An anterior limit of FGF/Erk signal activity marks the earliest future somite boundary in zebrafish.

Vertebrate segments called somites are generated by periodic segmentation of the anterior extremity of the presomitic mesoderm (PSM). During somite segmentation in zebrafish, mesp-b determines a future somite boundary at position B-2 within the PSM. Heat-shock experiments, however, suggest that an earlier future somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Here, we characterized fibroblast growth factor (FGF) signal activity within the PSM, and demonstrated that an anterior limit of downstream Erk activity corresponds to the future B-5 somite boundary. Moreover, the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. Our results provide the first molecular evidence of the future somite boundary at B-5, and we propose that clock-dependent cyclic inhibition of the FGF/Erk signal is a key mechanism in the generation of perfect repetitive structures in zebrafish development.

Identification of olfactory alarm substances in zebrafish.

Escaping from danger is one of the most fundamental survival behaviors for animals. Most freshwater fishes display olfactory alarm reactions in which an injured fish releases putative alarm substances from the skin to notify its shoaling company about the presence of danger. Here, we identified two small compounds in zebrafish skin extract, designated as ostariopterin and daniol sulfate. Ostariopterin is a pterin derivative commonly produced in many freshwater fishes belonging to the Ostariophysi superorder. Daniol sulfate is a novel sulfated bile alcohol specifically present in the Danio species, including zebrafish. Ostariopterin and daniol sulfate activate distinct glomeruli in the olfactory bulb. Zebrafish display robust alarm reactions, composed of darting, freezing, and bottom dwelling, only when they are concomitantly stimulated with ostariopterin and daniol sulfate. These results demonstrate that the fish alarm reaction is driven through a coincidence detection mechanism of the two compounds along the olfactory neural circuitry.

Preparation and in vitro/in vivo evaluations of dimpled poly(L-lactic acid) fibers mixed/coated with hydroxyapatite nanocrystals.

A novel hydroxyapatite (HAp)/poly(L-lactic acid) (PLLA) nanocomposite nonwoven fabric, which was coated and mixed with calcined HAp nanocrystals, and has submicron-sized dimples on its surface, was fabricated. First, HAp-mixed PLLA fabric was prepared by electrospinning a HAp nanocrystal dispersion in dichloromethane (DCM)-dissolved PLLA. It was found that most of the HAp nanocrystals were not exposed on the HAp-mixed PLLA fiber surface but covered with the PLLA matrix. A HAp-nanocrystal coating was applied onto the surface of the HAp-mixed PLLA fabric after corona discharge treatment followed by ethanol washing. The submicron-sized dimples were enlarged after the ethanol washing. After the HAp-nanocrystal coating, the HAp-mixed PLLA fabric surface was uniformly coated with the HAp nanocrystals. In vitro cell spread tests showed that the rat osteoblasts spread more on HAp-nanocrystal-coated fabrics than on non-HAp-coated fabrics. Upon covering calvarial defects, the in vivo hard tissue responses suggested earlier restoration of the defects with HAp-nanocrystal-coated fabrics than those with non-HAp-coated fabrics.

An Adenosine Receptor for Olfaction in Fish.

Nucleotides released from food sources into environmental water are supposed to act as feeding cues for many fish species. However, it remains unknown how fish can sensitively detect those nucleotides. Here we discover a novel olfactory mechanism for ATP sensing in zebrafish. Upon entering into the nostril, ATP is efficiently converted into adenosine through enzymatic reactions of two ecto-nucleotidases expressed in the olfactory epithelium. Adenosine subsequently activates a small population of olfactory sensory neurons expressing a novel adenosine receptor A2c that is unique to fishes and amphibians. The information is then transmitted to a single glomerulus in the olfactory bulb and further to four regions in higher olfactory centers. These results provide conclusive evidence for a sophisticated enzyme-linked receptor mechanism underlying detection of ATP as a food-derived attractive odorant linking to foraging behavior that is crucial and common to aquatic lower vertebrates.

Increase in cell adhesiveness on a poly(ethylene terephthalate) fabric by sintered hydroxyapatite nanocrystal coating in the development of an artificial blood vessel.

