The role of Atg16 in autophagy, anthocyanin biosynthesis, and programmed cell death in leaves of the lace plant (Aponogeton madagascariensis).

Aponogeton madagascariensis, commonly known as the lace plant, produces leaves that form perforations by programmed cell death (PCD). Leaf development is divided into several stages beginning with "pre-perforation" furled leaves enriched with red pigmentation from anthocyanins. The leaf blade is characterized by a series of grids known as areoles bounded by veins. As leaves develop into the "window stage", anthocyanins recede from the center of the areole towards the vasculature creating a gradient of pigmentation and cell death. Cells in the middle of the areole that lack anthocyanins undergo PCD (PCD cells), while cells that retain anthocyanins (non-PCD cells) maintain homeostasis and persist in the mature leaf. Autophagy has reported roles in survival or PCD promotion across different plant cell types. However, the direct involvement of autophagy in PCD and anthocyanin levels during lace plant leaf development has not been determined. Previous RNA sequencing analysis revealed the upregulation of autophagy-related gene Atg16 transcripts in pre-perforation and window stage leaves, but how Atg16 affects PCD in lace plant leaf development is unknown. In this study, we investigated the levels of Atg16 in lace plant PCD by treating whole plants with either an autophagy promoter rapamycin or inhibitors concanamycin A (ConA) or wortmannin. Following treatments, window and mature stage leaves were harvested and analyzed using microscopy, spectrophotometry, and western blotting. Western blotting showed significantly higher Atg16 levels in rapamycin-treated window leaves, coupled with lower anthocyanin levels. Wortmannin-treated leaves had significantly lower Atg16 protein and higher anthocyanin levels compared to the control. Mature leaves from rapamycin-treated plants generated significantly fewer perforations compared to control, while wortmannin had the opposite effect. However, ConA treatment did not significantly change Atg16 levels, nor the number of perforations compared to the control, but anthocyanin levels did increase significantly in window leaves. We propose autophagy plays a dual role in promoting cell survival in NPCD cells by maintaining optimal anthocyanin levels and mediating a timely cell death in PCD cells in developing lace plant leaves. How autophagy specifically affects anthocyanin levels remained unexplained.

Chemical Solution Deposition of Insulating Yttria Nanolayers as Current Flow Diverter in Superconducting GdBa2Cu3O7-δ Coated Conductors.

The primary benefit of a metallic stabilization/shunt in high temperature superconductor (HTS) coated conductors (CCs) is to prevent joule heating damage by providing an alternative path for the current flow during the HTS normal state transition (i.e., quench). However, the shunt presence in combination with unavoidable fluctuations in the critical current (I c) of the HTS film can develop a localized quench along the CC's length if the operational current is kept close to I c. This scenario, also known as the hot-spot regime, can lead to the rupture of the CC if the local quench does not propagate fast enough. The current flow diverter (CFD) is the CC architecture concept that has proven to increase the conductor's robustness against a hot-spot regime by simply boosting the quench velocity in the CC, which avoids the shunt compromise in some applications. This work investigates a practical manufacturing route for incorporating the CFD architecture in a reel-to-reel system via the preparation of yttrium oxide (Y2O3) as an insulating thin nanolayer (∼100 nm) on top of a GdBa2Cu3O7 (GdBCO) superconductor. Chemical solution deposition (CSD) using ink jet printing (IJP) is shown to be a suitable manufacturing approach. Two sequences of the experimental steps have been investigated, where oxygenation of the GdBCO layer is performed after or before the solution deposition and the Y2O3 nanolayer thermal treatment formation step. A correlated analysis of the microstructure, in situ oxygenation kinetics, and superconducting properties of the Ag/Y2O3/GdBCO trilayer processed under different conditions shows that a new customized functional CC can be prepared. The successful achievement of the CFD effect in the case of the preoxygenated customized CC was confirmed by measuring the current transfer length, thus demonstrating the effectiveness of the CSD-IJP as a processing method.

RNA-Seq analysis reveals potential regulators of programmed cell death and leaf remodelling in lace plant (Aponogeton madagascariensis).

