Search

Abstract Biological rhythms are involved in almost all types of biological processes, not only physiological processes but also morphogenesis. Currently, how periodic morphological patterns of tissues/organs in multicellular organisms form is not fully understood. Here, using mouse zigzag hair, which has 3 bends, we found that a change in the combination of hair progenitors and their micro-niche and subsequent bend formation occur every three days. Chimeric loss-of-function and gain-of-function of Ptn and Aff3, which are upregulated immediately before bend formation, resulted in defects in the downward movement of the micro-niche and the rhythm of bend formation in an in vivo hair reconstitution assay. Our study demonstrates the periodic change in the combination between progenitors and micro-niche, which is vital for the unique infradian rhythm.

Globally, the rapid aging of the population is predicted to become even more severe in the second half of the 21st century. Thus, it is expected to establish a growing expectation for innovative, non-invasive health indicators and diagnostic methods to support disease prevention, care, and health promotion efforts. In this study, we aimed to establish a new health index and disease diagnosis method by analyzing the minerals and free amino acid components contained in hair shaft. We first evaluated the range of these components in healthy humans and then conducted a comparative analysis of these components in subjects with diabetes, hypertension, androgenetic alopecia, major depressive disorder, Alzheimer's disease, and stroke. In the statistical analysis, we first used a student's t test to compare the hair components of healthy people and those of patients with various diseases. However, many minerals and free amino acids showed significant differences in all diseases, because the sample size of the healthy group was very large compared to the sample size of the disease group. Therefore, we attempted a comparative analysis based on effect size, which is not affected by differences in sample size. As a result, we were able to narrow down the minerals and free amino acids for all diseases compared to t test analysis. For diabetes, the t test narrowed down the minerals to 15, whereas the effect size measurement narrowed it down to 3 (Cr, Mn, and Hg). For free amino acids, the t test narrowed it down to 15 minerals. By measuring the effect size, we were able to narrow it down to 7 (Gly, His, Lys, Pro, Ser, Thr, and Val). It is also possible to narrow down the minerals and free amino acids in other diseases, and to identify potential health indicators and disease-related components by using effect size.

Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-β-lactamase (NDM)-1, and NDM-5. The strain harboring blaNDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing blaIMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN4 plasmid type. However, integrons found in the strains possessing blaIMP-6, which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae, suggesting the prevalence of identical integrons. Transposons containing blaNDM are similar or identical to the transposon structure of K. aerogenes harboring blaNDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens.

In Japan, nationwide epidemiological surveys on carbapenem-resistant Enterobacterales (CREs), including comprehensive information, are scarce, with most data available only through public reports. This study analyzed data on the Enterobacterales family collected from nationwide testing centers between January 2016 and December 2022, focusing on isolates that met the criteria for CRE in Japan based on drug susceptibility. We investigated 5,323,875 Enterobacterales isolates of 12 different species; among 4696 (0.09%) CRE strains, the proportion of major CRE isolates was as follows: Escherichia coli, 31.3%; Klebsiella pneumoniae, 28.0%; Enterobacter cloacae, 18.5%; and Klebsiella aerogenes, 6.7%. Moreover, over a 7-year period, Providencia rettgeri, E. cloacae, K. aerogenes, and K. pneumoniae demonstrated relatively high CRE percentages of 0.6% (156/26,185), 0.47% (869/184,221), 0.28% (313/110,371), and 0.17% (1314/780,958), respectively. The number of CRE strains isolated from different samples was as follows: urine, 2390; respiratory specimens, 1254; stool, 425; blood, 252; others, 375. In the broader context, including colonization, the predominant isolates of CREs collected at nationwide testing centers are E. coli and K. pneumoniae. Furthermore, recently, attention has been directed toward less common CRE species, such as Klebsiella oxytoca and Providencia rettgeri, and thus, it might be necessary to continue monitoring these less common species.

