Characterization and Demonstration of Mock Communities as Control Reagents for Accurate Human Microbiome Community Measurements.

Standardization and quality assurance of microbiome community analysis by high-throughput DNA sequencing require widely accessible and well-characterized reference materials. Here, we report on newly developed DNA and whole-cell mock communities to serve as control reagents for human gut microbiota measurements by shotgun metagenomics and 16S rRNA gene amplicon sequencing. The mock communities were formulated as near-even blends of up to 20 bacterial species prevalent in the human gut, span a wide range of genomic guanine-cytosine (GC) contents, and include multiple strains with Gram-positive type cell walls. Through a collaborative study, we carefully characterized the mock communities by shotgun metagenomics, using previously developed standardized protocols for DNA extraction and sequencing library construction. Further, we validated fitness of the mock communities for revealing technically meaningful differences among protocols for DNA extraction and metagenome/16S rRNA gene amplicon library construction. Finally, we used the mock communities to reveal varying performance of metagenome-based taxonomic profilers and the impact of trimming and filtering of sequencing reads on observed species profiles. The latter showed that aggressive preprocessing of reads may result in substantial GC-dependent bias and should thus be carefully evaluated to minimize unintended effects on species abundances. Taken together, the mock communities are expected to support a myriad of applications that rely on well-characterized control reagents, ranging from evaluation and optimization of methods to assessment of reproducibility in interlaboratory studies and routine quality control. IMPORTANCE Application of high-throughput DNA sequencing has greatly accelerated human microbiome research and its translation into new therapeutic and diagnostic capabilities. Microbiome community analyses results can, however, vary considerably across studies or laboratories, and establishment of measurement standards to improve accuracy and reproducibility has become a priority. The here-developed mock communities, which are available from the NITE Biological Resource Center (NBRC) at the National Institute of Technology and Evaluation (NITE, Japan), provide well-characterized control reagents that allow users to judge the accuracy of their measurement results. Widespread and consistent adoption of the mock communities will improve reproducibility and comparability of microbiome community analyses, thereby supporting and accelerating human microbiome research and development.

Proxy evaluation of dignity expectations and satisfaction of older patients with dementia by family members and nurses.

AIM: Develop a proxy evaluation questionnaire for patients' family members and nurses to evaluate dignity expectations and satisfaction of patients with dementia. DESIGN: Cross-sectional study. METHOD: A proxy questionnaire draft was prepared with 30 items on expectations for dignity and 23 items on satisfaction with dignity. And administered to three paired groups: 81 older patients with intact cognitive function, 75 family members, and 77 nurses. RESULTS: 18 of 30 items and 21 of 23 were correlated between patients and their family members' responses on dignity expectations and satisfaction, respectively. When limited to nurses with clinical experience of ≥20 years, there were significant correlations between patients' and nurses' responses (p < .05). Exploratory factor analysis of patient's responses to significantly correlated items extracted 3 factors with 13 items of expectations for dignity but no factors of satisfaction with dignity. Using a questionnaire provides insight for proxy evaluation of expectations for dignity.

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

BACKGROUND: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. RESULTS: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. CONCLUSIONS: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

Development of the Inpatient Dignity Scale Through Studies in Japan, Singapore, and the United Kingdom.

PURPOSE: The importance of human dignity in care is well-recognized. Care recipients' experiences with undignified care have been reported in many countries. However, few studies have measured these situations quantitatively, especially as there are no tools applicable to inpatients receiving ordinary daily care. This study aimed to develop a valid and reliable Inpatient Dignity Scale (IPDS) that can measure inpatients' expectations of and satisfaction with dignity in daily care. METHODS: We conducted a three-phase research project: item generation and a preliminary survey with 47 items related to patients' dignity in Japan, a main survey with 36 items with deliberate translation into English in Singapore, and a confirmatory survey with 35 items in England, with 442, 430, and 500 inpatients as participants in questionnaire surveys, respectively. Data from each survey were processed using factor analysis. RESULTS: Authors obtained a scale with a four-factor structure with acceptable reliability: (F1) respect as a human being, (F2) respect for personal feelings and time, (F3) respect for privacy, and (F4) respect for autonomy. CONCLUSION: The Inpatient Dignity Scale can be periodically used by hospital administrators or nurses to preserve inpatients' dignity in daily care by monitoring inpatients' views regarding their expectations of and satisfaction with dignity.

Information-sharing ethical dilemmas and decision-making for public health nurses in Japan.

