Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The β-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.     

Characterization of Malassezia microbiota in the human external auditory canal and on the sole of the foot

Of the fungal skin microbiota, the lipophilic yeast genus Malassezia predominates at all body sites. Of the members of this genus, M. globosa, M. restricta, and M. sympodialis are the most common on the face, limbs, and trunk. In the present study, the Malassezia microbiotas in the external auditory canal and on the sole of the foot were characterized. M. slooffiae was the most common species in both the external auditory canal and on the sole of the foot, followed by M. restricta. Principal component analysis further revealed that the Malassezia microbiota in the external auditory canal and on the sole of the foot constitute a different cluster from those on the scalp and cheek and in the nasal cavity. Additionally, five new Malassezia phylotypes were detected on the sole of the foot and in the external auditory canal. Our results suggest that a distinctive Malassezia microbiota is present in the external auditory canal and on the sole of the foot, although the clinical significance of this finding remains unknown.     

Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the ??toxin receptor mediated cell knockout?? method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 ??-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3?C7?days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.     

PCR method of detecting pork in foods for verifying allergen labeling and for identifying hidden pork ingredients in processed foods

A PCR method to detect porcine DNA was developed for verifying the allergen labeling of foods and for identifying hidden pork ingredients in processed foods. The primer pair, F2/R1, was designed to detect the gene encoding porcine cytochrome b for the specific detection of pork with high sensitivity. The amplified DNA fragment (130 bp) was specifically detected from porcine DNA, while no amplification occurred with other species such as cattle, chicken, sheep, and horse. When the developed PCR method was used for investigating commercial food products, porcine DNA was clearly detected in those containing pork in the list of ingredients. In addition, 100 ppb of pork in heated gyoza (pork and vegetable dumpling) could be detected by this method. This method is rapid, specific and sensitive, making it applicable for detecting trace amounts of pork in processed foods.     

Electron microscopic examination of the dormant spore and the sporulation of Paenibacillus motobuensis strain MC10

We previously reported a new species Paenibacillus motobuensis. The type strain MC10 was stained gram-negative, but had a gram-positive cell wall structure and its spore had a characteristic star shape. The spore and sporulation process of P. motobuensis strain MC10 were examined by electron microscopy using the technique of freeze-substitution in thin sectioning. The structure of the dormant spore was basically the same as that of the other Bacillus spp. The core of the spore was enveloped with two main spore components, the cortex and the spore coat. In thin section, the spore showed a star-shaped image, which was derived from the structure of the spore coat, which is composed of three layers, namely the inner, middle and outer spore coat. The middle coat was an electron-dense thick layer and had a characteristic ridge. By scanning electron microscopic observation, the ridges were seen running parallel to the long axis of the oval-shaped spore. The process of sporulation was essentially the same as that of the other Bacillus spp. The forespore was engulfed by the mother cell membrane, then the spore coat and the cortex were accumulated in the space between the mother cell membrane and forespore membrane. The mother cell membrane seemed to participate in the synthesis of the spore coat. MC10 strain showed almost identical heat resistance to that of B. subtilis.     

Evaluation of the levels of specific IgE against Cryptococcus diffluens and Cryptococcus liquefaciens in patients with atopic dermatitis

Cryptococcus diffluens and Cryptococcus liquefaciens, 2 basidiomycetous yeasts, frequently colonize the skin of patients with atopic dermatitis (AD). In this study, we investigated the presence of specific IgE antibodies against C. diffluens and C. liquefaciens in the sera of AD patients by using an enzyme immunoassay . Of the 122 AD serum samples tested, 43 (35.2%) and 50 (41.0%) were positive for specific IgE antibodies against C. diffluens and C. liquefaciens, respectively. The levels of specific IgE against the C. diffluens antigen and that against the C. liquefaciens antigen were strongly correlated (r=0.96). In contrast, no remarkable correlation was observed between the levels of specific IgE against the 2 Cryptococcus species and that of specific IgE against Malassezia restricta. Competitive enzyme-linked immunosorbent assay (ELISA) inhibition tests revealed that C. diffluens and C. liquefaciens shared common antigens. This finding was consistent with the IgE immunoblotting data which demonstrated that several IgE-binding proteins with molecular masses of 77, 54, and 30 kDa were recognized in both C. diffluens and C. liquefaciens antigens . These results suggest that fungal components from C. diffluens and C. liquefaciens may act as allergens and play a role in the pathogenesis of AD.     

