Creation of an experimental rearing environment for microbiome animal research using an individually ventilated cage system and bioBUBBLE enclosure

To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.     

Cardiac Arrest during Gamete Release in Chum Salmon Regulated by the Parasympathetic Nerve System

Cardiac arrest caused by startling stimuli, such as visual and vibration stimuli, has been reported in some animals and could be considered as an extraordinary case of bradycardia and defined as reversible missed heart beats. Variability of the heart rate is established as a balance between an autonomic system, namely cholinergic vagus inhibition, and excitatory adrenergic stimulation of neural and hormonal action in teleost. However, the cardiac arrest and its regulating nervous mechanism remain poorly understood. We show, by using electrocardiogram (ECG) data loggers, that cardiac arrest occurs in chum salmon (Oncorhynchus keta) at the moment of gamete release for 7.39±1.61 s in females and for 5.20±0.97 s in males. The increase in heart rate during spawning behavior relative to the background rate during the resting period suggests that cardiac arrest is a characteristic physiological phenomenon of the extraordinarily high heart rate during spawning behavior. The ECG morphological analysis showed a peaked and tall T-wave adjacent to the cardiac arrest, indicating an increase in potassium permeability in cardiac muscle cells, which would function to retard the cardiac action potential. Pharmacological studies showed that the cardiac arrest was abolished by injection of atropine, a muscarinic receptor antagonist, revealing that the cardiac arrest is a reflex response of the parasympathetic nerve system, although injection of sotalol, a β-adrenergic antagonist, did not affect the cardiac arrest. We conclude that cardiac arrest during gamete release in spawning release in spawning chum salmon is a physiological reflex response controlled by the parasympathetic nervous system. This cardiac arrest represents a response to the gaping behavior that occurs at the moment of gamete release.     

Glycyrrhizin and its derivatives promote hepatic differentiation via sweet receptor,Wnt, and Notch signaling

The acute liver disease is involved in aberrant release of high-mobility group box 1 (HMGB1). Glycyrrhizin (GL), a traditional Chinese medicine for liver disease, binds to HMGB1, thereby inhibits tissue injury. However the mode of action of GL for chronic liver disease remains unclear.We investigated the effects of glycyrrhizin (GL) and its derivatives on liver differentiation using human iPS cells by using a flow cytometric analysis.GL promoted hepatic differentiation at the hepatoblast formation stage. The GL derivatives, 3-O-mono-glucuronyl 18β-glycyrrhetinic acid (Mono) and 3-O-[glucosyl (1 → 2)-glucuronyl] 18β-glycyrrhetinic acid increased AFP⁺ cell counts and albumin⁺ cell counts. Glucuronate conjugation seemed to be a requirement for hepatic differentiation. Mono exhibited the most significant hepatic differentiation effect.We evaluated the effects of (±)-2-(2,4-dichlorophenoxy) propionic acid (DP), a T1R3 antagonist, and sucralose, a T1R3 agonist, on hepatic differentiation, and found that DP suppressed Mono-induced hepatic differentiation, while sucralose promoted hepatic differentiation. Thus, GL promoted hepatic differentiation via T1R3 signaling. In addition, Mono increased β-catenin⁺ cell count and decreased Hes5⁺ cell count suggesting the involvement of Wnt and Notch signaling in GL-induced hepatic differentiation.In conclusion, GL exerted a hepatic differentiation effect via sweet receptor (T1R3), canonical Wnt, and Notch signaling.     

Growth and distribution patterns of Roseobacter/Rhodobacter, SAR11, and Bacteroidetes lineages in the Southern Ocean

Roseobacter/Rhodobacter and SAR11, affiliated with Alphaproteobacteria, and the phylum Bacteroidetes constitute a large proportion of marine planktonic bacteria, but information about their growth and distribution patterns in the Southern Ocean is scarce. The aim of the present study is to determine patterns in the biomass and productivity of Roseobacter/Rhodobacter, SAR11, and Bacteroidetes groups along the steep temperature, salinity, and organic matter gradients in the Southern Ocean by using catalyzed reporter deposition-fluorescence in situ hybridization and bromodeoxyuridine (BrdU) immunocytochemistry FISH. We found that Roseobacter/Rhodobacter, SAR11, and Bacteroidetes are prominent contributors to total bacterial biomass and production. SAR11 bacteria were the predominant lineage, but their biomass was low in the coldest regions. In contrast, the biomasses of Roseobacter/Rhodobacter and Bacteroidetes lineages were positively correlated with organic matter concentrations. The Roseobacter/Rhodobacter had the highest proportion of BrdU-positive (i.e., actively growing) cells among the three phylotypes at all stations, despite their low abundance. The relative contribution of Bacteroidetes to the total bacterial productivity (number of active cells) was negatively correlated with temperature. These results suggest that the growth and distribution patterns of Roseobacter/Rhodobacter, SAR11, and Bacteroidetes were determined by different environmental gradients (e.g., organic matter concentrations or temperature) in the Southern Ocean.     

Automated screening and primer design of fish microsatellite DNA loci on pyrosequencing data

In recent years, massive sequencing approaches have allowed us to determine genomic structures of various organisms rapidly, raising novel applicability of the high-throughput sequence data obtained to various fields of biological studies. We present here a pipeline to search for microsatellite DNA and design PCR primers encompassing the microsatellites on genomic sequence data produced by 454 pyrosequencing. We tested this pipeline, called ‘Auto-primer’, on several fish genomic sequences and obtained many and various candidates for microsatellite DNA loci useful for detecting intraspecies genetic variability. This in silico search for microsatellite DNA is superior to conventional cloning methods, since any sequence patterns of repeat unit can be screened.     

Dipeptide synthesis catalyzed by aminoacyl-tRNA synthetases from Bacillus stearothermophilus

A new approach to enzymatic peptide synthesis by using aminoacyl-tRNA synthetase (ARS) as a catalyst has been investigated. Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus. By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide. In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions. TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D-amino acids were incorporated into peptides fairly easily in this enzymatic reaction. Preparative scale synthesis of L-Tyr-L-LeuNH2 was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.     

Indole–porphyrin hybrids produced by metagenomics

New indole–porphyrin hybrid molecules were isolated from Escherichia coli harboring metagenomic DNA from the Japanese marine sponge Discodermia calyx. The indole was appended to the reactive vinyl substituent of the harderoporphyrin chromophore, encoded by the insert DNA. Thus, the chimeric pathway between the heterologously expressed porphyrins and the endogenous indole generated new indole-conjugated chiral porphyrins in E. coli.