Search

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate., Competing Interests: Declaration of competing interest All the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Smart wearable devices are still powered by batteries requiring constant recharging, which is a key challenge faced in wearable technologies. Fabrication of flexible thermoelectric materials that can utilize body heat for wearable applications are an attractive alternative to batteries. Developing a thermoelectric material that is flexible, affordable, and has good performance remains a considerable challenge owing to the constrained thermoelectric efficiency of conducting polymers and the inherent rigidity of inorganic materials. Here, we have used a solvothermal technique to incorporate Ag 2-x Sn x S into conductive carbon fabric as a flexible thermoelectric material. To further enhance its thermoelectric performance, various concentrations of Ag 2-x Sn x S are grown on carbon fabric. The monoclinic phase of Ag 2 S on carbon fabric was verified by XRD analysis. After analyzing the thermoelectric characteristics of Ag 1.84 Sn 0.16 S, a maximum power factor of 110 μW/mK 2 was observed for the SSS8 sample. This value is four times higher than the percentage of pristine Ag 2 S-CF. The W-TEG device fabricated using 3 pair modules produced an output voltage ranging from 0.09 to 1.5 mV across a temperature gradient of 3 to 8 K., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The prss59.1 gene was identified as one of 11 genes that were highly upregulated during the induction of ovulation in zebrafish by using an in vivo ovulation assay. Previously, we conducted biochemical characterization of Prss59.1 and revealed it to be a trypsin-like proteolytic enzyme. In this study, we established a prss59.1 gene knockout strain using the CRISPR/Cas9 system. Phenotypic analysis of prss59.1 knockout fish showed that prss59.1 is associated with chorion elevation, a prominent event in egg activation during fertilization. The chorions of heterozygous and homozygous prss59.1 mutant zebrafish were smaller than those of the wild type. The results suggested that Prss59.1 is necessary for chorion expansion. The homozygous prss59.1 mutant strain, with a small chorion, showed an extremely low survival rate. Fiber-supported knob-like structures (KS) on the chorion showed an abnormal structure in prss59.1 mutants. Prss59.1 was detected in the KS on the chorion. The pores on the chorion were smaller in the prss59.1 mutants than in the wild type. Transmission electron microscopy (TEM) observations of the cross sections of the chorions showed abnormalities in the chorion structure in prss59.1 mutants. These results demonstrated that Prss59.1 is involved in chorion elevation and in proper formation of the chorion, which is necessary for embryo development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The thermoelectric (TE) performance of Molybdenum disulpide (MoS 2 ) can be improved by the incorporation of nanomaterials. MoS 2 has been reported as promising thermoelectric materials due to their large bandgap and low thermal conductivity. In the present work, n-type MoS 2 was successfully synthesized by facile hydrothermal route with an excellent thermoelectric performance by introducing rhenium (Re) dopant. The structural and morphological analyses confirmed the incorporation of Re into Mo (Molybdenum) lattice. The thermoelectric results showed that both the electrical conductivity (σ) and Seebeck coefficient (S) has been increased with the increase in Re content (2.5, 5, 7.5 and 10 at%) and temperature (303 K to 700 K), while the thermal conductivity (κ) was low. Doping with Re on MoS 2 enhances the electrical conductivity through band engineering, improving carrier concentration and shifting the Fermi level to the conduction band. Introducing a heavy atomic element can reduce the total thermal conductivity by facilitating mass fluctuation. The maximum Seebeck coefficient was obtained as -100 μVK -1 at 500 K for Re 5 at% sample, which is 3.7 times greater than undoped MoS 2 . In addition, introducing electrons through Re doping induced bipolar conduction. These enhancements have increased the power factor of 8 μWm -1 K -2 at 650 K., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The challenge of developing low-cost, highly flexible, and high-performance thermoelectric (TE) materials persists due to the low thermoelectric efficiency of conducting polymers and the inflexibility of inorganic materials. In this study, we successfully integrated Ag 2 Se and Ag 2 S with highly conductive carbon fabric (CF) to produce a flexible thermoelectric material. A facile one-step solvothermal method was employed to synthesize the Ag 2 Se-CF and Ag 2 S-CF, which were then subjected to X-ray analysis to confine the phase formation of Ag 2 Se and Ag 2 S on the carbon fabric. The analysis revealed that Ag 2 Se and Ag 2 S nanoparticles were tightly packed on the surface of carbon fabric, and compositional analysis confirmed the interaction between the material and carbon fabric. The thermoelectric properties of Ag 2 Se-CF and Ag 2 S-CF were significantly altered due to carrier concentration and mobility variations, resulting in a low power factor of 6.7 μW/mK 2 for Ag 2 Se-CF and a high-power factor of 24 μW/mK 2 at 373 K for Ag 2 S-CF. The growth of Ag 2 Se-CF and Ag 2 S-CF on carbon fabric led to an enhancement in their thermoelectric properties. Further, TE legs were fabricated using the Ag 2 Se-CF (p-type) and Ag 2 S-CF (n-type), and the fabricated legs exhibited an output voltage of ∼20 mV to ∼86.65 mV at a temperature gradient (ΔT) of 3-8 K. This work represents a cutting-edge approach to the fabrication of high-performance, wearable thermoelectric devices., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

