Consideration for the validation of clinical laboratory methods in nonclinical fields.

In new drug development, cells or animals are treated with the selected candidate compound to confirm its efficacy and safety in nonclinical studies. Clinical laboratory tests are carried out using samples from experimental animals in these studies. The clinical laboratory test method validation in nonclinical fields should be conducted keeping in mind that the circumstances differ from those in clinical settings. However, the validation procedures have not been systematically integrated into any standard. The considerations in this paper set out systematically practical guidance for the validation of quantitative analytical methods for fluid samples collected from animal studies, for the purpose of ensuring that laboratory test method validation is conducted in nonclinical fields at an enough level.

Multiple evolutionary origins of Australian soil-burrowing cockroaches driven by climate change in the Neogene.

Parallel evolution is the independent appearance of similar derived phenotypes from similar ancestral forms. It is of key importance in the debate over whether evolution is stochastic and unpredictable, or subject to constraints that limit available phenotypic options. Nevertheless, its occurrence has rarely been demonstrated above the species level. Climate change on the Australian landmass over the last approximately 20 Myr has provided conditions conducive to parallel evolution, as taxa at the edges of shrinking mesic habitats adapted to drier biomes. Here, we investigate the phylogeny and evolution of Australian soil-burrowing and wood-feeding blaberid cockroaches. Soil burrowers (subfamily Geoscapheinae) are found in relatively dry sclerophyllous and scrubland habits, whereas wood feeders (subfamily Panesthiinae) are found in rainforest and wet sclerophyll. We sequenced and analysed mitochondrial and nuclear markers from 142 specimens, and estimated the evolutionary time scale of the two subfamilies. We found evidence for the parallel evolution of soil-burrowing taxa from wood-feeding ancestors on up to nine occasions. These transitions appear to have been driven by periods of aridification during the Miocene and Pliocene across eastern Australia. Our results provide an illuminating example of climate-driven parallel evolution among species.

The homolog of Ciboulot in the termite (Hodotermopsis sjostedti): a multimeric beta-thymosin involved in soldier-specific morphogenesis.

BACKGROUND: Caste differentiation in social insects is a type of polyphenism that enables division of labor among members of a colony. This elaborate social integration has attracted broad interest, although little is known about its regulatory mechanisms, especially in Isoptera (termites). In this study, we analyzed soldier differentiation in the damp-wood termite Hodotermopsis sjostedti, focusing on a possible effector gene for caste development. The gene for an actin-binding protein, HsjCib, which shows a high level of expression in developing mandibles during soldier differentiation, is characterized in detail. RESULTS: To examine the HsjCib gene, full-length cDNAs were obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) and sequencing. Multiple isoforms were identified, and on the basis of the results of northern and Southern hybridization analyses, these isoforms were considered to be transcriptional variants from a single gene. On the basis of their sequence similarity to homologous genes of other organisms, functions in actin assembly were assumed to be different among isoforms. Expression analysis revealed high expression in the head during soldier differentiation, which was consistent with their allometric growth. Although isoform expression was observed in various tissues, different expression levels were observed among tissues, suggesting the possibility of tissue-specific morphogenetic regulation by HsjCib isoforms. CONCLUSION: This study revealed the characteristics and dynamics of the HsjCib gene during soldier differentiation as a potential representative of downstream effector genes in caste-specific morphogenesis. From the expression patterns observed, this gene is considered to be involved in cephalic morphogenesis and neural reorganization, resulting in the establishment of caste-specific morphology and behavior.

Extraction of PCBs and water from river sediment using liquefied dimethyl ether as an extractant.

We investigated whether polychlorinated biphenyls (PCBs) and water could be simultaneously removed from river sediment by solvent extraction using liquefied dimethyl ether (DME) as the extractant. DME exists in a gaseous state at normal temperature and pressure and can dissolve organic substances and some amount of water; therefore, liquefied DME under moderate pressure (0.6-0.8 MPa) at room temperature can be effectively used to extract PCBs and water from contaminated sediment, and it can be recovered from the extract and reused easily. First, we evaluated the PCB and water extraction characteristics of DME from contaminated sediment. We found that 99% of PCBs and 97% of water were simultaneously extracted from the sediment using liquefied DME at an extraction time of 4320 s and a liquefied DME/sediment ratio of 60 mL g(-1). The extraction rate of PCBs and water was expressed in terms of a pseudo-first-order reaction rate. Second, we estimated the amount of DME that was recovered after extraction. We found that 91-92% of DME could be recovered. In other words, approximately 5-10% of DME was lost during extraction and recovery. It is necessary to optimize this process in order to recover DME efficiently. The extraction efficiency of the recovered DME is similar to that of the pure DME. From the results, we conclude that solvent extraction using liquefied DME is suitable for extracting PCBs and water from contaminated sediment.

Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae).

The evolution of genome size has been discussed in relation to the evolution of various biological traits. In the present study, the genome sizes of 22 dictyopteran species were estimated by Feulgen image analysis densitometry and 6-diamidino-2-phenylindole (DAPI)-based flow cytometry. The haploid genome sizes (C-values) of termites (Isoptera) ranged from 0.58 to 1.90 pg, and those of Cryptocercus wood roaches (Cryptocercidae) were 1.16 to 1.32 pg. Compared to known values of other cockroaches (Blattaria) and mantids (Mantodea), these values are low. A relatively small genome size appears to be a (syn)apomorphy of Isoptera + Cryptocercus, together with their sociality. In some phylogenetic groups, genome size evolution is thought to be influenced by selective pressure on a particular trait, such as cell size or rate of development. The present results raise the possibility that genome size is influenced by selective pressures on traits associated with the evolution of sociality.

Relationship between dynamic change of copper and dioxin generation in various fly ash.

Only limited information on the chemical forms of copper in municipal solid waste incinerator (MSWI) fly ash is available in the literature. Therefore, we performed in situ X-ray absorption near-edge spectroscopy (XANES) experiments involving two types of real fly ash to confirm the behavior of copper in fly ash at secondary formation temperatures for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). CuCl(2).3Cu(OH)(2) was detected in both fly ash types before heating experiments. Cupric compounds in real fly ash were reduced to cuprous compounds or elemental copper at approximately 200 degrees C. The changes in the Cu XANES spectra observed for the two fly ash samples were similar to those observed previously. We also examined the behavior of copper in model fly ash using SiO(2) to investigate the effect of supporting materials and copper content in fly ash on the dynamic changes of copper and amount of dioxins generated. There was little difference between the use of boron nitride (BN) and SiO(2) as a supporting material. The formation mechanisms of PCDDs and PCDFs in both of the model fly ash types were basically the same. Thus, we conclude that the dynamic change of copper in MSWI fly ash during heating is a commonly observed feature, and no simple relationship between the composition of fly ash and the amount of PCDD/PCDF generated or dynamic change of copper is apparent. The dynamic change of copper explained the generated amount and homologue distribution of PCDDs and PCDFs.

Altricial development in subsocial wood-feeding cockroaches.

Species in the wood-feeding genus Salganea within the cockroach subfamily Panesthiinae (Blaberidae) typically live in biparental families; their first instars suffer high mortality when removed from adults, and in at least one species, adults are known to feed neonates on oral liquids. In the closely related gregarious wood-feeding genus Panesthia , no parental interaction with offspring is known. We compared the external morphology of first instars of these two genera and found that eye development and cuticular pigmentation at hatching are correlated with social structure. First instars of Panesthia have a dark cuticle and well-developed eyes. First instars of Salganea have a pale, transparent cuticle, and eyes significantly smaller than those of Panesthia relative to adult eye size. The body size of the first-instar of Salganea esakii is significantly smaller than that of Panesthia angustipennis spadica , relative to adult body size (24.0 and 27.4% of adult head-capsule width, respectively), but a more general survey suggests that, overall, neonate size may be similar in the two genera. We compared the first instars of these two taxa of Panesthiinae to those in the biparental, wood-feeding cockroach Cryptocercus (Cryptocercidae) and discuss how decreased investment in both integumentary and ocular development in subsocial cockroaches parallels that seen in altricial vertebrates.

Cloning and expression of a geranylgeranyl diphosphate synthase gene: insights into the synthesis of termite defence secretion.

In Nasutitermes takasagoensis, a termite in which soldiers perform specialized chemical defence, Nts19-1 gene is highly expressed exclusively in soldier head. In this study, two types of transcripts for this gene were obtained, and the full-length cDNAs were determined by rapid amplification of cDNA ends (RACE). These transcripts were putative homologues of the geranylgeranyl diphosphate (GGPP) synthase gene, involved in the condensation of dimethylallyl diphosphate with isopentenyl diphosphate in the isoprenoid biosynthetic pathway. The genes were thus termed NtGGPPS1. GGPP is a precursor of diterpenes in plants. In situ hybridization localized NtGGPPS1 expression to the epidermal secretory cells of the frontal gland reservoir where many kinds of diterpenes are produced, suggesting that NtGGPPS1 is involved in the biosynthesis of defence secretion.

