Prenatal test cohort of a modified rat comparative thyroid assay adding brain thyroid hormone measurements and histology but lowering group size appears able to detect disruption by sodium phenobarbital.

The Comparative Thyroid Assay (CTA, USEPA) is a screening test for thyroid hormone (TH) disruption in peripheral blood of dams and offspring. Recently, we began investigating feasible improvements to the CTA by adding examination of offspring brain TH concentrations and brain histopathology. In addition, we hypothesize that the number of animals required could be reduced by 50 % while still maintaining sensitivity to characterize treatment related changes in THs. Previously, we showed that the prenatal test cohort of the modified CTA could detect 1000 ppm sodium phenobarbital (NaPB)-induced suppression of brain T3 (by 9 %) and T4 (by 33 %) with no significant changes in serum T3 and T4 (less than 8 %). In the current study we expanded the dose response in a prenatal test cohort. Pregnant SD rats (N = 10/group) were exposed to 0, 1000 or 1500 ppm NaPB in the diet from gestational days (GD) 6 to GD20. Serum THs concentrations in GD20 dams together with serum/brain THs concentrations and brain histopathology in the GD20 fetuses were examined. NaPB dose-dependently suppressed serum T3 (up to -26 %) and T4 (up to -44 %) in dams, with suppression of T3 in serum (up to -26 %) and brain (up to -18 %) and T4 in serum (up to -26 %) and brain (up to -29 %) of fetuses but without clear dose dependency. There were no remarkable findings that deviated significantly from controls in GD20 fetal brain by qualitative histopathology. Overall, the present study suggests that the prenatal test cohort of this modified CTA is able to detect the expected fetal TH disruptions by prenatal exposure to NaPB, while also reducing the number of animals used by 50 %, consistent with the results of our previous study. These findings add to the suggestion that lowering group sizes and adding endpoints may be a useful alternative to the original CTA design.

Comparison of the sensitivity of histopathological and immunohistochemical analyses and blood hormone levels for early detection of antithyroid effects in rats treated with thyroid peroxidase inhibitors.

Although measurements of blood triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) levels in rodent toxicity studies are useful for detection of antithyroid substances, assays for these measurements are expensive and can show high variability depending on blood sampling conditions. To develop more efficient methods for detecting thyroid disruptors, we compared histopathological and immunohistochemical findings in the thyroid and pituitary glands with blood hormone levels. Six-week-old male and female Sprague-Dawley rats (five rats per group) were treated with multiple doses of the thyroid peroxidase inhibitors propylthiouracil (PTU) and methimazole by gavage for 28 days. Significant decreases in serum T3 and T4 and increases in TSH were observed in the ≥1 mg/kg PTU and ≥3 mg/kg methimazole groups. An increase in TSH was also detected in male rats in the 0.3 mg/kg PTU group. Histopathological and immunohistochemical analyses revealed that follicular cell hypertrophy and decreased T4 and T3 expressions in the thyroid gland were induced at doses lower than doses at which significant changes in serum hormone levels were observed, suggesting that these findings may be more sensitive than blood hormone levels. Significant increases in thyroid weights, Ki67-positive thyroid follicular cell counts, and TSH-positive areas in the pituitary gland were detected at doses comparable with those at which changes in serum T4 and TSH levels were observed, indicating that these parameters may also be useful for evaluation of antithyroid effects. Combining these parameters may be effective for detecting antithyroid substances without relying on hormone measurements.

Physico-chemical and Textural Properties of 3D Printed Plant-based and Hybrid Soft Meat Analogs.

This study investigated the physico-chemical and textural properties of 3D-printed pea protein-only and pea protein-chicken-based hybrid meat analogs. Both pea protein isolate (PPI)-only and hybrid cooked meat analogs had a similar moisture content of approximately 70%, which was similar to that of chicken mince. However, the protein content increased significantly with the amount of chicken in the hybrid paste undergoing 3D printing and cooking. Significant differences were observed in the hardness values of the non-printed cooked pastes and the 3D printed cooked counterparts, suggesting that the 3D printing process reduces the hardness of the samples and is a suitable method to produce a soft meal, and has significant potential in elderly health care. Scanning electron microscopy (SEM) revealed that adding chicken to the plant protein matrix led to better fiber formation. PPI itself was not able to form any fibers merely by 3D printing and cooking in boiling water. Protein-protein interactions were also studied through the protein solubility test, which indicated that hydrogen bonding was the major bonding that contributed to the structure formation in cooked printed meat analogs. In addition, disulfide bonding was correlated with improved fibrous structures, as observed through SEM.