BACKGROUND: The lace plant (Aponogeton madagascariensis) is an aquatic monocot that develops leaves with uniquely formed perforations through the use of a developmentally regulated process called programmed cell death (PCD). The process of perforation formation in lace plant leaves is subdivided into several developmental stages: pre-perforation, window, perforation formation, perforation expansion and mature. The first three emerging "imperforate leaves" do not form perforations, while all subsequent leaves form perforations via developmentally regulated PCD. PCD is active in cells called "PCD cells" that do not retain the antioxidant anthocyanin in spaces called areoles framed by the leaf veins of window stage leaves. Cells near the veins called "NPCD cells" retain a red pigmentation from anthocyanin and do not undergo PCD. While the cellular changes that occur during PCD are well studied, the gene expression patterns underlying these changes and driving PCD during leaf morphogenesis are mostly unknown. We sought to characterize differentially expressed genes (DEGs) that mediate lace plant leaf remodelling and PCD. This was achieved performing gene expression analysis using transcriptomics and comparing DEGs among different stages of leaf development, and between NPCD and PCD cells isolated by laser capture microdissection. RESULTS: Transcriptomes were sequenced from imperforate, pre-perforation, window, and mature leaf stages, as well as PCD and NPCD cells isolated from window stage leaves. Differential expression analysis of the data revealed distinct gene expression profiles: pre-perforation and window stage leaves were characterized by higher expression of genes involved in anthocyanin biosynthesis, plant proteases, expansins, and autophagy-related genes. Mature and imperforate leaves upregulated genes associated with chlorophyll development, photosynthesis, and negative regulators of PCD. PCD cells were found to have a higher expression of genes involved with ethylene biosynthesis, brassinosteroid biosynthesis, and hydrolase activity whereas NPCD cells possessed higher expression of auxin transport, auxin signalling, aspartyl proteases, cysteine protease, Bag5, and anthocyanin biosynthesis enzymes. CONCLUSIONS: RNA sequencing was used to generate a de novo transcriptome for A. madagascariensis leaves and revealed numerous DEGs potentially involved in PCD and leaf remodelling. The data generated from this investigation will be useful for future experiments on lace plant leaf development and PCD in planta.

The role of auxin in developmentally regulated programmed cell death in lace plant.

PREMISE: Lace plant (Aponogeton madagascariensis) leaves are remodeled via developmental programmed cell death (PCD) to produce perforations located equidistantly between longitudinal and transverse veins. Auxin has been implicated in other developmental PCD processes in plants; however, the role of auxin in perforation formation in lace plant is unknown. Here the role of auxin in developmental PCD in lace plant was studied using two auxin inhibitors N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, and auxinole, a potent auxin antagonist. METHODS: Sterile cultures of lace plants were propagated and treated with NPA or auxinole. Leaf length, leaf width, and number of perforations were then analyzed. Vein patterning and perforation area were further examined in NPA-treated plants. Downstream PCD transduction events were investigated via spectrophotometric assays, histochemical staining, and immuno-probing. RESULTS: Lace plants treated with NPA or auxinole produced leaves with fewer perforations compared to their respective controls. Although NPA treatment was insufficient to completely alter vein patterning, NPA-treated leaves did have significantly more atypical areoles compared to control leaves. Events involved in perforation formation in lace plant leaves were altered following treatment with NPA, including anthocyanin production, reactive oxygen species (ROS) accumulation, and the release of mitochondrial cytochrome c. CONCLUSIONS: Our results indicated that inhibition of auxin signaling disrupts several downstream features of the lace plant PCD signaling cascade and results in fewer or no perforations. Therefore, we concluded that auxin signaling is important for developmentally regulated PCD in lace plant leaves.

The Function of Autophagy in Lace Plant Programmed Cell Death.

