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Abstract Urine is a complex liquid containing numerous small molecular metabolites. The ability to non-invasively test for cancer biomarkers in urine is especially beneficial for screening child patients. This study attempted to identify neuroblastoma biomarkers by comprehensively analysing urinary metabolite samples from children. A total of 87 urine samples were collected from 54 participants (15 children with neuroblastoma and 39 without cancer) and used to perform a comprehensive analysis. Urine metabolites were extracted using liquid chromatography/mass spectrometry and analysed by Metabolon, Inc. Biomarker candidates were extracted using the Wilcoxon rank sum test, random forest method (RF), and orthogonal partial least squares discriminant analysis (OPLS-DA). RF identified three important metabolic pathways in 15 samples from children with neuroblastoma. One metabolite was selected from each of the three identified pathways and combined to create a biomarker candidate (3-MTS, CTN, and COR) that represented each of the three pathways; using this candidate, all 15 cases were accurately distinguishable from the control group. Two cases in which known biomarkers were negative tested positive using this new biomarker. Furthermore, the predictive value did not decrease in cases with a low therapeutic effect. This approach could be effectively applied to identify biomarkers for other cancer types.

Abstract Background In cases of Hirschsprung disease, complete and reproducible resection of the aganglionic bowel is ideal to achieve good postoperative bowel function. Reliable identification of the upper margin of the surgical anal canal, which is the squamous-columnar junction, is necessary during transanal pull-through. Here, we describe a novel staining technique using Lugol’s iodine stain to visualize the upper margin of the surgical anal canal. Methods Lugol’s iodine staining was performed in five patients with Hirschsprung disease treated using a single-stage laparoscopic transanal pull-through modified Swenson procedure. In two of these patients, endocytoscopic observation with ultra-high magnification was performed using methylene blue and crystal violet to mark the border of the squamous epithelium at 1 week before surgery. The alignment between the incisional line, which was revealed using Lugol’s iodine staining and endocytoscopic marking, was evaluated. Complications, including postoperative bowel dysfunction, were evaluated. Results In all cases, Lugol’s iodine staining produced a well-demarcated line. The endocytoscopic marking of the upper margin of the surgical anal canal was aligned with the line revealed by Lugol’s iodine staining. There were no complications associated with the transanal pull-through procedure, including postoperative bowel dysfunction. Conclusions Lugol’s iodine staining could be a safe and practical method to visualize the upper margin of the surgical anal canal intraoperatively. This finding may be useful for surgeons to make a consistent removal of the aganglionic bowel during surgery for Hirschsprung disease.

Abstract Background The fundamental treatment for patients with pediatric malignant mediastinal tumors is chemotherapy. Therefore, accurate diagnosis is essential for selecting the appropriate chemotherapeutic regimen. However, malignant mediastinal tumors occasionally cause respiratory distress, and biopsies under general anesthesia are dangerous for such patients as invasive mechanical ventilation can aggravate airway obstruction caused by mass effect. In this study, we reviewed our 10-year diagnostic experience to evaluate the efficacy of our practices and confirm a safe diagnostic protocol for future patients. Methods We retrospectively reviewed medical records of children with malignant mediastinal tumors diagnosed at Nagoya University Hospital from 2007 to 2018 who demonstrated respiratory distress. Respiratory distress included dyspnea, massive pleural effusion, wheezing, and hypoxemia owing to tumors. Data on sex, age at onset, primary symptoms, location of tumor, management strategy (especially the method of diagnosis and definitive diagnosis), clinical course, prognosis during the acute phase (within 3 months from the onset of respiratory symptoms), and long-term outcome were collected. Results Twelve pediatric patients met the review criteria. There were seven anterior mediastinal tumors and five posterior mediastinal tumors. All anterior mediastinal tumors were diagnosed via bone marrow smear, thoracentesis, or core needle biopsy while maintaining spontaneous breathing. Regarding posterior tumors, two patients were diagnosed via a core needle biopsy and lymph node excisional biopsy under spontaneous breathing. Two cases were initially diagnosed solely using tumor markers. One patient with severe tracheal compression underwent tumor resection with extracorporeal membrane oxygenation stand-by. No patient died of diagnostic procedure-related complications. Conclusions In 11 of the 12 cases reviewed, safe and accurate tumor diagnosis was accomplished without general anesthesia. A diagnostic strategy without general anesthesia considering the tumor location proved to be useful.

