Water structure changes induced by ceramics can be detected by increased permeability through aquaporin

Aquporins are intrinsic membrane proteins that function as water channel to transport water and/or mineral nutrients across biological membranes. In this study, we aimed to clarify whether water structure can be changed by the presence of ceramics and whether such a change can be determined by aquaporin. First, we confirmed that ceramics could transform tap water into active tap water by increasing water permeability through aquaporin. We also found that this change in water permeability by treatment with ceramics occurred in distilled water. The distilled water was determined to exhibit the same aquaporin permeability as the original tap water. Our data indicate that the aquaporin permeability of water can be changed by severe physical shocks, such as slapping and sonication, which is consistent with the implication that the aquaporin permeability is closely related to the structure of the water. In this study, using aquaporins, we first reported that the treatment of water with ceramics can affect the structure of water, and the water can retain the structure for a given period under certain condition     

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.     

Microdose clinical trial: quantitative determination of fexofenadine in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry

A sample treatment procedure and high-sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for quantitative determination of fexofenadine in human plasma was developed for a microdose clinical trial with a cold drug, i.e., a non-radioisotope-labeled drug. Fexofenadine and terfenadine, as internal standard, were extracted from plasma samples using a 96-well solid-phase extraction plate (Oasis HLB). Quantitation was performed on an ACQUITY UPLC system and an API 5000 mass spectrometer by multiple reaction monitoring. Chromatographic separation was achieved on an XBridge C18 column (100 mm x 2.1 mm i.d., particle size 3.5 microm) using acetonitrile/2 mM ammonium acetate (91:9, v/v) as the mobile phase at a flow rate of 0.6 ml/min. The analytical method was validated in accordance with the FDA guideline for validation of bioanalytical methods. The calibration curve was linear in the range of 10-1000 pg/ml using 200 microl of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 1-500 ng/ml using 20 microl of plasma, was also conducted. Each method was successfully applied for making determinations in plasma using LC/ESI-MS/MS after administration of a microdose (100 microg solution) and a clinical dose (60 mg dose) in eight healthy volunteers.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

The process of cardiorespiratory autoresuscitation in intact newborn rats

To examine the process of spontaneous autoresuscitation and the recovery of the hypoxic ventilatory response (HVR) after prolonged anoxia, we monitored respiratory frequency (f, by body plethysmography) and heart rate (HR, by ECG) in intact newborn rats (n = 12, day 2-4) before, during, and after 100% N2 exposure. The rat before anoxia showed signs of HVR: f changes at acute hypoxia (10% O2) and hyperoxia (100% O2). During anoxia, the spontaneous respiratory movement "gasping" appeared for 21 min (mean). At O2 restoration (with 100% O2), gasping stopped and no respiratory flow was detected for 1 min. One rat failed to autoresuscitate and had heart arrhythmia during the transient apnea, but 11 rats recovered respiration after the HR acceleration. Despite the successful autoresuscitation, the rats did not show HVR at 10 min into the recovery period and the recovery of HVR required more than 30 min. The results indicate that O2 inhalation is useful to trigger autoresuscitation even when the rat has already been in a state of profound hypoxic depression, but the rat becomes transiently insensitive to HVR after autoresuscitation. We estimate that reform of the respiratory control system in newborn rats is not yet firmly established to track HVR early in the recovery phase after prolonged anoxia.     

Bovine milk lactoferrin modulates the proliferative lymphocyte response to phytohemagglutinin

Bovine milk lactoferrin (2 to 20 g ml^–1) changed enhancement of [³H]thymidine incorporation by phytohemagglutinin-stimulated rat spleen lymphocytes into suppression as their lactoferrin-withdrawal incorporation increased to greater than 10000 cpm culture^–1 under the present isotope-labeling conditions. The enhancement disappeared by 15-min delayed addition of lactoferrin after addition of lectin. There was no lactoferrin effect when the cells were stimulated with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin. Thus, lactoferrin has a certain extracellular effect on lymphocyte proliferation in response to the lectin.     

A GFP-transfected HFWT cell line,GHINK-1, as a novel target for non-RI activated natural killer cytotoxicity assay

An anchorage-dependent Wilms' tumor cell line, HFWT, has been found to be extremely susceptible to human natural killer (NK) cells. Here we established a transfectant of HFWT with the green fluorescence protein (GFP) gene, designated GHINK-1 cells, to apply to the activated NK cytotoxicity assay without radioisotope labeling. After being co-cultured with CD3 CD56+ NK cells, GHINK-1 cells released GFP into the medium. The intensity of the fluorescence from the released GFP correlated almost exactly with the radioactivity of a standard 51Cr-release assay performed with suspension-cultured K562 cells. Because it does not require separation of the remaining live target cells by centrifugation, the non-radioisotopic GFP release assay with GHINK-1 cells is a convenient alternative for monitoring human activated NK killing activity.     

A new assay using surface plasmon resonance (SPR) to determine binding of the Lactobacillus acidophilus group to human colonic mucin

A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcalpha1-3(Fucalpha1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.     

An engram found? Evaluating the evidence from fruit flies

Is it possible to localize a memory trace to a subset of cells in the brain? If so, it should be possible to show: first, that neuronal plasticity occurs in these cells. Second, that neuronal plasticity in these cells is sufficient for memory. Third, that neuronal plasticity in these cells is necessary for memory. Fourth, that memory is abolished if these cells cannot provide output during testing. And fifth, that memory is abolished if these cells cannot receive input during training. With regard to olfactory learning in flies, we argue that the notion of the olfactory memory trace being localized to the Kenyon cells of the mushroom bodies is a reasonable working hypothesis.