Feasibility study for a downsized comparative thyroid assay with measurement of brain thyroid hormones and histopathology in rats: Case study with 6-propylthiouracil and sodium phenobarbital at high dose.

Concern has been raised that thyroid hormone disruptors (THDs) may potentially interfere with the developing brain, but effects of mild suppression of maternal THs by environmental contaminants on neonatal brain development are not fully understood. The comparative thyroid assay (CTA) is a screening test for offspring THDs, but it requires several animals and is criticized that reliance on serum THs alone as predictive markers of brain malfunction is inadequate. To verify feasibility of the downsized CTA but additional examination of brain THs levels and histopathology, we commenced internal-validation studies. This paper presents the data of the study where 6-propylthiouracil (6-PTU, 10 ppm) and sodium phenobarbital (NaPB, 1000 ppm) were dosed by feeding from gestational days (GD)6-20, and from GD6 to lactation day 21. The modified CTA detected 6-PTU-induced severe (>70%) suppression of serum THs in dams, with >50% suppressed serum/brain TH levels in offspring and brain heterotopia in postnatal day 21 pups. The modified CTA also detected NaPB-induced mild (<35%) suppression of serum THs in dams, with mild (<35%) reduction of serum/brain TH levels in fetuses but not in pups. These findings suggest that the modified CTA may have a potential as a screening test for offspring THDs.

A new perspective on the evolution of "Kawara" roof tiles in Ryukyu: A multidisciplinary non-destructive analysis of roof tile transition at Shuri Castle, Ryukyu Islands, Japan.

A unique historical architecture was created at Shuri Castle (Shuri-jo) in the Ryukyu Islands by its "Kawara" roof tiles. After the 13th and 14th centuries, Kawara tiles were introduced to the Ryukyu Islands from several regions, including China, Korea, and mainland Japan, and evolved shapes and patterns that are unique to this island region. However, the transition of some internal features, such as the chemical components and microstructure, had not been analyzed. This study used a multi-faceted approach for such internal data and non-destructive quantitative methods to propose a new perspective on the evolution of historical Ryukyuan Kawara. We analyzed two styles of Ryukyuan Kawara from the 13th to 15th centuries and found that the material processing and firing conditions of the two styles were very similar, even though it had been suggested that they had different origins. A quantitative analysis of tiles from the 16th to 19th centuries revealed a transition in color tone to red, leading to the modern traditional Ryukyuan tiles; traces of changes in firing conditions were also found along with this transition. Finally, the study revealed that the evolution of Ryukyuan Kawara consisted of changed factors, e.g. surface color, and unchanged factors, e.g. paste density. Previous archaeological studies mainly focused on changing external characteristics, such as form and pattern; however, our analysis showed that the internal features changed, while the elemental composition and paste density remained constant from the appearance of the roof tiles until the 19th century. We propose that this is related to different responses of individual factors to external stressors, such as the social context, which may be common to other archaeological artifacts as well. Our study provides a new perspective on the evolution of Ryukyuan Kawara and presents a different discussion of and methods for the chronological study of material culture.

Population admixtures in medaka inferred by multiple arbitrary amplicon sequencing.

Cost-effective genotyping can be achieved by sequencing PCR amplicons. Short 3-10 base primers can arbitrarily amplify thousands of loci using only a few primers. To improve the sequencing efficiency of the multiple arbitrary amplicon sequencing (MAAS) approach, we designed new primers and examined their efficiency in sequencing and genotyping. To demonstrate the effectiveness of our method, we applied it to examining the population structure of the small freshwater fish, medaka (Oryzias latipes). We obtained 2987 informative SNVs with no missing genotype calls for 67 individuals from 15 wild populations and three artificial strains. The estimated phylogenic and population genetic structures of the wild populations were consistent with previous studies, corroborating the accuracy of our genotyping method. We also attempted to reconstruct the genetic backgrounds of a commercial orange mutant strain, Himedaka, which has caused a genetic disturbance in wild populations. Our admixture analysis focusing on Himedaka showed that at least two wild populations had genetically been contributed to the nuclear genome of this mutant strain. Our genotyping methods and results will be useful in quantitative assessments of genetic disturbance by this commercially available strain.

