Bio-Based Hydrogel and Aerogel Composites Prepared by Combining Cellulose Solutions and Waterborne Polyurethane.

Hydrogel composites can be prepared from cellulose-based materials and other gel materials, thus combining the advantages of both kinds of material. The aerogel, porous material formed after removing the water in the hydrogel, can maintain the network structure. Hydrogel and aerogel have high application potential. However, low mechanical strength and weight loss of cellulose hydrogel due to the water dehydration/absorption limit the feasibility of repeated use. In this study, cellulose hydrogels were prepared using microcrystalline cellulose (MC), carboxymethyl cellulose (CMC), and hydroxyethyl cellulose (HEC) as raw materials. Waterborne polyurethane (WPU) was added during the preparation process to form cellulose/WPU composite hydrogel and aerogel. The influence of the cellulose type and WPU addition ratio on the performance of hydrogel and aerogel were investigated. The results show that the introduction of WPU can help strengthen and stabilize the structure of cellulose hydrogel, reduce weight loss caused by water absorption and dehydration, and improve its reusability. The mixing of cellulose and WPU at a weight ratio of 90/10 is the best ratio to make the cellulose/WPU composite aerogel with the highest water swelling capacity and heat resistance.

Assessing developmental toxicity of caffeine and sweeteners in medaka (Oryzias latipes).

The use of artificial sweeteners (ASWs) has increased and become more widespread, and consequently ASWs have appeared in aquatic environments around the world. However, their safety to the health of humans and wildlife remains inconclusive. In this study, using medaka embryos (Oryzias latipes), we investigated developmental toxicity of aspartame (ASP) and saccharin (SAC). Since ASWs are often consumed with caffeine (CAF) and CAF with sucrose (SUC), we tested biological activities of these four substances and the mixtures of CAF with each sweetener. The embryos were exposed to ASP at 0.2 and 1.0 mM, SAC at 0.005 and 0.050 mM, CAF at 0.05 and 0.5 mM, or SUC at 29 and 146 mM, starting from less than 5 h post fertilization until hatch. Control embryos were treated with embryo solution only. Several endpoints were used to evaluate embryonic development. Some of the hatchlings were also tested for anxiety-like behavior with the white preference test. The results showed that all four substances and the mixtures of CAF with the sweeteners affected development. The most sensitive endpoints were the heart rate, eye density, and hatchling body length. The hatchlings of several treatment groups also exhibited anxiety-like behavior. We then used the Integrated Biological Response (IBR) as an index to evaluate the overall developmental toxicity of the substances. We found that the ranking of developmental toxicity was SAC > CAF > ASP > SUC, and there was a cumulative effect when CAF was combined with the sweeteners.

Developmental toxicity of cigarette butts - An underdeveloped issue.

Cigarette butts (CBs) littering is not just an unsightly nuisance but also a public health problem, because chemicals contained in cigarettes can leach into aquatic environments and pose a risk to the health of humans and wildlife. However, this risk is largely unrecognized or ignored by the public, and toxicological evidence of CBs is scarce. Therefore, we used medaka embryos (Oryzias latipes) to explore developmental toxicity of CBs. The embryos were exposed to various concentrations of leachates from smoked and unsmoked cigarette tobacco (ST and UST) and filters (SF and USF), and observed from 1 to 3 days post-fertilization. The images were recorded and several developmental endpoints analyzed. The values from these endpoints were then used to calculate the Integrated Biomarker Response and evaluate overall effects of the leachates. Some of the embryos were allowed to hatch, and the hatchlings were tested for anxiety-like behavior. Our results showed that low concentrations of the leachates from ST, UST, and SF raised the heart rate, accelerated development, and changed behavior, while high concentrations lowered the heart rate, suppressed development, and increased mortality. The lowest observed effect concentration for the leachates was ≤0.2piece (pc)/L. The USF leachate had no effect at the concentration of 20pc/L. Developmental toxicity of the leachates was ranked as: ST>UST>SF>USF. This study has demonstrated for the first time that CB leachates affect fish development, and provided toxicological evidence to better assess ecological impacts of CBs.

Using medaka embryos as a model system to study biological effects of the electromagnetic fields on development and behavior.

