimicking a resting state (i.e. in the absence of ADP) demonstrated that increasing carnitine from 25 μm to 5 mm to maximize palmitoyl-CoA oxidation resulted in a 3-fold increase in the rate of H₂O₂ emission. The combined inhibition of Trx reductase by auranofin (AF) and GSH reductase by carmustine (BCNU) increased H₂O₂ emission by almost 4-fold, demonstrating that the activity of mitochondrial peroxidases buffers >70% of H₂O₂ production driven by β-oxidation. In addition, H₂O₂ formation was shown to depend on a complete fatty acid oxidation, including acetyl-CoA buffering by carnitine acetyltransferase and/or acetyl CoA utilization by the TCA cycle. Notably, the highest rate of fatty acid oxidation obtained at 5 mm carnitine caused an increase in proton conductance that was prevented by AF/BCNU treatment. This interesting finding suggests that GSH and Trx reductases utilize NADPH(H⁺) produced by NNT, which in turn uses Δp generated by mitochondrial respiration. The authors validated this hypothesis comparing permeabilized fibers from B6J and B6N mice. Indeed, the increase in proton conductance was absent in B6J fibers that also were not affected by AF/BCNU addition. Notably, oxygen consumption was 18.6% lower in B6J samples. Therefore, a significant fraction of mitochondrial respiration supports NNT activity in mediating an optimal rate of ROS removal (Fig. 1).Open in a separate windowFigure 1.Schematic of the pathways involved in NNT coupling of mitochondrial ROS formation with oxygen consumption under resting conditions (i.e. no ATP synthesis). ROS removal requires NADPH(H⁺) provided by NNT in a process coupled with the utilization of the proton gradient generated by oxygen consumption. For the sake of simplicity, flavin nucleotides and thioredoxin are omitted, as well as the utilization of the proton gradient for ATP synthesis. FAO, fatty acid oxidation; GPX, GSH peroxidase; GR, GSH reductase; LCACoA, long-chain acyl-CoA; SOD, superoxide dismutase(s).The work conducted by Smith et al. suggests that NNT performs direct and indirect coupling activities that are tightly linked. NNT directly couples NADPH(H⁺) formation from NADH(H⁺) with mitochondrial proton uptake, as is well-established. Smith et al. (1) demonstrate that Δp is maintained by oxygen consumption, such that NNT-mediated ROS removal is physiologically coupled with mitochondrial respiration. However, it is worth noting that the current study does not include in situ or in vivo experiments, perhaps because the carnitine titration of β-oxidation along with the use of reductase and respiration inhibitors could not be applied to intact cells or organs. Thus, it will be important to extend this work to more intact models. Moreover, a word of caution should be mentioned for the use of B6N mice as control strain. A recent study demonstrated that B6N hearts are more prone to contractile failure because of the absence of MYLK3, a protein kinase required for actin assembly (7). However, this defect is unlikely to impact the findings of Smith et al. (1). Nevertheless, a proteomic analysis of BJ6 mitochondria is lacking. Future studies should investigate whether the lack of NNT is compensated by changes in mitochondrial proteins involved in substrate oxidation and ROS removal.

Funding and additional information—This work was supported by Leducq Transatlantic Network of Excellence Grant 16CVD04 and COST Action EU-CARDIOPROTECTION Grant CA16225.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

ROS

reactive oxygen species

ETC

electron transport chain

IMM

inner mitochondrial membrane

NNT

nicotinamide nucleotide transhydrogenase

TCA

tricarboxylic acid cycle

AF

auranofin

BCNU

carmustine.

