Method for Rapid Labeling of Waste Sludge from a Food Factory with 15N-Glycine and Evaluation of N Use Using Komatsuna (Brassica rapa Var. perviridis)

The waste sludge from food factories has rich nutrients and useful material for fertilizer or animal feed, but quick treatments and recycling of the waste sludge are difficult due to its higher water content. We have developed a rapid composting system to make sludge fertilizer using mix of waste sludge and shredded newspaper (Sludge Fertilizer Made by Paper Mixing Method, SF-PMM). The mixture was incubated in a box reactor, continuously aerated with warm air around 35°C, and changed to mature SF-PMM, in only two weeks. To search movement of N from the SF-PMM to plants, we developed a new method to label small amounts of SF-PMM with 15N-glycine. 50 L of wastewater from a food factory was incubated with 1 L of active sludge and 3 g of 15N-glycine (98 atom% 15N), and 175 g of labeled sludge was obtained in a day. This sludge was mixed with 25 g of newspaper chips, packed between two steel meshes, and placed at 20 cm depth in the reactor composting 200 kg of unlabeled sludge-paper mixture. Composting was restarted, and after about 7 days of reaction, 15N-labeled SF-PMM 7.03 atom% 15N was obtained. The surrounding unlabeled compost contained 4.0, 4.0, and 0.8% of N, P2O5, and K2O, respectively. C/N and pH were 10 and 7.4, respectively. Komatsuna (Brassica rapa var. perviridis) was cultivated in a pot with 50 and 100 mg N of SF-PMM, and healthy plants were obtained as in the Control experiments containing 50 mg N ammonium sulfate. No growth inhibition was observed in these experiments. Even in 100 mg SF-PMM, excellent growth of the roots was observed. About 56% of the N in the plant was shown to come from 15N-SF-PMM, and about 6% of the total15N in the 15N-SF-PMM was also shown to be incorporated into the plant.

Identification of Lymphatic and Hematogenous Routes of Rapidly Labeled Radioactive and Fluorescent Exosomes through Highly Sensitive Multimodal Imaging

There has been considerable interest in the clinical use of exosomes as delivery vehicles for treatments as well as for promising diagnostic biomarkers, but the physiological distribution of exosomes must be further elucidated to validate their efficacy and safety. Here, we aimed to develop novel methods to monitor exosome biodistribution in vivo using positron emission tomography (PET) and optical imaging. Exosomes were isolated from cultured mouse breast cancer cells and labeled for PET and optical imaging. In mice, radiolabeled and fluorescently labeled exosomes were injected both via lymphatic and hematogenous metastatic routes. PET and fluorescence images were obtained and quantified. Radioactivity and fluorescence intensity of ex vivo organs were measured. PET signals from exosomes in the lymphatic metastatic route were observed in the draining sentinel lymph nodes. Immunohistochemistry revealed greater exosome uptake in brachial and axillary versus inguinal lymph nodes. Following administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in the lungs, liver, and spleen. Exosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and fluorescence and were visualized via PET and optical imaging, suggesting that this simultaneous and rapid labeling method could provide valuable information for further exosome translational research and clinical applications.

Flow Chemistry System for Carbohydrate Analysis by Rapid Labeling of Saccharides after Glycan Hydrolysis

This study demonstrates the utilization of a flow chemistry system for continuous glycan hydrolysis and saccharide labeling to assist with the existing methods in glycan structural analysis. Acidic hydrolysis of glycans could be accelerated in a flow system. Aldoses and α-ketoacid-type saccharides were effectively labeled with naphthalene-2,3-diamine (NADA) at 60 °C for 10 min to form the fluorescent naphthimidazole (NAIM) and quinoxalinone (QXO) derivatives, respectively. The NADA-labeled derivatives improved the structural determination and composition analysis for their parent saccharides by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), liquid chromatography mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). Furthermore, this protocol was applied to determine the SA–Gal–Glc sequence of GM3-sugar out of six possible permutations.

Characterization and Biosynthesis of Lipids in Paulinella micropora MYN1: Evidence for Efficient Integration of Chromatophores into Cellular Lipid Metabolism

The chromatophores found in the cells of photosynthetic Paulinella species, once believed to be endosymbiotic cyanobacteria, are photosynthetic organelles that are distinct from chloroplasts. The chromatophore genome is similar to the genomes of α-cyanobacteria and encodes about 1,000 genes. Therefore, the chromatophore is an intriguing model of organelle formation. In this study, we analyzed the lipids of Paulinella micropora MYN1 to verify that this organism is a composite of cyanobacterial descendants and a heterotrophic protist. We detected glycolipids and phospholipids, as well as a betaine lipid diacylglyceryl-3-O-carboxyhydroxymethylcholine, previously detected in many marine algae. Cholesterol was the only sterol component detected, suggesting that the host cell is similar to animal cells. The glycolipids, presumably present in the chromatophores, contained mainly C16 fatty acids, whereas other classes of lipids, presumably present in the other compartments, were abundant in C20 and C22 polyunsaturated fatty acids. This suggests that chromatophores are metabolically distinct from the rest of the cell. Metabolic studies using isotopically labeled substrates showed that different fatty acids are synthesized in the chromatophore and the cytosol, which is consistent with the presence of both type I and type II fatty acid synthases, supposedly present in the cytosol and the chromatophore, respectively. Nevertheless, rapid labeling of the fatty acids in triacylglycerol and phosphatidylcholine by photosynthetically fixed carbon suggested that the chromatophores efficiently provide metabolites to the host. The metabolic and ultrastructural evidence suggests that chromatophores are tightly integrated into the whole cellular metabolism.