Identification and characterization of a fibroblast growth factor gene in the planarian Dugesia japonica.

Planarians belong to the phylum Platyhelminthes and can regenerate their missing body parts after injury via activation of somatic pluripotent stem cells called neoblasts. Previous studies suggested that fibroblast growth factor (FGF) signaling plays a crucial role in the regulation of head tissue differentiation during planarian regeneration. To date, however, no FGF homologues in the Platyhelminthes have been reported. Here, we used a planarian Dugesia japonica model and identified an fgf gene termed Djfgf, which encodes a putative secreted protein with a core FGF domain characteristic of the FGF8/17/18 subfamily in bilaterians. Using Xenopus embryos, we found that DjFGF has FGF activity as assayed by Xbra induction. We next examined Djfgf expression in non-regenerating intact and regenerating planarians. In intact planarians, Djfgf was expressed in the auricles in the head and the pharynx. In the early process of regeneration, Djfgf was transiently expressed in a subset of differentiated cells around wounds. Notably, Djfgf expression was highly induced in the process of head regeneration when compared to that in the tail regeneration. Furthermore, assays of head regeneration from tail fragments revealed that combinatorial actions of the anterior extracellular signal-regulated kinase (ERK) and posterior Wnt/ß-catenin signaling restricted Djfgf expression to a certain anterior body part. This is the region where neoblasts undergo active proliferation to give rise to their differentiating progeny in response to wounding. The data suggest the possibility that DjFGF may act as an anterior counterpart of posteriorly localized Wnt molecules and trigger neoblast responses involved in planarian head regeneration.

The molecular logic for planarian regeneration along the anterior-posterior axis.

The planarian Dugesia japonica can regenerate a complete individual from a head, trunk or tail fragment via activation of somatic pluripotent stem cells. About a century ago, Thomas Hunt Morgan attempted to explain the extraordinary regenerative ability of planarians by positing two opposing morphogenetic gradients of formative "head stuff" and "tail stuff" along the anterior-posterior axis. However, Morgan's hypothesis remains open to debate. Here we show that extracellular signal-related kinase (ERK) and Wnt/ß-catenin signalling pathways establish a solid framework for planarian regeneration. Our data suggest that ERK signalling forms a spatial gradient in the anterior region during regeneration. The fibroblast growth factor receptor-like gene nou-darake (which serves as an output of ERK signalling in the differentiating head) and posteriorly biased ß-catenin activity negatively regulate ERK signalling along the anterior-posterior axis in distinct manners, and thereby posteriorize regenerating tissues outside the head region to reconstruct a complete head-to-tail axis. On the basis of this knowledge about D. japonica, we proposed that ß-catenin signalling is responsible for the lack of head-regenerative ability of tail fragments in the planarian Phagocata kawakatsui, and our confirmation thereof supports the notion that posterior ß-catenin signalling negatively modulates the ERK signalling involved in anteriorization across planarian species. These findings suggest that ERK signalling has a pivotal role in triggering globally dynamic differentiation of stem cells in a head-to-tail sequence through a default program that promotes head tissue specification in the absence of posteriorizing signals. Thus, we have confirmed the broad outline of Morgan's hypothesis, and refined it on the basis of our proposed default property of planarian stem cells.

Usefulness of multiple chalk-based food colorings for inducing better gene silencing by feeding RNA interference in planarians.

Planarians have become widely recognized as one of the major animal models for regeneration studies in invertebrates. To induce RNA interference (RNAi) by feeding in planarians, the widely accepted protocol is one in which animals undergo two or three feedings of food containing double-stranded RNA (dsRNA) plus visible food coloring (e.g., blood) for confirmation of feeding by individual animals. However, one possible problem is that incorporated food coloring is often retained within the gut for several days, which makes it difficult to confirm the success of each round of dsRNA feeding based on the difference of the color density within the gut before and after feeding. As a consequence, the difference of appetite levels among individuals undergoing dsRNA feeding leads to phenotypic variability among them due to insufficient knockdown. In our attempts to overcome this problem, we have developed a novel method for achieving robust confirmation of the success of dsRNA feeding in individuals fed multiple times by means of including a combination of three different colored chalks (pink, yellow and blue) as food coloring. Notably, we found that this method is superior to the conventional method for positively marking individuals that actively consumed the dsRNA-containing food during four times of once-daily feeding. Using these selected animals, we obtained stable and sufficiently strong RNAi-induced phenotypes. We termed this improved multi-colored chalk-spiked method of feeding RNAi "Candi" and propose its benefits for gene function analysis in planarians.

