We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

It is well known that maternal stress during the gestation and lactation periods induces abnormal behavior in the offspring and causes a lowering of the offspring's body weight. Various causes of maternal stress during the lactation period, relating to, for example, maternal nutritional status and reduced maternal care, have been considered. However, little is known about the effects on milk of maternal stress during the lactation period. The current study aimed to determine whether free amino acids, with special reference to sulfur-containing amino acids in milk, are altered by restraint stress in lactating mice. The dams in the stress group were restrained for 30 min at postnatal days 2, 4, 6, 8, 10 and 12. Restraint stress caused a reduction in the body weight of lactating mice. The concentration of taurine and cystathionine in milk was significantly higher in the stress group, though stress did not alter their concentration in maternal plasma. The ratio of taurine concentration in milk to its concentration in maternal plasma was significantly higher in the stress group, suggesting that stress promoted taurine transportation into milk. Furthermore, taurine concentration in milk was positively correlated with corticosterone levels in plasma. In conclusion, restraint stress in lactating mice caused the changes in the metabolism and in the transportation of sulfur-containing amino acids and resulted in higher taurine concentration in milk. Taurine concentration in milk could also be a good parameter for determining stress status in dams.

The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.

The growth of offspring is affected not only by the protein in maternal milk but also by the free amino acids (FAAs) contained in it. L-Serine (L-Ser) is known as an important FAA for the development of the central nervous system and behavioral activity. However, it is not clear whether L-Ser is transported into the pool of FAAs contained in milk and thereby affects the growth of offspring. Using mice, the current study investigated the effects of dietary L-Ser during pregnancy and lactation on milk and plasma FAA composition, as well as on growth, behavior, and plasma FAAs of offspring. Dietary L-Ser did not significantly affect the maternal, anxiety-like, or cognitive behaviors of either the dam or the offspring. The FAA composition notably differed between plasma and milk in dams. In milk, dietary L-Ser increased free L-Ser levels, while glutamic acid, L-alanine, D-alanine and taurine levels were decreased. The body weight of the offspring was lowered by dietary L-Ser. The concentrations of plasma FAAs in 13-day-old offspring (fed only milk) were not altered, but 20-day-old offspring (fed both milk and parental diet) showed higher plasma L-Ser and D-Ser concentrations as a result of the dietary L-Ser treatment. In conclusion, the present study found that dietary L-Ser transported easily from maternal plasma to milk and that dietary L-Ser treatment could change the FAA composition of milk, but that an enhanced level of L-Ser in milk did not enhance the plasma L-Ser level in the offspring.

We have isolated a basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed zath3, from zebrafish. zath3 is expressed in neurons of the central nervous system and in subsets of cranial ganglia. Zebrafish mindbomb (mib) mutants have a higher density of zath3 expressing cells and narrowminded (nrd) mutants lack zath3 expression in a domain corresponding to primary sensory neurons showing that the expression of zath3 is regulated by both mib and nrd. Injection of synthetic zath3 RNA into zebrafish embryos expands the neural plate size, promotes ectopic expression of neuronal markers, and partially rescues the deficit of sensory neurons seen in nrd mutants. Interfering with zath3 function using antisense morpholino oligonucleotides (MO) has no significant effect on early neurogenesis. However, a double knock down of zath3 and neurogenin1 (ngn1), another atonal homologue, with morpholinos (MOs) leads to more severe defects in neurogenesis than are seen with ngn1 MO alone: a subtle reduction of motor and inter-neurons, and an almost complete loss all cranial ganglia. This study suggests that zath3 and ngn1 have partially overlapping roles in early neurogenesis.

This study aimed to investigate the effects of Brazilian propolis on body fat mass and levels of adiponectin and reactive oxygen species among community-dwelling elderly females. This was a double-blind randomized placebo-controlled trial. Altogether, 78 females aged 66-84 years were randomly assigned to the propolis (PRO; n = 39) or placebo (PLA; n = 39) group. For 12 weeks, the PRO group were given three capsules containing 227 mg of propolis twice a day. Meanwhile, the PLA group were given daily placebo capsules. Of 78 participants, 53 (PLA group: n = 28, PRO group: n = 25) completed the study. Although no changes were observed in absolute or relative fat mass in the PLA group, they showed a significant decline in the PRO group. The level of serum adiponectin in the PLA group did not change, although that of the PRO group significantly increased. The level of d-ROMs in the PLA group significantly increased, whereas that of the PRO group significantly decreased. The serum SOD activity in the PLA group significantly decreased, whereas that of the PRO group tended to increase. These results suggest that propolis supplementation may decrease body fat mass and oxidative stress among community-dwelling elderly females.

The behavioral effects of ethanol have been studied in multiple animal models including zebrafish. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear. Here we examined whether the intrinsic properties of neurons play roles in this resistance. In order to minimize the contribution of metabolism and diffusional barriers, larvae were hemisected and the anterior half immersed in a range of ethanol concentrations thereby ensuring the free access of bath ethanol to the brain. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular [Ca(2+)] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Finally, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment abolished long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets.

