Monitoring Autophagy Flux and Activity: Principles and Applications

Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi-step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome-dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been developed. In these assays, autophagosome marker proteins, microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) and gamma-aminobutyric acid receptor-associated protein-II (GABARAP-II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3-II and/or GABARAP-II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure.     

Decrease in bacterial production over the past three decades in the north basin of Lake Biwa,Japan

Limnology - In Lake Biwa, the largest lake in Japan, external pollutant loads have decreased since the 1980s, leading to improved water quality, such as reduction in biochemical oxygen demand (BOD)...     

Invasive legume management strategies differentially impact mutualist abundance and benefit to native and invasive hosts

Determining the best management practices for plant invasions is a critical, but often elusive goal. Invasive removals frequently involve complex and poorly understood biotic interactions. For example, invasive species can leave potent legacies that influence the success of native species restoration efforts, and positive plant‐microbial feedbacks may promote continued reinvasion by an exotic species following restoration. Removal methods can vary in their effects on plant–soil feedbacks, with consequences for restoration of native species. We determined the effects of invasion by a leguminous shrub (French broom; Genista monspessulana) on the density and community composition of, and benefit conferred by, its microbial mutualists in its invading range. Densities of soil‐dwelling rhizobia were much higher in areas invaded by G. monspessulana relative to uninvaded areas, and this increased density of rhizobia fed back to increase seedling growth of both the invader and native legumes. We further compared how three techniques for removing G. monspessulana affected the densities of rhizobia relative to areas where G. monspessulana was still present. Removal by hand‐pulling reduced soil rhizobial densities, and reduced growth of one native legume, while having no effect on the growth of the invader. Overall, our results show that the consequences of restoration techniques, both above‐ and belowground, could be critical for the successful removal of an invasive legume and restoration of native species.     

Enhancing coral larval supply and seedling production using a special bundle collection system “coral larval cradle” for large‐scale coral restoration

Larval recruitment is essential for sustaining coral communities and a fundamental tool in some interventions for reef restoration. To improve larval supply and post‐settlement survival in sexually assisted coral restoration efforts, an integrated in situ collector system, the larval cradle, was designed to collect spawned gametes then culture the resulting larvae until settled on artificial substrates. The final design of the larval cradle was cylindrical, a nylon mesh structure with a volume of 9 m³, suspended in the sea and extending vertically toward the seabed. We found three key design features that improved the efficiency of the apparatus: (1) an open area of sea surface and mesh size of less than 100 μm produced high fertilization and optimal survival (>90%), (2) a special skirt‐shaped net (3 m in diameter) with a connection hose for attaching the cradle to collect bundles from many adult colonies over a wide area and at various depths, and (3) adding short square tube pieces, called square hollow sections, as a substrate for enhancing larval settlement and survival, to a larval cradle at 4 days after spawning was optimal for uniform settlement. This system allowed not only the collection of several million eggs, but also subsequent production of several thousand settled juvenile corals, without land facilities. Our design achieved several hundred times higher survival for early life stages of Acropora tenuis compared to nature.     

Proteomic Study to Understand Promotive Effects of Plant-Derived Smoke on Soybean (Glycine max L.) Root Growth Under Flooding Stress

Plant-derived smoke plays a key role in plant growth. Proteomic technique was used for underlying mechanisms of plant-derived smoke on the growth of soybean (Glycine max L.) under flooding stress. The length and weight of soybean root increased with 2000 parts per million plant-derived smoke under flooding stress within 4 days. Altered proteins by plant-derived smoke treatment under flooding stress were mainly related to protein metabolism, stress, and redox. Furthermore, proteins related to mitochondrial electron transport chain decreased by flooding stress; however, they increased by addition of plant-derived smoke under flooding stress. Based on the results of proteomic analysis, confirmation experiments were performed. ATPase abundance and ATP content increased with the treatment of plant-derived smoke under flooding stress. Furthermore, the ascorbate/glutathione cycle was activated with the treatment of plant-derived smoke under flooding stress. These results suggest that plant-derived smoke improves the root growth of soybean with energy production and reactive oxygen scavenging even if it is under flooding stress, which might positively regulate soybean tolerance towards flooding stress.

     

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.     

Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H₂O₂, the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.