A circular cell culture system using microalgae and mammalian myoblasts for the production of sustainable cultured meat.

For sustainable production of cultured meat, we propose a novel circular cell culture (CCC) system in which microalgae are used as nutrient supply for the mammalian cell culture and as a waste-medium recycler. Chlorococcum littorale, RL34 hepatocytes, and C2C12 myoblasts were used as cell sources for microalgae, growth factor-producing cells, and muscle cells, respectively. In the first cycle, C2C12 cells were amplified 4.0-fold after 48 h of culture in an RL34 cell-conditioned medium. In the second cycle, C2C12 cells were cultured in the C. littorale culture waste medium to which the C. littorale-derived nutrients were added. The proliferation rates of C. littorale and C2C12 and the nutrient extraction efficiency from C. littorale were the same in the first and second cycles. Therefore, this CCC system, which works without additional grain-derived nutrients and animal sera, will help drastically reduce environmental load, resource/energy consumption, and costs in future cultured meat production.

Nanostructure and Molecular-Level Characterization of Aminoalkyl Methacrylate Copolymer and the Impact on Drug Solubilization Ability.

The effects of pH changes and saccharin (SAC) addition on the nanostructure and mobility of the cationic aminoalkyl methacrylate copolymer Eudragit E PO (EUD-E) and its drug solubilization ability were investigated. Small-angle X-ray scattering performed using synchrotron radiation and atomic force microscopy showed that the EUD-E nanostructure, which has a size of approximately several nanometers, changed from a random coil structure at low pH (pH 4.0-5.0) to a partially folded structure at high pH (pH 5.5-6.5). The EUD-E also formed a partially folded structure in a wide pH range of 4.5-6.5 when SAC was present, and the coil-to-globule transition was moderate with pH increase, compared with that when SAC was absent. The equilibrium solubility of the neutral drug naringenin (NAR) was enhanced in the EUD-E solution and further increased as the pH increased. The enlargement of the hydrophobic region of EUD-E in association with the coil-to-globule transition led to efficient solubilization of NAR. The interaction with SAC enhanced the mobility of the EUD-E chains in the hydrophobic region of EUD-E, resulting in changes in the drug-solubilizing ability. 1H high-resolution magic-angle spinning NMR measurements revealed that the solubilized NAR in the partially folded structure of EUD-E showed higher molecular mobility in the presence of SAC than in the absence of SAC. This study highlighted that solution pH and the presence of SAC significantly changed the drug solubilization ability of EUD-E, followed by changes in the EUD-E nanostructure, including its hydrophobic region.

Stable Thiele's Hydrocarbon Derivatives Exhibiting Near-Infrared Absorption/Emission and Two-Step Electrochromism.

We report synthesis and characterization of near-infrared (NIR)-absorbing/emitting Thiele's hydrocarbon derivatives, in which four aryl groups are bridged to a quinodimethane skeleton. The quinoid structure of the bridged-tetra-aryl- p-quinodimethanes (BTAQs) was confirmed by spectroscopic, X-ray crystallographic, and computational methods. Although quinodimethane derivatives with a small HOMO-LUMO energy gap often exhibit biradical character, BTAQs showed no biradical character. Instead, they exhibited two-step near-infrared electrochromism. The donor/acceptor properties of the aryl groups were found to play a key role in the unique properties of BTAQs.

Core-fucosylation plays a pivotal role in hepatitis B pseudo virus infection: a possible implication for HBV glycotherapy.

The functions of cell surface proteins, such as growth factor receptors and virus/bacteria-entry receptors, can be dynamically regulated by oligosaccharide modifications. In the present study, we investigated the involvement of glycosylation in hepatitis B virus (HBV) entry into hepatoma cells. Infection of oligosaccharide-remodeling hepatoma cells with a pseudo virus of HBV, bio-nanocapsule (BNC), was evaluated by flow cytometry and confocal microscopy. Among various experiments using several hepatoma cells, marked difference was observed between Huh6 cells and HB611 cells, which were established by HBV gene transfection into hepatoma cells. Comprehensive oligosaccharide analysis showed dramatic increases of core fucosylation in HB611 cells, compared with Huh6 cells. Knock down of fucosyltransferase 8 (FUT8) reduced BNC entry into HB611 cells. In contrast, overexpression of FUT8 in Huh6 cells increased BNC entry. Although expression of sodium taurocholate cotransporting polypeptide (NTCP), which is one of HBV receptors was very similar between Huh6 and HB611 cells, proteins coprecipitated with NTCP were dependent on levels of core-fucosylation, suggesting that core-fucosylation regulates BNC entry into hepatoma cells. Our findings demonstrate that core-fucosylation is an important glycosylation for HBV infection of hepatoma cells through HBV-receptor-mediated endocytosis. Down-regulation of core-fucosylation may be a novel target for HBV therapy.