We identified the bullfrog Rana catesbeiana sulfotransferase 1 (SULT1) family from the BLAST search tool of the public databases based on the SULT1 families of Nanorana parkeri, Xenopus laevis, and Xenopus tropicalis as queries, revealing the characteristics of the anuran SULT1 family. The results showed that the anuran SULT1 family comprises six subfamilies, four of which were related to the mammalian SULT1 subfamily. Additionally, the bullfrog has two SULT1Cc subfamily members that are consistent with the characteristics of the expanded Xenopus SULT1C subfamily. Several members of the bullfrog SULT1 family were suggested to play important roles in sulfation during metamorphosis. Among these, cDNAs encoding SULT1Cc1 and SULT1Y1 were cloned, and the sulfation activity was analyzed using recombinant proteins. The affinity for 2-naphthol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the enzymatic reaction rate were higher in SULT1Cc1 than in SULT1Y1. Both the enzymes showed inhibitory effect of many thyroid hormones (THs) analogs on the sulfation of 2-naphthol. The potency of sulfation activities of SULT1Cc1 and SULT1Y1 against T 4 indicated their possible role in the intracellular T 4 clearance during metamorphosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bismuth Selenide is a Tellurium free topological insulator in V-VI compounds with an excellent thermoelectric performance from room temperature to mid-temperature region. Herein, hydrothermally prepared polycrystalline Bi 2 Ag x Se 3 nanostructures have been reported for thermoelectric application. The crystal structure identification and morphology with the elemental presence were analyzed by XRD (X-ray diffraction), HR-SEM with EDS (High resolution scanning electron microscope with energy dispersive X-ray), and HR-TEM (High-resolution transmission electron microscope) measurements. The reduced lattice thermal conductivity and enhanced electrical transport properties synergistically boost the thermoelectric properties through the highly-dense stacking faults with the presence of dislocations. The IFFT (Inverse Fast Fourier Transform) pattern reveals the existence of stacking faults and dislocations. These highly dense stacking faults and dislocations act as active phonon scattering centers, which can contribute to effective phonon scattering resultsin extremely low lattice thermal conduction of 0.3 W/mK at 543 K. On the other hand, the involvement of phonon-phonon scattering primarily reduced the lattice thermal conductivity at elevated temperatures. In addition, phonon-carrier scattering was less compared to phonon-phonon scattering at elevated temperature region. Moreover, the enhancement of electrical conductivity and controlled reduction of the Seebeck coefficient plays a vital role in achieving the maximum power factor of 335 μW/mK 2 at 543 K due to the energy filtering effect. The synergistic combination of low thermal conduction and the maximum power factor helps to achieve the high peak zT of 0.3 at 543 K., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The design and construction of state-of-the-art wearable thermoelectric materials are important for the development of self-powered wearable thermoelectric generators (WTEGs). Molybdenum disulfide (MoS 2 ) has been reported as a noteworthy thermoelectric (TE) material because of its large intrinsic bandgap and high carrier mobility. In this work, Cu-doped two-dimensional layered MoS 2 nanosheets were grown on carbon fabric (CF) via a hydrothermal method. The electrical conductivity, Seebeck coefficient, and power factor for the Cu-doped MoS 2 were found to increase with increasing temperature. The maximum Seebeck coefficient was obtained for a MoS 2 sample doped with 4 at% of Cu (CM4) was ∼10 μV/K at 303 K and ∼13 μV/K at 373 K. The enhancement in the Seebeck coefficient was attributed to an energy-filtering effect caused by the interfacial barrier between MoS 2 and Cu. In addition, a thermoelectric device was designed with four pairs of TE materials, where CM4 (4 at%) was used as a p-type material and Cu wire was used as an n-type material. These p- and n-type materials were connected electrically in series and thermally in parallel to generate a voltage of 190.7 μV at a temperature gradient of 8 K., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The thermoelectric (TE) performance of conducting polymers can be improved by the incorporation of carbon nanomaterials. In this work, the impact of carbon black (CB) on polypyrrole (PPy) and polypyrrole/polyaniline (PPy/PANI) binary composite have been investigated. Herein, PPy/PANI binary composite was initially prepared through chemical oxidative polymerization and then solution mixed with CB to form PPy/PANI/CB ternary nanocomposite. The structural and morphological analyses confirmed the formation of composites, and the strong interaction present between polymer matrix and CB. This was further confirmed by theoretical study, which showed strong noncovalent interaction and high complex stability between the materials. The thermoelectric results showed that both the electrical conductivity (σ) and Seebeck coefficient (S) has been increased with the increase in CB content (from 10 wt% to 30 wt%) and temperature (303 K to 373 K), while the thermal conductivity (κ) increase was low. The ternary nanocomposite involving 30 wt% of CB was found to be the most promising material which showed an enhanced power factor (PF) of 0.0251 μW/mK 2 and high figure of merit (ZT) of 4.37x10 -5 at 370 K. The enhancement in ZT for PPy/PANI/CB ternary composite is 2 times, 316 times, 17.3 times, 3.97 times, 11.7 times, and 6.8 times greater than other samples. The enhancement in power factor and ZT was due to energy filtering effect and strong non-covalent interactions between the homopolymers and CB., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Individuals who sleep poorly report spending more time mind wandering during the day. However, past research has relied on self-report measures of sleep or measured mind wandering during laboratory tasks, which prevents generalization to everyday contexts. We used ambulatory assessments to examine the relations between several features of sleep (duration, fragmentation, and disturbances) and mind wandering (task-unrelated, stimulus-independent, and unguided thoughts). Participants wore a wristband device that collected actigraphy and experience-sampling data across 7 days and 8 nights. Contrary to our expectations, task-unrelated and stimulus-independent thoughts were not associated with sleep either within- or between-persons (n = 164). Instead, individual differences in unguided thoughts were associated with sleep disturbances and duration, suggesting that individuals who more often experience unguided train-of-thoughts have greater sleep disturbances and sleep longer. These results highlight the need to consider the context and features of mind wandering when relating it to sleep., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Lingulidae are often considered living fossils, because they have shown little morphological change since the Paleozoic. Limited morphological variation has also made the taxonomic study of living lingulids challenging. We investigated species diversity and phylogenetic relationships of extant lingulids and show that they are substantially more diverse than realized, demonstrating that morphological stasis was commonly accompanied by speciation. Species delimitation based on cytochrome c oxidase subunit I (COI) gene sequences from 194 specimens sampled from East Asia, Australia, Oceania, and the Americas suggested 14-22 species in the lingulids (9-17 species in Lingula and 4-5 species in Glottidia), in contrast to the 11-12 species currently recognized globally in the family. Four-gene phylogenetic analyses supported the sister relationship between Lingula and Glottidia. Within Lingula, L. adamsi, which possesses large, brownish shells, was recovered as sister to all remaining Lingula species, which have more or less greenish shells. Within the greenish Lingula clade, the 'L. anatina' complex was sister to the clade that includes the 'L. reevei' complex. The 'L. anatina' complex was further separated into two major clades with partly separate ranges centered on (i) temperate East Asia, and (ii) the tropical west-central Pacific. Within Glottidia, Pacific species were nested within Atlantic species. Time-calibrated phylogenetic analyses suggested that Lingula likely originated in the early Cretaceous contrary to a previously proposed hypothesis advocating a Cenozoic origin. The separation of Lingula and Glottidia appears to date from the Mesozoic, not from the Carboniferous, contrary to a previous hypothesis. Overall, our results uncovered substantial cryptic diversity in lingulids, which will form the basis for conservation and further taxonomic revision., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Rous sarcoma virus-like particles (RSV-LPs) displaying hemagglutinins of H1N1 (A/New Caledonia/20/99) (H1) and H5N1 (A/Vietnam/1194/2004) (H5) of the influenza A virus were produced. The H1 has its transmembrane domain, but the H5 was fused with the transmembrane domain of glycoprotein 64 (BmGP64) from Bombyx mori nucleopolyhedrovirus (BmNPV). H1 and RSV Gag protein were coexpressed in the hemolymph of silkworm larvae, copurified, and confirmed RSV-LP displaying H1 (VLP/H1). Similarly, the RSV-LP displaying H5 (VLP/H5) production was also achieved. Using fetuin agarose column chromatography, RSV Gag protein-coexpressed H1 and H5 in silkworms were copurified from the hemolymph. By immuno-TEM, H1 and H5 were observed on the surface of an RSV-LP, indicating the formation of bivalent RSV-LP displaying two HAs (VLP/BivHA) in the hemolymph of silkworm larvae. VLP/H1 induced the hemagglutination of red blood cells (RBCs) of chicken and rabbit but not sheep, while VLP/H5 induced the hemagglutination of RBCs of chicken and sheep but not rabbit. Additionally, VLP/BivHA allowed the hemagglutination of RBCs of all three animals. Silkworm larvae can produce RSV-LPs displaying two HAs and is a promising tool to produce the bivalent enveloped VLPs for the vaccine platform., (Copyright © 2022 Elsevier Inc. All rights reserved.)