Purpose: Experimental models of laparoscopic surgery generally use large animals owing to a sufficient abdominal working space. We developed a novel laparoscopic surgery model in rats. We performed intestinal anastomosis to demonstrate the feasibility and reliability of this model. Materials and Methods: We designed a device for rats that expanded the abdominal working space and allowed us to manipulate the intraperitoneal organs by hand under direct vision with pneumoperitoneum. We performed small bowel resection and intestinal anastomosis in rats using this model. To elucidate the effects of pneumoperitoneum and skin incision length, rats were randomly divided into four groups with differing surgical techniques: small incision group, large incision group, small incision + pneumoperitoneum group, and large incision + pneumoperitoneum group. Intraoperative abdominal pressure and postoperative cytokines were measured. Results: One experimenter completed small bowel resection and hand-sewn anastomosis under direct vision without any difficulties or assistance. Carbon dioxide pneumoperitoneum was maintained at 8–10 mmHg during surgery in both pneumoperitoneum groups. Necropsies revealed no evidence of anastomotic leakage at 24 h after surgery. The interleukin-6 and C-reactive protein concentrations were significantly greater in large incision group than in small incision group, but were not significantly different between small incision + pneumoperitoneum group and small incision group. These cytokines concentrations were the greatest in large incision + pneumoperitoneum group. Conclusions: Our laparoscopic surgery model in rats is a simple and reliable experimental model. The length of skin incision might be a more influential determinant of surgical invasiveness than pneumoperitoneum.

Background The Japanese healthcare system currently faces numerous challenges, including a super-aging society and an excessive burden on medical workers; therefore, the need for innovative solutions from healthcare ventures to tackle these issues has increased. Meanwhile, as physicians play important roles in healthcare ventures, the need for Japanese physician entrepreneurs is more important than ever. Given the lack of research examining barriers to physicians starting ventures and what skills, knowledge, and surrounding environments act as facilitators, this study aimed to identify the facilitators and barriers faced by physicians to start ventures. Methods Between September and November 2019 and in May 2021, qualitative interviews were conducted with 33 participants, which included eight physician entrepreneurs; two administrative officers at the Ministry of Health, Labour and Welfare and the Ministry of Economy, Trade and Industry; three faculty members at Tokyo Medical and Dental University (in-depth interviews); and 20 medical students (focus group discussions). The interviews were deductively coded based on the social ecological model. The inductive approach was applied to coding any knowledge necessary to start a business. We conducted member checking with three physician entrepreneurs and seven medical students to improve our results’ credibility. Results The factors influencing a physician’s decision to launch a new business include their willingness to contribute to society, the unique environment in which an individual is placed while in medical school and afterward, negative aspects of the lack of diversity in physicians’ careers, the financial stability provided by a medical license, and self-efficacy. Conclusions Our study revealed facilitators and barriers to physicians’ entrepreneurial ventures. Knowledge about these factors might be useful in supporting physicians to launch or become involved in healthcare ventures.

Background Misdiagnoses are associated with various cognitive biases, which are difficult to reduce even if physicians think about clinical cases deliberately. We propose a new "2-Dimensional Approach" that combines two perspectives in diagnostic reasoning: organs (brain, heart, lung, intestine/liver, kidney) and systems (metabolic, endocrine, blood/tumor, infection, immune, circulatory, nervous systems). Systems involve throughout the whole body and can be pathogenesis of diseases. As a result of diseases, organs show abnormal vital signs and symptoms. We investigated: (1) whether each individual resident has a bias for either organs or systems when they diagnose clinical cases, and (2) whether the 2D Approach can reduce such a bias. Methods We randomly assigned PGY1 residents (n=105) to either the 2D Approach group (n=45) or a control group (n=60). After attending either a lecture on the 2D Approach or a non-related lecture, residents were asked to diagnose two clinical cases. We divided each diagnosis into one of the two categories, either "organs" or "systems". We investigated whether each resident would diagnose the two cases into the same category, either organs or systems (i.e., a bias for either organs or systems). Results The participants in the control group tended to diagnose the two cases into the same category, either organs or systems (OR: 5.63, 95% CI: 1.62-21.7, p=0.0030, Fisher's exact test). In the 2D Approach group, the category of diagnoses for the two cases were not related to each other (OR: 2.14, 95% CI: 0.50-9.81, p=0.33). Conclusion There is a bias for either organs or systems when residents diagnose clinical cases, suggesting that organs and systems are different perspectives in diagnostic reasoning. By combining these 2 different perspectives in a 2-dimensional matrix, the 2D Approach reduces this bias.