     

Monitoring,treatment and control of blood glucose and lipids in Ontario First Nations people with diabetes

BACKGROUND:Indigenous people worldwide are disproportionately affected by diabetes and its complications. We aimed to assess the monitoring, treatment and control of blood glucose and lipids in First Nations people in Ontario.METHODS:We conducted a longitudinal population-based study using administrative data for all people in Ontario with diabetes, stratified by First Nations status. We assessed age- and sex-specific rates of completion of recommended monitoring for low-density lipoprotein (LDL) and glycated hemoglobin (A_1c) from 2001/02 to 2014/15. We used data from 2014/15 to conduct a cross-sectional analysis of rates of achievement of A_1c and LDL targets and use of glucose-lowering medications.RESULTS:The study included 22 240 First Nations people and 1 319 503 other people in Ontario with diabetes. Rates of monitoring according to guidelines were 20%–50% for A_1c and 30%–70% for lipids and were lowest for younger First Nations men. The mean age- and sex-adjusted A_1c level was higher among First Nations people than other people (7.59 [95% confidence interval (CI) 7.57 to 7.61] v. 7.03 [95% CI 7.02 to 7.03]). An A_1c level of 8.5% or higher was observed in 24.7% (95% CI 23.6 to 25.0) of First Nations people, compared to 12.8% (95% CI 12.1 to 13.5) of other people in Ontario. An LDL level of 2.0 mmol/L or less was observed in 60.3% (95% CI 59.7 to 61.6) of First Nations people, compared to 52.0% (95% CI 51.1 to 52.9) of other people in Ontario. Among those aged 65 or older, a higher proportion of First Nations people than other Ontarians were using insulin (28.1% v. 15.1%), and fewer were taking no medications (28.3% v. 40.1%).INTERPRETATION:As of 2014/15, monitoring and achievement of glycemic control in both First Nations people and other people in Ontario with diabetes remained suboptimal. Interventions to support First Nations patients to reach their treatment goals and reduce the risk of complications need further development and study.

Diabetes and its related complications are major contributors to morbidity and mortality worldwide.^1–3 Indigenous populations in Canada and around the world are disproportionately affected by diabetes owing to the complex relations among colonization, social disadvantage, stress, trauma and metabolic health.^4–7 In addition to our own work showing persistently higher rates of peripheral vascular disease, stroke, cardiac disease, renal dysfunction and ophthalmologic complications in Ontario First Nations,^8–12 other Canadian and international studies also showed higher complication rates in diverse Indigenous populations.^6,7,13–15Glycemic control is fundamental to the management of diabetes and the prevention of complications.¹⁶ Glycated hemoglobin (A_1c) is a reliable way to estimate the average level of glucose in the blood.¹⁷ Since A_1c levels higher than 7.0% have been associated with an increased risk of microvascular complications,^18–20 treatment guidelines suggest A_1c should be measured every 3–6 months to ensure that glycemic goals are being met or maintained.²¹ Since people with diabetes also have an elevated risk for cardiovascular disease,^22–24 management and control of cardiovascular risk factors, particularly lipids such as low-density lipoprotein (LDL) cholesterol, are also important.^25–27 Guidelines further recommend that a full lipid profile be measured every 1–3 years, depending on cardiovascular risk, and suggest that LDL be consistently less than 2.0 mmol/L.²⁸ Control of A_1c and lipids has been shown to be associated with reduced morbidity and mortality in patients with diabetes.^18,29–32One possible reason for the high burden of complications among Indigenous people with diabetes may be failure to achieve control of these 2 key clinical parameters. We examined differences between Status First Nations people with diabetes in Ontario and all other Ontario residents with diabetes in rates of monitoring of A_1c and lipids, achievement of targets for A_1c and LDL outlined in clinical guidelines, and patterns of medication use to help attain these targets.     

An estimator of the Opportunity for Selection that is independent of mean fitness

Variation among individuals in number of offspring (fitness, k) sets an upper limit to the evolutionary response to selection. This constraint is quantified by Crow's Opportunity for Selection (I), which is the variance in relative fitness (I = σ²_k/(u_k)²). Crow's I has been widely used but remains controversial because it depends on mean offspring number in a sample (). Here, I used a generalized Wright-Fisher model that allows for unequal probabilities of producing offspring to evaluate behavior of Crow's I and related indices under a wide range of sampling scenarios. Analytical and numerical results are congruent and show that rescaling the sample variance (s²_k) to its expected value at a fixed removes dependence of I on mean offspring number, but the result still depends on choice of . A new index is introduced, Δ_I = Π– E(Î_drift) = Π– 1/, which makes Î independent of sample without the need for variance rescaling. Δ_I has a straightforward interpretation as the component of variance in relative fitness that exceeds that expected under a null model of random reproductive success. Δ_I can be used to directly compare estimates of the Opportunity for Selection for samples from different studies, different sexes, and different life stages.     