The electromagnetic fields (EMFs) of anthropogenic origin are ubiquitous in our environments. The health hazard of extremely low frequency and radiofrequency EMFs has been investigated for decades, but evidence remains inconclusive, and animal studies are urgently needed to resolve the controversies regarding developmental toxicity of EMFs. Furthermore, as undersea cables and technological devices are increasingly used, the lack of information regarding the health risk of EMFs to aquatic organisms needs to be addressed. Medaka embryos (Oryzias latipes) have been a useful tool to study developmental toxicity in vivo due to their optical transparency. Here we explored the feasibility of using medaka embryos as a model system to study biological effects of EMFs on development. We also used a white preference test to investigate behavioral consequences of the EMF developmental toxicity. Newly fertilized embryos were randomly assigned to four groups that were exposed to an EMF with 3.2kHz at the intensity of 0.12, 15, 25, or 60µT. The group exposed to the background 0.12µT served as the control. The embryos were exposed continually until hatch. They were observed daily, and the images were recorded for analysis of several developmental endpoints. Four days after hatching, the hatchlings were tested with the white preference test for their anxiety-like behavior. The results showed that embryos exposed to all three levels of the EMF developed significantly faster. The endpoints affected included the number of somites, eye width and length, eye pigmentation density, midbrain width, head growth, and the day to hatch. In addition, the group exposed to the EMF at 60µT exhibited significantly higher levels of anxiety-like behavior than the other groups did. In conclusion, the EMF tested in this study accelerated embryonic development and heightened anxiety-like behavior. Our results also demonstrate that the medaka embryo is a sensitive and cost-efficient in vivo model system to study developmental toxicity of EMFs.

Screening estrogenic activity of environmental contaminants and water samples using a transgenic medaka embryo bioassay.

Many natural or synthetic chemicals may act as exogenous estrogens and affect the reproductive health of humans and wildlife. Since these xenoestrogens are ubiquitous, it is essential to monitor their presence in the environment. Hence, we developed a bioassay using the transgenic medaka (Oryzias latipes) embryo, in which the green fluorescent protein (GFP) was placed under the control of the gnrh3 promoter, one of the three paralogous gonadotropin-releasing hormone (GnRH) genes that regulate reproductive function and behavior. As medaka embryos are transparent, the fluorescent expression of GFP can be easily observed in vivo during development. We exposed newly fertilized medaka embryos to varying solutions of bisphenol A (BPA), nonylphenol (NP), 17ß-estradiol (E2), or a river water sample, and monitored their development. During embryonic development, the mRNA levels of GnRHs, GnRH receptors, and estrogen receptors (ERs) were measured with quantitative real-time reverse transcription-PCR. Our results showed that the chemicals and the river water significantly decreased the fluorescent intensity of the GnRH3 neurons, postponed the eye development, and retarded the growth of the embryos. The three xenoestrogens also lowered the heart rate, lengthened the time to hatch, suppressed the expression of the three GnRH genes, and up-regulated the ERα mRNA level. In addition, the GnRH3 mRNA level was significantly correlated with the fluorescence intensity of the GnRH neurons. We concluded that the transgenic medaka embryo is a rapid and sensitive bioassay for screening environmental water samples. We also found that xenoestrogens had significant effects on GnRH gene expression and embryonic development.

Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio).

There are multiple populations of gonadotropin-releasing hormone (GnRH) neurons that have distinct physiological and behavioral functions. Teleost fish have a population of GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb that is thought to play a neuromodulatory role in multiple physiological systems, including olfactory, visual, and reproductive. We used transgenic zebrafish in which the GnRH3 promoter drives expression of a green fluorescent protein to identify GnRH3 neurons during development in live embryos. Unlike with hypophysiotropic GnRH neurons of zebrafish, TN-GnRH3 neurons are of neural crest origin and are one of the first populations of GnRH neurons to develop in the early embryo. Using a combination of optical imaging and electrophysiology, we showed that during the first 3 days post-fertilization, TN-GnRH3 neurons increase in number, extend neural projections, move in association with tissue expansion, and acquire an adult-pattern of spontaneous action potential firing. Early during development, about half of the neurons were quiescent/non-firing. Later, at 3 days post-fertilization, there was an increase in the proportion of neurons showing action potential firing and an increase in the number of neurons that showed an adult-like tonic or beating pattern of action potential firing with a firing frequency similar to that seen in adult TN-GnRH3 neurons. This study represents the first neurophysiological investigation of developing GnRH neurons in live embryos--an important advancement in understanding their potential non-reproductive roles during embryogenesis.

A reproductive toxicology study of phenanthrene in medaka (Oryzias latipes).

Environmental contaminants, such as polycyclic aromatic hydrocarbons (PAHs), can disrupt the endocrine system and affect reproductive function of humans and wildlife. In this study, we exposed Japanese medaka (Oryzias latipes) to water-borne or food-spiked phenanthrene, an ubiquitous PAH, and investigated the chronic effects of the chemical on medaka reproduction. The results showed that phenanthrene significantly prolonged the time to hatch for embryos. Other parameters such as fecundity and fertility, organ-somatic ratios, hepatic vitellogenin production, and the histology of testes and ovaries were not different between the control and phenanthrene-treated groups. This suggests that the time to hatch in embryos might be a more sensitive biomarker for environmental contaminants.

Novolak PF resins prepared from phenol liquefied Cryptomeria japonica and used in manufacturing moldings.

