Air channels create a directional light signal to regulate hypocotyl phototropism.

In plants, light direction is perceived by the phototropin photoreceptors, which trigger directional growth responses known as phototropism. The formation of a phototropin activation gradient across a photosensitive organ initiates this response. However, the optical tissue properties that functionally contribute to phototropism remain unclear. In this work, we show that intercellular air channels limit light transmittance through various organs in several species. Air channels enhance light scattering in Arabidopsis hypocotyls, thereby steepening the light gradient. This is required for an efficient phototropic response in Arabidopsis and Brassica. We identified an embryonically expressed ABC transporter required for the presence of air channels in seedlings and a structure surrounding them. Our work provides insights into intercellular air space development or maintenance and identifies a mechanism of directional light sensing in plants.

Correlated cryo-SEM and CryoNanoSIMS imaging of biological tissue.

BACKGROUND: The development of nanoscale secondary ion mass spectrometry (NanoSIMS) has revolutionized the study of biological tissues by enabling, e.g., the visualization and quantification of metabolic processes at subcellular length scales. However, the associated sample preparation methods all result in some degree of tissue morphology distortion and loss of soluble compounds. To overcome these limitations an entirely cryogenic sample preparation and imaging workflow is required. RESULTS: Here, we report the development of a CryoNanoSIMS instrument that can perform isotope imaging of both positive and negative secondary ions from flat block-face surfaces of vitrified biological tissues with a mass- and image resolution comparable to that of a conventional NanoSIMS. This capability is illustrated with nitrogen isotope as well as trace element mapping of freshwater hydrozoan Green Hydra tissue following uptake of 15N-enriched ammonium. CONCLUSION: With a cryo-workflow that includes vitrification by high pressure freezing, cryo-planing of the sample surface, and cryo-SEM imaging, the CryoNanoSIMS enables correlative ultrastructure and isotopic or elemental imaging of biological tissues in their most pristine post-mortem state. This opens new horizons in the study of fundamental processes at the tissue- and (sub)cellular level. TEASER: CryoNanoSIMS: subcellular mapping of chemical and isotopic compositions of biological tissues in their most pristine post-mortem state.

ABCG transporters export cutin precursors for the formation of the plant cuticle.

The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs, forming a diffusion barrier and protecting the plant against various abiotic and biotic stresses. Cutin, the structural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors. Here, we characterize SlABCG42 of tomato representing an ortholog of AtABCG32 in Arabidopsis. SlABCG42 expression in Arabidopsis complements the cuticular deficiencies of the Arabidopsis pec1/abcg32 mutant. RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32 (SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomato fruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 and SlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursor in vivo. Interestingly, also free ω-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported, furthering the research on the identification of cutin precursors in vivo and the respective mechanisms of their integration into the cutin polymer.

Drosophila melanogaster cloak their eggs with pheromones, which prevents cannibalism.

Oviparous animals across many taxa have evolved diverse strategies that deter egg predation, providing valuable tests of how natural selection mitigates direct fitness loss. Communal egg laying in nonsocial species minimizes egg predation. However, in cannibalistic species, this very behavior facilitates egg predation by conspecifics (cannibalism). Similarly, toxins and aposematic signaling that deter egg predators are often inefficient against resistant conspecifics. Egg cannibalism can be adaptive, wherein cannibals may benefit through reduced competition and added nutrition, but since it reduces Darwinian fitness, the evolution of anticannibalistic strategies is rife. However, such strategies are likely to be nontoxic because deploying toxins against related individuals would reduce inclusive fitness. Here, we report how D. melanogaster use specific hydrocarbons to chemically mask their eggs from cannibal larvae. Using an integrative approach combining behavioral, sensory, and mass spectrometry methods, we demonstrate that maternally provisioned pheromone 7,11-heptacosadiene (7,11-HD) in the eggshell's wax layer deters egg cannibalism. Furthermore, we show that 7,11-HD is nontoxic, can mask underlying substrates (for example, yeast) when coated upon them, and its detection requires pickpocket 23 (ppk23) gene function. Finally, using light and electron microscopy, we demonstrate how maternal pheromones leak-proof the egg, consequently concealing it from conspecific larvae. Our data suggest that semiochemicals possibly subserve in deceptive functions across taxa, especially when predators rely on chemical cues to forage, and stimulate further research on deceptive strategies mediated through nonvisual sensory modules. This study thus highlights how integrative approaches can illuminate our understanding on the adaptive significance of deceptive defenses and the mechanisms through which they operate.

A targeted 3D EM and correlative microscopy method using SEM array tomography.

Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography for asymmetric samples and model organisms (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio). We modified a diamond knife to simplify serial section array acquisition with minimal artifacts. After array acquisition, the arrays were transferred to a glass coverslip or silicon wafer support. Using light microscopy, the arrays were screened rapidly for initial recognition of global anatomical features (organs or body traits). Then, using SEM, an in-depth study of the cells and/or organs of interest was performed. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.

Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis.

The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense.

The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis.