Contribution of multi-antimicrobial resistance to the population of antimicrobial resistant Escherichia coli isolated from apparently healthy pigs in Japan

The aim of this study was to clarify the involvement of tetracycline usage in resistance rates against other antimicrobials. Antimicrobial susceptibility testing was carried out on 545 porcine Escherichia coli isolates throughout Japan. As the result of analyzing by regions, resistance rates against kanamycin, oxytetracycline and trimethoprim in the Kanto/Koshinetu district were higher than those in some other districts. High resistance rates against kanamycin or trimethoprim in oxytetracycline-resistant isolates were also observed in the Kanto/Koshinetu district. The prevalence of multi-antimicrobial resistance through co-selection of resistances against kanamycin or trimethoprim by tetracycline usage could be the cause of regional differences in these resistances in porcine E. coli. By a communicative surveillance, kanamycin- and trimethoprim-resistance rates were likely to be elevated with tetracycline usage. Thus, usage of specific antimicrobial(s) is a remarkable viewpoint to control antimicrobial resistant bacteria.     

Gene expression analysis of human induced pluripotent stem cells cryopreserved by vitrification using StemCell Keep

In recent years, regenerative medicine research using human somatic and induced pluripotent stem cells has advanced considerably, promoting clinical applications. However, it is essential that these cells are cryopreserved safely and effectively. Most cryopreservation solution agents contain dimethyl sulfoxide (DMSO), which exhibits strong toxicity and can potentially promote cell differentiation. Hence, it is important to explore substitutes for DMSO in cryoprotectant solutions. One such alternative is StemCell Keep (SCK), a DMSO-free solution that has been reported to effectively cryopreserve human induced pluripotent stem cells (hiPS cells). To clarify the effect of cryopreservation agents on cells, DNA microarray analysis is useful, as it can identify a large number of gene expression differences in cryopreserved cells, as well as functional increases in gene groups. In this study, we performed gene expression analysis of SCK-cryopreserved hiPS cells using a DNA microarray gene chip. The hiPS cells vitrified with SCK or DMSO-based vitrification solutions were thawed and cultured on Matrigel under feeder-free conditions, and RNA was extracted for DNA microarray analysis. Genes obtained from DNA microarray data were classified by the keywords of Gene Ontology Biological Process Term, and their relationships were analyzed using DAVID or the GeneMANIA database.SCK-cryopreserved hiPS cells expressed several anti-apoptotic genes, as well as genes related to cell adhesion or proliferation at levels that were nearly equivalent to those of non-frozen hiPS cells. Gene enrichment analysis with selected genes of SCK-cryopreserved hiPS cells whose expression differences were superior to those of DAP-cryopreserved showed strong interactions of negative regulation of apoptotic process, cell adhesion and positive regulation of cell proliferation in DAVID analysis. We demonstrated that SCK successfully maintained the key functions of hiPS cells, including anti-apoptosis, cell adhesion, and cell proliferation, during cryopreservation.     

Immature male gibbons produce female-specific songs

Gibbons are apes that are well known to produce characteristic species-specific loud calls, referred to as “songs.” Of particular interest is the sex specificity of the “great calls” heard in gibbon songs. However, little is known about the development of such calls. While great calls are given by female gibbons of various ages, they have never been recorded from males. Here, we report two observations of immature male gibbons from two different species, wild Hylobates agilis and captive H. lar, which spontaneously sang female-specific great calls. Based on the video clips, we conclude that immature males also have the potential to produce great calls. Our observations led us to propose a new hypothesis for the development of sexual differentiation in the songs of gibbons, and its implications for the general issue of sex-specific behavior in primates.