BACKGROUND:Group-based trajectory modeling is a useful tool for categorizing students' academic trajectories and their determinants. Using insights gained from the analyses, we can identify students at risk for poor academic performance and monitor them to provide support. To date, studies investigating the associations between demographic factors and academic performance trajectories among medical students are scarce. The study objective was to examine the associations between demographic factors and academic performance trajectories in medical students using group-based trajectory modeling. METHODS:Participants included all medical students admitted to Tokyo Medical and Dental University in Japan in 2013 and 2014 (n = 202). Academic performance was evaluated by biannual grade point average (GPA) scores in preclinical years. We used group-based trajectory modeling to categorize students into GPA trajectories. Multinomial logistic regression was used to examine the association between the odds of being in a certain GPA trajectory group and demographic factors such as high school type, high school geographical area, admission test type, high school graduation year, whether the student was a biology major, and sex. RESULTS:Students' GPA trajectories were classified into four trajectory groups as well as another group that consisted of students who withdrew or repeated years. We found that students whose high school geographical area was outside the National Capital Region were 7.2 times more likely to withdraw or repeat years in comparison with students whose school was inside the National Capital Region (OR: 7.21, 95% CI: 1.87, 27.76). In addition, admission test type, high school graduation year, and sex were associated with GPA trajectories. CONCLUSIONS:High school geographical area, admission test type, high school graduation year, and sex were associated with GPA trajectories. These findings provide important insights into identifying students at risk for poor academic performance and strategies for monitoring them to provide adequate and timely support.

Abstract Background Emergency rescue lung resection is rarely performed to treat congenital lung cysts (CLCs) in neonates. Many reports have described fetal CLC treatment; however, prenatal predictors for postnatal respiratory failure have not been characterized. We hypothesized that fetal imaging findings are useful predictors of emergency surgery. Methods We retrospectively studied patients with CLC who underwent lung surgery during the neonatal period in our hospital between January 2001 and December 2015. The demographic data, fetal imaging findings, and intra- and postoperative courses of patients who underwent emergency surgery (Em group) were compared with those of patients who received elective surgery, i.e., non-emergency surgery (Ne group). Results The Em group and Ne group included 7 and 11 patients, respectively. No significant difference was noted in gestational age, time at prenatal diagnosis, birth weight, and body weight at surgery. The volumes of contralateral lung per thoracic volume were significantly smaller in the Em group than in the Ne group (p = 0.0188). Mediastinal compression was more common in the Em group (7/7) than in the Ne group (4/11) (p = 0.0128). Conclusions This is the report describing neonatal emergency lobectomy in patients with CLC evaluated by fetal MRI using the lung volume ratio and mediastinal shift. In patients with CLC, mediastinal shift and significant decreases in contralateral lung volumes during the fetal stages are good prenatal predictors of postnatal emergency lung resection.

Background: Clinical reasoning is an essential competence of a physician. Particularly, pathophysiological understanding is the key for novices to organize their knowledge and improve clinical reasoning. To this end, we propose a "2-Dimensional (2D) Approach" that visualizes pathophysiology by using a matrix of organs and systems. Methods: This study was a prospective observational study of 100 residents (PGY-1), who attended a lecture on the 2D Approach. They underwent a pre- and post-test that assessed their ability to list differentials for various symptoms and to choose the most likely diagnosis for a case vignette. We stratified the participants according to their baseline knowledge level on the pretest into 2 groups (high-knowledge and low-knowledge). In each of the 2 groups, we compared the change in the pre- and post-test scores between participants who used the 2D Approach during the posttest and those who did not. Results: In the high-knowledge group (n=59), the change in the pre- and post-test scores was significantly larger in the 2D Approach users than non-users (p=0.046), while in the low-knowledge group (n=41), there was no significant difference in the change of the scores between the 2D Approach users and non-users. Conclusions: On condition that residents have sufficient knowledge, the 2D Approach helps them organize their knowledge and improve their clinical reasoning skills, through visualization of pathophysiology on a matrix of organs and systems.

We describe a simple and feasible procedure for single-incision laparoscopic repair of a pediatric epigastric hernia using a novel suture-assisting needle. A multichannel port was inserted through the umbilical vertical incision. After the orifice of the hernia was identified, a suture-assisting needle, which can hold a suture at its tip, with a 2-0 thread was pierced through the skin into one side of the rectus muscle sheath into the abdominal cavity. Next, after releasing the thread, the needle was pulled out to the subcutis and pierced through another side of the rectus muscle sheath. The needle, grasping the thread again, was subsequently pulled out through the abdominal wall outside, and the thread was tied extracorporeally. This knot was buried subcutaneously. Operative procedures of single-incision laparoscopic repair of an epigastric hernia have become simple and feasible with the use of a novel suture-assisting needle with an excellent cosmetic result.

