A fatty acid anabolic pathway in specialized-cells sustains a remote signal that controls egg activation in Drosophila.

Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation.

A simple and efficient process for the synthesis of 2D carbon nitrides and related materials.

We describe here a new process for the synthesis of very high quality 2D Covalent Organic Frameworks (COFs), such a C2N and CN carbon nitrides. This process relies on the use of a metallic surface as both a reagent and a support for the coupling of small halogenated building blocks. The conditions of the assembly reaction are chosen so as to leave the inorganic salts by-products on the surface, to further confine the assembly reaction on the surface and increase the quality of the 2D layers. We found that under these conditions, the process directly returns few layers material. The structure/quality of these materials is demonstrated by extensive cross-characterizations at different scales, combining optical microscopy, Scanning Electron Microscopy (SEM)/Transmission Electron Microscopy (TEM) and Energy Dispersive Spectroscopy (EDS). The availability of such very large, high-quality layers of these materials opens interesting perspectives, for example in photochemistry and electronics (intrinsic transport properties, high gap substrate for graphene, etc...).

Diclofenac prodrugs nanoparticles: An alternative and efficient treatment for rheumatoid arthritis?

We have synthesized new lipidic prodrugs of diclofenac by grafting aliphatic chains (C10, C12, C16 and C18) to diclofenac through an ester bond. Their molecular formulas were confirmed through HR-MS and the formation of ester bond by FTIR and NMR spectroscopy. Nanoparticles of the different prodrugs were successfully formulated using emulsion evaporation method and DSPE-PEG2000 as the only excipient. All nanoparticles were spherical and had a size between 110 and 150 nm, PdI ≤ 0.2 and negative Zeta potential values from -30 to -50 mV. In addition, they were stable upon storage at 4 °C up to 30-35 days. The encapsulation efficiency of the prodrug was above 90 % independently of the aliphatic chain length grafted. Nanoparticles did not induce any toxicity on LPS-activated THP-1 cells up to a concentration of 100 µg/mL (equivalent diclofenac) whereas diclofenac sodium salt IC50 was around 20 µg/mL. Following incubation of nanoparticles with LPS-activated THP-1 cells, a dose dependent inhibition of TNF-α was observed comparable to standard diclofenac sodium. Based on in vitro studies representative nanoparticles, Prodrug 3 NPs (C16 aliphatic chain) were selected for further in vitro and in vivo studies. Upon incubation in murine plasma, Prodrug 3 NPs underwent an enzymatic cleavage and almost 70 % of diclofenac was released from nanoparticles in 8 h. In vivo studies on a collagen induced arthritis murine model showed contrasted results: on one hand Prodrug 3 NPs led to a significant decrease of arthritis score and of paw volume compared to PBS after the second injection, on the other hand the third injection induced an important hepatic toxicity with the death of half of the mice from the NP group. To promote the reduction of inflammation while avoiding hepatic toxicity using NPs would require to precisely study the No Observable Adverse Effect Level and the schedule of administration in the future.

Caveolin-1ß promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles inSpodoptera frugiperda21 insect cells.

The heterologous expression in Spodoptera frugiperda 21 (Sf21) insect cells of the ß isoform of canine caveolin-1 (caveolin-1ß), using a baculovirus-based vector, resulted in intracellular vesicles enriched in caveolin-1ß. We investigated whether these vesicles could act as membrane reservoirs, and promote the production of an active membrane protein (MP) when co-expressed with caveolin-1ß. We chose hMGST1 (human microsomal glutathione S-transferase 1) as the co-expressed MP. It belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral MPs, and, as a phase II detoxification enzyme, it catalyzes glutathione conjugation of lipophilic drugs present in the lipid membranes. In addition to its pharmaceutical interest, its GST activity can be conveniently measured. The expression of both MPs were followed by Western blots and membrane fractionation on density gradient, and their cell localization by immunolabeling and transmission electron microscopy. We showed that caveolin-1ß kept its capacity to induce intracellular vesicles in the host when co-expressed with hMGST1, and that hMGST1 is in part addressed to these vesicles. Remarkably, a fourfold increase in the amount of active hMGST1 was found in the most enriched membrane fraction, along with an increase of its specific activity by 60% when it was co-expressed with caveolin-1ß. Thus, heterologously expressed caveolin-1ß was able to induce cytoplasmic vesicles in which a co-expressed exogenous MP is diverted and sequestered, providing a favorable environment for this cargo.

NEM6, KBTBD13-Related Congenital Myopathy: Myopathological Analysis in 18 Dutch Patients Reveals Ring Rods Fibers, Cores, Nuclear Clumps, and Granulo-Filamentous Protein Material.

Nemaline myopathy type 6 (NEM6), KBTBD13-related congenital myopathy is caused by mutated KBTBD13 protein that interacts improperly with thin filaments/actin, provoking impaired muscle-relaxation kinetics. We describe muscle morphology in 18 Dutch NEM6 patients and correlate it with clinical phenotype and pathophysiological mechanisms. Rods were found in in 85% of biopsies by light microscopy, and 89% by electron microscopy. A peculiar ring disposition of rods resulting in ring-rods fiber was observed. Cores were found in 79% of NEM6 biopsies by light microscopy, and 83% by electron microscopy. Electron microscopy also disclosed granulofilamentous protein material in 9 biopsies. Fiber type 1 predominance and prominent nuclear internalization were found. Rods were immunoreactive for α-actinin and myotilin. Areas surrounding the rods showed titin overexpression suggesting derangement of the surrounding sarcomeres. NEM6 myopathology hallmarks are prominent cores, rods including ring-rods fibers, nuclear clumps, and granulofilamentous protein material. This material might represent the histopathologic epiphenomenon of altered interaction between mutated KBTBD13 protein and thin filaments. We claim to classify KBTBD13-related congenital myopathy as rod-core myopathy.

Dp71 contribution to the molecular scaffold anchoring aquaporine-4 channels in brain macroglial cells.

Intellectual disability in Duchenne muscular dystrophy has been associated with the loss of dystrophin-protein 71, Dp71, the main dystrophin-gene product in the adult brain. Dp71 shows major expression in perivascular macroglial endfeet, suggesting that dysfunctional glial mechanisms contribute to cognitive impairments. In the present study, we investigated the molecular alterations induced by a selective loss of Dp71 in mice, using semi-quantitative immunogold analyses in electron microscopy and immunofluorescence confocal analyses in brain sections and purified gliovascular units. In macroglial pericapillary endfeet of the cerebellum and hippocampus, we found a drastic reduction (70%) of the polarized distribution of aquaporin-4 (AQP4) channels, a 50% reduction of ß-dystroglycan, and a complete loss of α1-syntrophin. Interestingly, in the hippocampus and cortex, these effects were not homogeneous: AQP4 and AQP4ex isoforms were mostly lost around capillaries but preserved in large vessels corresponding to pial arteries, penetrating cortical arterioles, and arterioles of the hippocampal fissure, indicating the presence of Dp71-independent pools of AQP4 in these vascular structures. In conclusion, the depletion of Dp71 strongly alters the distribution of AQP4 selectively in macroglial perivascular endfeet surrounding capillaries. This effect likely affects water homeostasis and blood-brain barrier functions and may thus contribute to the synaptic and cognitive defects associated with Dp71 deficiency.

Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria.

The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature ultramicrotomy and (ii) high-pressure freezing followed by cryo-ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant.

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids.

S6 kinase 1 is required for rapamycin-sensitive liver proliferation after mouse hepatectomy.

Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.