Chemo- and Regioselective Rhodium(I)-Catalyzed [2+2+2] Cycloaddition of Allenynes with Alkynes.

A highly chemo- and regioselective partially intramolecular rhodium(I)-catalyzed [2+2+2] cycloaddition of allenynes with alkynes is described. A range of diverse polysubstituted benzene derivatives could be synthesized in good to excellent yields, in which the allenynes served as synthetic equivalent to the diynes. A high regioselectivity could be observed when allenynes were treated with unsymmetrical alkynes.

Fused-Fluoran Leuco Dyes with Large Color-Change Derived from Two-Step Equilibrium: iso-Aminobenzopyranoxanthenes.

Fluorans are popular leuco dyes that are used in various applications, such as carbonless-copy papers and thermal papers. Here, we describe unique leuco dyes in which two fluoran units are fused into a C2h structure (iso-aminobenzopyranoxanthenes; iso-ABPXs). iso-ABPXs exhibited a large two-step color change (colorless/pink and pink/blue-green) due to opening-closing of two spirolactone rings. The two-step equilibrium among the colorless, pink, and blue-green forms could be well controlled by adjusting acid concentration, solvent, and/or temperature.

Effects of Exercise on Functional Recovery in Patients with Bipolar Depression: A Study Protocol for a Randomized Controlled Trial.

Treatment of bipolar disorder is prone to prolongation despite various treatments, including medication. The efficacy of exercise treatment (i.e., interventions involving physical exercise and sports intervention) for major depressive disorders has been reported for depressive symptoms, cognitive function, and sleep disturbances. However, its efficacy for bipolar disorder has yet to be established. We designed a randomized, controlled, double-blind clinical trial that includes 100 patients with bipolar disorder aged 20-65 years. This will be a cluster-randomized, two-group trial that will be conducted in ten psychiatric hospitals. The hospitals will be randomly assigned to an exercise intervention + treatment as usual (exercise) group or a placebo exercise intervention (stretching) + treatment as usual (control) group. Patients will be assessed using an extensive battery of clinical tests, physical parameters, sleep status, biological parameters (cytokines, neurotrophic factors), and genetic parameters (DNA and RNA) at baseline after a 6-week intervention period, at 10-week follow-up, and at 6-month follow-up. This innovative study may provide important evidence for the effectiveness of exercise in the treatment of bipolar depression based on clinical, biological, genetic, and physiological markers.

Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat.

Recently, cultured meat obtained from livestock-derived cells is being considered as a sustainable food source that reduces the use of natural resources. This study aimed to show that nutrients extracted from Chlorella vulgaris were beneficial in the culture of primary bovine myoblasts (PBMs), a major cell source for cultured meat production. Nutrients (glucose, amino acids, and vitamins) present in the animal-cell culture media were effectively recovered from C. vulgaris using acid hydrolysis treatment. On culture in nutrient-free inorganic salt solution, cell death was induced in most PBMs after 6 days of cultivation. However, the addition of C. vulgaris extract (CVE) significantly improved PBM viability, which was comparable to the viability in conventional culture medium (Dulbecco's modified Eagle's medium). Furthermore, by adding horse serum to induce differentiation, the formation of myotubes was confirmed when CVE were used. Together, the results showed that CVE could be used as an alternative to the conventional culture medium for PBMs. These findings will not only lower the environmental risks associated with the establishment of this eco-friendly cell culture system, but also highlight microalgae as a potent nutrient source that can replace conventional grain-dependent nutrient sources.

Mammalian cell cultivation using nutrients extracted from microalgae.

