Melatonin Attenuates RANKL-Induced Osteoclastogenesis via Inhibition of Atp6v0d2 and DC-STAMP through MAPK and NFATc1 Signaling Pathways

Melatonin is a hormone secreted by the pineal gland that is involved in the biorhythm of reproductive activities. The present study investigated the inhibitory effects of melatonin on osteoclastogenesis in RAW 264.7 cells according to changes in V-ATPase and the corresponding inhibition of the MAPK and NFATc1 signaling processes. Methods: the cytotoxic effect of melatonin was investigated by MTT assay. Osteoclast differentiation and gene expression of osteoclast-related factors were confirmed via TRAP staining, pit formation assay, immunofluorescence imaging, western blot, and real-time PCR. Results: melatonin was found to inactivate the p38 and JNK of MAP kinase in RAW264.7 cells treated with RANKL and treated with a combination RANKL and melatonin for 1, 3, and 5 days. The melatonin treatment group showed a reduction in osteoclastogenesis transcription factors and ATP6v0d2 gene expression. Conclusions: melatonin inhibits osteoclast differentiation and cell fusion by inhibiting the expression of Atp6v0d2 through the inactivation of MAPK and NFATc1 signaling in RANKL-stimulated RAW264.7 macrophages. The findings of the present study suggest that melatonin could be a suitable therapy for bone loss and imply a potential role of melatonin in bone health.

Development and retinal remodeling in the brown anole lizard (Anolis sagrei)

Background. The fovea, a pit in the retina, is believed to be important for high-acuity vision and is a feature found in the eyes of humans and a limited number of vertebrate species that include certain primates, birds, lizards, and fish. At present, model systems currently used for ocular research lack a foveated retina and studies investigating fovea development have largely been limited to histological and molecular studies in primates. As a result, progress towards understanding the mechanisms involved in regulating fovea development in humans is limited and is completely lacking in other, non-primate, vertebrates. To address this knowledge gap, we provide here a detailed histological atlas of retina and fovea development in the bifoveated Anolis sagrei lizard, a novel reptile model for fovea research. We also further test the hypothesis that retinal remodeling, which leads to fovea formation and photoreceptor cell packing, is related to asymmetric changes in eye shape. Results. Anole retina development follows the conventional spatiotemporal patterning observed in most vertebrates, where retina neurogenesis begins within the central retina, progresses throughout the temporal retina, and concludes in the nasal retina. One exception to this general rule is that areas that give rise to the fovea undergo retina differentiation prior to the rest of the retina. We find that retina thickness changes dynamically during periods of ocular elongation and retraction. During periods of ocular elongation, the retina thins, while during retraction it becomes thicker. Ganglion cell layer mounding is also observed in the temporal fovea region just prior to pit formation. Conclusions. Anole retina development parallels that of humans, including the onset and progression of retinal neurogenesis followed by changes in ocular shape and retinal remodeling that leads to pit formation in the retina. We propose that anoles are an excellent model system for fovea development research.

Bordered Pit Formation in Cell Walls of Spruce Tracheids

The process of pit formation in plants still has various questions unaddressed and unknown, which opens up many interesting and new research opportunities. The aim of this work was elucidation of the mechanism for the formation of bordered pits of the spruce (Picea abies (L.) Karst.) tracheid with exosomes participation and mechanical deformation of the cell wall. Sample sections were prepared from spruce stem samples after cryomechanical destruction with liquid nitrogen. The study methods included scanning electron microscopy and enzymatic treatment. Enzymatic treatment of the elements of the bordered pit made it possible to clarify the localization of cellulose and pectin. SEM images of intermediate stages of bordered pit formation in the radial and tangential directions were obtained. An asynchronous mechanism of formation of bordered-pit pairs in tracheids is proposed. The formation of the pit pair begins from the side of the initiator cell and is associated with enzymatic hydrolysis of the secondary cell wall and subsequent mechanical deformation of the primary cell walls. Enzymatic hydrolysis of the S1 layer of the secondary cell wall is carried out by exosome-delivered endoglucanases.