Histopathological and immunohistochemical evaluation for detecting changes in blood hormone levels caused by endocrine disruptors in a 28-day repeated-dose study in rats.

Although measurements of blood hormone levels in rodent toxicological studies can provide important information on the mechanisms of toxicity and extrapolation to humans, there are several difficulties such as large individual differences and limited sample volume. To develop a more simplified method that does not depend solely on blood samples, we examined the possible application of immunohistochemistry for detecting endocrine disruptors in short-term studies. Aminotriazole (AMT), propylthiouracil (PTU), phenobarbital, aminoglutethimide (AGT), estradiol, and vitamin D3 were administered orally to 6-week-old male and female SD rats (five/group) for 28 days. Measurements of serum hormone levels revealed decreases in triiodothyronine (T3) and thyroxine (T4) in the AMT and PTU groups, an increase in thyroid stimulating hormone (TSH) in the AMT, PTU, and AGT groups, and an increase in adrenocorticotrophic hormone in the AGT group. Increased thyroid, pituitary, and adrenal gland weights; histopathological lesions, including follicular hypertrophy/hyperplasia, hypertrophy/vacuolation of anterior pituitary cells, and increased adrenocortical vacuolation were observed in association with the hormone level changes. Immunohistochemical analysis revealed a decreased T4 level in the thyroid gland of the AMT and PTU groups and an increased area of TSH positive immunostaining in the pituitary gland of the AMT, PTU, and AGT groups, consistent with the changes in serum T4 and TSH levels, respectively. These results suggest that histopathological analysis and immunohistochemistry for T4 and TSH might be useful and sensitive methods of detecting thyroid dysfunction, and that combining organ weight measurements is a reliable parameter of detecting endocrine disruptors.

3D Printing of Textured Soft Hybrid Meat Analogues.

Meat analogue is a food product mainly made of plant proteins. It is considered to be a sustainable food and has gained a lot of interest in recent years. Hybrid meat is a next generation meat analogue prepared by the co-processing of both plant and animal protein ingredients at different ratios and is considered to be nutritionally superior to the currently available plant-only meat analogues. Three-dimensional (3D) printing technology is becoming increasingly popular in food processing. Three-dimensional food printing involves the modification of food structures, which leads to the creation of soft food. Currently, there is no available research on 3D printing of meat analogues. This study was carried out to create plant and animal protein-based formulations for 3D printing of hybrid meat analogues with soft textures. Pea protein isolate (PPI) and chicken mince were selected as the main plant protein and meat sources, respectively, for 3D printing tests. Then, rheology and forward extrusion tests were carried out on these selected samples to obtain a basic understanding of their potential printability. Afterwards, extrusion-based 3D printing was conducted to print a 3D chicken nugget shape. The addition of 20% chicken mince paste to PPI based paste achieved better printability and fibre structure.

Oxidative Stress as a Common Key Event in Developmental Neurotoxicity.

The developing brain is extremely sensitive to many chemicals. Perinatal exposure to neurotoxicants has been implicated in several neurodevelopmental disorders, including autism spectrum disorder, attention-deficit hyperactive disorder, and schizophrenia. Studies of the molecular and cellular events related to developmental neurotoxicity have identified a number of "adverse outcome pathways," many of which share oxidative stress as a key event. Oxidative stress occurs when the balance between the production of free oxygen radicals and the activity of the cellular antioxidant system is dysregulated. In this review, we describe some of the developmental neurotoxins that target the antioxidant system and the mechanisms by which they elicit stress, including oxidative phosphorylation in mitochondria and plasma membrane redox system in rodent models. We also discuss future directions for identifying adverse outcome pathways related to oxidative stress and developmental neurotoxicity, with the goal of improving our ability to quickly and accurately screen chemicals for their potential developmental neurotoxicity.