Mammalian cells have been used in various research fields. More recently, cultured cells have been used as the cell source of "cultured meat." Cell cultivation requires media containing nutrients, of which glucose and amino acids are the essential ones. These nutrients are generally derived from grains or heterotrophic microorganisms, which also require various nutrients derived from grains. Grain culture, in turn, requires many chemical fertilizers and agrochemicals, which can cause greenhouse gas emission and environmental contamination. Furthermore, grain production is greatly influenced by environmental changes. In contrast, microalgae efficiently synthesize various nutrients using solar energy, water, and inorganic substances, which are widely used in the energy sector. In this study, we aimed to apply nutrients extracted from microalgae in the culture media for mammalian cell cultivation. Glucose was efficiently extracted from Chlorococcum littorale or Arthrospira platensis using sulfuric acid, whereas 18 of the 20 proteinogenic amino acids were efficiently extracted from Chlorella vulgaris using hydrochloric acid. We further investigated whether nutrients present in the algal extracts could be used in mammalian cell cultivation. Although almost all C2C12 mouse myoblasts died during cultivation in a glucose- and amino acid-free medium, the cell death was rescued by adding algal extract(s) into the nutrient-deficient media. This indicates that nutrients present in algal extracts can be used for mammalian cell cultivation. This study is the first step toward the establishment of a new cell culture system that can reduce environmental loads and remain unaffected by the impact of environmental changes.

The feeding behavior of dogs correlates with their responses to commands.

Motivation is one of the most important factors in dog training. To generate motivation, people use various reinforcer mechanisms. In particular, many pet owners use food because it is simple and convenient. The aim of this study was to investigate the association between dogs' level of interest in food and their responsiveness to commands. Thirty-four dogs were divided into three groups based on their feeding patterns (Fast, Slow, and Leftover). The fast group (n=15) had the highest interest in food and showed a high response to commands when food was used as a reinforcer, rather than praise/stroking. The leftover group (n=6) showed the least interest in food and had a low reaction to commands, regardless of the reinforcer. In contrast, the slow group (n=13) had modest interest in food and maintained the same response to commands regardless of the reinforcer. Results of this study indicate that dogs' feeding patterns are indicative of their level of interest in food, and may be useful in determining the optimal training reinforcer. This can help dog owners improve their relationships with their dogs.

Post-flood social support networks and morbidity in Jôsô City, Japan.

Social support networks are considered beneficial for post-disaster survivor mental health. However, there are family and non-family networks, and support can be received or provided. Therefore, their complex contribution to wellbeing requires analysis. Researching elderly residents of Jôsô City NE of Tokyo (N = 1182 [female: n = 618], Age M = 69.76y, SD = 6.10y) who experienced severe flooding in September 2015 investigated data for mental health outcomes of depression (K6), trauma (IES-R), and existence of recent worry from evacuation and house damage. An original instrument tapped support source and direction, controlled to examine mental health symptom changes. House damage was a higher mental health predictor (ηp2 = .10-.16) than evacuation (ηp2 = .033-.093). Results indicated family social support may buffer mental health outcomes, but non-family social support may burden them. Overall support network size also indicated burdening compared to social support receiving-providing imbalance.

Cell surface N-glycan alteration in HepAD38 cell lines expressing Hepatitis B virus.

Hepatitis B virus (HBV) is the smallest partially double-stranded DNA virus known to infect humans. Worldwide, more than 50% of hepatocellular carcinoma (HCC) cases are related to chronic Hepatitis B. Development of HCC from normal liver cells is characterized by changes in cell surface N-glycans, which can promote the invasive behavior of tumor cells, leading ultimately to the progression of cancer. However, little is understood about the cell surface N-glycans of HBV-infected liver cells. We try to address this by taking advantage of the HepAD38 cell line, which can replicate HBV in the absence of tetracycline [tet(-)] in growth medium. In the presence of tetracycline [tet(+)], this cell line is free from the virus due to the repression of pregenomic (pg) RNA synthesis. In culture medium without tetracycline, cells express viral pgRNA and start to secrete virions into the supernatant. Here we studied the expression of glycosyltransferases and the cell surface N-glycan composition of tet(+) and tet(-) HepAD38. Among the glycosyltransferases upregulated by the expression of HBV were GnT-II, GnT-IVa, ST6Gal1, and GnT-V, whereas GnT-I, GnT-III, ß4GalT1, and FUT8 were downregulated. About one-third of the total cell surface N-glycans found on tet(-)HepAD38 were sialylated. As for tet(+)HepAD38, sialylation was 6% lower compared to the tet(-) cells. Neither treatment changed the cell surface N-glycans expression of the total complex type or the total fucosylated type, which were about 50% or 60%, respectively. Our results showed that the expression of HBV triggers higher sialylation in HepAD38 cells. Altogether, the results show that HBV expression triggered the alteration of the cell surface N-glycosylation pattern and the expression levels of glycosyltransferases of HepAD38 cells.