A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes

The evolution of Na⁺-selective four-domain voltage-gated channels (4D-Na_vs) in animals allowed rapid Na⁺-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na⁺-selective voltage-gated channels (BacNa_v), they typically exhibit much slower kinetics than 4D-Na_vs, and are not thought to have crossed the prokaryote–eukaryote boundary. As such, the capacity for rapid Na⁺-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na⁺-selective, single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores, Emiliania huxleyi and Scyphosphaera apsteinii. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Na_v blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na⁺-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Na_vs. The EukCatB channels therefore represent an independent evolution of fast Na⁺-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae.

Electrical signals trigger rapid physiological events that underpin an array of fundamental processes in eukaryotes, from contractile amoeboid locomotion (Bingley and Thompson, 1962), to the action potentials of mammalian nerve and muscle cells (Hodgkin and Huxley, 1952). These events are mediated by voltage-gated ion channels (Brunet and Arendt, 2015). In excitable animal cells, Ca²⁺- or Na⁺-selective members of the four-domain voltage-gated cation channel family (4D-Ca_v/Na_v) underpin well-characterized signaling processes (Catterall et al., 2017). The 4D-Ca_v/Na_v family is broadly distributed across eukaryotes, contributing to signaling processes associated with motility in some unicellular protist and microalgal species (Fujiu et al., 2009; Lodh et al., 2016), although these channels are absent from land plants (Edel et al., 2017). It is likely that the ancestral 4D-Ca_v/Na_v channel was Ca²⁺-permeable, with Na⁺-selective channels arising later within the animal lineage (Moran et al., 2015). This shift in ion selectivity represented an important innovation in animals, allowing rapid voltage-driven electrical excitability to be decoupled from intracellular Ca²⁺ signaling processes (Moran et al., 2015).Na⁺-selective voltage-gated channels have not been described in other eukaryotes, although a large family of Na⁺-selective channels (BacNa_v) is present in prokaryotes (Ren et al., 2001; Koishi et al., 2004). BacNa_v are single-domain channels that form homotetramers, resembling the four-domain architecture of 4D-Ca_v/Na_v. Studies of BacNa_v channels have provided considerable insight into the mechanisms of gating and selectivity in voltage-dependent ion channels (Payandeh et al., 2012; Zhang et al., 2012). The range of activation and inactivation kinetics of native BacNa_v are generally slower than observed for 4D-Na_v, suggesting that the concatenation and subsequent differentiation of individual pore-forming subunits may have enabled 4D-Na_v to develop specific properties such as fast inactivation, which is mediated by the conserved intracellular Ile–Phe–Met linker between domains III and IV (Fig. 1A; Irie et al., 2010; Catterall et al., 2017).Open in a separate windowFigure 1.EukCatBs represent a novel class of single-domain channels. A, Schematic diagram of a single-domain EukCatB channel. The voltage-sensing module (S1–S4, blue), including conserved positively charged (++) residues of segment (S4) that responds to changes in membrane potential, is shown. The pore module (S5–S6, red) is also indicated, including the SF motif (Ren et al., 2001). The structure of a 4D-Na_v (showing the SF of rat 4D-Na_v1.4 with canonical “DEKA” locus of Na⁺-selective 4D-Na_v1s) is also displayed (right). The Ile–Phe–Met motif of the fast inactivation gate is indicated (West et al., 1992) B, Maximum likelihood phylogenetic tree of single-domain, voltage-gated channels including BacNa_v and the three distinct classes of EukCat channels (EukCatA–C). Representatives of the specialized family of single-domain Ca²⁺ channels identified in mammalian sperm (CatSpers) are also included. SF for each sequence is shown (right). “Position 0” of the high-field–strength site that is known to be important in determining Na⁺ selectivity (Payandeh et al., 2011), is colored red. Channel sequences selected for functional characterization in this study are shown in bold. EukCatA sequences previously characterized (Helliwell et al., 2019) are also indicated, as is NaChBac channel from B. halodurans (Ren et al., 2001). Maximum likelihood bootstrap values (>70) and Bayesian posterior probabilities (>0.95) are above and below nodes, respectively. Scanning electron micrographs of coccolithophores E. huxleyi (scale bar = 2 μm) and S. apsteinii, (scale bar = 10 μm) are shown.We recently identified several classes of ion channel (EukCats) bearing similarity to BacNa_v in the genomes of eukaryotic phytoplankton. Characterization of EukCatAs found in marine diatoms demonstrated that these voltage-gated channels are nonselective (exhibiting permeability to both Na⁺ and Ca²⁺) and play a role in depolarization-activated Ca²⁺ signaling (Helliwell et al., 2019). Two other distinct classes of single-domain channels (EukCatBs and EukCatCs) were also identified that remain uncharacterized. These channels are present in haptophytes, pelagophytes, and cryptophytes (EukCatBs), as well as dinoflagellates (EukCatCs; Helliwell et al., 2019). Although there is a degree of sequence similarity between the distinct EukCat clades, the relationships between clades are not well resolved, and there is not clear support for a monophyletic origin of EukCats. The diverse classes of EukCats may therefore exhibit significant functional differences. Characterization of these different classes of eukaryote single-domain channels is thus vital to our understanding of eukaryote ion channel structure, function, and evolution, and to our gaining insight into eukaryote membrane physiology more broadly.Notably, EukCatB channels were found in ecologically important coccolithophores, a group of unicellular haptophyte algae that represent major primary producers in marine ecosystems. Coccolithophores are characterized by their ability to produce a cell covering of ornate calcium carbonate platelets (coccoliths; Fig. 1B; Taylor et al., 2017). The calcification process plays an important role in global carbon cycling, with the sinking of coccoliths representing a major flux of carbon to the deep ocean. Patch-clamp studies of coccolithophores indicate several unusual aspects of membrane physiology, such as an inwardly rectifying Cl⁻ conductance and a large outward H⁺ conductance at positive membrane potentials, which may relate to the increased requirement for pH homeostasis associated with intracellular calcification. Here we report that EukCatB channels from two coccolithophore species (Emiliania huxleyi and Scyphosphaera apsteinii) act as very fast Na⁺-selective voltage-gated channels that exhibit many similarities to the 4D-Na_vs, which underpin neuronal signaling in animals. Thus, our findings demonstrate that the capacity for rapid Na⁺-based signaling has evolved in certain photosynthetic eukaryotes, contrary to previous widely held thinking.     