Histological analysis of fat body development and molting events during soldier differentiation in the damp-wood termite, Hodotermopsis sjostedti (Isoptera, Termopsidae).

The caste system of termites is well defined, with a high degree of polyphenism among colony members. Polyphenic caste characteristics are hormonally regulated, and juvenile hormone (JH) is particularly involved in caste determination, as is the case with many other social insects. In the present study, soldier differentiation in the damp-wood termite, Hodotermopsis sjostedti, was induced by treatment with a JH analog (pyriproxyfen) in order to establish the chronology of tissular modifications appearing in response to the hormone. The fat body is involved in the physiological events that prepare the insect for the molting transition. The development of the fat body started within three days after hormonal treatment, and it filled the entire abdominal cavity for about four days prior to the molt to presoldier, maintaining this state until the next molt to soldier. Fat body development was accompanied by the accumulation of protein granules in the cytoplasm, but these granules disappeared during the few days preceding the molt to presoldier. The timing of consumption of these storage proteins corresponded to the window of epidermal growth, which was conspicuous about 14 days after hormonal treatment, and synthesis of the new cuticle, which was initiated 10 days after treatment. We summarize the chronology of the histological events under hormonal control.

Application of microwave-assisted extraction to the analysis of PCBs and CBzs in fly ash from municipal solid waste incinerators.

Polychlorinated biphenyls (PCBs) and chlorobenzenes (CBzs) are two classes of dioxin precursors formed in municipal solid waste incinerators (MSWIs) producing negative health effects similar to those of dioxins. Reducing the analytical time required for determining the concentrations of these compounds in MSWIs is important for quickly evaluating their importance and associated health risks. In the present study, microwave-assisted extraction (MAE) is compared with traditional Soxhlet extraction (SE) to determine the extraction efficiencies attained for PCB and CBz analysis. The efficiencies of MAE are compared with those of SE under various experimental conditions, using fly ash spiked with standards. Water is used as a safe and environmentally friendly solvent in MAE for PCB and CBz analyses and MAE has high extraction efficiency for spiked fly ash compared with that of SE. Furthermore, the extraction time and organic solvent consumption are reduced with MAE compared with SE. The optimum conditions for MAE established in this study are using a 30-ml volume of toluene/acetone (1/1) or a 15-ml volume of toluene, samples with less than 60% water content (WC), and an irradiation time of 15 min.

Kinetics on the decomposition of polychlorinated biphenyls with activated carbon-supported iron.

The process of destroying polychlorinated biphenyls (PCBs) generates exhaust gases that contain low quantities of PCBs, which cannot be disposed of easily. Activated carbon (AC) can be used to adsorb residual PCBs after disposal of high-level PCBs. We examined the chemical reactivity of AC-supported iron as a catalyst to decompose PCB-153, and varied three decomposition parameters (temperature, time and iron concentration) under an atmosphere of either air or N(2). We measured the Brunauer-Emmett-Teller (BET) surface area and pore volume of AC to assess the adsorption capacity of AC before and after decomposition. At low temperatures the adsorption process was more important than the decomposition process. The decomposition process was completed within 30 and 60 min under air and N(2), respectively. The efficiency of PCB-153 decomposition at 350 degrees C for 120 min was approximately 100.0% and 97.1% under air and N(2), respectively. Analysis of inorganic chloride ions revealed that PCB-153 was effectively destroyed during decomposition. The differences between decomposition under air and N(2) reflected differences in BET surface and pore volume.

Phylogenetic analyses of fat body endosymbionts reveal differences in invasion times of blaberid wood-feeding cockroaches (Blaberidae: Panesthiinae) into the Japanese archipelago.

Cockroaches have endosymbiotic bacteria in their fat bodies. Recent molecular phylogenetic analyses on both hosts and endosymbionts have revealed that co-evolution has occurred throughout the history of cockroaches and termites. Co-cladogenesis was also shown among closely related taxa (woodroach genus Cryptocercus; Cryptocercidae), and thus endosymbiont data are likely to be useful for biogeographical analyses. To test the possibility of co-cladogenesis among inter-and intraspecific taxa, as well as the utility of endosymbiont data for inferring biogeographical scenarios, we analyzed rRNA genes of endosymbionts of Japanese and Taiwanese Panesthiinae (Salganea and Panesthia; Blaberidae), on which phylogenetic analyses previously had been performed based on the mitochondrial genes. Statistical analyses on the topologies inferred from both endosymbiont and host mitochondria genes showed that co-cladogenesis has occurred. The endosymbiont sequences examined appear to have evolved in a clock-like manner, and their rate of evolution based on the host fossil data showed a major difference in the time of invasion of the two Japanese genera, that is congruent with the recent analyses of their mitochondrial genes.