Structural elucidation of a novel transglycosylated compound α-glucosyl rhoifolin and of α-glucosyl rutin by NMR spectroscopy.

We report the full assignment of 1H and 13C NMR signals belonging to α-glucosyl rhoifolin (Rhf-G), a novel transglycosylated compound synthesized from a flavone glycoside, rhoifolin, as well as its chemical structure. Furthermore, we report the complete NMR signal assignment for another transglycosylated compound, α-glucosyl rutin (Rutin-G), as the signals corresponding to its sugar moieties had not been identified. Electrospray ionization-mass spectrometry along with multiple NMR methods revealed that Rhf-G possesses three sugar moieties in its chemical structure. The additional glucose was bound directly via a transglycosylation to rhoifolin at position 3a of the sugar moiety. Interestingly, intramolecular hydrogen bonds in the basic Rhf-G and Rutin-G skeletons were confirmed by HMBC experiments. These findings will be helpful for comprehensive NMR studies on transglycosylated compounds in food, cosmetic, and pharmaceutical fields.

Increased Feeding Speed Is Associated with Higher Subsequent Sympathetic Activity in Dogs.

Although the domestication process has altered the feeding behavior of dogs, some breeds still demonstrate a remarkable ability to gorge, and will eat exceptionally large quantities of food whenever it is available. Lesions in the ventromedial hypothalamus increase appetite and lead to obesity, suggesting that the autonomic nervous system plays an important role in feeding. Focusing on the autonomic activities closely involved in food intake, we investigated sympathetic activities before and after feeding in dogs. The subjects were 56 healthy dogs of 21 different breeds (29 males and 27 females). Based on feeding habits, the 56 dogs were divided into three groups: Fast (n = 19), Slow (n = 24) and Leftover (n = 13). The feeding speed and the amount of food per mouthful of the Fast dogs were significantly greater than those of the Slow and the Leftover dogs. The plasma norepinephrine level in dogs of the Fast group was significantly increased after feeding, while those in the Slow and Leftover groups were significantly decreased after feeding, compared with the pre-feeding concentrations. The low frequency/high frequency ratio of heart rate variability is a good indicator of sympathetic activity and was also significantly higher in the Fast group than in the other groups. Delayed feeding using automatic feeding equipment decreased the plasma norepinephrine concentration and low frequency/high frequency ratio observed after feeding in dogs of the Fast group. In conclusion, dogs eating rapidly with less chewing, which indicates increased sympathetic activity during feeding, may benefit from delayed feeding. The slow eating may activate the parasympathetic nervous system after feeding, which could enhance the activity of the digestive system.

Accelerometry-based gait analysis predicts falls among patients with a recent fracture who are ambulatory: a 1-year prospective study.

The aim of this prospective study was to assess whether accelerometry-based gait analysis is associated with the incidence of falls among individuals who are ambulatory and recently had a fracture, and to compare the ability to discriminate body function and structure and activity measures. A total of 100 patients who fulfilled our inclusion criteria were enrolled. At hospital discharge, the following outcomes were assessed: timed-up-and-go test, five-times-sit-to-stand test, one-leg standing test, 5-m gait test, and gait analysis using a wireless three-axis accelerometer. Root mean square (RMS) and autocorrelation (AC) values were analyzed on the basis of the acceleration waveform. Follow-up assessment of falls and subsequent fractures was performed 12 months after the fracture. Eighty-five percent of the participants completed the follow-up at 12 months. During the 12 months of follow-up, 61 falls were reported by 34 patients (31 women, three men), for a fall rate of 40.0% (34/85). The fall group had significantly poorer body function and structure and activity measures compared with the no-fall group. In accelerometry-based gait analysis, RMS and AC in the vertical axis and RMS in the anteroposterior axis were significantly lower in the fall group compared with the no-fall group. Multivariate logistic regression analysis showed that only AC in the vertical axis was a significant predictor of falls. This finding suggests that trunk acceleration AC in the vertical axis showed good discriminative ability for predicting the incidence of falls among patients who are ambulatory and recently had a fracture.