White spot syndrome virus (WSSV) is one of the most concerning pathogens in penaeid shrimp and can cause severe loss in shrimp aquaculture worldwide. Among the WSSV structural proteins, VP15, a DNA-binding protein located in the WSSV nucleocapsid, is an antiviral protein candidate to protect kuruma shrimp (Marsupenaeus japonicus) from WSSV infection. We identified that the truncated VP15, VP15 (26-57) , is responsible for the protective effect against the WSSV. This study attempts to develop an immunizing agent against WSSV using silkworm pupa as a delivery vector through oral administration. The VP15, VP15 (26-57) , and SR11 peptide derived from VP15 (26-57) were expressed in silkworm pupae. Oral administration of feed mixed with the powdered pupae that expressed VP15-derived constructs enhanced the survivability of kuruma shrimp with an overall relative percent survival (RPS) higher than 70%. There is no death for the group receiving pupa/VP15 (26-57) , and the RPS is 100%. In addition, we also investigated the relative mRNA expression levels of immune-related genes by qPCR at different time points. Our results indicate that the oral administration of pupa/VP15-derived products could provide a high protective effect against WSSV and be a practical approach for controlling WSSV in aquaculture., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Recombinantly expressed VP1 of norovirus self-assembled and formed norovirus-like particles (NoV-LPs). This native VP1 was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) expression system in silkworm larva. NoV-LPs were collected from silkworm fat body lysate by density gradient centrifugation. To improve the purity of the NoV-LP, the proteins were further purified using immobilized metal affinity chromatography based on the surface exposed side chain of histidine residues. The additional purification led to a highly purified virus-like particle (VLP). The morphology and size of the purified VLPs were examined using a transmission electron microscope, and dynamic light scattering revealed a monodispersed spherical morphology with a diameter of 34 nm. The purified product had a purity of >90% with a recovery yield of 48.7% (equivalent to 930 μg) from crude lysate, obtained from seven silkworm larvae. In addition, the purified VLP could be recognized by antibodies against GII norovirus in sandwich enzyme-linked immunosorbent assay, which indicated that the silkworm-derived VLP is biologically functional as a NoV-LP in its native state, is structurally correct, and exerts its biological function. Our results suggest that the silkworm-derived NoV-LP may be useful for subsequent applications, such as in a vaccine platform. Moreover, the silkworm-based expression system is known for its robustness, facile up-scalability, and relatively low expense compared to insect cell systems., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In plasma, thyroid hormone (TH) is bound to several TH distributor proteins (THDPs), constituting a TH delivery/distribution network. Extensive studies of THDPs from tetrapods has proposed an evolutionary scenario concerning structural and functional changes in THDPs, especially for transthyretin (TTR). When assessing, in an evolutionary context, the roles of THDPs as a component constituting part of the vertebrate thyroid system, the data from fish THDPs are critical. In this review the phylogenetic distributions, spatiotemporal expression patterns and binding properties of THDPs in fish are described, and the question of whether the evolutionary hypotheses proposed in tetrapod THDPs can be applied to fish THDPs is assessed. The phylogenetic distributions of THDPs are highly variable among fish groups. Analysis in this review reveals that the evolutionary hypotheses proposed in tetrapod THDPs cannot be applied to fish THDPs, and that the role of plasma lipoproteins as THDPs grows in importance in fish groups. In primitive fish, zinc is an import factor in TH binding to TTR, and high zinc content may facilitate the acquisition of high TH binding activity during the early evolution of TTR. Finally, the possible roles of THDPs in the vertebrate thyroid system are discussed., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The hybrid of organic conducting polymers and inorganic materials with ultralow thermal conductivity, which is a promising strategy for the realization of polymer based effective thermoelectric (TE) applications. In this work, ultrathin layered molybdenum disulphide (MoS 2 ) nanosheets/PANI nanocomposites are prepared by hydrothermal route. The effect of varying PANI wt% in the nanocomposites and its interface effect on thermoelectric properties are well investigated. The successful incorporation of PANI between the MoS 2 layers confirmed by high resolution transmission electron microscope (HRTEM). The significantly enhanced potential difference of MoS 2 / PANI nanocomposites with increasing PANI content is well clarified by the increased Seebeck value. The variable range hopping property is identified and conductivity is raised up highly due to insertion of PANI in layered van der Waal's gap of MoS 2 . The effective interface facilitates charge for fast transport. The reduced thermal conductivity is observed of about 0.248 W*m -1* K -1 for 2.5 wt% addition of PANI. The key factor is that the stability of the sample is improved for MoS 2 / PANI nanocomposites than pristine MoS 2 . Our work paved a new approach to improve TE performance by preparing TE MoS 2 material through simple chemical route., (Copyright © 2020. Published by Elsevier Inc.)

Although the insulin-like peptide hormone INSL3 and its cognate receptor RXFP2 (relaxin-family peptide receptor 2) have existed throughout chordate evolution, their physiological diversification appears to be linked closely with mammalian emergence and radiation. In contrast, they have been lost in birds and reptiles. Both hormone and receptor are expressed from autosomal genes which have maintained their synteny across vertebrate evolution. Whereas the INSL3 gene comprises only two exons closely linked to the JAK3 gene, RXFP2 is normally encoded by 18 exons. Both genes, however, are subject to alternative splicing to yield a variety of possibly inactive or antagonistic molecules. In mammals, the INSL3-RXFP2 dyad has maintained a probably primitive association with gametogenesis, seen also in fish, whereby INSL3 promotes the survival, growth and differentiation of male germ cells in the testis and follicle development in the ovary. In addition, however, the INSL3/RXFP2 system has adopted a typical 'neohormone' profile, essential for the promotion of internal fertilisation and viviparity; fetal INSL3 is essential for the first phase of testicular descent into a scrotum, and also appears to be associated with male phenotype, in particular horn and skeletal growth. Circulating INSL3 is produced exclusively by the mature testicular Leydig cells in male mammals and acts as a potent biomarker for testis development during fetal and pubertal development as well as in ageing. As such it can be used also to monitor seasonally breeding animals as well as to investigate environmental or lifestyle conditions affecting development. Nevertheless, most information about INSL3 and RXFP2 comes from a very limited selection of species; it will be especially useful to gain further information from a more diverse range of animals, especially those whose evolution has led them to express unusual reproductive phenotypes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