Clathrin regulates the β-arrestin pathway regardless of ligand bias

μ-opioid receptors (MOP) are thought to activate the G protein-mediated analgesic pathway and β-arrestin 2-mediated side effect pathway; however, ligands that recruit β-arrestin 2 only minimally to MOP may also cause opioid side effects. Such side effects are also induced in mutant mice lacking β-arrestin 2 or expressing phosphorylation-deficient MOP that do not recruit β-arrestin 2. These findings critically questioned whether β-arrestin 2 recruitment to MOP triggers side effects. Here, we show that β-arrestin 1 partially compensates for the lack of β-arrestin 2 in a neuronal cell line and thus might be involved in triggering such side effects in β-arrestin 2-null mice. Moreover, the magnitude of β-arrestin-mediated signals is not correlated with β-arrestin recruitment to MOP via phosphorylation of the carboxyl-terminal of MOP, which has long been used to evaluate β-arrestin bias of a ligand. Instead, β-arrestin activates downstream signals by binding with the clathrin heavy chain in the process of clathrin-coated pit formation. Our findings provide not only a novel insight into G protein-coupled receptor-mediated signalling to overcome opioid side effects but also an unexpected concept that the accumulation of molecules required for endocytosis is a key for activating the intracellular signalling.

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

SUMMARYAs the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

Ebulin l Is Internalized in Cells by Both Clathrin-Dependent and -Independent Mechanisms and Does Not Require Clathrin or Dynamin for Intoxication

Ebulin l is an A-B toxin, and despite the presence of a B chain, this toxin displays much less toxicity to cells than the potent A-B toxin ricin. Here, we studied the binding, mechanisms of endocytosis, and intracellular pathway followed by ebulin l and compared it with ricin. COS-1 cells and HeLa cells with inducible synthesis of a mutant dynamin (K44A) were used in this study. The transport of these toxins was measured using radioactively or fluorescently labeled toxins. The data show that ebulin l binds to cells to a lesser extent than ricin. Moreover, the expression of mutant dynamin does not affect the endocytosis, degradation, or toxicity of ebulin l. However, the inhibition of clathrin-coated pit formation by acidification of the cytosol reduced ebulin l endocytosis but not toxicity. Remarkably, unlike ricin, ebulin l is not transported through the Golgi apparatus to intoxicate the cells and ebulin l induces apoptosis as the predominant cell death mechanism. Therefore, after binding to cells, ebulin l is taken up by clathrin-dependent and -independent endocytosis into the endosomal/lysosomal system, but there is no apparent role for clathrin and dynamin in productive intracellular routing leading to intoxication.

Ocular elongation and retraction in foveated reptiles

BackgroundPronounced asymmetric changes in ocular globe size during eye development have been observed in a number of species ranging from humans to lizards. In contrast, largely symmetric changes in globe size have been described for other species like rodents. We propose that asymmetric changes in the three-dimensional structure of the developing eye correlate with the types of retinal remodeling needed to produce areas of high photoreceptor density. To test this idea, we systematically examined three-dimensional aspects of globe size as a function of eye development in the bifoveated brown anole, Anolis sagrei.ResultsDuring embryonic development, the anole eye undergoes dynamic changes in ocular shape. Initially spherical, the eye elongates in the presumptive foveal regions of the retina and then proceeds through a period of retraction that returns the eye to its spherical shape. During this period of retraction, pit formation and photoreceptor cell packing are observed. We found a similar pattern of elongation and retraction associated with the single fovea of the veiled chameleon, Chamaeleo calyptratus.ConclusionsThese results, together with those reported for other foveated species, support the idea that areas of high photoreceptor packing occur in regions where the ocular globe asymmetrically elongates and retracts during development.Key FindingsThe eyes of the brown anole, Anolis sagrei, and veiled chameleon, Chamaeleo calyptratus undergo dynamic asymmetrical changes in ocular shape during development.In both species, asymmetric elongation and retraction of the ocular globe is associated with fovea morphogenesis.Pit formation and photoreceptor cell packing in the foveal area occur when the corresponding region of the ocular globe is retracting relative to adjacent regions.

Effects of MOVPE Growth Conditions on GaN Layers Doped with Germanium

The effect of growth temperature and precursor flow on the doping level and surface morphology of Ge-doped GaN layers was researched. The results show that germanium is more readily incorporated at low temperature, high growth rate and high V/III ratio, thus revealing a similar behavior to what was previously observed for indium. V-pit formation can be blocked at high temperature but also at low V/III ratio, the latter of which however causing step bunching.

Effects of MOVPE Growth Conditions on GaN Layers Doped with Germanium

The effect of growth temperature and precursor flows on the doping level and surface morphology of Ge-doped GaN layers was researched. The results show that germanium is more readily incorporated at low temperature, high growth rate and high V/III ratio, thus revealing a similar behavior to what was previously observed for indium. V-pit formation can be blocked at high temperature but also at low V/III ratio, the latter of which however causing step bunching.