The Amidohydrolases IAR3 and ILL6 Contribute to Jasmonoyl-Isoleucine Hormone Turnover and Generate 12-Hydroxyjasmonic Acid Upon Wounding in Arabidopsis Leaves

Jasmonates (JAs) are a class of signaling compounds that mediate complex developmental and adaptative responses in plants. JAs derive from jasmonic acid (JA) through various enzymatic modifications, including conjugation to amino acids or oxidation, yielding an array of derivatives. The main hormonal signal, jasmonoyl-l-isoleucine (JA-Ile), has been found recently to undergo catabolic inactivation by cytochrome P450-mediated oxidation. We characterize here two amidohydrolases, IAR3 and ILL6, that define a second pathway for JA-Ile turnover during the wound response in Arabidopsis leaves. Biochemical and genetic evidence indicates that these two enzymes cleave the JA-Ile signal, but act also on the 12OH-JA-Ile conjugate. We also show that unexpectedly, the abundant accumulation of tuberonic acid (12OH-JA) after wounding originates partly through a sequential pathway involving (i) conjugation of JA to Ile, (ii) oxidation of the JA-Ile conjugate, and (iii) cleavage under the action of the amidohydrolases. The coordinated actions of oxidative and hydrolytic branches in the jasmonate pathway highlight novel mechanisms of JA-Ile hormone turnover and redefine the dynamic metabolic grid of jasmonate conversion in the wound response.     

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for ¹⁵N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.     

Phenoscope: an automated large‐scale phenotyping platform offering high spatial homogeneity

Increased phenotyping accuracy and throughput are necessary to improve our understanding of quantitative variation and to be able to deconstruct complex traits such as those involved in growth responses to the environment. Still, only a few facilities are known to handle individual plants of small stature for non‐destructive, real‐time phenotype acquisition from plants grown in precisely adjusted and variable experimental conditions. Here, we describe Phenoscope, a high‐throughput phenotyping platform that has the unique feature of continuously rotating 735 individual pots over a table. It automatically adjusts watering and is equipped with a zenithal imaging system to monitor rosette size and expansion rate during the vegetative stage, with automatic image analysis allowing manual correction. When applied to Arabidopsis thaliana, we show that rotating the pots strongly reduced micro‐environmental disparity: heterogeneity in evaporation was cut by a factor of 2.5 and the number of replicates needed to detect a specific mild genotypic effect was reduced by a factor of 3. In addition, by controlling a large proportion of the micro‐environmental variance, other tangible sources of variance become noticeable. Overall, Phenoscope makes it possible to perform large‐scale experiments that would not be possible or reproducible by hand. When applied to a typical quantitative trait loci (QTL) mapping experiment, we show that mapping power is more limited by genetic complexity than phenotyping accuracy. This will help to draw a more general picture as to how genetic diversity shapes phenotypic variation.     

Restricted gene flow within and between rapidly diverging Neotropical plant species

Speciation involves the evolution of traits and genetic differences that contribute to reproductive isolation and the cessation of gene flow, and studying closely related species and divergent populations gives insight into how these phenomena proceed. Here, we document patterns of gene flow within and between two members of a rapid Neotropical species radiation, Costus pulverulentus and Costus scaber (Costaceae). These species co‐occur in the tropical rainforest and share pollinators, but are reproductively isolated by a series of prezygotic barriers, some of which show evidence of reinforcement at sympatric sites. Here, we genotype microsatellite markers in plants from eight sites that span the geographical range of both species, including four sympatric sites. We also genotype putative hybrids found at two sympatric sites. We find high levels of genetic isolation among populations within each species and low but detectable levels of introgression between species at sympatric sites. Putative hybrids identified by morphology are consistent with F1 or more advanced hybrids. Our results highlight the effectiveness of prezygotic isolating mechanisms at maintaining species boundaries in young radiations and provide empirical data on levels of gene flow consistent with reinforcement.     

Is Rapoport's rule a recent phenomenon? A deep time perspective on potential causal mechanisms

Macroecology strives to identify ecological patterns on broad spatial and temporal scales. One such pattern, Rapoport''s rule, describes the tendency of species'' latitudinal ranges to increase with increasing latitude. Several mechanisms have been proposed to explain this rule. Some invoke climate, either through glaciation driving differential extinction of northern species or through increased seasonal variability at higher latitudes causing higher thermal tolerances and subsequently larger ranges. Alternatively, continental tapering or higher interspecific competition at lower latitudes may be responsible. Assessing the incidence of Rapoport''s rule through deep time can help to distinguish between competing explanations. Using fossil occurrence data from the Palaeobiology Database, we test these hypotheses by evaluating mammalian compliance with the rule throughout the Caenozoic of North America. Adherence to Rapoport''s rule primarily coincides with periods of intense cooling and increased seasonality, suggesting that extinctions caused by changing climate may have played an important role in erecting the latitudinal gradients in range sizes seen today.     

