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ABSTRACT The high specificity of the human skin microbiome is expected to provide a new marker for personal identification. Metagenomic sequencing of clustered regularly interspaced short palindromic repeats (CRISPRs), which we call metaCRISPR typing, was shown to achieve personal identification accurately. However, the intra-individual variability observed in previous studies, which may be due to poor DNA yields from skin samples, has resulted in non-reproducible results. Furthermore, whether metaCRISPR typing can assist in the forensic human DNA analysis of low-biomass samples, from which the information obtained is insufficient, is unknown. In the present study, we sequenced serially diluted control streptococcal CRISPRs cloned into plasmids to determine the minimum copy number required to obtain reproducible results from metaCRISPR typing. We found that at least 102 copies of CRISPRs are necessary to obtain reproducible results. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA typing. When the DNA extracted from the skin swabs was diluted, no information was obtained from six out of eight samples by human DNA typing. On the other hand, beta diversity indices of spacer sequences compared with reference samples were below 0.8 for three out of six samples, for which no information was obtained from human DNA analysis, indicating that the spacers observed in these samples were similar to those in the references. These results indicate that metaCRISPR typing may contribute to the identification of individuals from whom the samples were obtained, even in cases where human DNA yields are insufficient to perform human DNA analysis.IMPORTANCEPrevious studies have developed new personal identification methods utilizing personal differences in the skin microbiome. However, intra-individual diversity of skin microbiome may preclude the application of microbiome-based personal identification. Moreover, no study has compared microbiome-based personal identification and practical human DNA analysis. Here, we revealed that the results of metaCRISPR typing, a previously developed microbiome-based personal identification method, are stable if the copy number of the marker gene is sufficient. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA analysis. Our results indicate that metaCRISPR typing may provide additional information for personal identification using low-biomass samples that cannot be used for conventional human DNA analysis.

The one-dimensional thermo-mechanical behaviour of two peats was investigated through consolidation tests, including incremental loadings and constant-rate-of-strain loadings, under a variety of temperature-stress conditions. As an appropriate expression of highly compressible materials, the natural strain was employed instead of conventional engineering strain, which eliminates the apparent stress dependency of the compression index and the coefficient of secondary compression. Test results suggest the applicability of an isotach approach to the two peats used in this study. The temperature dependency of the coefficient of secondary compression defined with natural strain, λ*α, is very small. All the results summarized on the effective stress-strain plane show that there exists a unique Normal Compression Line (NCL) corresponding to each combination of temperature and strain rate. The NCLs are parallel to each other. Finally, in an attempt to construct a constitutive model, the temperature dependency of the NCL was quantified by introducing a constant, λ*T. By using the two parameters, λ*α and λ*T, which describe the strain rate- and temperature-dependent characteristics, respectively, a 1-D thermo-mechanical behaviour observed in this study will be modelled in a simple manner.

The human skin microbiome, a complex microbial community harbored by the human skin surface, has been proposed as a new forensic genetic marker for human identification to complement short tandem repeat typing. A recent study utilized Streptococcus clustered regularly interspaced short palindromic repeat (CRISPR) polymorphism of the skin microbiome to characterize individuality of the skin microbiome; however, further validation is required for forensic purposes. Optimization of the DNA extraction protocol is critical for both forensic human DNA and microbiome analyses. Previous reports have demonstrated that DNA extraction protocols may alter the results of microbiome analysis, suggesting careful selection of DNA extraction protocols. In this study, we evaluated protocols for DNA extraction from skin swab samples by comparing two DNA extraction protocols, EZ1 DNA Investigator Kit and DNeasy PowerSoil Kit, in order to optimize the metagenomic CRISPR typing method. Quantification of 16S rRNA gene copy numbers and qualification of CRISPR diversities suggested that microbial DNA yield and composition of the CRISPRs were not altered by the two DNA extraction methods. Furthermore, the accuracy of the individual prediction models built using each sample set was comparable. Thus, our results support that the EZ1 platform, an automated DNA extraction protocol used in forensic laboratories, can also be used for metagenomic CRISPR typing intended for forensic human identification.

The article presents research on the use of x-ray examination and pulmonary function tests among toner-handling workers. The study shows no evidence of side effects on chest x-rays and pulmonary function indices as well as obvious fibrotic findings in chest x-ray findings associated with toner exposure in workers.

The article investigates the health impact of toner exposure on toner-handling workers in terms of biomarkers. The study shows no evidence of excessive allergic, inflammatory or oxidative stress reaction in workers exposed to toxic substances or materials. Low concentrations in toner exposure is also reported.