The lace plant (Aponogeton madagascariensis) is an aquatic monocot that utilizes programmed cell death (PCD) to form perforations throughout its mature leaves as part of normal development. The lace plant is an emerging model system representing a unique form of developmental PCD. The role of autophagy in lace plant PCD was investigated using live cell imaging, transmission electron microscopy (TEM), immunolocalization, and in vivo pharmacological experimentation. ATG8 immunostaining and acridine orange staining revealed that autophagy occurs in both healthy and dying cells. Autophagosome-like vesicles were also found in healthy and dying cells through ultrastructural analysis with TEM. Following autophagy modulation, there was a noticeable increase in vesicles and vacuolar aggregates. A novel cell death assay utilizing lace plant leaves revealed that autophagy enhancement with rapamycin significantly decreased cell death rates compared to the control, whereas inhibition of autophagosome formation with wortmannin or blocking the degradation of cargoes with concanamycin A had an opposite effect. Although autophagy modulation significantly affected cell death rates in cells that are destined to die, neither the promotion nor inhibition of autophagy in whole plants had a significant effect on the number of perforations formed in lace plant leaves. Our data indicate that autophagy predominantly contributes to cell survival, and we found no clear evidence for its direct involvement in the induction of developmental PCD during perforation formation in lace plant leaves.

Epitaxially stabilized thin films of ε-Fe2O3 (001) grown on YSZ (100).

Epsilon ferrite (ε-Fe2O3) is a metastable phase of iron(III) oxide, intermediate between maghemite and hematite. It has recently attracted interest because of its magnetocrystalline anisotropy, which distinguishes it from the other polymorphs, and results in a gigantic coercive field and a natural ferromagnetic resonance frequency in the THz range. Moreover, it possesses a polar crystal structure, making it a potential ferroelectric, hence a potential multiferroic. Due to the need of size confinement to stabilize the metastable phase, ε-Fe2O3 has been synthesized mainly as nanoparticles. However, to favor integration in devices, and take advantage of its unique functional properties, synthesis as epitaxial thin films is desirable. In this paper, we report the growth of ε-Fe2O3 as epitaxial thin films on (100)-oriented yttrium-stabilized zirconia substrates. Structural characterization outlined the formation of multiple in-plane twins, with two different epitaxial relations to the substrate. Transmission electron microscopy showed how such twins develop in a pillar-like structure from the interface to the surface. Magnetic characterization confirmed the high magnetocrystalline anisotropy of our film and revealed the presence of a secondary phase which was identified as the well-known magnetite. Finally, angular analysis of the magnetic properties revealed how the presence of twins impacts their azimuthal dependence.

Remodelling of lace plant leaves: antioxidants and ROS are key regulators of programmed cell death.

MAIN CONCLUSION: Antioxidants and reactive oxygen species are integral for programmed cell death signaling during perforation formation in the lace plant ( Aponogeton madagascariensis ). The lace plant is an excellent model system for studying developmentally regulated programmed cell death (PCD). During early lace plant leaf development, PCD systematically deletes cells resulting in a perforated leaf morphology that is unique in planta. A distinct feature in young lace plant leaves is an abundance of anthocyanins, which have antioxidant properties. The first sign of PCD induction is the loss of anthocyanin pigmentation in cells that are targeted for destruction, which results in a visible gradient of cell death. The cellular dynamics and time course of lace plant PCD are well documented; however, the signals involved in the pathway remain elusive. This study investigates the roles of antioxidants and ROS in developmental PCD signaling during lace plant perforation formation. The involvement of antioxidants and ROS in the pathway was determined using a variety of techniques including pharmacological whole plant experimentation, long-term live cell imaging, the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid anti-radical activity assay, and western blot analysis. Results indicate that antioxidants and ROS are key regulators of PCD during the remodelling of lace plant leaves.

Analysis of eight glycols in serum using LC-ESI-MS-MS.

A liquid chromatography coupled with electrospray tandem mass spectrometry method was developed for the analysis of ethylene glycol, diethylene glycol, triethylene glycol, 1,4-butanediol, 1,2-butanediol, 2,3-butanediol, 1,2-propanediol and 1,3-propanediol, in serum after a Schotten-Baumann derivatization by benzoyl chloride. Usual validation parameters were tested: linearity, repeatability and intermediate precision, limits of detection and quantification, carry over and ion suppression. Limits of detection were between 0.18 and 1.1 mg/L, and limits of quantification were between 0.4 and 2.3 mg/L. Separation of isomers was possible either chromatographically or by selecting specific multiple reaction monitoring transitions. This method could be a useful tool in case of suspected intoxication with antifreeze agents, solvents, dietary supplements or some medical drug compounds.