25th anniversary of the Berlin workshop on developmental toxicology: DevTox database update, challenges in risk assessment of developmental neurotoxicity and alternative methodologies in bone development and growth.

25 years after the first Berlin Workshop on Developmental Toxicity this 10th Berlin Workshop aimed to bring together international experts from authorities, academia and industry to consider scientific, methodologic and regulatory aspects in risk assessment of developmental toxicity and to debate alternative strategies in testing developmental effects in the future. Proposals for improvement of the categorization of developmental effects were discussed as well as the update of the DevTox database as valuable tool for harmonization. The development of adverse outcome pathways relevant to developmental neurotoxicity (DNT) was debated as a fundamental improvement to guide the screening and testing for DNT using alternatives to animal methods. A further focus was the implementation of an in vitro mechanism-based battery, which can support various regulatory applications associated with the assessment of chemicals and mixtures. More interdisciplinary and translation research should be initiated to accelerate the development of new technologies to test developmental toxicity. Technologies in the pipeline are (i) high throughput imaging techniques, (ii) models for DNT screening tests, (iii) use of computer tomography for assessment of thoracolumbar supernumerary ribs in animal models, and (iv) 3D biofabrication of bone development and regeneration tissue models. In addition, increased collaboration with the medical community was suggested to improve the relevance of test results to humans and identify more clinically relevant endpoints. Finally, the participants agreed that this conference facilitated better understanding innovative approaches that can be useful for the identification of developmental health risks due to exposure to chemical substances.

Age-dependent changes of the mandible bone throughout the lifespan in female F344/N rat.

Age-dependent changes of the mandible bone in female F344/N rats, aged 22-1196 days, were analyzed using physiological bone properties and morphology. Bone weight, bone area, bone mineral components, and bone mineral density were assessed using dual-energy X-ray absorptiometry. The bone weight, bone area, bone mineral components, and bone mineral density increased rapidly until approximately 150 days of age, increased gradually thereafter, and then stabilized or decreased after 910 days of age. The ratio of bone mineral components to bone weight (bone mineral ratio) increased rapidly until approximately 43 days of age and stabilized thereafter. Size of the mandible, which was measured at 13 points on mandible surface, increased with age, and the rate of change showed a similar pattern to the other parameters. From a principal component analysis on morphometric measurements, principal component 1 (size factor) increased proportionally with age, whereas principal component 2 (shape factor) decreased until approximately 88 days of age and then increased after 365 days of age. As a result, the scatter plots for principal component 1 and principal component 2 were V-shaped, which indicates that the mandible developed in size, with deformation at younger ages, and recovered its original shape later in life. Our results revealed the occurrence of inflection points at approximately 43, 88, 150, 365, and 910 days of age. Some of these ages corresponded to transition points revealed by the age-dependent changes of the occlusal mandibular condyle and tooth wear in the same rat.

Dietary Intake of Ascorbic Acid Attenuates Lipopolysaccharide-Induced Sepsis and Septic Inflammation in ODS Rats.

The aim of this study was to verify the protective effects of ascorbic acid (AsA) against lipopolysaccharide (LPS)-induced sepsis. The study was conducted using osteogenic disorder Shionogi (ODS) rats, which are unable to synthesize AsA. Male ODS rats (6 wk old) were fed either an AsA-free diet (AsA-deficient group), a diet supplemented with 300 mg/kg AsA (control group), or a diet supplemented with 3,000 mg/kg AsA (high-AsA group) for 8 d. On day 8, all the rats were intraperitoneally injected with LPS (15 mg/kg body weight). Forty-eight hours after the injection, the survival rates of the rats in the control (39%) and the high-AsA (61%) groups were significantly higher than that in the AsA-deficient group (5.5%). Next, we measured several inflammatory parameters during 10 h after administering LPS. At 6 h, elevated serum levels of markers for hepatic and systemic injuries were suppressed in rats fed AsA. Similarly, 10 h after LPS injection, the elevation in the serum levels of markers for renal injury were also suppressed proportionally to the amount of AsA in the diet. The elevated serum concentrations of TNFα and IL-1ß by LPS in the AsA-deficient group decreased in groups fed AsA. Hematic TNFα mRNA levels at 6 h after the LPS injection were also lowered by feeding AsA. These results demonstrated that the dietary intake of AsA improved the survival rates and suppressed the inflammatory damage, in a dose-dependent manner, caused during sepsis induced by LPS in ODS rats.

