Fgf10 mutant newts regenerate normal hindlimbs despite severe developmental defects.

In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb development, but whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established Fgf10 mutant lines in the newt Pleurodeles waltl which has amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs fail to develop and downregulate FGF target genes. Despite these developmental defects, Fgf10 mutants were able to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype. Together, our results demonstrate an important role for FGF10 in hindlimb formation, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

CRISPR/Cas9-based simple transgenesis in Xenopus laevis.

Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.

A simple and practical workflow for genotyping of CRISPR-Cas9-based knockout phenotypes using multiplexed amplicon sequencing.

Founder animals carrying high proportions of somatic mutation induced by CRISPR-Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR-Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype-phenotype correlations in CRISPR-Cas9-based loss-of-function screening of numerous target genes in various organisms.

Molecular characterization and antimicrobial resistance of group A streptococcus isolates in streptococcal toxic shock syndrome cases in Japan from 2013 to 2018.

Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock, multi-organ failure, and puerperal sepsis and shows high mortality. Its primary cause is group A streptococcus (GAS, Streptococcus pyogenes). In this study, we genotyped the cell-surface M virulence protein gene (emm) from 621 GAS isolates obtained from patients with STSS in Japan in 2013-2018 and performed antimicrobial susceptibility testing using the broth microdilution method. The predominant emm type was found to be 1, followed by 89, 12, and 3, which were identified in more than 70 % of STSS isolates. The proportions of emm3 and emm89 increased from 2.4 % and 12.0 %, respectively, during 2010-2012 to 5.6 % and 23.3 % during 2013-2018. In contrast, the proportion of emm1 decreased from 60.6 % to 39.3 % during the same two periods. Some emm types showed increasing proportions and were not isolated from patients with STSS in 2010-2012. Among these, an emm76 type increased in prevalence and was not included in the 30-valent M protein-based vaccine. Continual investigation of changes in the epidemiology of GAS which causes STSS can provide useful monitoring information such as future vaccination strategies and the emergence status of antimicrobial-resistant bacteria.

T serotyping of group a streptococcus isolated from patients with pharyngitis or streptococcal toxic shock syndrome in Japan between 2005 and 2017.

Streptococcus pyogenes (group A streptococcus; GAS) is an important gram-positive human pathogen capable of causing diseases ranging from mild superficial skin and pharyngeal infections to more severe invasive diseases, including streptococcal toxic shock syndrome (STSS). GAS produces a T protein, and T serotyping has considerable discriminatory power for epidemiological characterization of GAS. To clarify the relationship between STSS and pharyngitis in Japan, we examined the T serotypes of GAS strains isolated from clinical specimens of streptococcal infections (STSS, 951 isolates; pharyngitis, 16268 isolates) from 2005 to 2017. The most prevalent T serotype from pharyngitis isolates was T12, followed by T1, T4, and TB3264. The most prevalent T serotype from STSS isolates was T1, followed by TB3264. Trend of increase and decrease in the frequency of T1 or TB3264 isolation from pharyngitis was correlated with that of STSS patients. The increase of T1 or TB3264 strain-infection in pharyngitis patients may increase the probability of causing STSS, indicating that careful monitoring of GAS serotypes is essential for the prediction of rapid increase of STSS in time to develop effective management strategies.

Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration.

Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl, a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase, pax6, tbx5); notably, all tyrosinase Cas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F0 generation. In addition, we demonstrated the generation of tyrosinase null F1 offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders.

Reactivation of larval keratin gene (krt62.L) in blastema epithelium during Xenopus froglet limb regeneration.

Limb regeneration is considered a form of limb redevelopment because of the molecular and morphological similarities. Forming a regeneration blastema is, in essence, creating a developing limb bud in an adult body. This reactivation of a developmental process in a mature body is worth studying. Xenopus laevis has a biphasic life cycle that involves distinct larval and adult stages. These distinct developmental stages are useful for investigating the reactivation of developmental processes in post-metamorphic frogs (froglets). In this study, we focused on the re-expression of a larval gene (krt62.L) during Xenopus froglet limb regeneration. Recently renamed krt62.L, this gene was known as the larval keratin (xlk) gene, which is specific to larval-tadpole stages. During limb regeneration in a froglet, krt62.L was re-expressed in a basal layer of blastema epithelium, where adult-specific keratin (Krt12.6.S) expression was also observable. Nerves produce important regulatory factors for amphibian limb regeneration, and also play a role in blastema formation and maintenance. The effect of nerve function on krt62.L expression could be seen in the maintenance of krt62.L expression, but not in its induction. When an epidermis-stripped limb bud was grafted in a froglet blastema, the grafted limb bud could reach the digit-forming stage. This suggests that krt62.L-positive froglet blastema epithelium is able to support the limb development process. These findings imply that the developmental process is locally reactivated in an postmetamorphic body during limb regeneration.

Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis.

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation.