Dynamic change of copper in fly ash during de novo synthesis of dioxins.

Although many researchers have reported that copper chloride is an important catalyst that generates relatively large amounts of dioxins in heat experiments involving model fly ash, details on the behavior of copper during the process are still unavailable. In this study, we used in situ XANES experiments involving one type of real fly ash, which originated from a municipal solid-waste incinerator (MSWI), and two fly ash models to investigate the behavior of copper in fly ash at temperatures that are suitable for de novo synthesis, which is the major formation route for dioxins during waste incineration and thermal processes. Cupric compounds in real fly ash and model fly ash A(CuCl2.2H2O + activated carbon (AC) + boron nitride (BN)) were reduced to cuprous compounds or elemental copper at low temperatures. The changes in the Cu XANES spectra of real fly ash were similar to those of model fly ash A and those of an oxychlorination catalyst. In model fly ash B (CuO + AC + KCl + BN), CuO did not vary dramatically in the temperature range studied. In this study, we found strong evidence that oxychlorination, the key mechanistic step in the formation of dioxins, occurred in both real MSWI and model fly ash.

Application of water as a solvent in microwave-assisted extraction for analysis of PCBs and CBzs in fly ash.

Polychlorinated biphenyls (PCBs) and chlorobenzenes (CBzs) are two classes of dioxin precursors formed in municipal solid waste incinerators (MSWIs); they produce negative health effects similar to those of dioxins. Reducing the analytical time required for determining the concentrations of these compounds in MSWIs is important for quickly evaluating their importance and assessing associated health risks. In the present study, water is used as a safe and environmentally friendly solvent in microwave-assisted extraction (MAE) for PCB and CBz analyses. MAE is compared with traditional Soxhlet extraction (SE) to determine the extraction efficiencies. The evaluation of extraction efficiencies shows that MAE has a high extraction efficiency compared with that of SE when water content is lower than 60%. Furthermore, the extraction time and organic solvent consumption are reduced with MAE compared with SE.

The effect of copper speciation on the formation of chlorinated aromatics on real municipal solid waste incinerator fly ash.

A limited amount of information exists regarding the relationship between the chemical form of copper and the formation of chlorinated aromatics in fly ash. To understand the effects of the various forms of copper on the formation of chlorinated aromatics in real fly ash, we determined the chemical forms of copper present in various types of real fly ash using X-ray absorption near edge structure (XANES) and evaluated the relationship between the chemical forms of copper and the formation of chlorinated aromatics. Copper chloride hydroxide (CuCl2 x 3Cu(OH)2) and cuprous chloride (CuCl) were the predominant copper species found in real fly ash. Although pure cupric chloride (CuCl2) is known to be the most active catalyst for the formation of chlorinated aromatics under experimental conditions with synthetic fly ash, CuCl2 was not found in every real fly ash sample. The amount of copper chloride hydroxide was positively correlated with the formation of chlorinated aromatics in real fly ash and is, consequently, considered to be one of the key species involved in the formation of chlorinated aromatics.

Identification of soldier caste-specific protein in the frontal gland of nasute termite Nasutitermes takasagoensis (Isoptera: Termitidae).

The termite soldier is unique because of its defensive task in a colony. In Nasutitermitinae (family Termitidae), soldiers use in their defense frontal glands, which contain various chemical substances. To isolate the gene products related to the chemical defense, we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of soldier heads with those of workers of the nasute termite Nasutitermes takasagoensis. We identified a 26-kDa soldier-specific protein (Ntsp1) that exists most abundantly in the dorsal head including the frontal gland. We determined the N-terminal amino acid sequence of Ntsp1, and then cloned the Ntsp1 cDNA by rapid amplification of the cDNA ends-polymerase chain reaction (RACE-PCR). A putative signal peptide was detected upstream of the N-terminus and the Ntsp1 protein showed sequence homologies with known insect secretory carrier proteins, which bind to hydrophobic ligands such as juvenile hormone, suggesting that Ntsp1 belongs to this class of proteins. Northern blot analysis confirmed that the expression level of Ntsp1 was high only in the soldier head. In addition, the localization of Ntsp1 expression was limited in epithelial cells of the frontal gland reservoir, suggesting that this protein binds to some terpenoid(s) preserved in the frontal gland reservoir.