BACKGROUND: Information sharing is one of the most important means of public health nurses collaborating with other healthcare professionals and community members. There are complicated ethical issues in the process. RESEARCH OBJECTIVES: To describe the ethical dilemmas associated with client information sharing that Japanese public health nurses experience in daily practice and to clarify their decision-making process to resolve these dilemmas. RESEARCH DESIGN: Data were collected using a three-phase consensus method consisting of semi-structured interviews, self-administered questionnaires and a group interview. PARTICIPANTS AND RESEARCH CONTEXT: We surveyed administrative public health nurses in Shizuoka Prefecture, Japan. The semi-structured interviews were carried out with 12 administrative public health nurses, and the self-administered questionnaires were sent to all 899 administrative public health nurses. The group interview was carried out with eight administrative public health nurses. ETHICAL CONSIDERATIONS: Ethical approval was granted by the ethics committee of the School of Health Sciences, Nagoya University, Japan (8-158, 9-130). FINDINGS: Information-sharing ethical dilemmas occurred most often when clients' decisions did not coincide with the nurses' own professional assessments, particularly when they faced clinical issues that were inherently ambiguous. In their decision-making processes, nurses prioritised 'protection of health and life'. DISCUSSION: These findings suggest that, above all, they sought to address urgent risks to clients' lives while upholding the principle of client autonomy as much as possible. In such cases, the nurses made decisions regarding whether to share information about the client depending on the individual situation. CONCLUSION: Public health nurses should protect the client's health while taking into consideration their relationship with the client.

Development of a whole community genome amplification-assisted DNA microarray method to detect functional genes involved in the nitrogen cycle.

A novel DNA microarray analysis targeting key functional genes involved in most nitrogen cycling reactions was developed to comprehensively analyze microbial populations associated with the nitrogen cycle. The developed microarray contained 876 oligonucleotide probes based on the nucleotide sequences of the nif, amo, hao/hzo, nap, nar, nirK, nirS, nrf, cnor, qnor and nos genes. An analytical method combining detection by the designed microarray with whole community genome amplification was then applied to monitor the nitrogen cycling microorganisms in river water and wastewater treatment sludge samples. The developed method revealed that nitrogen cycling microorganisms in river water appeared to become less diverse in response to input of effluent from municipal wastewater treatment plants. Additionally, the nitrogen cycling community associated with anaerobic ammonium oxidation and partial nitrification reactors could be reasonably analyzed by the developed method. However, the results obtained for two activated sludge samples from municipal wastewater treatment plants with almost equivalent wastewater treatment performance differed greatly from each other. These results suggested that the developed method is useful for comprehensive analysis of nitrogen cycling microorganisms, although its applicability to complex samples with abundant untargeted populations should be further examined.

Bacterial community dynamics in a full-scale municipal wastewater treatment plant employing conventional activated sludge process.

To elucidate the bacterial community dynamics in a full-scale wastewater treatment plant (WWTP) and the relatedness among bacterial communities in the influent, effluent and sludge, the structure and metabolic ability of the bacterial community throughout a full-scale WWTP employing a conventional activated sludge process was investigated during a period of 10 months. The bacterial community structure was analyzed by terminal-restriction fragment length polymorphism targeting eubacterial 16S rRNA genes, while a Biolog assay was applied to assess the metabolic ability of the activated sludge. Influent bacterial community structure was generally stable. In contrast, the bacterial community structure in the effluent was similar to that in the influent in some cases, while in other cases it was unique and differed greatly from that in the influent and sludge. These results suggest that temporal variations of the effluent bacterial community may be useful to predict the wastewater treatment performance and settleability of activated sludge. The bacterial community structure in the sludge was relatively stable and was rarely impacted by the influent populations. Biolog assay also revealed that activated sludge maintained a remarkably similar metabolic potential of organic compounds over time due to functional redundancy, in which the minor populations played a significant role.

Plasmid-mediated bioaugmentation of sequencing batch reactors for enhancement of 2,4-dichlorophenoxyacetic acid removal in wastewater using plasmid pJP4.

Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100 % removal against 200 mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0 % on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10 %, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100 % after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500 mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95 %. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65 % on day 76, almost 100 % removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.

Educational content in nurse education in Japan: a Delphi study.