Outbreak of Legionnaire's Disease Caused by Legionella pneumophila Serogroups 1 and 13.

In Japan, hot springs and public baths are the major sources of legionellosis. In 2015, an outbreak of Legionnaires' disease occurred among 7 patients who had visited a spa house. Laboratory investigation indicated that L. pneumophila serogroup 1 and 13 strains caused the outbreak and that these strains were genetically related.

Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.

In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration.

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.

Cold sensitivity among female clinical nurses in Japan: A nationwide study.

Objectives: This study aimed to clarify the nature of cold sensitivity in female nurses working in hospitals in Japan. Design: A cross-sectional post survey research design. Settings: The questionnaire targeted nurses working in 14 hospitals across ten prefectures throughout Japan. Participants: 1,138 female nurses. Methods: The survey captured participants' characteristics, work environment, health, and lifestyle. The factors investigated were perceived cold sensitivity, cold sensitivity according to the Hiesho Sensation Scale, and cold sensitivity during nursing care. The Diagnostic Inventory of Health and Life Habits (DIHAL.2) assessed health and lifestyle habits. Results: Of the respondents, 44.3 % were aware of daily cold sensitivity and 21.4 % had a cold sensitivity disorder. The majority of respondents (63.1 %) felt cold in their hands when providing nursing care, and 28.1 % felt discomfort due to the coldness of their hands. Individuals diagnosed with cold sensitivity disorders exhibited poorer overall health compared to those without such disorders. However, there was no statistically significant difference in the overall health status between individuals who reported perceiving cold sensitivity and those who did not. The group with perceived cold sensitivity had significantly lower scores on exercise behavior, meal regularity, rest, sleep regularity, and sleep sufficiency. The proportion of female nurses working in Japanese hospitals who experienced cold sensitivity was similar to that of Japanese women with the same sensitivity. Most female nurses experienced cold in their hands while providing care. Nearly 30 % of the respondents experienced discomfort owing to cold hands. Conclusions: The study underscores the widespread issue of cold sensitivity among female nurses in Japanese hospitals, emphasizing the critical need for targeted interventions to improve their comfort and optimize care provision.

Assisted bathing of older adults with dementia: a mixed methods systematic review update.

OBJECTIVE: This review update aimed to determine the best strategies for assisted bathing or showering for older adults with dementia. INTRODUCTION: Assisted bathing is a high-risk activity, as it can trigger agitated behaviors. Assisted bathing of older adults with dementia can create caregiver challenges and stress. INCLUSION CRITERIA: This review update considered quantitative, qualitative, and mixed methods studies that investigated, firstly, older adults with dementia who required assistance with bathing and, secondly, their caregivers and family members who provided this assistance. The quantitative component considered randomized controlled trials and quasi-experimental studies testing interventions for reducing agitated behaviors in older adults with dementia during bathing, as well as perceived confidence or satisfaction in caregivers. The qualitative component considered studies that reported on experiences of clients or caregivers during the bathing process. METHODS: A JBI mixed methods review was conducted following the convergent segregated approach. The review considered studies published between 1990 and March 11, 2022. The databases searched were PubMed, CINAHL, and Embase. Gray literature was also searched. Two independent reviewers screened titles and abstracts. Full texts were retrieved for studies that met the inclusion criteria and were assessed further for eligibility. Two reviewers independently assessed the quality of included studies and extracted data using the standardized JBI tools. Due to methodological and clinical heterogeneity, the results were presented narratively in the quantitative section. For the qualitative component, meta-synthesis was conducted following the JBI approach of meta-aggregation. Finally, evidence from the 2 components was integrated following the convergent segregated approach. RESULTS: Ten quantitative and 4 qualitative studies were included. The methodological quality was poor to moderate in the quantitative studies and moderate to high in the qualitative studies. Results from 3 quantitative studies suggested that providing training to caregivers on person-centered bathing reduced agitated behaviors in older adults with dementia. Other interventions did not show conclusive evidence of their effectiveness in any outcomes of interest. Two synthesized findings highlighted i) the importance of working within each person's reality by having the skills and knowledge required to deliver person-centered assistance and ii) the challenges experienced by caregivers, such as lack of support, time pressure, and safety-related fears. The integrated evidence showed that the quantitative and qualitative components complemented each other to promote the training of caregivers to deliver person-centered bathing. DISCUSSION: Integrated findings can help inform an evidence-based strategy utilizing a person-centered bathing approach to reduce agitated behaviors in older adults with dementia. Due to the limited number of eligible studies, and the clinical and methodological heterogeneity of included quantitative studies, no statistical pooling was possible. More studies are needed, particularly intervention studies with high methodological quality. CONCLUSIONS: This review update suggests that providing caregivers with person-centered bathing training should be encouraged prior to bathing older adults with dementia. Caregivers should have the knowledge and skills, such as relevant assessment and communication skills, enabling them to provide effective bathing experiences to older adults living with dementia. Organizations should provide caregivers with appropriate resources and training for bathing older adults with dementia. REVIEW REGISTRATION: PROSPERO CRD42020208048. SUPPLEMENTAL DIGITAL CONTENT: A Japanese-language version of the abstract of this review is available as supplemental digital content 1: http://links.lww.com/SRX/A37 .

CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl.

Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool.

[Clinical study of smoking cessation clinic subjects].

The Cigarette-associated diseases will be decreased only by stopping smoking. At a smoking cessation clinic, we evaluated 22 subjects for smoking cessation after a three-month smoking cessation program. The program was completed by 20 subjects, or 90%, of whom 15, or 68%, quit smoking. Varenicline tartrate was used to treat 90% of subject, of whom 8, or 40%, suffered nausea. This was treated using an anti-nausea agent, which relieved symptoms immediately.

[The relationship of empathic-affective responses toward others' positive affect with prosocial behaviors and aggressive behaviors].

The purposes of this study were to develop and validate the Empathic-Affective Response Scale, and to examine the relationship of empathic-affective responses with prosocial behaviors and aggressive behaviors. Undergraduate students (N = 443) participated in a questionnaire study. The results of factor analysis indicated that empathic-affective responses involved three factors: (a) sharing and good feeling toward others' positive affect, (b) sharing of negative affect and (c) sympathy toward others' negative affect. Correlations with other empathy-related scales and internal consistency suggested that this scale has satisfactory validity and reliability. Cluster analysis revealed that participants were clustered into four groups: high-empathic group, low-empathic group, insufficient positive affective response group and insufficient negative affective response group. Additional analysis showed the frequency of prosocial behaviors in high-empathic group was highest in all groups. On the other hand, the frequency of aggressive behaviors in both insufficient positive affective response group and low-empathic group were higher than others' groups. The results indicated that empathic-affective responses toward positive affect are also very important to predict prosocial behaviors and aggressive behaviors.

Effect of nursing procedures on the hand skin temperature of female hospital nurses.

AIMS: Nurses' hand temperature may affect patient comfort but has not been investigated. This study aimed to determine female hospital nurses' hand skin temperature and clarify the effects of measurement site, time, nursing procedures, and environment. DESIGN: An observational study. METHODS: The middle finger, thenar eminence, hypothenar eminence, and medial forearm skin temperature of 29 female hospital nurses was measured at four time points during a day shift and before and after nursing procedures (hand disinfection, hand washing, taking vital signs, hygiene care, and positioning). RESULTS: Mean hand skin temperature was in the range of 29-32°C with interpersonal variations. Mean skin temperature at the medial forearm was 31.94-32.35°C (SD 0.87-1.52°C) and at the middle finger, 29.73-31.07°C (SD > 3°C). Time-dependent skin temperature fluctuations were confirmed on the middle finger (p = .022), and thenar (p = .005) and hypothenar eminence (p = .001). There were weak correlations between skin temperature and environmental factors, including ambient temperature (ρ = .194-.266), humidity (ρ = -.309 to -.319), and hospital room wind speed (ρ = .253-.314). The skin temperature dropped significantly after hand disinfection and handwashing at all measurement sites (middle finger: -1.30 and -3.56°C, respectively; thenar eminence: -1.19 and -3.32°C; hypothenar eminence: -0.80 and -3.39°C; medial forearm: -0.21 and -1.60°C). CONCLUSION: These findings may raise nurses' awareness of their skin temperature. Moreover, our study highlights the need to develop countermeasures to ensure optimal nurses' skin temperature and patient comfort.

Influence of epinastine hydrochloride, an H1-receptor antagonist, on the function of mite allergen-pulsed murine bone marrow-derived dendritic cells in vitro and in vivo.

There is established concept that dendritic cells (DCs) play essential roles in the development of allergic immune responses. However, the influence of H(1) receptor antagonists on DC functions is not well defined. The aim of the present study was to examine the effect of epinastine hydrochloride (EP), the most notable histamine H(1) receptor antagonists in Japan, on Dermatophagoides farinae (Der f)-pulsed mouse bone marrow-derived DCs in vitro and in vivo. EP at more than 25 ng/mL could significantly inhibit the production of IL-6, TNF-alpha and IL-10 from Der f-pulsed DCs, which was increased by Der f challenge in vitro. On the other hand, EP increased the ability of Der f-pulsed DCs to produce IL-12. Intranasal instillation of Der f-pulsed DCs resulted in nasal eosinophilia associated with a significant increase in IL-5 levels in nasal lavage fluids. Der f-pulsed and EP-treated DCs significantly inhibited nasal eosinophila and reduced IL-5. These results indicate that EP inhibits the development of Th2 immune responses through the modulation of DC functions and results in favorable modification of clinical status of allergic diseases.

A comprehensive reference transcriptome resource for the Iberian ribbed newt Pleurodeles waltl, an emerging model for developmental and regeneration biology.

Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).