Abstract In mammals, organ induction occurs only during embryonic development except for hair follicles (HFs). However, HF-resident epithelial stem cells (HFSCs), which are responsible for repetitive HF regeneration, are not fully characterized. Here, we establish in vitro culture systems that are capable of controlling the ability of HFSCs to regenerate HFs. Based on a method that precisely controlled the number of HFs for regeneration, functional analysis revealed that CD34/CD49f/integrin β5 (Itgβ5)-triple-positive (CD34+/CD49f+/Itgβ5+) cells have multipotency and functional significance for continual hair regeneration. In native HFs, these cells reside in the uppermost area of the bulge region, which is surrounded by tenascin in mice and humans. This study unveils the subpopulation of HFSCs responsible for long-term hair cycling of HFs regenerated from bioengineered HF germ, suggesting the presence of functional heterogeneity among bulge HFSCs and the utility of our culture system to achieve HF regenerative therapy.

ABSTRACT The genus Pseudomonas, a complex Gram-negative genus, includes species isolated from various environments, plants, animals, and humans. We compared whole-genome sequencing (WGS) with clinical bacteriological methods and evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify Pseudomonas species. Clinical isolates (N = 42) identified as P. putida or P. fluorescens by a bacterial identification system based on biochemical properties were reexamined by another identification system based on biochemical properties, two systems based on MALDI-TOF MS, and WGS. WGS revealed that 30 of the 42 isolates belonged to one of 14 known Pseudomonas species, respectively. The remaining 12 belonged to one of 9 proposed novel Pseudomonas species, respectively. MALDI-TOF MS analysis showed that the 9 novel species had unique major peaks. These results suggest that WGS is the optimal method to identify Pseudomonas species and that MALDI-TOF MS may complement WGS in identification. Based on their morphologic, physiologic, and biochemical properties, we propose nine novel Pseudomonas species. IMPORTANCE Most of the clinical isolates, identified as P. putida or P. fluorescens, were misidentified in clinical laboratories. Whole-genome sequencing (WGS) revealed that these isolates belonged to different Pseudomonas species, including novel species. WGS is a gold-standard method to identify Pseudomonas species, and MALDI-TOF MS analysis has the potential to complement WGS to reliably identify them.

Objective: To provide data on the molecular characteristics of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae clinical isolates in Japan. Methods: A total of 100 clinical isolates of ESBL-producing K. pneumoniae collected throughout Japan between June and July 2018 were studied. ESBL genes were analyzed using PCR and DNA sequencing. Transferability of ESBL genes was investigated by conjugation experiments. Plasmid replicon types, virulence genes (rmpA, rmpA2, iucA, iroB, and peg-344) associated with hypervirulent K. pneumoniae (hvKp), and capsule types were detected using PCR. Genotyping was performed using multilocus sequence typing. Results: All ESBL-producing isolates carried blaCTX-M genes. The most predominant CTX-M-type identified was CTX-M-15 (n = 55). We identified 24 sequence types (STs) among the CTX-M-15 producers, with ST25 (n = 8) being the most common. Most of the transconjugants carrying blaCTX-M-15 contained the FIIk replicon. Of the 100 ESBL-producing isolates, 31 were hvKp defined by the presence of the virulence genes. These ESBL-producing hvKp isolates belonged to eight STs (STs 23, 25, 36, 65, 86, 268, 412, and 4492), with five capsule types (K1, K2, K20, K57, and undefined). Conclusions: CTX-M-15 was the predominant ESBL among K. pneumoniae isolates from Japan. This study shows that ESBL-producing hvKp strains comprising various clones are emerging in Japan.