The purpose of this study was to clarify the minimum necessary educational content in the area of nursing ethics in a basic nursing education program, and the level of students' mastery of this content, based on a Delphi study in both educational and clinical settings. A Delphi study was conducted in three rounds with faculty members who teach nursing ethics at all 158 four-year nursing universities in Japan as targeted panelists. In this study the opinions on nursing ethics of nursing instructors responsible for hospital education at all 82 special functioning hospitals were reflected in the panelists' opinions. Consensus was obtained on 41 items from a total of 63 items in 4 proposed frameworks. There were 20 items related to the Concept of nursing ethics, 7 items related to Ethical codes, 13 items related to Ethical issues and methods to resolve them, and 1 item related to Efforts and issues in practical and educational settings. Consensus as to desired level of mastery was reached on a total of 40 items. This agreed-upon level involved understanding of the concept for 22 items, the ability to explain the concept for 16 items, and the ability to act based on the concept for 2 items.

Transfer of plasmid pJP4 from Escherichia coli and Pseudomonas putida to bacteria in activated sludge developed under different sludge retention times.

To add the knowledge on the plasmid-mediated bioaugmentation in the activated sludge process, conjugative transfer of a self-transmissible, broad host range, mercury resistance, and partial 2,4-dichlorophenoxyactic acid (2,4-D)-degrading plasmid pJP4 from Escherichia coli HB101 or Pseudomonas putida KT2440 to activated sludge bacteria were examined by filter mating. Activated sludge samples were collected from an actual wastewater treatment plant and laboratory reactors operated under different sludge retention times (SRTs) of 5, 10, and 15 days. Transfer of pJP4 was observed in all combinations of donor and activated sludge (recipient) with the transfer frequency from 6.5 x 10⁻8 to 2.5 x 10⁻4 transconjugants per recipient, except from E. coli HB101 to the activated sludge sample of SRT 5 days. Although plasmid pJP4 was transferred mainly to dominant bacteria belonging to Pseudomonas, the host range of pJP4 in the activated sludge varied depending on the donor strain. Phenotypically various transconjugants were isolated from activated sludge sampled from the reactors with longer SRT operations. All transconjugants exhibited high mercury resistance equivalent to the donor strains; some were capable of degrading 2,4-D. These results suggest that the behavior of plasmids can be controlled partially through selection of appropriate donor strains and operational conditions of the activated sludge process.

Research on the societal impacts of nanotechnology: a preliminary comparison of USA, Europe and Japan.

We initiate some comparisons between Japan, Europe and USA on how far there is governmental support for the ethical, legal, social and environmental dimensions of nanotechnology development. It is evident that in the USA and Europe nanotechnology is now firmly embedded in the consideration of ELSI. Yet Japan has not yet adequately recognized the importance of these dimensions. The history of bioethics in Japan is short. In Europe, as early as 2004, a nanotechnology report by the UK's Royal Society referred to the possibility of some nanotubes and fibres having asbestos-like toxicity. The negative history of asbestos in Europe and USA is not yet fully identified as a Japanese problem. Japan is therefore in the process of seeking how best to address societal aspects of nanotechnology. Should the precautionary principle be applied to Japan's nanotechnology initiative as in Europe? Should 5-10% of the government's nanotechnology budget be allocated to ELSI research and measures? We propose that the government and industrial sector in Japan play a much more proactive part in the regional and international growth of research into the wider risk assessment, social, health and environmental context of nanotechnologies, not simply try to borrow lessons from the West at a later date.

Nurses' perception on information privacy in Japan.

This study explores how nurses perceive patients' privacy and with whom they share the patients' information. A descriptive study was conducted on 710 nurses from three hospitals. The results show 1) nurses think of patients' privacy more importantly than their own privacy, 2) nurses hesitated somewhat to share information on "family structure" and "elimination habit" with other nurses, 3) nurses shared more limited information with the staff on the hospital food service unit than with other health professionals. Compared to the previous study conducted three years ago, this study showed nurses have become more sensitive to patient privacy in Japan.

HIV/AIDS and professional freedom of expression in Japan.

A senior physician with a government role in Japan made a widely reported and misleading statement about Thailand's policy on HIV/AIDS patients. He claimed that in Thailand the policy is to spend public money on the prevention of HIV infection while allowing AIDS patients to die untreated. The author, a community nursing specialist in Japan with first-hand knowledge of HIV/AIDS policy in Thailand, thought that this statement would influence attitudes negatively in Japan. However, speaking out about this misrepresentation of the facts carried certain risks. Although freedom of expression is valued in Japan, in practice it is not easy to contradict senior medical professionals. The author uses his experience of this difficult professional situation to teach nurses how to approach speaking out in the public interest.