The cuticle is an essential diffusion barrier on aerial surfaces of land plants whose structural component is the polyester cutin. The PERMEABLE CUTICLE1/ABCG32 (PEC1) transporter is involved in plant cuticle formation in Arabidopsis. The gpat6 pec1 and gpat4 gapt8 pec1 double and triple mutants are characterized. Their PEC1-specific contributions to aliphatic cutin composition and cuticle formation during plant development are revealed by gas chromatography/mass spectrometry and Fourier-transform infrared spectroscopy. The composition of cutin changes during rosette leaf expansion in Arabidopsis. C16:0 monomers are in higher abundance in expanding than in fully expanded leaves. The atypical cutin monomer C18:2 dicarboxylic acid is more prominent in fully expanded leaves. Findings point to differences in the regulation of several pathways of cutin precursor synthesis. PEC1 plays an essential role during expansion of the rosette leaf cuticle. The reduction of C16 monomers in the pec1 mutant during leaf expansion is unlikely to cause permeability of the leaf cuticle because the gpat6 mutant with even fewer C16:0 monomers forms a functional rosette leaf cuticle at all stages of development. PEC1/ABCG32 transport activity affects cutin composition and cuticle structure in a specific and non-redundant fashion.

Transmission Fourier transform infrared microspectroscopy allows simultaneous assessment of cutin and cell-wall polysaccharides of Arabidopsis petals.

A procedure for the simultaneous analysis of cell-wall polysaccharides, amides and aliphatic polyesters by transmission Fourier transform infrared microspectroscopy (FTIR) has been established for Arabidopsis petals. The combination of FTIR imaging with spectra derivatization revealed that petals, in contrast to other organs, have a characteristic chemical zoning with high amount of aliphatic compounds and esters in the lamina and of polysaccharides in the stalk of the petal. The hinge region of petals was particular rich in amides as well as in vibrations potentially associated with hemicellulose. In addition, a number of other distribution patterns have been identified. Analyses of mutants in cutin deposition confirmed that vibrations of aliphatic compounds and esters present in the lamina were largely associated with the cuticular polyester. Calculation of spectrotypes, including the standard deviation of intensities, allowed detailed comparison of the spectral features of various mutants. The spectrotypes not only revealed differences in the amount of polyesters in cutin mutants, but also changes in other compound classes. For example, in addition to the expected strong deficiencies in polyester content, the long-chain acyl CoA synthase 2 mutant showed increased intensities of vibrations in a wavelength range that is typical for polysaccharides. Identical spectral features were observed in quasimodo2, a cell-wall mutant of Arabidopsis with a defect in pectin formation that exhibits increased cellulose synthase activity. FTIR thus proved to be a convenient method for the identification and characterization of mutants affected in the deposition of cutin in petals.

PPARgamma in placental angiogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in diverse biological processes including adipocyte differentiation, glucose homeostasis, and inflammatory responses. Analyses of PPARγ knockout animals have been so far preempted by the early embryonic death of PPARγ-/- embryos as a consequence of the severe alteration of their placental vasculature. Using Sox2Cre/PPARγL2/L2 mice, we obtained fully viable PPARγ-null mice through specific and total epiblastic gene deletion, thereby demonstrating that the placental defect is the unique cause of PPARγ-/- embryonic lethality. The vasculature defects observed in PPARγ-/- placentas at embryonic d 9.5 correlated with an unsettled balance of pro- and antiangiogenic factors as demonstrated by increased levels of proliferin (Prl2c2, PLF) and decreased levels of proliferin-related protein (Prl7d1, PRP), respectively. To analyze the role of PPARγ in the later stage of placental development, when its expression peaks, we treated pregnant wild-type mice with the PPARγ agonist rosiglitazone. This treatment resulted in a disorganization of the placental layers and an altered placental microvasculature, accompanied by the decreased expression of proangiogenic genes such as Prl2c2, vascular endothelial growth factor, and Pecam1. Together our data demonstrate that PPARγ plays a pivotal role in controlling placental vascular proliferation and contributes to its termination in late pregnancy.

The Smartcanula: a new tool for remote access perfusion in limited access cardiac surgery.

Devices for venous cannulation have seen significant progress over time: the original, rigid steel cannulas have evolved toward flexible plastic cannulas with wire support that prevents kinking, very thin walled wire wound cannulas allowing for percutaneous application, and all sorts of combinations. In contrast to all these rectilinear venous cannula designs, which present the same cross-sectional area over their entire intravascular path, the smartcanula concept of "collapsed insertion and expansion in situ" is the logical next step for venous access. Automatically adjusting cross-sectional area up to a pre-determined diameter or the vessel lumen provides optimal flow and ease of use for both, insertion and removal. Smartcanula performance was assessed in a small series of patients (76 +/- 17 kg) undergoing redo procedures. The calculated target pump flow (2.4 L/min/m2) was 4.42 +/- 61 L/ min. Mean pump flow achieved during cardiopulmonary bypass was 4.84 +/- 87 L/min or 110% of the target. Reduced atrial chatter, kink resistance in situ, and improved blood drainage despite smaller access orifice size, are the most striking advantages of this new device. The benefits of smart cannulation are obvious in remote cannulation for limited access cardiac surgery, but there are many other cannula applications where space is an issue, and that is where smart cannulation is most effective.