White spot syndrome virus (WSSV) is known as one of the most lethal pathogenic viruses in shrimp causing massive damage to shrimp aquaculture industries. To date, no effective treatment or prevention has been found. In this study, five recombinant viral proteins VP15, VP19, VP24, VP26, and VP28 were expressed and purified in E. coli, which were employed as candidates against WSSV in Kuruma shrimp Marsupenaeus japonicus. In vivo antiviral assay in this study newly revealed that VP15 of major nucleocapsid protein, being known as a DNA-binding protein provided the substantial protection against the viral infection when pre-injected into shrimps. Furthermore, we also verified the immunogenic effects of purified VP15 and VP19 proteins produced in a silkworm-bacmid expression system. Taken together, our study identified VP15 as an effective candidate against WSSV infection in the Kuruma shrimp. It is interesting to uncover why and how VP15 is involved in the immune memory in shrimp in the future study., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Biological activities in ectothermic vertebrates depend to a great extent on ambient temperature. Adapting their biological systems to annual or short-term alterations in temperature may play an important role in thermal resistance or overwintering survival. Using SDS-PAGE and western blot, we examined plasma proteins in bullfrog (Rana catesbeiana) tadpoles that were seasonally acclimatized (winter vs. summer) or thermally acclimated (4 °C vs. 21 °C) and identified two season-responsive proteins. The first, transthyretin (TTR), is a plasma thyroid hormone distributor protein that was abundant in summer, and the second is a protein containing C-type lectin-like domain (CTLD) that was abundant in winter and cold acclimation of 4 weeks. Sequence analysis revealed that the C-terminal carbohydrate recognition domain of this CTLD protein (termed collectin X) was highly similar to those of the collectin family members, which participate in complement activation of the innate immune system; however, it lacked most of collagen-like domain. Among the hepatic genes involved in the thyroid system, ttr and dio3 were up-regulated, whereas thra and thrb were down-regulated, in summer acclimatization or warm acclimation. In contrast, the collectin X gene (colectx), as well as colect10 and colect11 in the collectin family involved in the innate immune system, were down-regulated during warm acclimation, although fcn2 in the ficolin family was up-regulated during summer acclimatization and warm acclimation. These findings indicate that seasonal acclimatization and thermal acclimation differentially affect some components of the thyroid and innate immune systems at protein and transcript levels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this review article, information about the development of the hypothalamo-hypophyseal axis, endocrine control of metamorphosis, and hormonal and pheromonal involvements in reproductive behavior in some amphibian species is assembled from the works conducted mainly by our research group. The hypothalamic and pituitary development was studied using Bufo embryos and larvae. The primordium of the epithelial hypophysis originates at the anterior neural ridge and migrates underneath the brain to form a Rathke's pouch-like structure. The hypothalamo-hypophyseal axis develops under the influence of thyroid hormone (TH). For the morphological and functional development of the median eminence, which is a key structure in the transport of regulatory hormones to the pituitary, contact of the adenohypophysis with the undeveloped median eminence is necessary. For the development of proopiomelanocortin-producing cells, contact of the pituitary primordium with the infundibulum is required. The significance of avascularization in terms of the function of the intermediate lobe of the pituitary was evidenced with transgenic Xenopus frogs expressing a vascular endothelial growth factor in melanotropes. Metamorphosis progresses via the interaction of TH, adrenal corticosteroids, and prolactin (PRL). We emphasize that PRL has a dual role: modulation of the speed of metamorphic changes and functional development of organs for adult life. A brief description about a novel type of PRL (1B) that was detected was made. A possible reason why the main hypothalamic factor that stimulates the release of thyrotropin is not thyrotropin-releasing hormone, but corticotropin-releasing factor is considered in light of the fact that amphibians are poikilotherms. As regards the reproductive behavior in amphibians, studies were focused on the courtship behavior of the newt, Cynops pyrrhogaster. Male newts exhibit a unique courtship behavior toward sexually developed conspecific females. Hormonal interactions eliciting this behavior and hormonal control of the courtship pheromone secretion are discussed on the basis of our experimental results., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Prolactin (PRL) is one of the major hormones that control amphibian metamorphosis. Recently, a PRL (PRL1B) gene that is different from the known PRL (PRL1A) gene has been found in the genomes of several amphibian species. In order to ascertain whether the PRL1B gene is expressed in the bullfrog (Rana catesbeiana) pituitary, cloning of cDNA encoding PRL1B in the pituitary of the premetamorphic bullfrog tadpole was attempted. The bullfrog PRL1B amino acid sequence predicted from the obtained cDNA showed 62% identity with those of Xenopus PRL1Bs that have been presumed from the genome sequences, whereas the sequence identity between bullfrog PRL1A and PRL1B was 48%. A molecular phylogenetic tree showed that bullfrog PRL1B is most appropriately grouped with amphibian PRL1Bs. Quantitative PCR analysis revealed that the mRNA expression levels of bullfrog PRL1B in the pituitary were high during pre- and prometamorphosis, sharply declined at metamorphic climax and became undetectable after metamorphosis. In contrast, PRL1A mRNA levels were relatively low during pre- and prometamorphosis, rose at climax and remained high after metamorphosis. Immunohistochemical study using antibodies against partial peptides of PRL1A and PRL1B revealed that most of the PRL1A- and PRL1B-immunoreactive cells in the larval pituitary were distributed separately, but that some of the cells immunoreactive with both antibodies were also present. Western blot analysis with the larval pituitary extract indicated that PRL1B-immunoreactive band appeared at the position of molecular weight ca. 22.1 kDa and PRL1A-immunoreactive band at the position of ca. 22.8 kDa. The results obtained in this experiment suggest the possibility that PRL1B plays as-yet-unknown role(s) during the pre-climactic period of metamorphosis. This is the first report on the existence of PRL1B as a protein in the amphibian larval pituitary., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transpiration plays a critical role in the water cycle of terrestrial ecosystems, especially for arid ecosystems in which water availability is typically the main constraint for plant growth. Although remote sensing has provided insights into transpiration across different temporal and spatial scales, its potential has not been fully exploited. This is due to a lack of synchronous observations of fluxes and reflectance. Only a few models have attempted to incorporate both radiative transfer and physiological processes. In this study, we calibrated the newly developed Soil, Canopy Observation, Photochemistry and Energy fluxes (SCOPE) model to trace synchronous fluxes of water, energy and reflectance, and thus, their interplays, in a typical arid ecosystem dominated by Haloxylon ammodendron based on long-term continuous field measurements. An initial global sensitivity analysis is conducted to identify parameters that have the greatest impact on model output before subsequent calibration with field data. The resulting calibrated model gives insight into the interplay between reflectance, energy and water fluxes in an arid ecosystem. The calibrated model is thus a useful tool to understand land surface fluxes and radiative transfer processes theoretically, from which additional reflectance information can be exploited to trace the physiological status of ecosystems., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Herein, we reported the photo-catalytic degradation/anti-bacterial property of Ag-doped ZnO nanoparticles (SDZO Nps) prepared by a facile gel-combustion technique and its self-cleaning/self-refreshing/self-disinfectant behaviour while on impregnating as pigment into the alkyd resin based coating. The influence of doping Ag (1% & 2%) with ZnO has been evaluated in terms of crystal structure, morphology, optical properties, etc. using X-ray diffraction analysis, Field Emission Scanning Electron Microscope, UV-Vis analysis, and Photoluminescence spectra. The photo-catalytic degradation of crystal violet solution by SDZO Nps is spectroscopically followed employing UV-Vis spectroscopy. From the obtained results, the rate of degradation of 1% SDZO Nps is found higher than that of other samples under sunlight illumination; degrading 1 mg of crystal violet in 30 min. Thus, implementing the synergic effect of nano ZnO and the doped Ag provides a suitable pathway for the development of high efficient photo-catalyst. Further, alkyd resin based self-cleaning coating is formulated using 1% SDZO Nps as pigment along with other additives; the contents are milled to form a homogeneous mixture by high energy ball milling technique. Crystal violet solution coated over dried alkyd coating gets decolorized on exposure to sunlight indicating the mineralization of pollutants and proves the fact that the as obtained coating possess self-cleaning nature. Besides the self-cleaning property, the coating exhibits self-refreshing property which is essential for the long lasting self-cleaning activity. Further, the disinfectant properties of 1% SDZO Nps and 1% SDZO Nps impregnated coating have been evaluated against gram negative Escherichia coli bacterial strain. The acquired experimental outcomes suggest the potential use of self-cleaning coating to keep the environment clean and hygienic economically., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chlorophyll fluorescence can be used to quantify the efficiency of photochemistry and heat dissipation. While several instruments such as Pulse-Amplitude-Modulation (PAM) fluorometers are available for taking direct measurements of parameters related to chlorophyll fluorescence, large-scale instantaneous ecosystem monitoring remains difficult. Several hyperspectral indices have been claimed to be closely related to some chlorophyll fluorescence parameters (e.g. photosystem II quantum yield (Yield), qP, NPQ), which may pave a way for efficient large-scale monitoring of fluorescence parameters. In this study, we have examined 30 published hyperspectral indices for their possible use in tracing chlorophyll fluorescence parameters. The comparison is based on a series of unique datasets with synchronous measurements of reflected hyperspectra and seven fluorescence parameters (i.e., F m , F 0 , F s , F m ', Yield, qP and NPQ) from leaves of Fagus crenata and other six broadleaf species sampled in Mt. Naeba, Japan. Among them, the first dataset is composed of seasonal canopy field measurements of Fagus crenata leaves, while the second is composed of field measurements of other deciduous species including Lindera umbellate, Clethra barbinervis, Viburnum furcatum, Eleutherococcus sciadophylloides, Quercus crispula and Acer japonicum. Furthermore, an additional dataset composed of data resulting from various controlled experiments using inhibitors has been applied for improving physiological interpretations of indices. Results revealed that PRI had higher coefficients of determination and lower root mean square errors than other indices evaluated with a set of chlorophyll fluorescence parameters. However, this pattern was seen only for beech leaves and performed poorly across other species. As a result, no specific indices that are currently available are recommended for tracing fluorescence parameters., (Copyright © 2018. Published by Elsevier Ltd.)