Abstract Background The spread of Enterobacteriaceae producing both carbapenemases and Mcr, encoded by plasmid-mediated colistin resistance genes, has become a serious public health problem worldwide. This study describes three clinical isolates of Enterobacter cloacae complex co-harboring bla IMP-1 and mcr-9 that were resistant to carbapenem but susceptible to colistin. Methods Thirty-two clinical isolates of E. cloacae complex non-susceptible to carbapenems were obtained from patients at 14 hospitals in Japan. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods and E-tests. Their entire genomes were sequenced by MiSeq and MinION methods. Multilocus sequence types were determined and a phylogenetic tree constructed by single nucleotide polymorphism (SNP) alignment of whole genome sequencing data. Results All 32 isolates showed MICs of ≥2 μg/ml for imipenem and/or meropenem. Whole-genome analysis revealed that all these isolates harbored bla IMP-1, with three also harboring mcr-9. These three isolates showed low MICs of 0.125 μg/ml for colistin. In two of these isolates, bla IMP-1 and mcr-9 were present on two separate plasmids, of sizes 62 kb and 280/290 kb, respectively. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr-9. In the third isolate, however, both bla IMP-1 and mcr-9 were present on the chromosome. Conclusion The mcr-9 is silently distributed among carbapenem-resistant E. cloacae complex isolates, of which are emerging in hospitals in Japan. To our knowledge, this is the first report of isolates of E. cloacae complex harboring both bla IMP-1 and mcr-9 in Japan.

Various carbapenemases have been identified in the Enterobacteriaceae. However, the induction and corresponding regulator genes of carbapenemase NmcA has rarely been detected in the Enterobacter cloacae complex (ECC). The NmcA-positive isolate ECC NR1491 was first detected in Japan in 2013. It was characterized and its induction system elucidated by evaluating its associated regulator genes nmcR, ampD, and ampR. The isolate was highly resistant to all β-lactams except for third generation cephalosporins (3GC). Whole-genome analysis revealed that blaNmcA was located on a novel 29-kb putatively mobile element called EludIMEX-1 inserted into the chromosome. The inducibility of β-lactamase activity by various agents was evaluated. Cefoxitin was confirmed as a strong concentration-independent β-lactamase inducer. In contrast, carbapenems induced β-lactamase in a concentration-dependent manner. All selected 3GC-mutants harboring substitutions on ampD (as ampR and nmcR were unchanged) were highly resistant to 3GC. The ampD mutant strain NR3901 presented with a 700 × increase in β-lactamase activity with or without induction. Similar upregulation was also observed for ampC and nmcA. NR1491 (pKU412) was obtained by transforming the ampR mutant (135Asn) clone plasmid whose expression increased by ∼100×. Like NR3901, it was highly resistant to 3GC. Overexpression of ampC, rather than nmcA, may have accounted for the higher MIC in NR1491. The ampR mutant repressed nmcA despite induction and it remains unclear how it stimulates nmcA transcription via induction. Future experiments should analyze the roles of nmcR mutant strains.

ABSTRACT Carbapenemase-producing Enterobacteriaceae represent a serious public health threat worldwide. Carbapenemase genes, harbored on a transferable plasmid, have been isolated globally with distinct geographical features. Klebsiella pneumoniae, included in Enterobacteriaceae, also produces carbapenemase and often shows hypervirulence. Overlapping carbapenem resistance and hypervirulence in K. pneumoniae have been reported, but such strains have not yet been found in Japan. Here, we screened 104 carbapenemase-producing K. pneumoniae isolates collected from 37 hospitals and outpatient clinics in Japan between September 2014 and July 2015. PCR and DNA sequencing demonstrated IMP-1 in 21 isolates and IMP-6 in 83 isolates, 77 of which coharbored CTX-M-2. Most of the isolates showed low MICs toward imipenem and meropenem but high MICs toward penicillin and cephalosporins. Conjugation experiments with an Escherichia coli J53 recipient showed that most of the plasmids in IMP-6 producers were transferable, whereas only one-half of the plasmids in IMP-1 producers were transferable. PCR-based replicon typing and multiplex PCR identified five isolates belonging to the CG258 non-tonB79 cluster and no isolate belonging to the CG258-tonB79 cluster or sequence type 307 (ST307). Four K1-ST23 isolates, 10 K2-ST65 isolates, and 7 K2-ST86 isolates were detected that harbored virulence genes. The resistance genes in 85 isolates were transferable, but the virulence genes were not transferred. These results demonstrate the acquisition of IMP-type carbapenemase genes and CTX-M-type genes among hypervirulence isolates in Japan, warranting further attention and countermeasures. In this study, we have determined the molecular characteristics and epidemiology of IMP-6 producers that coharbored various CTX-M genes in Japan. IMPORTANCE Carbapenems serve as a last resort for the clinical treatment of multidrug-resistant infections. Therefore, the rapid spread of carbapenemase-producing strains represents a serious public health threat, further limiting antibiotic choices. The current findings of hypervirulent carbapenemase-producing Klebsiella pneumoniae clinical isolates in Japan demonstrate the potential broad spread and transfer of these genes, necessitating close surveillance.