ABSTRACT The highly personalized human skin microbiome may serve as a viable marker in personal identification. Amplicon sequencing resolution using 16S rRNA cannot identify bacterial communities sufficiently to discriminate between individuals. Thus, novel higher-resolution genetic markers are required for forensic purposes. The clustered regularly interspaced short palindromic repeats (CRISPRs) are prokaryotic genetic elements that can provide a history of infections encountered by the bacteria. The sequencing of CRISPR spacers may provide phylogenetic information with higher resolution than other markers. However, using spacer sequencing for discrimination of personal skin microbiome is difficult due to limited information on CRISPRs in human skin microbiomes. It remains unclear whether personal microbiome discrimination can be achieved using spacer diversity or which CRISPRs will be forensically relevant. We identified common CRISPRs in the human skin microbiome via metagenomic reconstruction and used amplicon sequencing for deep sequencing of spacers. We successfully reconstructed 24 putative CRISPR arrays using metagenomic data sets. A total of 1,223,462 reads from three CRISPR arrays revealed that spacers in the skin microbiome were highly personalized, and conserved repeats were commonly shared between individuals. These individual specificities observed using CRISPR typing were confirmed by comparing the CRISPR diversity to microbiome diversity assessed using 16S rRNA amplicon sequencing. CRISPR typing achieved 95.2% accuracy in personal classification, whereas 16S rRNA sequencing only achieved 52.6%. These results suggest that sequencing CRISPRs in the skin microbiome may be a more powerful approach for personal identification and ecological studies compared to conventional 16S rRNA sequencing. IMPORTANCE Microbial community diversity analysis can be utilized to characterize the personal microbiome that varies between individuals. CRISPR sequences, which reflect virome structure, in the human skin environment may be highly personalized similar to the structures of individual viromes. In this study, we identified 24 putative CRISPR arrays using a shotgun metagenome data set of the human skin microbiome. The findings of this study expand our understanding of the nature of CRISPRs by identifying novel CRISPR candidates. We developed a method to efficiently determine the diversity of three CRISPR arrays. Our analysis revealed that the CRISPR spacer diversity in the human skin microbiome is highly personalized compared with the microbiome diversity assessed by 16S rRNA sequencing, providing a new perspective on the study of the skin microbiome.

In order to clarify the genetic factors in alcohol-related chronic pancreatitis among Japanese, we determined the genotype of two major alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). The restriction fragment-length polymorphisms of the ADH2 and the ALDH2 genes were analyzed in 47 normal subjects and 31 patients with alcoholic pancreatitis. No significant difference between the patient and control groups was found in the ADH2 genotypes. A significant genetic difference between the two groups was found in the ALDH2 locus. The frequency of the ALDH2*1 allele was found to be 0.681 and that of the ALDH2*2 allele was 0.319 in the controls, while these values were 0.935 and 0.065 in the patients, respectively. Most of the patients (27 of 31) were ALDH2*1/2*1, only four were ALDH2*1/2*2, and none of the patients were ALDH2*2/2*2. These results indicate that genetic polymorphism of the ALDH2 gene influences the risk of developing alcoholic pancreatitis in Japanese. [ABSTRACT FROM AUTHOR]

The anthrax toxin is a virulence factor produced by the bacterium Bacillus anthracis. Transcription of anthrax toxin genes is controlled by the transcription factor AtxA. Thus, AtxA is thought to be a key factor for the pathogenicity of B. anthracis. Despite its important role in B. anthracis infection, the molecular mechanism by which AtxA controls expression of anthrax toxin remains unclear. This study aimed to characterize the molecular mechanism of AtxA-mediated regulation of protective antigen (PA), a component of anthrax toxin encoded by the pagA gene. First, the interaction between the upstream region of pagA and AtxA was evaluated in vivo by constructing a transcriptional fusion of the upstream region with an auxotrophic marker. The results showed that (i) the upstream region of pagA suppressed transcription of the downstream gene and (ii) AtxA recovered suppressed transcription. Second, in vitro analysis using a gel mobility shift assay was performed to evaluate binding specificity of the AtxA–DNA interaction. The result showed sequence-independent binding of AtxA to DNA. Taken together, our findings suggest that the expression of PA was suppressed by the upstream region of pagA and that an interaction of AtxA and the upstream region releases the suppression.

Copyright of Canadian Journal of Anaesthesia / Journal Canadien d'Anesthésie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Canadian Journal of Anaesthesia / Journal Canadien d'Anesthésie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Surface-reaction film-formation technology of epitaxial Si and polcrystalline silicon using free-jet molecular flow is proposed. High-rate (∼0.5 μm/min or higher) growth of homoepitaxial Si films with high crystallographic perfection has been achieved at temperatures as low as 600 °C without the chemical by-product deposition on the inner surface of the reaction chamber. This result also implies that this system has the cleaning-free function. The film-formation mechanism appears to be dominated by the chemical reaction on the substrate surface without the vapor phase reaction. [ABSTRACT FROM AUTHOR]

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.