High throughput screening of ultrafiltration and diafiltration processing of monoclonal antibodies via the ambr® crossflow system

As the biopharmaceutical industry moves toward high concentration of monoclonal antibody drug substance, additional development is required early on when material is still limited. A key constraint is the availability of predictive high-throughput low-volume filtration screening systems for bioprocess development. This particularly impacts final stages such as ultrafiltration/diafiltration steps where traditional scale-down systems need hundreds of milliliters of material per run. Recently, the ambr® crossflow system has been commercialized by Sartorius Stedim Biotech (SSB) to meet this need. It enables parallel high throughput experimentation by only using a fraction of typical material requirements. Critical parameters for predictive filtration systems include loading, mean transmembrane pressure (Δ$\overline{P}$ _TMP), and crossflow rate (Q_F). While axial pressure drop (ΔP_axial) across the cartridge is a function of these parameters, it plays a key role and similar values should result across scales. The ambr® crossflow system is first presented describing typical screening experiments. Its performance is then compared to a traditional pilot-scale tangential flow filtration (TFF) at defined conditions. The original ambr® crossflow (CF) cartridge underperformed resulting in ~20x lower ΔP_axial than the pilot-scale TFF flat-sheet cassette. With an objective to improve the scalability of the system, efforts were made to understand this scale difference. The ambr® CF cartridge was successfully modified by restricting the flow of the feed channel, and thus increasing its ΔP_axial. Additional studies across a range of loading (100–823 gm⁻²); Δ$\overline{P}$ _TMP (12–18 psi); and Q_F (4–8 L/min/m²) were conducted in both scales. Comparable flux and aggregate levels were achieved.     