It has been shown that the single-domain intrabody 2H9-L against the hepatitis C virus (HCV) capsid (core) protein inhibits the viral propagation and NF-κB promoter activity induced by the HCV core. In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1-123 amino acid residues (C123), 1-152 amino acid residues (C152), 1-177 amino acid residues (C177) and 1-191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E. coli and then purified. Approximately 175 and 132 μg of the intrabody were purified from 100 ml of E. coli culture and 10 silkworm pupae, respectively, by affinity chromatography. The C123, C152, C177 and C191 HCV core protein variants were purified to approximately 152, 127, 103 and 155 μg, respectively, from 100 ml of E. coli culture. An ELISA in which the intrabodies were immobilized revealed that the intrabodies purified from both hosts were bound to all HCV core protein variants. However, their binding to the C191 appeared to be weak compared to their bindings to the other HCV core protein variants. When C152 was immobilized in the ELISA, the binding of each intrabody to the core protein was also observed. These purified intrabodies can be used in biochemical analyses of the inhibitory mechanism of HCV propagation and as protein interference reagents, thus providing a potential pathway to developing a new type of antiviral drug., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Scanning ion conductance microscopy (SICM), which belongs to the family of scanning probe microscopy, regulates the tip-sample distance by monitoring the ion current through the use of an electrolyte-filled nanopipette as the probing tip. Thus, SICM enables "contact-free" imaging of cell surface topography in liquid conditions. In this paper, we applied hopping mode SICM for obtaining topographical images of convoluted tissue samples such as trachea and kidney in phosphate buffered saline. Some of the SICM images were compared with the images obtained by scanning electron microscopy (SEM) after drying the same samples. We showed that the imaging quality of hopping mode SICM was excellent enough for investigating the three-dimensional surface structure of the soft tissue samples. Thus, SICM is expected to be used for imaging a wide variety of cells and tissues - either fixed or alive- at high resolution under physiologically relevant liquid conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Transthyretin (TTR) is a vertebrate-specific protein involved in thyroid hormone distribution in plasma, and its gene is thought to have emerged by gene duplication from the gene for the ancient TTR-related protein, 5-hydroxyisourate hydrolase, at some early stage of chordate evolution. We investigated the molecular and hormone-binding properties of the brown hagfish Paramyxine atami TTR. The amino acid sequence deduced from the cloned hagfish TTR cDNA shared 33-50% identities with those of other vertebrate TTRs but less than 24% identities with those of vertebrate and deuterostome invertebrate 5-hydroxyisourate hydrolases. Hagfish TTR, as well as lamprey and little skate TTRs, had an N-terminal histidine-rich segment, allowing purification by metal-affinity chromatography. The affinity of hagfish TTR for 3,3',5-triiodo-L-thyronine (T3) was 190 times higher than that for L-thyroxine, with a dissociation constant of 1.5-3.9nM at 4°C. The high-affinity binding sites were strongly sensitive to metal ions. Zn 2+ and Cu 2+ decreased the dissociation constant to one-order of magnitude, whereas a chelator, o-phenanthroline, increased it four times. The number of metal ions (mainly Zn 2+ and Cu 2+ ) was approximately 12/TTR (mol/mol). TTR was also a major T3-binding protein in adult hagfish sera and its serum concentration was approximately 8μM. These results suggest that metal ions and the acquisition of N-terminal histidine-rich segment may cooperatively contribute to the evolution toward an ancient TTR with high T3 binding activity from either 5-hydroxyisourate hydrolase after gene duplication., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Preparation of suprastructure assemblies with unique colloidal and optical properties remains challenging. Non-uniform covering of magnetic nanoparticles (NPs) with an external inert Au shell has been attempted to protect the magnetic core against oxidation as well as to produce multifunctional supraparticles (SPs) possessing respective optical and magnetic properties. In this study, a concave Au NP coating was deposited on magnetic nanoparticles (MNPs) with precise control of the shell thickness and roughness through a layer-by-layer (LbL) assisted ionic reduction method termed ion-reducible LbL (IR-LbL) method. Surface enhanced Raman spectra were obtained using graphene quantum dots (GQDs) on the magnetically aligned structure of the prepared core-shell SPs. It is probable that this synthesis method and the generated SPs are essential for characterizing the merge of electronics and magnetism in the nano-regime and may be applicable for further electronics, magnetic storage, and biomedical applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glycoside hydrolases are divided into two groups, known as inverting and retaining enzymes, based on their hydrolytic mechanisms. Glycoside hydrolase family 63 (GH63) is composed of inverting α-glycosidases, which act mainly on α-glucosides. We previously found that Escherichia coli GH63 enzyme, YgjK, can hydrolyze 2-O-α-d-glucosyl-d-galactose. Two constructed glycosynthase mutants, D324N and E727A, which catalyze the transfer of a β-glucosyl fluoride donor to galactose, lactose, and melibiose. Here, we determined the crystal structures of D324N and E727A soaked with a mixture of glucose and lactose at 1.8- and 2.1-Å resolutions, respectively. Because glucose and lactose molecules are found at the active sites in both structures, it is possible that these structures mimic the enzyme-product complex of YgjK. A glucose molecule found at subsite -1 in both structures adopts an unusual 1 S 3 skew-boat conformation. Comparison between these structures and the previously determined enzyme-substrate complex structure reveals that the glucose pyranose ring might be distorted immediately after nucleophilic attack by a water molecule. These structures represent the first enzyme-product complex for the GH63 family, as well as the structurally-related glycosidases, and it may provide insight into the catalytic mechanism of these enzymes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In a previous study, we showed that corticotropin-releasing factor (CRF) is the major thyroid-stimulating hormone (TSH)-releasing factor in the bullfrog (Rana catesbeiana) hypothalamus. Our findings prompted us to ascertain whether CRF or arginine vasotocin (AVT), a known adrenocorticotropic hormone (ACTH) secretagogue in several vertebrates, is the main stimulator of the release of ACTH from the bullfrog pituitary. Both the frog CRF and AVT stimulated the release of immunoassayable ACTH from dispersed anterior pituitary cells in vitro in a concentration-dependent manner. AVT, however, exhibited far more potent ACTH-releasing activity than CRF. Although CRF by itself weakly stimulated ACTH release, it acted synergistically with AVT to enhance the release of ACTH markedly. Mesotocin and AVT-related peptides such as hydrin 1 and hydrin 2 showed relatively weak ACTH-releasing activity. Subsequently, cDNAs encoding the bullfrog AVT V1a-type and V1b-type receptors were molecularly cloned. Reverse transcriptase-PCR using specific primers revealed that the anterior lobe of the pituitary predominantly expressed AVT V1b-type receptor mRNA but scarcely expressed AVT V1a-type receptor mRNA. Abundant signals for V1b-type receptor mRNA in the corticotropes were also detected by in situ hybridization. The results obtained