Germination of the fully myco-heterotrophic orchid Cyrtosia septentrionalis is characterized by low fungal specificity and does not require direct seed-mycobiont contact

We examined the level of specificity exhibited by seeds of Cyrtosia septentrionalis for germination-inducing fungi. Three wood-decomposing Armillaria species (A. gallica, A. mellea subsp. nipponica and A. tabescens) known to colonize adult plants and an unidentified species of Polyporales isolated from naturally growing protocorms were tested. Xylobolus annosus, a free-living decomposer of Russulales, was included as a control. Seed germination occurred in a sawdust-based medium in sealed and unsealed containers in the presence of all fungi, indicating low mycorrhizal specificity in germination. Moreover, germination occurred even in modified containers in which the fungus was physically isolated from the orchid seeds, indicating that direct seed-fungus contact is not required. Higher germination percentages were observed in sealed containers in which a modified atmosphere, consisting of a lower O₂ and a higher CO₂ concentration, had been established in the air above the inoculated medium as a result of the saprophytic activity of the fungus. In nature, atmospheric conditions more effective for seed germination might be established by the action of mycobionts in decomposing wood. Seeds germinated in the presence of the unknown Polyporales species underwent further growth and development, as compared with the other fungi tested, when covered with soil.     

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP‐170

AMP‐activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP‐170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell–cell junction present between cardiomyocytes. A contractile inhibitor, MYK‐461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK‐461, suggesting that the localization of AMPK is regulated by mechanical stress. Time‐lapse imaging analysis revealed that the inhibition of CLIP‐170 Ser‐311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK‐461 increased the individual cell area of cardiomyocytes in CLIP‐170 phosphorylation‐dependent manner. Moreover, heart‐specific CLIP‐170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP‐170 at the intercalated disks.     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.     

Thermodynamic and Kinetic Analyses of DNA Triplex Formation: Application of Filter-Binding Assay

Abstract

We have developed a simple and efficient method for studying equilibrium thermodynamics and kinetics of DNA triplex formation, which utilizes a filter-binding procedure. The application of this method to the triplex formation between a double-stranded homopurine-homopyrimidine and a single-stranded homopyrimidine oligonucleotides has demonstrated its ability in the quantitative estimation of equilibrium binding constants and rate constants under various conditions. Thus, this simple method can serve as a powerful tool for the systematic analysis of sequence and environmental effects on the equilibrium and kinetic quantities in the triplex formation.     

Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca²⁺ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8–14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca²⁺-sensor, the Ca²⁺ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca²⁺ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca²⁺. It is suggested that the photo-activation of ChRFR facilitated the release of Ca²⁺ from intracellular Ca²⁺ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.     

Crucial Role for CD69 in the Pathogenesis of Dextran Sulphate Sodium-Induced Colitis

CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In this study, we used CD69-deficient (CD69 KO) mice to assess the role of CD69 in the pathogenesis of dextran sulphate sodium (DSS)-induced acute and chronic colitis. The severity of colitis was assessed by the survival rate, clinical signs, colon length, histological examination and the expression of cytokines and chemokines in the large intestines. Both acute and chronic colitis were attenuated in the CD69 KO mice, as reflected by the lower lethality, weight loss, clinical signs, and improved histological findings. CD69⁺ cells infiltrated extensively into the inflamed mucosa of the colon in WT mice after DSS treatment. Experiments with the transfer of WT CD4 T cells into CD69 KO mice restored the induction of colitis. The administration of an anti-CD69 antibody also inhibited the induction of the DSS-induced colitis. These results indicate that CD69 expressed on CD4 T cells plays an important role in the pathogenesis of DSS-induced acute and chronic colitis, and that CD69 could be a possible therapeutic target for colitis.     

Effects of sampling seasons and locations on fish environmental DNA metabarcoding in dam reservoirs

Environmental DNA (eDNA) analysis has seen rapid development in the last decade, as a novel biodiversity monitoring method. Previous studies have evaluated optimal strategies, at several experimental steps of eDNA metabarcoding, for the simultaneous detection of fish species. However, optimal sampling strategies, especially the season and the location of water sampling, have not been evaluated thoroughly. To identify optimal sampling seasons and locations, we performed sampling monthly or at two‐monthly intervals throughout the year in three dam reservoirs. Water samples were collected from 15 and nine locations in the Miharu and Okawa dam reservoirs in Fukushima Prefecture, respectively, and five locations in the Sugo dam reservoir in Hyogo Prefecture, Japan. One liter of water was filtered with glass‐fiber filters, and eDNA was extracted. By performing MiFish metabarcoding, we successfully detected a total of 21, 24, and 22 fish species in Miharu, Okawa, and Sugo reservoirs, respectively. From these results, the eDNA metabarcoding method had a similar level of performance compared to conventional long‐term data. Furthermore, it was found to be effective in evaluating entire fish communities. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of fish species inhabiting the reservoirs. In addition, the number of detected species was significantly higher in shore, compared to offshore samples in the Miharu reservoir, and a similar tendency was found in the other two reservoirs. Based on these results, we can conclude that the efficiency of species detection by eDNA metabarcoding could be maximized by collecting water from shore locations during the breeding seasons of the inhabiting fish. These results will contribute in the determination of sampling seasons and locations for fish fauna survey via eDNA metabarcoding, in the future.