The wood of Japanese cedar (Cryptomeria japonica) was liquefied in phenol with H2SO4 and HCl as catalysts. The liquefied wood was reacted with formalin to prepare the novolak PF resin. The results showed that the reaction of liquefied Japanese cedar with formalin was an exothermic reaction, and formed a solid-like resin without extra heating. Two novolak PF resins were prepared from the liquefied wood which were identified as SF and CF that using the liquefied wood with H2SO4 and HCl as catalyst respectively. The novolak PF powder displayed thermo-melting characteristic. The resins of SF and CF had weight average molecular weight of 3638 and 3941 respectively and melting temperature of 149.4 degrees C and 127.5 degrees C respectively. Both of the novolak resins could be used to make moldings with good performance by mixing the novolak resin with wood powder, hardener and zinc stearate at the weight ratio of 60:30:10:1 and hot-pressed under 200 degrees C for 10min.

Cellular and molecular analysis of neural development of glycosyltransferase gene knockout mice.

Recent studies demonstrate that carbohydrates synthesized by specific glycosyltransferases play important roles in the development of the central nervous system. Among these carbohydrates, polysialic acid is a unique glycan that modulates functions of the neural cell adhesion molecule (NCAM) by attenuating NCAM-mediated interaction between neural cells. During brain development, polysialic acid is synthesized in a specific spatiotemporal pattern by two polysialyltransferases, ST8SiaII and ST8SiaIV. To study in vivo the roles of polysialic acid synthesized by each respective enzyme, we generated ST8SiaII and ST8SiaIV knockout mice. Single knockout ST8SiaII or ST8SiaIV mice show polysialic acid expression patterns differing from wild type, and those patterns indicate different roles of each gene during neural development. In this chapter, we discuss methods used to analyze polysialyltransferase knockout mice using immunohistochemistry of brain and primary cultures of neurons.

Properties of resorcinol-tannin-formaldehyde copolymer resins prepared from the bark extracts of Taiwan acacia and China fir.

Resorcinol-tannin-formaldehyde copolymer resins (RTF) were prepared by using the bark extracts of Taiwan acacia (Acacia confusa) and China fir (Cunninghamia lanceolata) to substitute part of the resorcinol. From the results, the content of reactive phenolic materials in Taiwan acacia and China fir bark extracts were 51.6% and 46.5%, respectively. Aromatic compounds were the main components in the bark extracts showed by FT-IR analysis. The conventional synthesis condition used for RF resin was certainly not suitable for the RTF copolymer resin. It should be formed the novolak RF prepolymer by reacting the resorcinol with formaldehyde at the first stage, and then the bark extracts added and underwent the copolymerization reaction under acidic condition at the second-stage. The RTF copolymer resins prepared had cold-setting capability. They had higher viscosity, shorter gel time as compared with the RF resin. The RTF copolymer resins could be carried out the gluing application immediately after the hardener was added and had bonding strength the same as RF resin. But the RTF copolymer resins had worse stability and shorter shelf life than RF resin.

Sialyltransferase ST8Sia-II assembles a subset of polysialic acid that directs hippocampal axonal targeting and promotes fear behavior.

Polysialic acid (PSA) is a post-translational protein modification that is widely expressed among neural cell types during development. Found predominantly on the neural cell adhesion molecule (NCAM), PSA becomes restricted to regions of neurogenesis and neuroplasticity in the adult. In the mammalian genome, two polysialyltransferases termed ST8Sia-II and ST8Sia-IV have been hypothesized to be responsible for the production of PSA in vivo. Approaches to discover PSA function have involved the application of endoneuraminidase-N to remove PSA and genetic manipulations in the mouse to deplete either NCAM or ST8Sia-IV. Here we report the production and characterization of mice deficient in the ST8Sia-II polysialyltransferase. We observed alterations in brain PSA expression unlike those observed in mice lacking ST8Sia-IV. This included a PSA deficit in regions of neurogenesis but without changes in the frequency of mitotic neural progenitor cells. In further contrast with ST8Sia-IV deficiency, loss of ST8Sia-II did not impair hippocampal synaptic plasticity but instead resulted in the misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus. Consistent with studies of animal models bearing these morphological changes, ST8Sia-II-deficient mice exhibited higher exploratory drive and reduced behavioral responses to Pavlovian fear conditioning. PSA produced by the ST8Sia-II polysialyltransferase modifies memory and behavior processes that are distinct from the neural roles reported for ST8Sia-IV. This genetic partitioning of PSA formation engenders discrete neurological processes and reveals that this post-translational modification forms the predominant basis for the multiple functions attributed to the NCAM glycoprotein.

Secretion of locally synthesized neurohormone from neurites of peptidergic neurons.