by the experiments with the bullfrog pituitary indicate that AVT acts as the main ACTH-releasing factor through the AVT V1b-type receptor and that CRF acts synergistically with AVT to enhance the release of ACTH., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In contrast to many anuran amphibians, water is not reabsorbed from the urinary bladder in aquatic Xenopus, thereby helping to prevent excessive water influx. However, little is known about the molecular mechanisms for this process. In the present study, we have identified urinary bladder-type aquaporin, AQP-x2, in Xenopus laevis by cDNA cloning. The predicted amino acid sequence contained six putative transmembrane domains and the two conserved Asn-Pro-Ala motifs, characteristic of AQPs. The sequence also contained a putative N-glycosylation site and phosphorylation motifs for protein kinase A and protein kinase C. The oocyte swelling assay showed that AQP-x2 facilitated water permeability. Reverse transcription-PCR analysis indicated that AQP-x2 mRNA was expressed in the urinary bladder and lung, and faintly in the kidney. Immunomicroscopical study further localized AQP-x2 protein to the cytoplasm of granular cells in the luminal epithelium of the urinary bladder whilst AQP3 was observed along the basolateral side of these cells. In vitro stimulation of the urinary bladder with 10(-8)M vasotocin (AVT), 10(-8)M hydrin 1, or 10(-8)M hydrin 2 had no clear effect on the subcellular distribution of AQP-x2. When the AVT concentration was increased to 10(-6)M, however, AQP-x2 was partially transferred to the apical plasma membrane. The treatment with hydrin 1 or hydrin 2 at the same concentration failed to induce the translocation to the apical membrane. On the other hand, AQP3 remained along the basolateral side even after the treatment with vasotocin or hydrins. The results suggest that the poor responsiveness of AQP-x2 to neurohypophyseal peptides may be a main cause for the little water permeability of the urinary bladder of X. laevis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nucleoid-associated proteins (NAPs) play a role in changing the shape of microbial DNA, making it more compact and affecting the regulation of transcriptional networks in host cells. Genes that encode NAPs include H-NS family proteins (H-NS, Ler, MvaT, BpH3, Bv3F, HvrA, and Lsr2), FIS, HU, IHF, Lrp, and NdpA, and are found in both microbial chromosomes and plasmid DNA. In the present study, NAP genes were distributed among 442 plasmids out of 4602 plasmid sequences, and many H-NS family proteins, and HU, IHF, Lrp, and NdpA were found in plasmids of Alpha-, Beta-, and Gammaproteobacteria, while HvrA, Lsr2, HU, and Lrp were found in other classes including Actinobacteria and Bacilli. Larger plasmids frequently carried multiple NAP genes. In addition, NAP genes were more frequently found in conjugative plasmids than non-transmissible plasmids. Several host cells carried the same types of H-NS family proteins on both their plasmids and chromosome(s), while this was not observed for other NAPs. Recent studies have shown that NAP genes on plasmids and chromosomes play important roles in the physical and regulatory integration of plasmids into the host cell., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Transthyretin (TTR) diverged from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some early stage of chordate evolution. To clarify how TTR had participated in the thyroid system as an extracellular thyroid hormone (TH) binding protein, TH binding properties of recombinant little skate Leucoraja erinacea TTR was investigated. At the amino acid level, skate TTR showed 37-46% identities with the other vertebrate TTRs. Because the skate TTR had a unique histidine-rich segment in the N-terminal region, it could be purified by Ni-affinity chromatography. The skate TTR was a 46-kDa homotetramer of 14.5kDa subunits, and had one order of magnitude higher affinity for 3,3',5-triiodo-l-thyronine (T3) and some halogenated phenols than for l-thyroxine. However, the skate TTR had no HIUHase activity. Ethylenediaminetetraacetic acid (EDTA) treatment inhibited [(125)I]T3 binding activity whereas the addition of Zn(2+) to the EDTA-treated TTR recovered [(125)I]T3 binding activity in a Zn(2+) concentration-dependent manner. Scatchard analysis revealed the presence of two classes of binding site for T3, with dissociation constants of 0.24 and 17nM. However, the high-affinity sites were completely abolished with 1mM EDTA, whereas the remaining low-affinity sites decreased binding capacity. The number of zinc per TTR was quantified to be 4.5-6.3. Our results suggest that skate TTR has tight Zn(2+)-binding sites, which are essential for T3 binding to at least the high-affinity sites. Zn(2+) binding to the N-terminal histidine-rich segment may play an important role in acquisition or reinforcement of TH binding ability during early evolution of TTR., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Nkx2 homeodomain transcription factors are involved in various developmental processes and cell specification: e.g. in mammals, NKX2-1 is essential for thyroid-specific gene expression and thyroid morphogenesis. Among Nkx2 proteins, information is still very limited for Nkx2-4. In the present study, we have identified three distinct cDNAs encoding Nkx2-4 isoforms (Nkx2-4a, -b, and -c) from the rainbow trout thyroid tissue, and characterized their transcriptional properties. The trout Nkx2-4 proteins were all predicted to conserve three characteristic domains: the tinman-like amino terminal decapeptide, the NK2 homeodomain, and the NK2-specific domain, and also share 75-89% amino acid similarity. It was shown by dual luciferase assay that Nkx2-4a and Nkx2-4b, but not Nkx2-4c, significantly activated transcription from a cotransfected rat thyroglobulin (TG) promoter. An electrophoretic mobility shift assay indicated that all the Nkx2-4 isoforms could bind to the TG promoter, implying that the faint transcriptional activity of Nkx2-4c might result from some critical amino acid substitution(s) outside the homeodomain. RT-PCR analysis revealed similar tissue distribution patterns for Nkx2-4a and Nkx2-4b mRNAs. Both mRNAs were expressed abundantly in the thyroid, and weakly in the testis. On the other hand, Nkx2-4c mRNA was detected in the ovary as well as in the thyroid. The expression sites of Nkx2-4c mRNA were localized, by in situ hybridization histochemistry, to the ovarian granulosa cells and to the thyroid follicular cells. The results suggest that in the rainbow trout, Nkx2-4a and Nkx2-4b might play a major role in TG gene transcription whereas Nkx2-4c might have some functions in the ovary as well as the thyroid., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) initiate infection by sialic acid binding. Here, we investigated sialic acid linkage specificities for binding and infection of hPIV1 and hPIV3 by using sialic acid linkage-modified cells treated with sialidases or sialyltransferases. The hPIV1 is bound to only α2,3-linked sialic acid residues, whereas hPIV3 is bound to α2,6-linked sialic acid residues in addition to α2,3-linked sialic acid residues in human red blood cells. α2,3 linkage-specific sialidase treatment of LLC-MK2 cells and A549 cells decreased the infectivity of hPIV1 but not that of hPIV3. Treatment of A549 cells with α2,3 linkage-specific sialyltransferase increased infectivities of both hPIV1 and hPIV3, whereas α2,6 linkage-specific sialyltransferase treatment increased only hPIV3 infectivity. Clinical isolates also showed similar sialic acid linkage specificities. We concluded that hPIV1 utilizes only α2,3 sialic acid linkages and that hPIV3 makes use of α2,6 sialic acid linkages in addition to α2,3 sialic acid linkages as viral receptors., (Copyright © 2014. Published by Elsevier Inc.)