Floral development of Dieffenbachia and the occurrence of atypical flowers in Araceae.

BACKGROUND: The floral development of Dieffenbachia seguine (N. J. Jacquin) Schott is analysed in relation to the molecular phylogeny of the Spathicarpae by Cusimano et al. (Am J Bot 98:654-668, 2011). RESULTS: The initiation of discoid floral primordia occurs acropetally on the surface of the spadix. Female flowers, atypical bisexual flowers, sterile male flowers, and male flowers share the same phyllotactic spirals on the spadix. Four or five stamen primordia are initiated simultaneously on the periphery of the male floral meristem. During early stages of stamen initiation, individual stamen primordia are connate at their base. In a synandrium, stamen fusion occurs very early during their developmental cycle with the stamens being already united laterally when longitudinal elongation occurs. The staminodes are also initiated on the periphery of the discoid floral primordium, and their number varies from four to six. The development of the fused staminodes will eventually form a longitudinal cavity in the center of the mature synandrode. The atypical flowers located in the intermediate zone range in morphology from aborted female flowers to rudimentary sterile male flowers with incomplete staminodes. CONCLUSIONS: The development of the female flower of Dieffenbachia Schott presents some similarities with atypical bisexual flowers of Cercestis Schott representing one of three types of aberrant flower forms in the family. From a developmental point of view and in the context of a phylogeny of the group, we believe that the presence of staminodes in the female zone constitutes a plesiomorphy in the tribe Spathicarpeae.

Determination of levels of current drugs in hospital and urban wastewater.

A rapid, sensitive and highly specific HPLC-MS/MS method with direct on-line preparation was applied for the determination of 20 common pharmaceuticals in hospital and urban wastewater. Median drug concentrations were quite similar in the majority of samples, cerca 1 µg L⁻¹ ranging from 0.06 to 2.67 µg L⁻¹ in both water. Pharmaceutical hospital contribution, below 1 %, was negligible, as compared to the huge amount in the municipal plant flow. Due to only partial elimination in the plant, hundreds of kilograms of harmful waste per year are discharged in the River Seine. Therefore, to reduce potential human and environmental exposure, a topic of major concern, an efficient drug treatment procedure should be used at the municipal plant stage in order to reduce urban wastewater pollution. The HPLC-MS/MS method could be a very useful tool to optimize the pharmaceutical wastewater treatment process.

Establishment of a database of metformin plasma concentrations and erythrocyte levels in normal and emergency situations.

BACKGROUND AND OBJECTIVES: Knowledge of blood metformin concentrations may be crucial for adapting the dose to the patient's kidney function and investigating a putative link between this drug and lactic acidosis. We therefore established the first database of blood metformin concentrations. METHODS: In order to provide cross-sectional data from clinical practice in a university medical centre, the authors retrospectively reviewed all available metformin plasma concentrations and erythrocyte level results (all of which were measured using the same assay technique). The assays had been requested for either dose adjustment in relation to the patient's renal status or screening for potential metformin accumulation, metformin overdose or lactic acidosis. RESULTS: A total of 798 metformin assays were performed in 467 patients. The mean ± SD (range) plasma concentrations and erythrocyte levels were 2.7 ± 7.3 (0-113) mg/L and 2.0 ± 4.4 (0-61) mg/L, respectively (published therapeutic values: 0.5 ± 0.4 mg/L and 0.8 ± 0.4 mg/L, respectively). Four plasma concentration categories were defined on a pragmatic basis: undetectable (9.7%), therapeutic range (40.6%), slight-to-moderate elevation (42.6%) and marked elevation (7.1%). CONCLUSION: Greater knowledge of blood metformin concentrations is required to adjust treatment and avoid wrongly incriminating the drug in lactic acidosis. Establishment of the first database of blood metformin concentrations may help physicians to better evaluate metformin use and dosage levels (particularly in elderly patients and those with poor kidney function), determine the presence and intensity of metformin accumulation and, ultimately, establish the prognosis of the latter condition.