Combined MRI and 31P-MRS Investigations of the ACTA1(H40Y) Mouse Model of Nemaline Myopathy Show Impaired Muscle Function and Altered Energy Metabolism

Nemaline myopathy (NM) is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. Mutations in the skeletal muscle α-actin gene (ACTA1) account for ∼25% of all NM cases and are the most frequent cause of severe forms of NM. So far, the mechanisms underlying muscle weakness in NM patients remain unclear. Additionally, recent Magnetic Resonance Imaging (MRI) studies reported a progressive fatty infiltration of skeletal muscle with a specific muscle involvement in patients with ACTA1 mutations. We investigated strictly noninvasively the gastrocnemius muscle function of a mouse model carrying a mutation in the ACTA1 gene (H40Y). Skeletal muscle anatomy (hindlimb muscles and fat volumes) and energy metabolism were studied using MRI and ³¹Phosphorus magnetic resonance spectroscopy. Skeletal muscle contractile performance was investigated while applying a force-frequency protocol (from 1–150 Hz) and a fatigue protocol (80 stimuli at 40 Hz). H40Y mice showed a reduction of both absolute (−40%) and specific (−25%) maximal force production as compared to controls. Interestingly, muscle weakness was associated with an improved resistance to fatigue (+40%) and an increased energy cost. On the contrary, the force frequency relationship was not modified in H40Y mice and the extent of fatty infiltration was minor and not different from the WT group. We concluded that the H40Y mouse model does not reproduce human MRI findings but shows a severe muscle weakness which might be related to an alteration of intrinsic muscular properties. The increased energy cost in H40Y mice might be related to either an impaired mitochondrial function or an alteration at the cross-bridges level. Overall, we provided a unique set of anatomic, metabolic and functional biomarkers that might be relevant for monitoring the progression of NM disease but also for assessing the efficacy of potential therapeutic interventions at a preclinical level.     

How Does Circadian Rhythm Impact Salt Sensitivity of Blood Pressure in Mice? A Study in Two Close C57Bl/6 Substrains

BackgroundMouse transgenesis has provided the unique opportunity to investigate mechanisms underlying sodium kidney reabsorption as well as end organ damage. However, understanding mouse background and the experimental conditions effects on phenotypic readouts of engineered mouse lines such as blood pressure presents a challenge. Despite the ability to generate high sodium and chloride plasma levels during high-salt diet, observed changes in blood pressure are not consistent between wild-type background strains and studies.MethodsThe present work was designed in an attempt to determine guidelines in the field of salt-induced hypertension by recording continuously blood pressure by telemetry in mice submitted to different sodium and potassium loaded diets and changing experimental conditions in both C57BL/6N and C57BL/6J mice strain (Normal salt vs. Low salt vs. High-salt/normal potassium vs. High salt/low potassium, standard vs. modified light cycle, Non-invasive tail cuff blood pressure vs. telemetry).ResultsIn this study, we have shown that, despite a strong blood pressure (BP) basal difference between C57BL/6N and C57BL/6J mice, High salt/normal potassium diet increases BP and heart rate during the active phase only (dark period) in the same extent in both strains. On the other hand, while potassium level has no effect on salt-induced hypertension in C57BL/6N mice, high-salt/low potassium diet amplifies the effect of the high-salt challenge only in C57BL/6J mice. Indeed, in this condition, salt-induced hypertension can also be detected during light period even though this BP increase is lower compared to the one occurring during the dark period. Finally, from a methodological perspective, light cycle inversion has no effect on this circadian BP phenotype and tail-cuff method is less sensitive than telemetry to detect BP phenotypes due to salt challenges.ConclusionsTherefore, to carry investigations on salt-induced hypertension in mice, chronic telemetry and studies in the active phase are essential prerequisites.     

Identification of Tenrec ecaudatus,a Wild Mammal Introduced to Mayotte Island,as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis

Leptospirosis is a bacterial zoonosis of major concern on tropical islands. Human populations on western Indian Ocean islands are strongly affected by the disease although each archipelago shows contrasting epidemiology. For instance, Mayotte, part of the Comoros Archipelago, differs from the other neighbouring islands by a high diversity of Leptospira species infecting humans that includes Leptospira mayottensis, a species thought to be unique to this island. Using bacterial culture, molecular detection and typing, the present study explored the wild and domestic local mammalian fauna for renal carriage of leptospires and addressed the genetic relationships of the infecting strains with local isolates obtained from acute human cases and with Leptospira strains hosted by mammal species endemic to nearby Madagascar. Tenrec (Tenrec ecaudatus, Family Tenrecidae), a terrestrial mammal introduced from Madagascar, is identified as a reservoir of L. mayottensis. All isolated L. mayottensis sequence types form a monophyletic clade that includes Leptospira strains infecting humans and tenrecs on Mayotte, as well as two other Malagasy endemic tenrecid species of the genus Microgale. The lower diversity of L. mayottensis in tenrecs from Mayotte, compared to that occurring in Madagascar, suggests that L. mayottensis has indeed a Malagasy origin. This study also showed that introduced rats (Rattus rattus) and dogs are probably the main reservoirs of Leptospira borgpetersenii and Leptospira kirschneri, both bacteria being prevalent in local clinical cases. Data emphasize the epidemiological link between the two neighbouring islands and the role of introduced small mammals in shaping the local epidemiology of leptospirosis.     

Inhibition of the amino‐acid transporter LAT1 demonstrates anti‐neoplastic activity in medulloblastoma

Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo‐ and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L‐type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1‐specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD‐MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long‐term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.