Comparative palatability of orally disintegrating tablets (ODTs) of Praziquantel (L-PZQ and Rac-PZQ) versus current PZQ tablet in African children: A randomized,single-blind,crossover study

BackgroundPraziquantel (PZQ) is currently the only recommended drug for infection and disease caused by the schistosome species that infects humans; however, the current tablet formulation is not suitable for pre-school age children mainly due to its bitterness and the large tablet size. We assessed the palatability of two new orally disintegrating tablet (ODT) formulations of PZQ.MethodologyThis randomized, single-blind, crossover, swill-and-spit palatability study ({"type":"clinical-trial","attrs":{"text":"NCT02315352","term_id":"NCT02315352"}}NCT02315352) was carried out at a single school in Tanzania in children aged 6–11 years old, with or without schistosomiasis infection as this was not part of the assessment. Children were stratified according to age group (6–8 years or 9–11 years) and gender, then randomized to receive each formulation in a pre-specified sequence. Over 2 days, the children assessed the palatability of Levo-Praziquantel (L-PZQ) ODT 150 mg and Racemate Praziquantel (Rac-PZQ) ODT 150 mg disintegrated in the mouth without water on the first day, and L-PZQ and Rac-PZQ dispersed in water and the currently available PZQ 600 mg formulation (PZQ-Cesol) crushed and dispersed in water on the second day. The palatability of each formulation was rated using a 100 mm visual analogue scale (VAS) incorporating a 5-point hedonic scale, immediately after spitting out the test product (VAS_{t = 0} primary outcome) and after 2–5 minutes (VAS_{t = 2–5}).Principal findingsIn total, 48 children took part in the assessment. Overall, there was no reported difference in the VAS_{t = 0} between the two ODT formulations (p = 0.106) without water. Higher VAS_{t = 0} and VAS_{t = 2–5} scores were reported for L-PZQ ODT compared with Rac-PZQ ODT in older children (p = 0.046 and p = 0.026, respectively). The VAS_{t = 0} and VAS_{t = 2–5} were higher for both ODT formulations compared with the standard formulation (p<0.001 for both time points). No serious adverse events were reported.Conclusions/SignificanceThe new paediatric-friendly formulations dispersed in water were both found to be more palatable than the existing standard formulation of PZQ. There may be gender and age effects on the assessment of palatability. Further research is needed for assessing efficacy and tolerability of the newly ODTs Praziquantel drug in younger children.Trial registrationThe trial was registered on ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT02315352","term_id":"NCT02315352"}}NCT02315352) and in the Pan African Clinical Trials Registry (PACTR201412000959159).     

Zur Physiologischen Spezialisierung vonPhytophthora infestans de Bary

Zusammenfassung Die vorliegende Arbeit berichtet über Versuche mit 4 verschiedenen Einsporangienlinien vonPhytophthora infestans.Die Kultur- und Infektionsmethoden werden beschrieben. Es werden für diese Versuche Kulturen auf Malzextraktagar, Kartoffelknollen und Kartoffellaub neben- und nacheinander benutzt, um einerseits die Vermischungsgefahr weitgehend zu vermeiden und andererseits die Infektionstüchtigkeit der Sporen auf dem Optimum zu halten.Die Ergebnisse von Infektionsversuchen mit den 4 Linien des Pilzes auf 246 Kartoffelklonen werden besprochen. Es handelt sich umF ₁,F ₂,F₂,F₃,F₄, Klone der KreuzungSolanum demissum x Solanum tuberosum. Nach ihrem Verhalten gegenüber den 4 Phytophthoralinien zerfallen diese 246 Klone in 5 Klongruppen: A, W, K, M, Z. Das Verhalten der 4 Linien auf diesen Gruppen wird dargestellt.Die Bedeutung der verschiedenen Linien des Pilzes für die züchterische Arbeit wird diskutiert. Nicht die geographische Verbreitung einer Pilzrasse, sondern ihre Aggressivität gegenüber dem Zuchtmaterial des Züchters bestimmt ihre Bedeutung für unsere züchterischen Arbeiten.Ein Testsortiment zur Charakterisierung der 4 Linien wird angegeben.Die Möglichkeiten der Entstehung und der Auffindung weiterer Pilzrassen werden besprochen.Mit Unterstützung der Deutschen Forschungsgemeinschaft.