Genome Sequence of Lactobacillus paracasei Strain LC-Ikematsu, Isolated from a Pineapple in Okinawa, Japan.

The draft genome sequence of Lactobacillus paracasei strain LC-Ikematsu, isolated from a pineapple in Okinawa, was determined. The total length of the 87 contigs was 3.08 Mb with a G+C content of 46.2% and 2,946 coding sequences. The genome analysis revealed its biosynthetic ability of 11 amino acids.

Complete Genome Sequence of the Intracellular Bacterial Symbiont TC1 in the Anaerobic Ciliate Trimyema compressum.

A free-living ciliate, Trimyema compressum, found in anoxic freshwater environments harbors methanogenic archaea and a bacterial symbiont named TC1 in its cytoplasm. Here, we report the complete genome sequence of the TC1 symbiont, consisting of a 1.59-Mb chromosome and a 35.8-kb plasmid, which was determined using the PacBio RSII sequencer.

A quantitative protocol for DNA metabarcoding of springtails (Collembola).

We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.

Gene expression analysis of disabled and re-induced isoprene emission by the tropical tree Ficus septica before and after cold ambient temperature exposure.

Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase (ispS). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity (R(2) = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature.

Complete mitochondrial genome of Cacospongia mycofijiensis (Dictyoceratida: Demospongiae): the first report for the sponge family Thorectidae.

We sequenced a complete mitochondrial genome of the marine sponge, Cacospongia mycofijiensis, which is the first report for the family Thorectidae in the class Demospongiae. The mitogenome was obtained from a de novo assembly of shotgun genome sequencing using Illumina Miseq technology, which reconstructed a circular genome with 97 × of sequence coverage. The assembled mitochondrial genome consisting of 16,227 bp includes 14 protein-coding genes, 2 rRNAs and 2 tRNAs. This complete mitogenome sequence will be useful especially for the phylogenic studies of Demospongiae.

Predominant expression and activity of vacuolar H(+)-ATPases in the mixed segment of the wood-feeding termite Nasutitermes takasagoensis.

The mixed segment is a unique part of the gut present only in the most apical lineage of termites and consists of a complex of overlapping mesenteric and proctodeal epithelia. In spite of its unique structure, the physiological functions of the mixed segment have been poorly studied. We performed transcriptome analysis to identify functional enzymes acting in the mixed segment of the wood-feeding higher termite Nasutitermes takasagoensis. We sequenced the transcripts (4563 isotigs) of the mixed segment and compared them with those of the midgut (4813 isotigs) and the first proctodeal segment (3629 isotigs). We found that vacuolar H(+)-ATPase (V-ATPase) subunits were predominant in the mixed segment, which was confirmed by RT-qPCR analysis. The V-ATPase activity in these three tissues was in a good agreement with the expression patterns, suggesting that V-ATPase is a prevalent enzyme in the mixed segment of the termites. The results confirmed the proposed role of the mixed segment as a transporting epithelium.

Polygenic expression of teratozoospermia and normal fertility in B10.MOL-TEN1 mouse strain.

Subfertility and infertility are two major reproductive health problems in human and domestic animals. The contribution of the genotype to these conditions is poorly understood. To examine the genetic basis of male subfertility, we analyzed its relationship to sperm morphology in B10.MOL-TEN1 mice, which shows high-frequencies (about 50%) of morphologically abnormal sperm. Drastic histological changes were also found in the testis of the B10.MOL-TEN1. Segregation analysis showed that the abnormal sperm phenotype in B10.MOL-TEN1 was inherited and was predictably controlled by at least three loci. We also found that male fertility of this strain was normal. These findings indicate a complicated relationship between sperm morphology and male subfertility.