Nano-scaled sintered hydroxyapatite (HAp) crystals were covalently linked onto a poly(ethylene terephthalate) (PET) fabric substrate chemically modified by graft polymerization with gamma-methacryloxypropyl triethoxysilane (MPTS) for development of an artificial blood vessel. The weight gain of graft polymerization with poly(MPTS) on PET in benzyl alcohol containing H2O2 as an initiator increased as increasing the reaction time and finally reached a plateau value of about 3.5 wt%. The surface characterization of surface modification with poly(MPTS)-grafting was conducted by x-ray photoelectron spectroscopy. HAp nanocrystals of approximately 50 nm in diameter, monodispersed in pure ethanol, were coupled with alkoxysilyl groups of the poly(MPTS)-grafted PET substrate. The HAp nanocrystals were uniformly and strongly coated on the surface of the PET fabrics, although HAp particles adsorbed physically on the original PET without poly(MPTS) grafting were almost removed by ultrasonic wave treatment. More human umbilical vein endothelial cells adhered to the HAp/PET composite fabric compared with original PET after only 4 hours of initial incubation, and the same was observed on the collagen-coated PET. The coating of sintered HAp nanocrystals imparted bioactivity to the polyester substrate, which is a widely used biomedical polymer, without a coating of adhesion proteins derived from animals, such as collagen or gelatin. A prototype of an artificial blood vessel was finally fabricated by use of HAp/PET composite.

Olfactory receptor for prostaglandin F2α mediates male fish courtship behavior.

Pheromones play vital roles for survival and reproduction in various organisms. In many fishes, prostaglandin F2α acts not only as a female reproductive hormone, facilitating ovulation and spawning, but also as a sex pheromone inducing male reproductive behaviors. Here, we unravel the molecular and neural circuit mechanisms underlying the pheromonal action of prostaglandin F2α in zebrafish. Prostaglandin F2α specifically activates two olfactory receptors with different sensitivities and expression in distinct populations of ciliated olfactory sensory neurons. Pheromone information is then transmitted to two ventromedial glomeruli in the olfactory bulb and further to four regions in higher olfactory centers. Mutant male zebrafish deficient in the high-affinity receptor exhibit loss of attractive response to prostaglandin F2α and impairment of courtship behaviors toward female fish. These findings demonstrate the functional significance and activation of selective neural circuitry for the sex pheromone prostaglandin F2α and its cognate olfactory receptor in fish reproductive behavior.

Group A streptococcal antigen in the glomeruli of children with Henoch-Schönlein nephritis.

BACKGROUND: Although the pathogenesis of Henoch-Schönlein nephritis (HSN) remains unclear, there is substantial evidence that it is an immune complex-mediated disease. HSN is preceded by upper-respiratory tract infection in 30% to 50% of patients, but there is no evidence that group A streptococcal (GAS) infection has a pathogenetic role in this disease. Recently, nephritis-associated plasmin receptor (NAPlr), a GAS antigen, was found primarily in the glomerular mesangium of patients with early-stage acute poststreptococcal glomerulonephritis. METHODS: To determine the possible role of NAPlr in HSN, expression of the receptor was determined in glomeruli using fluorescein isothiocyanate-labeled rabbit polyclonal anti-NAPIr antibody, and serum antistreptolysin O (ASO) titers were measured in children with HSN. RESULTS: Ten of 33 patients (30%) with HSN showed segmental or global mesangial staining with NAPlr antibody, whereas only 4 of 120 patients (3%) with other renal diseases were positive (P < 0.001, Fisher's exact test). Patients with HSN also showed significantly greater ASO titers than patients with other renal diseases (P = 0.03, Mann-Whitney U test). Serum ASO titers were significantly greater in patients with HSN with than without glomerular NAPlr antigen (P = 0.03, Mann-Whitney U test). CONCLUSION: These findings suggest that the deposition of NAPlr in the mesangium, induced by GAS infection, may have a role in the pathogenesis of HSN in some patients. Am J Kidney Dis 41:366-370.

Olfactory projectome in the zebrafish forebrain revealed by genetic single-neuron labelling.