Does large oversizing of self-expandable endoprostheses compensate for aortic growth?

PURPOSE: There is experimental evidence that self-expandable endoprostheses are appropriate for compensation of aortic growth. A potential future application includes their use in the treatment of aortic coarctation. Yet their behavior is poorly investigated. The present study evaluates the performance of largely oversized self-expandable endoprostheses (EPs) in the growing porcine aorta and the biologic response toward them. METHODS: Twenty oversized EPs (Talent, TalentLoPro, Stenway, and Wallstent; nominal lumen area 314 mm(2) [diameter 20 mm]) were implanted in the descending thoracic aorta of juvenile pigs. Four nonoversized EPs (TalentLoPro; nominal lumen area 154 mm(2) [diameter 14 mm]) served as controls. Cross sections of aorta and EPs were measured by intravascular ultrasound (IVUS) at implantation and 3 months follow-up. The histologic response was assessed by microscopy. RESULTS: At implantation, the EPs were 99 +/- 41% oversized relative to the aortic area [48 +/- 22% in diameter]. At follow-up, the area of the aortic lumen increased 60 +/- 50%, P <.001 [29 +/- 23% in diameter]. The Stenway EPs demonstrated the largest prosthetic lumen with 80 +/- 10% of their nominal area (P =.001). However, the prosthetic lumen area was reduced by intimal hyperplasia in all devices. The largest perfused lumen was obtained by the TalentLoPro EPs due to their progressive expansion. Nonoversized EPs resulted in a significantly smaller perfused lumen (P =.012). The inflammatory response to polyester was moderate, whereas polyurethane evoked an extensive body-foreign reaction. CONCLUSION: Large oversizing of self-expandable EPs is essential in order to maintain a large perfused lumen during aortic growth, thereby minimizing the adverse effect of intimal hyperplasia. This was best achieved by the TalentLoPro EPs.

A prototype paediatric venous cannula with shape change in situ.

During cardiopulmonary bypass (CPB), venous drainage may be impeded due to small vessel and cannula size or chattering, thus, blood return to the heart-lung machine is reduced. We designed a self-expandable prototype cannula, which is able to maintain the vein open and overcome this problem and analysed its performance capability. This prototype and several other cannulae were tested using an access vessel diameter of 7 mm. An in vitro circuit was set up with a 10 mm penrose latex tube simulating the patient's vein placed between the patient preload reservoir and the cannula, encasing the cannula's inlet(s). Maximum flow rate was determined for passive venous drainage (PVD) at preloads (P) of 2 and 4 mmHg. We compared these results to three classic single-stage venous cannulae: basket tip, thoracic drain and percutaneous tip. By comparing the other cannulae to the prototype, under PVD conditions and a central venous pressure (CVP) of 2 mmHg, the prototype cannula's flow rate (1.32 +/- 0.04 L/min) outperformed the basket type (the best performing comparator) (1.02 +/- 0.08 L/min) by 23% (p < 0.005). When the preload was increased to 4 mmHg under PVD conditions, the same trend was noted with the prototype cannula (1.65 +/- 0.05 L/min), outperforming the basket cannula's value (1.26 +/- 0.05 L/min) by 24% (p < 0.001). This new cannula design provides superior flow characteristics, under all test conditions, compared to the classic single-stage venous cannulae used for paediatric CPB surgery.

Biologic fixation of polyester- versus polyurethane-covered stents in a porcine model.

PURPOSE: Migration of endoprostheses remains a concern in endovascular aneurysm treatment. Biologic fixation is supposed to enhance anchorage, but the diseased atherosclerotic aorta in humans has demonstrated a limited capacity to incorporate an endoprosthesis by cellular proliferation. The biologic response of two different types of endoprostheses was evaluated in the porcine aorta. MATERIALS AND METHODS: Two types of endoprostheses--four polyurethane-covered (PUC) stents with a macroporous polyurethane covering and four polyester-covered (PEC) stents with a woven polyester covering--were implanted in eight infrarenal porcine aortas for 6 weeks. Electron microscopy and qualitative and quantitative microscopy were performed on serial cross sections. RESULTS: The PUC stents demonstrated an increase in diameter (from 8 mm +/- 1 to 10 mm +/- 1, 12.5%; P = .009), whereas the PEC stents persisted in their original dimensions (8 mm +/- 1, 0%). PUC and PEC stents were covered by continuous thrombus-free neointima (269 microm +/- 51 vs 575 microm +/- 113, respectively; P < .01). The PUC stents demonstrated firm attachment to the aorta as a consequence of a granulation tissue with ingrowth into the pores of the polyurethane covering. The PEC stents remained in loose contact with the aorta in the absence of tissue ingrowth. CONCLUSIONS: Enhanced biologic fixation was achieved by extensive granulation tissue invading the pores of PUC endoprostheses. This finding can modify the design of future devices.