We have identified two distinct Pax8 (a and b) mRNAs from the thyroid gland of the rainbow trout (Oncorhynchus mykiss), which seemed to be generated by alternative splicing. Both Pax8a and Pax8b proteins were predicted to possess the paired domain, octapeptide, and partial homeodomain, while Pax8b lacked the carboxy-terminal portion due to an insertion in the coding region of the mRNA. RT-PCR analysis showed each of Pax8a and Pax8b mRNAs to be abundantly expressed in the thyroid and kidney. In situ hybridization histochemistry further detected the expression of Pax8 mRNA in the epithelial cells of the thyroid follicles of the adult trout and in the thyroid primordial cells of the embryo. The functional properties of Pax8a and Pax8b were investigated by dual luciferase assay. The transcriptional regulation by the rat thyroid peroxidase (TPO) promoter was found to be increased by Pax8a, but not by Pax8b. Pax8a further showed synergistic transcriptional activity with rat Nkx2-1 for the human TPO upstream region including the enhancer and promoter. On the other hand, Pax8b decreased the synergistic activity of Pax8a and Nkx2-1. Electrophoretic mobility shift assay additionally indicated that not only Pax8a but also Pax8b can bind to the TPO promoter and enhancer, implying that the inhibitory effect of Pax8b might result from the lack of the functional carboxy-terminal portion. Collectively, the results suggest that for the trout thyroid gland, Pax8a may directly increase TPO gene expression in cooperation with Nkx2-1 while Pax8b may work as a non-activating competitor for the TPO transcription., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24h at 41°C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12-18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37-50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37°C after the 24h incubation at 41°C under 5% CO2 in air. It survived up to 2days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX-100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24h incubation at 41°C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The pars distalis (PD) and the pars intermedia (PI) have the same embryonic origin, but their morphological and functional characteristics diverge during development. The PD is highly vascularized, whereas the highly innervated PI is essentially non-vascularized. Based on our previous finding that vascular endothelial growth factor-A (VEGF-A) is involved in vascularization of the rat PD, attempt was made to generate transgenic Xenopus expressing VEGF-A specifically in the melanotrope cells of the PI as a model system for studying the significance of vascularization or avascularization for the functional differentiation of the pituitary. The PI of the transgenic frogs, examined after metamorphosis, were distinctly vascularized but poorly innervated. The experimentally induced vascularization in the PI resulted in a marked increase in tissue volume and a decrease in the expression of both alpha-melanophore-stimulating hormone (α-MSH) and prohormone convertase 2, a cleavage enzyme essential for generating α-MSH. The transgenic animals had low plasma α-MSH concentrations and displayed incomplete adaptation to a black background. To our knowledge, this is the first report indicating that experimentally induced angiogenesis in the PI may bring about functional as well as structural alterations in this tissue., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Oocyte maturation (OM) in goldfish is induced by the maturation inducing hormone (MIH) via its membrane receptor. Previously, we described the cloning of the membrane progesterone receptor alpha (mPRα or paqr7b) cDNA from a goldfish ovarian cDNA library and obtained experimental evidence that the mPRα protein is an intermediary in MIH induction of OM in goldfish. Three mPR subtypes have been identified in fish by cDNA cloning or by in silico analysis of genome sequence databases. In order to investigate the potential roles of the mPR subtypes in oocyte maturation, we cloned additional mPRs from a goldfish ovarian cDNA library. RACE amplification, and screening of the cDNA library identified one β (paqr8) and two γ subtypes (paqr5) (hereafter referred to as γ-1 and γ-2), respectively. Tissue distribution of mPR subtypes showed differential expression pattern. However, in addition to mPRα, the β, γ-1 and γ-2 subtypes were also expressed in follicle-enclosed oocytes. Cell lines expressing the β, γ-1 and γ-2 genes were established and their steroid binding properties compared. The β subtype exhibited higher binding affinity than the γ subtypes for 17,20β-DHP, the MIH in goldfish. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRβ blocked the induction of oocyte maturational competence, whereas injection of antisense oliogonucleotides to mPRγ-1 and γ-2 were ineffective. These results suggest that the goldfish mPRβ protein acts as an intermediary during MIH induction of OM in goldfish, in a manner similar to that described previously for mPRα., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The whitefly, Aleurocanthus camelliae Kanmiya and Kasai (Hemiptera: Aleyrodidae), is an invasive species in Japan that was first discovered in 2004 on tea in Kyoto. Soon after its arrival epizootics of an entomopathogenic fungus were observed in populations of the whitefly in many tea-growing regions. Here we identify this fungus as Paecilomyces cinnamomeus (Petch) Samson and W. Gams (Hypocreales: Clavicipitaceae) based on morphological characteristics and molecular analyses. This is the first record of P. cinnamomeus in Japan and also the first time it has been recorded from the genus Aleurocanthus. A isolate of P. cinnamomeus caused greater than 50% and 90% infection in whitefly nymphs at 1×10(6) and 1×10(7)conidia/ml respectively, while the commercial mycoinsecticides Preferd® (Isaria fumosorosea) and Mycotal® (Lecanicillium muscarium) caused <10% infection at their recommended field rates (5×10(6) and 9×10(6)conidia/ml, respectively), suggesting that P. cinnamomeus may be more useful as a control agent than the currently available mycoinsecticides. Optimum and upper limit temperatures for in vitro growth of P. cinnamomeus isolates were 22.5-25°C and 32.5°C, respectively. At field rates, the fungicide thiophanate-methyl caused some inhibition of in vitro growth of P. cinnamomeus isolates, and the bactericide copper oxychloride and the insecticides tolfenpyrad and methidathion were strongly inhibitory. The findings obtained in this study will be useful in the development of microbial control programs using P. cinnamomeus against A. camelliae., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Detailed exploratory and mechanistic investigations on spontaneous formation of dye-functionalized gold nanoparticles (GNPs) using dye-based reverse micellar systems are described in this publication. The accumulated results from spectroscopic and microscopic investigations demonstrated that water molecules confined within nanoscopic enclosure of the self-assembled reverse micelles played critical role in the redox processes of aurate ions to produce GNPs, which are assumed to have approximately constant size distributions. The resulting dye-functionalized GNPs were found to offer their absorption and fluorescence emission tunability by changing the medium polarity as well as to exhibit excellent film-forming properties to give optically homogeneous polystyrene thin films. These key findings in addition to broad applicability of the self-assembling process with a variety of dye analogues have led to a conclusion that the protocol presented here serves as a versatile synthetic method to provide a potential convenience for future development of new organic-inorganic hybrid nanomaterials., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have investigated the sulfation of thyroid hormones (THs) in the cytosol from Rana catesbeiana tadpole tissues. Sulfation of 3,3',5-triiodothyronine (T(3)) by the liver cytosol, which was dependent on protein amount, incubation time, and temperature, suggested the presence of TH sulfotransferases (SULTs) in the liver. The apparent Michaelis-Menten constant (K(m)) of the liver cytosol was 0.22 microM for T(3), and the apparent maximum velocity (V(max)) of the liver cytosol was 7.65 pmol/min/mg protein for T(3). Iodothyronine sulfating activity in the liver cytosol was increased in tadpoles at premetamorphic (stages IX-X) and metamorphic climax (stage XX) stages, and in adult frogs. The substrate preference of iodothyronine sulfation for the liver cytosol from tadpoles (stage X) was: 3,3',5'-triiodothyronine>T(3)>3,3',5,5'-tetraiodothyroacetic acid>3,3',5-triiodothyroacetic acid, T(4), 3-iodothyronine>3,5-diiodothyronine. Several halogenated phenols were potent inhibitors (IC(50)=0.15-0.21 microM). The substrate preference for T(3) was gradually lost by the onset of metamorphic climax stages. These enzymatic characteristics of iodothyronine sulfation in the liver cytosol from tadpoles resembled those of mammalian phenol SULTs, except that the tadpole cytosol had a higher affinity (one or two orders of magnitude) for T(3) than mammalian SULTs. These results suggested that an enzyme homologous to mammalian phenol SULT (SULT1) may be involved in TH metabolism in tadpoles., ((c) 2009 Elsevier Inc. All rights reserved.)