Lobe-generating centres in the simple leaves of Myriophyllum aquaticum: evidence for KN1-like activity.

BACKGROUND AND AIMS: The mature morphology of most plants can usually be said to consist of three mutually exclusive organs: leaves, stems, and roots. The vast majority of mature morphologies may be easily grouped into one of these mutually exclusive categories. However, during very early stages of development and in many instances from inception, the division between organ categories becomes fuzzy due to the overlap in developmental processes that are shared between the aforementioned mutually exclusive categories. One such overlap has been described at the gene level where KNOXI homologues, transcription factors responsible for maintaining indeterminate cell fate, are expressed in the shoot apical meristem and during early stages of compound leaf development. This study characterizes the occurrence and spatial localization of mRNA of a KNOXI homologue, MaKN1, during the early stages of development in the simple leaves of Myriophyllum aquaticum, an aquatic angiosperm from the family Haloragaceae exhibiting pentamerous whorls of finely lobed leaves. METHODS: A 300-bp KNOXI fragment was sequenced from M. aquaticum and used in an RNA localization study to determine the temporal and spatial expression of KNOXI during the early stages of leaf lobe development in M. aquaticum. The developmental sequence of leaves of M. aquaticum was also described using scanning electron microscopy. KEY RESULTS: Lobe development of M. aquaticum occurs in two very distinct regions at the leaf base in an alternating fashion reminiscent of a distichous shoot system. It was discovered that MaKN1 expression is localized to both the shoot apical meristem and early stages of leaf primordia development (P1-P7). Initially, MaKN1 is expressed ubiquitously throughout primordia (P1-P3); however, as lobes develop, MaKN1 becomes localized to recently emerged lobe primordia, and disappears as lobes develop basipetally. CONCLUSIONS: The pattern of gene expression is indicative of shared developmental processes during early development between shoots, compound leaves, highly lobed simple leaves and unifoliate simple leaves which lack KNOXI expression. These findings are supportive of Arber's less rigid 'partial shoot' theory, which conceptualizes compound leaves as having shoot-like elements.

Fatal poisoning due to snorting buprenorphine and alcohol consumption.

High dosage buprenorphine (Subutex(®)) has been prescribed as a replacement therapy for major opioid dependencies in France since 1996. However, several studies have underlined its lethal risk, especially when administered intravenously, or when combined with benzodiazepines, alcohol or other central nervous system depressants. We report three fatal buprenorphine-related poisonings after snorting, among outside protocol individuals, observed at the Forensic Medicine Unit of Caen University Hospital. Medico-legal autopsies and complementary examinations were performed. The results are presented and discussed. Lethal poisoning after snorting buprenorphine was considered the most probable cause of death. These observations illustrate the risk of fatal poisoning by buprenorphine per-nasal route, which has rarely been reported in the literature although snorting is particularly prized by individuals outside the substitution therapy. We also observed the combination of buprenorphine and alcohol. By evaluating the pharmacological characteristics of this substance, as well as the data previously published in the literature, we have attempted to explain the pathophysiological mechanisms of this particular mode of poisoning that can easily be fatal.

[Drug-facilitated crime: a public health problem?]. / La soumission chimique: un problème de santé publique?

Drug-facilitated crime (DFC) is well known to the public, yet general practitioners and other physicians are unfamiliar with this issue, largely because toxicology is not part of the medical curriculum. This often leads to diagnostic errors. The frequency of DFC is underestimated, often owing to late examination and analytical problems. On 24 December 2002 the French authorities issued a circular defining DFC as "the administration of a psychoactive drug without the victim's knowledge, as a means of aggression"; and listing places where victims can be managed On 19 July 2005, the French Agency for Health Product Safety (Afssaps) sent a letter to all professionals potentially concerned by this issue, offering guidelines for both medical personnel and laboratory staff conducting toxicological investigations. One difficulty in drug identification is that the doses administered are often low. Toxicology laboratories need sophisticated equipment and expertise to ensure that the perpetrator is prosecuted or, alternatively, to rule out DFC. More information is needed, not only for the public but also for physicians and toxicologists. Benzodiazepines and related compounds are identified in about 75% of DFC cases.