Invasive Streptococcus agalactiae (GBS) infections are increasingly common among neonates and the elderly. Therefore, GBS surveillance for better antibiotic treatment and prophylaxis strategies are needed. We retrospectively evaluated the clinical aspects of invasive infections and the phenotypic and genetic diversity of infectious isolates from Nara, Japan, collected between 2007 and 2016, by using information from hospital records. GBS strains collected from the blood and cerebrospinal fluid cultures were evaluated for capsular types, multi-locus sequence typing (MLST), antibiotic susceptibility, antibiotics resistance gene, and pulsed-field gel electrophoresis. Forty GBS isolates (10 from children and 30 from adults) were analyzed, and the distribution of molecular serotype and allelic profiles varied between children and adults. We found the rates of early-onset disease in neonates with birth complications to be higher than that of previous reports, indicating that there could be relevance between complications at birth and early-onset disease. Standard antibiotic prophylaxis strategies may need to be reconsidered in patients with birth complications. In adults, the mean age of the patients was 68 years (male: 63%). Primary bacteremia was the most common source of infection. In the neonates, six had early-onset diseases and four had late-onset diseases. The most frequently identified strains were molecular serotype Ia ST23 (40%) and molecular serotype Ib ST10 (20%) in children and molecular serotype Ib ST10 (17%), molecular serotype VI ST1 (13%), and molecular serotype V ST1 (13%) in adults. Levofloxacin-resistant molecular serotype Ib strains and molecular serotypes V and VI ST1 were common causes of GBS infection in adults but were rarely found in children. Furthermore, pulsed-field gel electrophoresis in our study showed that specific clone isolates, that tend to have antibiotics resistance were widespread horizontally for a decade. Continuous surveillance and molecular investigation are warranted to identify the transmission route and improve antibiotic treatment strategies.

Functional salivary glands have not been generated from embryonic stem cells (mESCs) to date. Here the authors demonstrate directed in vitro differentiation of mESCs to oral ectoderm and salivary gland rudiments that form mature, functional salivary glands after orthotopic transplantation.

In this decade, substantial progress in the fields of developmental biology and stem cell biology has ushered in a new era for three-dimensional organ regenerative therapy. The emergence of novel three-dimensional cell manipulation technologies enables the effective mimicking of embryonic organ germ formation using the fate-determined organ-inductive potential of epithelial and mesenchymal stem cells. This advance shows great potential for the regeneration of functional organs with substitution of complete original function in situ. Organoids generated from multipotent stem cells or tissue stem cells via establishment of an organ-forming field can only partially recover original organ function owing to the size limitation; they are considered ‘mini-organs’. Nevertheless, they hold great promise to realize regenerative medicine. In particular, regeneration of a functional salivary gland and an integumentary organ system by orthotopic and heterotopic implantation of organoids clearly points to the future direction of organ regeneration research. In this review, we describe multiple strategies and recent progress in regenerating functional three-dimensional organs, focusing on ectodermal organs, and discuss their potential and future directions to achieve organ replacement therapy as a next-generation regenerative medicine.

The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses.

Open cultivation systems to monoculture microorganisms are promising for the commercialization of low-value commodities because they reduce the cultivation cost. However, contamination from biological pollutants frequently impedes the process. Here we propose a cultivation method using hollow hydrogel microfibers encapsulating microorganisms. Due to the pore size, hydrogels allow nutrients and waste to pass through while preventing invading microorganisms from entering the microfiber. Experimental cultivation shows the growth of target bacteria inside the alginate hydrogel microfiber during exposure to invading bacteria. The membrane thickness of the microfiber greatly affects the bacterial growth due to changes in membrane permeability. The enhancement of mechanical toughness is also demonstrated by employing a double-network hydrogel for long-term cultivation. The hollow hydrogel microfiber has the potential to become a mainstream solution for mass-cultivation of microorganisms in an open system.