The aim of this study is the improvement of the photostability of several natural anionic dyes, carmine (CM), carthamus yellow (CY), and annatto dye (ANA), by complexation with hydrotalcite. The composite of the dyes and hydrotalcite is prepared by the coprecipitation method. CM is successfully intercalated in the hydrotalcite layer when the amount of introduced CM is large. The photostability of CM in CM/HT composites is superior to the CM adsorbed on silica surface. The effect of the stability enhancement is larger when the amount of introduced CM exceeds 0.23 g/g-host, or when the layer charge density of the hydrotalcite is larger. CY is also stabilized by complexation with hydrotalcite, whereas ANA is not stabilized by complexation with hydrotalcite. The photostability of an anionic natural dye can be improved by intercalation into the hydrotalcite layer, if the dye has a hydrophilic nature and a rather planar structure. The intercalated dye is stabilized by the protection from the attack of the atmospheric oxygen. In addition, contribution of the electrostatic interaction between the positively charged hydrotalcite layer and the intercalated anionic dye is also proposed.

We propose a new diversity threshold theory which states that the specific CTLs to the viral strain become inactivated (that is, some HIV strain can escape from its specific immune response) when the diversity of HIV strains exceeds some threshold number. We call this number "immune diversity threshold". Our theory can explain the inactivation of specific immune response and a limit of maximum immune diversity. We can conclude that the accumulation of viral diversity eventually leads to AIDS.

The renin-angiotensin (RA) system is important for the regulation of blood pressure and electrolyte balance, and renin is the rate-limiting enzyme in this system. The recent discovery of (pro)renin receptor (PRR) has reinforced the functional role of the RA system. PRR non-proteolytically activates prorenin and its role has attracted the attention of researchers towards the RA system. However, there is insufficient information on the biochemical structure and biological functioning of PRR due to the difficulty of measuring PRR expression. In this work, human PRR (hPRR) with intact transmembrane and C-terminal domain (hPRR-wTM) and PRR without this domain (hPRR-w/oTM) were expressed in insect cells using baculovirus expression system (BES). Both hPRR-wTM and hPRR-w/oTM were fused with FLAG peptide by its N-terminus. Most of the hPRR-wTM was expressed in cell fraction and hPRR-w/oTM was secreted into the culture medium. hPRR-wTM was solubilized from the membrane fraction of recombinant baculovirus-infected cells by various detergents, suggesting that hPRR-wTM might be a transmembrane protein. hPRR-wTM was purified from the solubilized fraction using anti-FLAG M2 antibody agarose. Binding of purified hPRR-wTM to renin immobilized onto sensor chips was directly proportional to the hPRR-wTM concentration. Approximately 225 microg of functional hPRR-wTM was purified from 80 ml of baculovirus-infected cell culture. Scale-up of this system will lead to mass production and crystallization of hPRR-wTM and determination of its biochemical structure and biological function.

Thyroid system-disrupting activity in effluents from municipal domestic sewage treatment plants was detected using three in vitro assays and one in vivo assay. Contaminants in the effluents were extracted by solid-phase extraction (SPE) and eluted stepwise with different organic solvents. The majority of the thyroid system-disrupting activity was detected in the dichloromethane/methanol (1/1) fraction after SPE in all three in vitro assays: competitive assays of 3,3',5-[(125)I]triiodo-L-thyronine ([(125)I]T(3)) binding to the plasma protein transthyretin (TTR assay) and thyroid hormone receptor (TR assay) and T(3)-dependent luciferase assay (Luc assay). Subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) of the dichloromethane/methanol (1/1) fraction separated contaminants potent in the TR and Luc assays from those potent in the TTR assay. The contaminants potent in the TR and Luc assays were also potent in an in vivo short-term gene expression assay in Xenopus laevis (Tadpole assay). The present study demonstrated that the effluents from domestic sewage treatment plants contain contaminants with T(3)-like activity of approximately 10(-10) M T(3)-equivalent concentration (T(3)EQ) and that the TR and Luc assays are powerful in vitro bioassays for detecting thyroid system-disrupting activity in effluents. The availability and applicability of these bioassays for screening contaminants with thyroid system-disrupting activity in the water environment are discussed.

Nkx2.1 (TTF-1), a homeodomain-containing transcription factor essential for specific gene expression in thyroid follicular cells, exists also in the thyroidal C cells that secrete calcitonin (CT). In this report, we examined the effect of Nkx2.1 on the CT gene transcription. Luciferase reporter assay using the 2kbp promoter sequence of rat CT/CGRP gene revealed that Nkx2.1 induced a significant increase in the promoter transcription. Furthermore, we detected Pax1 and/or Pax9 gene expression in mammalian medullary thyroid carcinoma cell lines, rat rMTC and human TT cells, and in mammalian thyroid glands by RT-PCR. The Pax9 mRNA, expressed in the TT cells and rat thyroid, was then isolated by cDNA cloning. Sequence analysis showed that both rat and human Pax9 proteins contained characteristic domains: i.e. the paired domain and octapeptide motif. Alternative transcripts encoding Pax9 isoforms were not identified in the rat thyroid or TT cells. Dual luciferase assay indicated that Pax9 did not increase transcription from the CT/CGRP promoter. Pax9 also showed no cooperative effects when it was co-transfected with Nkx2.1. The results suggest that CT gene expression could be directly activated by Nkx2.1, whereas Pax9 is not involved in transcription from the 2kbp CT promoter.

Previously, a cDNA clone encoding a protein that satisfies the criteria for its designation as a membrane progestin receptor, mPRalpha, was discovered in spotted seatrout ovaries. Moreover, preliminary evidence was obtained for a role for mPRalpha in maturation-inducing hormone (MIH) induction of oocyte maturation in this species. Here, we describe the cloning of the mPRalpha cDNA from a goldfish ovarian cDNA library. Northern blot analysis indicates the presence of a major 2.6kb transcript in ovaries that encodes a 354 amino acid protein which shows high sequence identity with seatrout (81%), zebrafish (93%), and human (55%) mPRalphas. Western blot analysis using a polyclonal goldfish mPRalpha antibody shows a major immunoreactive band of the predicted molecular weight (40kDa) in goldfish ovarian membranes. Computer modeling predicts that the deduced protein has seven transmembrane domains, typical of G protein-coupled receptors. Treatment of full grown, late vitellogenic stage follicle-enclosed oocytes in vitro with gonadotropin increased mPRalpha protein levels. A correlation between mPRalpha protein levels and the ability of oocytes to undergo GVBD in response to the MIH (maturational competence) was observed after treatment with gonadotropin. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRalpha blocked both the induction of oocyte maturational competence and mPRalpha protein upregulation by gonadotropin. These results with the goldfish mPRalpha protein are similar to those obtained previously with spotted seatrout, further supporting the hypothesis that the mPRalpha acts as an intermediary in MIH induction of oocyte maturation in teleosts.

We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3'-l-triiodothyronine (T(3)) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T(3)-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T(3) that significantly induced the luciferase gene expression was 10(-11)M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10(-6)M, had a weak T(3)-agonist activity, whereas trichlorobisphenol A, at 10(-8) - 10(-6)M had a weak T(3)-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T(3)-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals.

Seasonal changes in LHbeta and FSHbeta mRNA levels were examined in the pituitary gland of the adult male newt, Cynops pyrrhogaster, using in situ hybridization histochemistry and a quantitative real-time RT-PCR method. The annual fluctuation of LHbeta mRNA and FSHbeta mRNA levels in the pituitary gland displayed a close relationship with seasonal changes in testicular function. The values obtained by both methods showed similar fluctuation. The levels of LHbeta mRNA were always exceeded those of FSHbeta. The present immunoelectron microscopic observations support the data on the gene expression levels of the beta-subunits of LH and FSH. Gonadectomy in the summer increased the LHbeta and FSHbeta mRNA levels. Testosterone replacement inhibited the expression of LHbeta mRNA, but not of FSHbeta mRNA, suggesting that the expression of FSHbeta is regulated by some non-steroid factor, probably inhibin. In the case of gonadectomy during any other season, the LHbeta mRNA level increased, but not to the same extent as in summer, and androgen concentrations decreased to the minimum of the year. This finding provides new information about the regulation of annual changes in LHbeta and FSHbeta expression in the pituitary gonadotrophs.