Screening of genes expressed in developing mandibles during soldier differentiation in the termite Hodotermopsis sjostedti.

We investigated the morphological changes accompanying soldier differentiation in the damp-wood termite Hodotermopsis sjostedti. Genes expressed in the developing mandibles, which undergo the most remarkable morphological changes during soldier differentiation, were screened using fluorescent differential display. Database searches for sequence similarities were conducted and the relative expression levels were then quantified by real-time polymerase chain reaction. Among the identified candidate genes, 12 genes were upregulated during soldier differentiation. These included genes for cuticle proteins, nucleic acid binding proteins, ribosomal proteins and actin-binding protein, which were inferred to be involved in caste-specific morphogenesis in termites.

Major alteration of the expression site of endogenous cellulases in members of an apical termite lineage.

Termites are among the most important cellulose-digesting animals on earth, and are well-known for the symbiotic relationship they have with cellulolytic trichomonad and oxymonad flagellates (unicellular eukaryotes). Perhaps less well-known is the fact that approximately 75% of the approximately 2600 described termite species -- those belonging to the family Termitidae -- do not harbour such flagellates. Unlike most termites from other families, the majority of termitids do not consume wood, feeding instead on soil, leaf litter, fungi, grass, or lichen. Recent years have seen the characterization of the endogenous cellulase enzymes that help termites digest cellulose, from one flagellate-harbouring species (Reticulitermes speratus), as well as one termitid (Nasutitermes takasagoensis). The genes encoding the enzymes in these two termites are similar. However, their site of expression differs markedly -- the salivary glands in R. speratus and the midgut in N. takasagoensis. To investigate this difference further, we performed a comparative study of cellulase expression in various termitid and flagellate-harbouring species, using enzyme assays and reverse transcription polymerase chain reactions. Taxa from phylogenetically basal lineages were consistently found to express endogenous genes specifically in the salivary glands, whilst those from a relatively apical lineage containing termitids expressed cellulases solely in the midgut. Relatively low levels of cellulase activity were found in nonwood-feeding species, while the wood-feeding Coptotermes formosanus -- arguably the most destructive pest species world-wide -- was found to have high levels of activity in all parts of the gut when compared to all other termites. In the light of these results, as well as recently accumulated phylogenetic data, we discuss scenarios for the evolution of cellulose digestion in termites.

Wing disc development during caste differentiation in the ant Pheidole megacephala (Hymenoptera: Formicidae).

The genus Pheidole has three distinct castes in females: queen, major, and minor workers. It has been believed that the larvae of major workers have prominent mesothoracic wing discs, although the minor worker larvae lack them. Here we conducted histological examinations of wing discs during larval development in P. megacephala. We show that all three castes have mesothoracic wing discs, at least in their early stage of the final larval instar, and that the wings degenerate differently in the dimorphic worker castes. The minute wing discs of minor workers neither grow nor metamorphose but disappear during the prepupal stage. On the contrary, the wing discs of major workers evaginate at the onset of the prepupal stage but subsequently degenerate by apoptotic cell death. This apoptotic wing degeneration in the prepupal stage was contradistinguished from wing degeneration in some lepidopteran insects, in which apoptosis occurs in the pupal wing buds. Our results suggest that each worker caste shows a different degeneration process to express the wingless character and that apoptotic degeneration has been adopted in association with the evolution of worker dimorphism.

Soldier-like intercastes in the rotten-wood termite Hodotermopsis sjostedti (Isoptera: Termopsidae).

All termite species possess a distinct sterile-soldier caste in their colonies, although reproductive soldiers, with soldier characteristics and reproductive ability, have been reported from several species of the family Termopsidae. Such intercastes have been considered the primitive-soldier caste, and based on this many researchers have discussed the evolutionary origin of termite soldiers. We investigated whether such soldier-reproductive intercastes also exist in the Japanese rotten-wood termite Hodotermopsis sjostedti. Abnormal individuals with soldier-like characteristics were found and designated as soldier-like intercastes, which appeared to have both soldier and reproductive characteristics. Based on our morphometric analyses and histological examinations, we suggest that the developmental origin of this intercaste is a pseudergate, nymph or sixth-instar larva. In addition, the intercaste was found to have relatively well-developed gonads, although mature oocytes and spermatozoa were not found in female ovaries and male seminal vesicles, respectively. We conclude that the soldier-like intercaste of H. sjostedti does not have reproductive ability, which is different from all other known soldier-like intercastes in Termopsidae.