The prevalence of ESBL has been increasing worldwide. In this study, we investigated the molecular characteristics of ESBL among clinical isolates of Escherichia coli from a Japanese tertiary hospital. A total of 71 consecutive and nonduplicate clinical isolates of ESBL-positive E. coli collected at Tohoku University Hospital between January 2008 and March 2011 were studied. The antimicrobial susceptibility profile of these strains was determined. PCR and sequencing were performed to identify genes for β-lactamase (bla(TEM), bla(SHV), bla(OXA-1-like), and bla(CTX-M)) and plasmid-mediated quinolone resistance determinants (PMQR). The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Of the 71 strains, 68 were positive for CTX-M, 28 were positive for TEM, four were positive for OXA-1, and one was positive for SHV. Sequencing revealed that CTX-M-14 was the most prevalent (31/71), followed by CTX-M-27 (21/71) and then CTX-M-15 (9/71). Of the 28 TEM-positive strains, one was TEM-10 and the rest were TEM-1. One SHV-positive strain was SHV-12. The 21 CTX-M-27-producing isolates were divided into 14 unique PFGE types, while the 9 CTX-M-15 producers were divided into 8 types. Based on MLST, 9 CTX-M-14 procedures, 19 CTX-M-27 procedures, and 8 CTX-M-15 producers belonged to ST131. Thirty-five (94.6%) of the 37 ST131 E. coli strains showed resistance to levofloxacin, which was a higher rate than among non-ST131 strains (63.6%). Among ESBL-producing isolates, one, two, and six possessed qnrB, qnrS, qepA, and aac(6')-Ib-cr, respectively. Of the 6 isolates with aac(6')-Ib-cr, 4 carried the CTX-M-15 gene. Our data suggest that CTX-M-15-producing E. coli ST131 has emerged as a worldwide pandemic clone, while CTX-M-27 (a variant of CTX-M-14) is also spreading among E. coli ST131 in Japan.

We propose mental fatigue measurement using a wearable eye detection system. The system is capable of acquiring movement of the pupil and blinking from the light reflected from the eye. The reflection is detected by dye-sensitized photovoltaic cells. Since these cells are patterned onto the eyeglass and do not require external input power, the system is notable for its lightweight and low power consumption and can be combined with other wearable devices, such as a head mounted display. We performed experiments to correlate information obtained by the eye detection system with the mental fatigue of the user. Since it is quite difficult to evaluate mental fatigue objectively and quantitatively, we assumed that the National Aeronautics and Space Administration Task Load Index (NASA-TLX) had a strong correlation with te mental fatigue. While a subject was requested to conduct calculation tasks, the eye detection system collected his/her information that included position, velocity and total movement of the eye, and amount and frequency of blinking. Multiple regression analyses revealed the correlation between NASA-TLX and the information obtained for 3 out of 5 subjects.

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.

Donor organ transplantation is currently an essential therapeutic approach to the replacement of a dysfunctional organ as a result of disease, injury or aging in vivo. Recent progress in the area of regenerative therapy has the potential to lead to bioengineered mature organ replacement in the future. In this proof of concept study, we here report a further development in this regard in which a bioengineered tooth unit comprising mature tooth, periodontal ligament and alveolar bone, was successfully transplanted into a properly-sized bony hole in the alveolar bone through bone integration by recipient bone remodeling in a murine transplantation model system. The bioengineered tooth unit restored enough the alveolar bone in a vertical direction into an extensive bone defect of murine lower jaw. Engrafted bioengineered tooth displayed physiological tooth functions such as mastication, periodontal ligament function for bone remodeling and responsiveness to noxious stimulations. This study thus represents a substantial advance and demonstrates the real potential for bioengineered mature organ replacement as a next generation regenerative therapy.