Role of MEKK1 in the anterior-posterior patterning during planarian regeneration.

Planarians have established a unique body pattern along the anterior-posterior (AP) axis, which consists of at least four distinct body regions arranged in an anterior to posterior sequence: head, prepharyngeal, pharyngeal (containing a pharynx), and tail regions, and possess high regenerative ability. How they reconstruct the regional continuity in a head-to-tail sequence after amputation still remains unknown. We use as a model planarian Dugesia japonica head regeneration from tail fragments, which involves dynamic rearrangement of the body regionality of preexisting tail tissues along the AP axis, and show here that RNA interference of the gene D. japonica mek kinase 1 (Djmekk1) caused a significant anterior shift in the position of pharynx regeneration at the expense of the prepharyngeal region, while keeping the head region relatively constant in size, and accordingly led to development of a relatively longer tail region. Our data suggest that DjMEKK1 regulates anterior extracellular signal-regulated kinase (ERK) and posterior ß-catenin signaling pathways in a positive and negative manner, respectively, to establish a proper balance resulting in the regeneration of planarian's scale-invariant trunk-to-tail patterns across individuals. Furthermore, we demonstrated that DjMEKK1 negatively modulates planarian ß-catenin activity via its serine/threonine kinase domain, but not its PHD/RING finger domain, by testing secondary axis formation in Xenopus embryos. The data suggest that Djmekk1 plays an instructive role in the coordination between the establishment of the prepharyngeal region and posteriorizing of pharynx formation by balancing the two opposing morphogenetic signals along the AP axis during planarian regeneration.

Simple blood-feeding method for live imaging of gut tube remodeling in regenerating planarians.

Live cell imaging is a powerful technique to study cellular dynamics in vivo during animal development and regeneration. However, few live imaging methods have been reported for studying planarian regeneration. Here, we developed a simple method for steady visualization of gut tube remodeling during regeneration of a living freshwater planarian, Dugesia japonica. When planarians were fed blood several times, gut branches were well-visualized in living intact animals under normal bright-field illumination. Interestingly, tail fragments derived from these colored planarians enabled successive observation of the processes of the formation of a single anterior gut branch in the prepharyngeal region from the preexisting two posterior gut branches in the same living animals during head regeneration. Furthermore, we combined this method and RNA interference (RNAi) and thereby showed that a D. japonica raf-related gene (DjrafA) and mek-related gene (DjmekA) we identified both play a major role in the activation of extracellular signal-regulated kinase (ERK) signaling during planarian regeneration, as indicated by their RNAi-induced defects on gut tube remodeling in a time-saving initial screening using blood-feeding without immunohistochemical detection of the gut. Thus, this blood-feeding method is useful for live imaging of gut tube remodeling, and provides an advance for the field of regeneration study in planarians.

Bridging the Language Gap: The Role of Human-Mediated Translation in Japanese Medical Settings.

Introduction Foreign residents in Japan often face challenges accessing healthcare due to language barriers, potentially leading to health inequities. This study aimed to assess the utilization and impact of human-mediated translation services in a specific medical setting in Tokyo. Methods A retrospective investigation was conducted on medical records of foreign patients who utilized human-mediated translation services at Navitas Clinic Tachikawa (Tachikawa, Tokyo, Japan) from November 2017 to December 2021. Data on age, gender, language used, department visited, diagnosis, insurance status, and booking methods were analyzed. Results Out of the 124 foreign patients who utilized the human-mediated translation services during the study period, 69 (56%) were male, and 55 (44%) were female. The median age was 35 years, with a range from 3 to 61 years. English was the predominant language used by 34 patients (59%), followed by Chinese for nine patients (16%) and Spanish for four patients (7%). The majority, 107 patients (86%) visited the internal medicine department, nine patients (7%) consulted dermatology, and six patients (5%) visited pediatrics. Regarding insurance status, 47 patients (81%) were insured, three patients (5%) were uninsured by the Japanese national health insurance system, and eight patients (14%) were self-pay. The primary mode of appointment booking was at the reception desk, with 112 patients (90%) using this method, while 12 patients (10%) made reservations online. Conclusions The findings of this study underscore the importance of human-mediated translation services for improving healthcare accessibility for foreign residents in Japan, emphasizing the need to address language barriers and promote health equity in clinical settings. Future studies should also explore challenges faced in patient-physician interactions from a linguistic perspective and potential technological solutions to enhance these services.