Mutations in AChR subunits, expressed as pentamers in neuromuscular junctions (NMJs), cause various types of congenital myasthenic syndromes. In AChR pentamers, the adult ε subunit gradually replaces the embryonic γ subunit as the animal develops. Because of this switch in subunit composition, mutations in specific subunits result in synaptic phenotypes that change with developmental age. However, a mutation in any AChR subunit is considered to affect the NMJs of all muscle fibers equally. Here, we report a zebrafish mutant of the AChR δ subunit that exhibits two distinct NMJ phenotypes specific to two muscle fiber types: slow or fast. Homozygous fish harboring a point mutation in the δ subunit form functional AChRs in slow muscles, whereas receptors in fast muscles are nonfunctional. To test the hypothesis that different subunit compositions in slow and fast muscles underlie distinct phenotypes, we examined the presence of ε/γ subunits in NMJs using specific antibodies. Both wild-type and mutant larvae lacked ε/γ subunits in slow muscle synapses. These findings in zebrafish suggest that some mutations in human congenital myasthenic syndromes may affect slow and fast muscle fibers differently.

Heat stress is an increasing concern in poultry industry as it can cause a rise in the body temperature of chickens. Recently, we reported that l-citrulline (l-Cit) is a potential hypothermic agent that could improve thermotolerance in chicks. However, synthetic l-Cit has not yet been approved for inclusion in animal diets. l-Cit was first isolated from watermelon. Watermelon rind (WR), an agricultural waste product, contains more l-Cit than the flesh of the fruit. In the current study, the chemical composition and l-Cit content of WR dried powder (WRP) were determined. WRP was mixed with water at a ratio of 4:5 (wt/v) to make WRP mash, and then mixed with a commercial starter diet to prepare a 9% WRP mash diet. The WRP mash diet was fed to 3- to 15-day-old chicks and daily food intake, body weight, and changes in rectal temperature were measured. At the end of the experiment, blood was collected from the chicks to analyze plasma l-Cit and other free amino acids. The chemical analysis of WRP revealed a variety of components including 19.1% crude protein. l-Cit was the most abundant free amino acid in WRP (3.18 mg/g). Chronic supplementation of the WRP mash diet significantly increased compensatory food intake, plasma l-Cit, l-ornithine, and l-tyrosine in chicks. WRP mash diet did not affect the body temperature of the chicks. In conclusion, WRP mash diet supplementation increased plasma l-Cit concentration in chicks. The increase in plasma l-Cit concentrations suggest that WR could be used as a natural source of l-Cit in chicks to ameliorate the adverse effects of heat stress.

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.

Gut eubiosis is essential for the host's health. In athletes, the gut microbiota can be altered by several factors, including diets. While eubiotic gut microbiota in elite rugby players has been reported, our survey found that university rugby players suffered from loose stools and frequent urgency to defecate. To establish the causes of the condition, the microbiota and the concentrations of organic acids in fecal samples of university male rugby players (URP) were analyzed and compared with those of age-matching, non-rugby playing males (control). Body mass indices were significantly (p < 0.05) different between groups. Chao1 index was significant (p < 0.05) lower in URP than in control. The relative abundances of phyla Firmicutes and Bacteroidetes were significantly (p < 0.05) higher and lower, respectively, in URP than in control. Potential pathobiont genera Collinsella, Enterobacter, and Haemophilus were significantly (p < 0.05) abundant, whereas beneficial Akkermansia was lower (p < 0.05) in URP than in control. Succinate, a potential causative of gut inflammation, was five-fold higher in URP than in controls. Our findings all but confirmed that the dysbiotic status of gut in URP.

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

This study targets the evaluation of melanin synthesis inhibition activity of the bamboo shoot skin as agro-waste. The total methanolic extract of bamboo peel extract was evaluated for its skin protective effects via measuring its melanin inhibitory activity and its suppression activity on the expression of tyrosinase mRNA levels. Results showed that bamboo peel extract has a good ability for the inhibition of melanin synthesis so further studies were performed for the isolation of its constituents. Twelve compounds have been isolated from the shoot skin of Phyllostachys pubescens. Their structures were elucidated based on extensive spectroscopic methods. The melanin inhibition potential of the isolates was tested with their collagen-production-promoting activity for the determination of active principles. Results showed that Betulinic acid, tachioside, and 1,2-dilinolenin significantly suppressed melanin production per cell compared to control. Triacontanol, tricin, and (+)-lyoniresinol 9'-O-glucoside also tended to decrease melanin production per cell. These findings indicated that the skin of bamboo shoots, a significant agricultural waste, is a useful natural source for further research on its potential for aging problems such hyperpigmentation and cognitive function impairment.