[The metallic profile: a new biological concept]. / Une nouvelle approche biologique: le profil métallique.

Considerable advances have been made in metals and metalloids analysis over the past decade. This analysis is a basic stage in deficiency or toxicity assessment. A recently introduced technique, inductively coupled plasma mass spectrometry (ICP-MS) is progressively replacing atomic absorption. This analysis permits multi-elementary determinations, many ten or so elements, among periodic classification, with an optimal gain in sensitivity in many biological matrices: i.e. whole blood, plasma, urine, hair, nail, and biopsy samples. Moreover, this method allows semi-quantitative determination with an additional thirty supplementary elements, which enables the toxicologist to sufficiently estimate the toxic levels and metal exposure. The authors demonstrate that the ICP-MS could be very useful for a wide range of clinical applications. Furthermore, this procedure offers new exploration possibilities in various fields such as clinical chemistry but also clinical toxicology, forensic toxicology as well as workplace testing or environmental exposure and permits epidemiologic studies. This analytical method in fact also provides a new biologic approach. To our knowledge we are the first to propose the metallic profile.

[Inductively coupled plasma: a promising new tool with application in human toxicology and public health]. / La torche a plasma: un nouvel equipement aux applications prometteuses au service de la toxicologie humaine et de la santé publique.

The authors describe the use of inductively coupled plasma to detect 32 metals and metalloids in blood, urine, hair and nails. They also report the first case of gadolinium overdose documented by blood analysis with this method Metal speciation, a new approach developed in our laboratory, can distinguish between toxic and non toxic metals.

Quantitative developmental analysis of homeotic changes in the inflorescence of Philodendron (Araceae).

BACKGROUND AND AIMS: The inflorescence of Philodendron constitutes an interesting morphological model to analyse the phenomenon of homeosis quantitatively at the floral level. The specific goals of this study were (1) to characterize and quantify the range of homeotic transformation in Philodendron billietiae, and (2) to test the hypothesis that the nature of flowers surrounding atypical bisexual flowers (ABFs) channel the morphological potentialities of atypical bisexual flowers. METHODS: Inflorescences of P. billietiae at different stages of development were observed using SEM. The number of appendices in male, female and sterile flowers were counted on 11 young inflorescences (5-6 flowers per inflorescence). The number of staminodes and carpels on ABFs were counted on 19 inflorescences (n = 143). These data were used for regression and ANOVA analyses. RESULTS: There was an average of 4.1 stamens per male flower, 9.8 carpels per female flower and 6.8 staminodes per sterile male flower. There was an average of 7.3 floral appendices per atypical flower. Staminodes and carpels are inserted on the same whorl in ABFs. A negative exponential relationship was found between the average number of staminodes and the number of carpels in ABFs. If only the ABFs consisting of less than six carpels are considered, there is a linear relationship between the number of carpels and the average number of staminodes. The value of the slope of the regression equation indicates that on average, in P. billietiae, 1.36 carpels are replaced by one staminode. CONCLUSIONS: In P. billietiae, the number of appendages in female flowers imposes a constraint on the maximum total number of appendages (carpels and staminodes) that can develop on ABFs. The quantitative analyses indicate that the average number of different types of floral appendages on an ABF and the number of organs involved in a homeotic transformation are two independent phenomena.

Determination of thiocyanate in plasma by ion chromatography and ultraviolet detection.

A specific and sensitive rapid high-performance liquid chromatographic method has been developed for determination of thiocyanate (SCN-) in plasma. This method is based on anion exchange chromatography after a simple ultrafiltration of plasma diluted in water. The detection of SCN- is carried out in ultraviolet (lambda = 210 nm). The proposed method is linear in the range 1-30 mg/L. Intra-assay and interassay accuracy and precision were maintained within the designated limit (< 20%). The total recovery of SCN- varied between 97 and 103.9%. The method described should be useful for clinical medicine. Moreover, this method was applied to the analysis of SCN- plasma in deceased subjects, within the context of fire, and could be of interest in forensic science as a useful additional measurement tool for cyanide determination in blood.