Geographical Profiles of COVID-19 Outbreak in Tokyo: An Analysis of the Primary Care Clinic-Based Point-of-Care Antibody Testing.

Introduction: The primary care clinic plays a major role in triage for coronavirus disease 2019 (COVID-19), where seroprevalence in the setting of primary care clinic remains less clear. As a point-of-care immunodiagnostic test for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the serosurvey represents an alternative to the polymerase chain reaction (PCR) test to measure the magnitude of COVID-19 outbreak in the communities lacking sufficient diagnostic capability for PCR testing. Methods: We assessed seropositivity for the SARS-CoV-2 IgG between April 21 and May 20, 2020, at 2 primary care clinics in Tokyo, Japan. Results: The overall positive percentage of SARS-CoV-2 IgG was 3.83% (95% confidence interval [CI]: 2.76-5.16) for the entire cohort (n = 1071). The 23 special wards of central Tokyo exhibited a significantly higher prevalence compared with the other areas of Tokyo after classification by residence (P = .02, 4.68% [3.08-6.79] vs 1.83 [0.68-3.95] in central and suburban Tokyo, respectively). In central Tokyo, the southern area showed the highest seroprevalence compared with the other areas (7.92% [3.48-15.01]), corresponding to the cumulative number of confirmed COVID-19 patients by PCR test reported by the Tokyo Metropolitan Government. Conclusion: The seroprevalence surveyed in this study was too low for herd immunity, suggesting the need for robust disease control and prevention. A regional-level approach, rather than state- or prefectural-level, could be of importance in ascertaining detailed profiles of the COVID-19 outbreak.

The pharyngeal nervous system orchestrates feeding behavior in planarians.

Planarians exhibit traits of cephalization but are unique among bilaterians in that they ingest food by means of goal-directed movements of a trunk-positioned pharynx, following protrusion of the pharynx out of the body, raising the question of how planarians control such a complex set of body movements for achieving robust feeding. Here, we use the freshwater planarian Dugesia japonica to show that an isolated pharynx amputated from the planarian body self-directedly executes its entire sequence of feeding functions: food sensing, approach, decisions about ingestion, and intake. Gene-specific silencing experiments by RNA interference demonstrated that the pharyngeal nervous system (PhNS) is required not only for feeding functions of the pharynx itself but also for food-localization movements of individual animals, presumably via communication with the brain. These findings reveal an unexpected central role of the PhNS in the linkage between unique morphological phenotypes and feeding behavior in planarians.

Demographic and geographical characteristics of pediatric patients presenting to a convenient clinic at a large railway station in a metropolitan area of Tokyo.

There is an increasing demand for medical provision systems that are friendly for working mothers with sick children in Japan. The aim of this cross-sectional, observational study was to analyze the demographic characteristics of pediatric patients presenting to a convenient care clinic, which was located in a large railway station and offered primary care with after-hours accessibility in a metropolitan area of Tokyo.We analyzed anonymous data for patients who had visited the pediatric department at a clinic between August 2013 and June 2016. Data regarding patients' sex, age, time of visit, waiting time, presence or absence of an appointment, diagnosis, and addresses were collected from electronic health and billing records.Overall, 8091 patients visited the department 45,388 times. The numbers of visits by patients who resided within 2, 5, and 10 miles of the clinic were 37,160 (84.6%), 42,336 (96.4%), and 43,399 (98.8%), respectively. No seasonal variation in the number of visits was observed. Male patients visited the clinic 23,742 times (52.3%) and the patients' median age was 3 years (interquartile range, 1-6). Most visits occurred on Mondays, and 5643 (15.2%) and 4790 (12.9%) patients visited the clinic when consultations began at 10 AM and 3 PM, respectively. Approximately 20% of weekday visits occurred after 6 PM, when other pediatricians' offices were typically closed. Children older than 7 years of age visited the clinic more frequently after 6 PM. The overall median waiting time was 650 seconds (interquartile range, 429-1020). The 3 most common diagnoses were upper respiratory tract infection (27,173), asthmatic bronchitis (23,744), and allergic rhinitis (10,556). The number of individuals who were referred to other medical institutions was 284 (0.6%).The majority of patients were children aged 1 to 4 years living near the clinic and 80% of visits were during the daytime. However, children older than 7 years of age visited the clinic more frequently after 6 PM. The convenience of the clinic contributed to the fulfillment of the medical needs of children with mild illnesses whose mothers were in full-time employment.