Chemotopic odour representations in the olfactory bulb are transferred to multiple forebrain areas and translated into appropriate output responses. However, a comprehensive projection map of bulbar output neurons at single-axon resolution is lacking in vertebrates. Here we unravel a projectome of the zebrafish olfactory bulb through genetic single-neuron tracing and image registration. We show that five major target regions receive distinct modes of projections from olfactory bulb glomeruli. The central portion of posterior telencephalon receives non-selective, interspersed inputs from all glomeruli, whereas the ventral telencephalon is diffusely innervated by axons from particular glomerular clusters. The right habenula and posterior tuberculum (diencephalic nuclei) receive convergent inputs from restricted and all glomerular clusters, respectively. The bulbar recurrent projections are coarsely topographic. Thus, the primary chemotopic organization is transformed into distinct sensory representations in higher olfactory centres. These findings provide a framework to understand general principles as well as species-specific features in decoding of odour information.

In vivo evaluation of hydroxyapatite nanocoating on polyester artificial vascular grafts and possibility as soft-tissue compatible material.

The efficacy of hydroxyapatite (HAp) nanocoating on polyester vascular grafts was investigated in animal experiments. The HAp nanocrystals were covalently bonded separately between hydroxyl groups on a nanocrystal and alkoxysilyl groups in gamma-methacryloxypropyl triethoxysilane graft polymerized on a polyester substrate. Twelve HAp-coated polyester grafts and 10 control grafts of 20, 30, or 50 mm in length were implanted in canine common carotid arteries. Serious complications or occlusions were not observed in any of the dogs after implantation. A histologic evaluation was conducted by staining with hematoxylin and eosin (HE), the von Willebrand factor (vWf), and alpha-smooth muscle actin (alpha-SMA) around the inner lumen of the grafts. The number of inflammation cells and giant cells in the HAp-coated group was significantly lower than that in the group receiving noncoated grafts (p < 0.05).

Cell adhesion and tissue response to hydroxyapatite nanocrystal-coated poly(L-lactic acid) fabric.

Cell adhesion and tissue response to poly(l-lactic acid) (PLLA) fabric coated with nanosized hydroxyapatite (HAp) crystals were studied. The HAp nanocrystals were prepared by the wet chemical process followed by calcination at 800 degrees C with an anti-sintering agent to prevent calcination-induced sintering. After the PLLA fabric was hydrolyzed with an alkaline aqueous solution, the HAp nanocrystals were coated via ionic interaction between the calcium ions on the HAp and the carboxyl groups on the alkali-treated PLLA. The PLLA surface uniformly coated with the HAp nanocrystals was observed by scanning electron microscope. The ionic interaction between the HAp and the PLLA was estimated by FT-IR. Improved cell adhesion to the HAp nanocrystal-coated surface was demonstrated by in vitro testing using a mouse fibroblast cell line L929. Furthermore, reduced inflammatory response to the HAp nanocrystal-coated PLLA fabric (as compared with a non-treated one) was confirmed by a subcutaneous implantation test with rats. Thus the HAp nanocrystal-coated PLLA developed has possible efficacy as an implant material in the fields of general and orthopedic surgery, and as a cell scaffold in tissue engineering.

Preparation of hydroxyapatite-nanocrystal-coated stainless steel, and its cell interaction.

Calcined nanocrystals of hydroxyapatite (HAp) having spherical or rod-shaped morphologies were coated through covalent linkage on a type 316L stainless steel substrate, which was chemically modified by the graft polymerization of gamma-methacryloxypropyl triethoxysilane (MPTS) at 70-110 degrees C. The grafting of poly(MPTS) on the substrate was confirmed by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR). In order to coat the substrate with the HAp crystals through covalent linkage, the reactionbetween the alkoxysilyl groupsin the poly (MPTS) grafted on the substrate and the OH groups on the HAp crystals was conducted at 80 degrees C. The poly(MPTS)-grafted substrate was strongly coated with the HAp nanocrystals, although the HAp crystals adsorbed physically on the original substrate without poly(MPTS) grafting were removed by ultrasonic treatment. Human umbilical vein endothelial cells (HUVEC) adhered in larger numbers on the HAp-coated stainless steel substrate as compared with the original substrate after 24 h of initial incubation. The number of HUVEC adhered on the rod-shaped HAp-coated substrate was not significantly different from that on the spherical HAp-coated substrate under the present conditions.