Local protein synthesis in neuronal processes is a common phenomenon and may play an important role in synaptic plasticity and hormonal regulation. We have used neuroendocrine bag cells of Aplysia californica as a model system to study local protein synthesis. In our previous work we found that bag cell neurites are capable of synthesizing and processing the prohormone of egg-laying hormone (pro-ELH). In the present study, we found that bag cell neurites are also capable of releasing locally synthesized pro-ELH and ELH-related products via both constitutive and regulated pathways. However, an electrical afterdischarge did not enhance local pro-ELH synthesis, as it does in the bag cell soma. This is the first evidence that isolated neurites are capable of secreting locally synthesized proteins.

Post-afterdischarge depolarization does not stimulate prolonged neurohormone secretion but is required for activity-dependent stimulation of neurohormone biosynthesis from peptidergic neurons.

Membrane depolarization plays a critical role in stimulating secretion of neuropeptides and can also be important in regulating transcriptional and translational events that control peptide biosynthesis. The purpose of this study was to test the hypothesis that persistent membrane depolarization after the end of an electrical afterdischarge plays an important role in stimulating both prolonged secretion of egg-laying hormone (ELH) and ELH synthesis from peptidergic bag cell neurons of Aplysia. Experimental preparations were treated with a low Na(+) solution to rapidly repolarize membrane potential (Vm). Compared with control preparations, the low Na(+) solution caused rapid membrane repolarization to resting levels, significant shortening of the duration of the afterdischarge, and significant decrease in the decay time constant of cytosolic Ca(2+) ([Ca(2+)](i)) concentrations, but no effect on peak [Ca(2+)](i), total [Ca(2+)](i) above baseline, or duration of elevated [Ca(2+)](i). Contrary to both theoretical expectations and findings in other cell types, low Na(+) treatment and the resulting premature repolarization of Vm did not inhibit ELH secretion. On the other hand, low Na(+) treatment that blocked prolonged depolarization, as well as inhibition of Ca(2+) influx, prevented the afterdischarge-induced stimulation of ELH synthesis. These findings provide support for membrane depolarization acting as a trigger mechanism, rather than a sustained driving force, for cellular events that control ELH secretion. It also demonstrates, for the first time, a critical role for postdischarge Vm in regulating an important aspect of neuroendocrine cell function-that of hormone synthesis.

Activity-dependent regulation of neurohormone synthesis and its impact on reproductive behavior in aplysia.

The bag cell neurons (BCNs) of the mollusk Aplysia californica provide a simple model system for investigating cellular and molecular events regulating synthesis and secretion of a reproductive neuropeptide and their impact on physiology and behavior. The BCNs secrete a large amount of egg-laying hormone (ELH) in response to an electrical afterdischarge. The afterdischarge also triggers cellular and molecular events leading to upregulation of ELH biosynthesis to replenish the supply of releasable hormone that was lost because of secretion. In the present review, we discuss signal-transduction events that link membrane excitability to ELH biosynthesis. We present evidence that the afterdischarge stimulates ELH synthesis by upregulating translation of ELH mRNA rather than by activating ELH gene transcription. This increase in ELH synthesis is accompanied by a decrease in total protein synthesis, suggesting that the synthetic machinery is being funneled selectively toward ELH. We also discuss work showing that afterdischarge-induced ELH synthesis uses a novel mechanism of translation initiation, one involving a switch from cap-dependent to cap-independent translation initiation that activates an internal ribosome entry site (IRES) located in the 5'-untranslated region of ELH mRNA. The IRES-regulated translation provides a unique cellular mechanism to selectively upregulate synthesis of a critical reproductive hormone at the expense of nonessential proteins.

Regional differences in processing of locally translated prohormone in peptidergic neurons of Aplysia californica.

Earlier work showed that cell bodies and neurites of the peptidergic bag cell neurons of Aplysia californica contain mRNA for egg-laying hormone. The purpose of the present study was to determine if egg-laying hormone synthesis and prohormone processing is similar in the pleurovisceral connective nerves (containing neurites of bag cell neurons) and the bag cell neuron clusters (containing both cell bodies and neurites of bag cell neurons). Initial experiments confirmed by RT-PCR and sequencing that egg-laying hormone mRNA was present in the pleurovisceral connective nerves. To investigate possible regional differences in translation of mRNA and prohormone processing, clusters were separated from connective nerves and newly synthesized egg-laying hormone-immunoreactive proteins were analyzed. Results showed that synthesis and processing of prohormone occurred in both the clusters and isolated connective nerves; however, the relative abundance of prohormone, processing intermediates, and egg-laying hormone was different. Pulse-chase experiments showed that prohormone was processed more slowly in the connective nerves than in the clusters. These results show that mRNA in isolated neural processes of neuroendocrine cells can be translated, and that the cellular machinery for protein synthesis is present, but processing of the ELH prohormone is significantly compromised.