Patients' demographics of a convenient clinic located in a large railway station in metropolitan Tokyo area.

Hidden barriers to visit a medical facility especially for young busy workers have been neglected in the aging society. The aim of this cross-sectional study is to analyze demographics of patients who had visited the first known convenient clinic located inside a railway station, which is adjusted to the lifestyle of working generations.We analyzed de-identified data of patients who had visited the department of internal medicine of a clinic, which is located inside a railway station building and offers primary care with after-hours accessibility in Tokyo, between August 2013 and June 2016. Data were collected on patients' sex, age, time of visit, waiting time, presence or absence of an appointment, diagnosis, and patients' addresses using the electronic health and billing records.Overall, 28,001 patients visited 87,126 times. Number of visits increased in winter season compared with the other seasons. Sixty-one percent were women and the median age of all patients was 38 years (range, 0-102). The number of visits on Mondays was the highest in a week and the most frequent visiting time was between 6 and 7 p.m. The number of visits of working generations (from 15 to 65 years old) and men increased after 6 p.m. and on weekends. The 3 most common diagnoses were upper respiratory tract infection (22,457), allergic rhinitis (20,916), and hypertension (4869). The number of individuals who were referred to other medical institutions was 1022 (1.2%). The median waiting time was 748 seconds (range, 2-5344). The number of visits from within 2-, 5-, and 10-mile radius from our clinic was 41,696 (50.6%), 63,190 (76.7%), and 75,015 (91.1%), respectively, and patients' addresses were mainly located along the railway network.The locational and temporal convenience of our clinic has attracted the unmet medical demands especially for young workers who have difficulty in visiting conventional medical institutions.

Unusually Large Number of Mutations in Asexually Reproducing Clonal Planarian Dugesia japonica.

We established a laboratory clonal strain of freshwater planarian (Dugesia japonica) that was derived from a single individual and that continued to undergo autotomous asexual reproduction for more than 20 years, and we performed large-scale genome sequencing and transcriptome analysis on it. Despite the fact that a completely clonal strain of the planarian was used, an unusually large number of mutations were detected. To enable quantitative genetic analysis of such a unique organism, we developed a new model called the Reference Gene Model, and used it to conduct large-scale transcriptome analysis. The results revealed large numbers of mutations not only outside but also inside gene-coding regions. Non-synonymous SNPs were detected in 74% of the genes for which valid ORFs were predicted. Interestingly, the high-mutation genes, such as metabolism- and defense-related genes, were correlated with genes that were previously identified as diverse genes among different planarian species. Although a large number of amino acid substitutions were apparently accumulated during asexual reproduction over this long period of time, the planarian maintained normal body-shape, behaviors, and physiological functions. The results of the present study reveal a unique aspect of asexual reproduction.

Concerns about unapproved meningococcal vaccination for eculizumab therapy in Japan.

An orphan medicinal product, eculizumab is approved in Japan and globally for treating paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab therapy can cause late complement pathway deficiencies that predispose patients to meningococcal infections. Although meningococcal vaccinations are typically considered mandatory for eculizumab therapy, no approved vaccine is available in Japan as of March, 2014. Advertising unapproved, privately imported pharmaceuticals is prohibited under Japanese pharmaceutical law; detailed information concerning the unapproved meningococcal vaccines is therefore not widely available. The situation jeopardizes the safety of patients receiving eculizumab therapy, and Japanese clinicians are advised caution when prescribing this therapy.