Surgical pancreatic duct (PD) drainage for chronic pancreatitis in children is relatively rare. It is indicated in cases of recurrent pancreatitis and PD dilatation that have not responded to medical therapy and therapeutic endoscopy. We performed laparoscopic side-to-side pancreaticojejunostomy for two paediatric patients with chronic pancreatitis. The main PD was opened easily by electrocautery after locating the dilated PD by intraoperative ultrasonography. The dilated PD was split longitudinally from the pancreatic tail to the pancreatic head by laparoscopic coagulation shears or electrocautery after pancreatography. A laparoscopic side-to-side pancreaticojejunostomy was performed by a one-layered technique using continuous 4-0 polydioxanone (PDS) sutures from the pancreatic tail to the pancreatic head. There were no intraoperative or postoperative complications or recurrences. This procedure has cosmetic advantages compared with open surgery for chronic pancreatitis. Laparoscopic side-to-side pancreaticojejunostomy in children is feasible and effective for the treatment of chronic pancreatitis.

A majority of facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of macrosatellite repeats called D4Z4 that are located in the subtelomeric region of human chromosome 4q35. Sequencing the FSHD locus has been technically challenging due to its long size and nearly identical nature of repeat elements. Here we report sequencing and partial assembly of a BAC clone carrying an entire FSHD locus by a single molecule real time (SMRT) sequencing technology which could produce long reads up to about 18 kb containing D4Z4 repeats. De novo assembly by Hierarchical Genome Assembly Process 1 (HGAP.1) yielded a contig of 41 kb containing all but a part of the most distal D4Z4 element. The validity of the sequence model was confirmed by an independent approach employing anchored multiple sequence alignment by Kalign using reads containing unique flanking sequences. Our data will provide a basis for further optimization of sequencing and assembly conditions of D4Z4.

Background: Recent studies reported that cerebral microbleeds (CMBs), i.e. small areas of signal loss on T2*-weighted gradient-echo (GE) imaging, could develop rapidly after acute ischemic stroke. We hypothesized that CMBs rapidly emerge after carotid artery stenting (CAS). Objective: We investigated the frequency of and predisposing factors for CMBs after CAS. Methods: We retrospectively examined MRI before and after CAS in 88 consecutive patients (average age: 71.7 ± 7.2 years, average rates of carotid stenosis: 72.6 ± 12.8%) who underwent CAS for carotid artery stenosis between March 1, 2009, and September 30, 2010. We defined new CMBs as signal losses that newly appeared on the follow-up GE. We examined the association of new CMBs with demographics, risk factors, and baseline MBs. Results: Among 88 patients, 18 (20.5%) had CMBs initially, and 7 (8.0%) developed new CMBs right after CAS. New CMBs appeared on the same side of CAS in all of the 7 patients. New CMBs appeared significantly more frequently in the CMB-positive group than in the CMB-negative one (22% vs. 4%, p = 0.03) on the pre-CAS MRI. Multivariate analysis also revealed that the presence of CMBs before CAS was an independent predictor of new development of CMBs after CAS (odds ratio: 8.09, 95% confidence interval: 1.39–47.1). Conclusion: CMBs can develop rapidly after CAS, especially in patients with pre-existing CMBs. Since the existence of CMBs prior to CAS suggests a latent vascular damage which is vulnerable to hemodynamic stress following CAS, particular attention should be paid to the prevention of intracerebral hemorrhage due to hyperperfusion after CAS.

Pneumoperitoneum in a neonate is a serious condition for which bowel perforation is indicated in most cases. Because the transdiaphragmatic air dissection could occur in some ventilated neonates without peritonitis, making the right diagnosis is sometimes difficult, and exploratory laparotomy is often necessary. Here, we report the first case of neonatal pneumoperitoneum caused by a perforated Meckel′s diverticulum in which diagnostic laparoscopy was useful in achieving minimal access surgery. Using a multiuse single-site port on the umbilicus could enable the extraction of a certain length of a small intestine with good cosmetic results. This method would decrease the hurdles of early surgical interventions for the suspected perforation of unknown aetiology and shorten the fasting period, which is beneficial for the neurodevelopment of small neonates.

Stress-inducible transcription factors play a pivotal role in cellular adaptation to environment to maintain homeostasis and integrity of the genome. Activating transcription factor 3 (ATF3) is induced by a variety of stress and inflammatory conditions and is over-expressed in many kinds of cancer cells. However, molecular mechanisms underlying pleiotropic functions of ATF3 have remained elusive. Here we employed systems analysis to identify genome-wide targets of ATF3 that is either induced by an alkylating agent methyl methanesulfonate (MMS) or over-expressed in a prostate tumour cell line LNCaP. We show that stress-induced and cancer-associated ATF3 is recruited to 5,984 and 1,423 targets, respectively, in the human genome, 89% of which are common. Notably, ATF3 targets are highly enriched for not only ATF/CRE motifs but also binding sites of several other stress-inducible transcription factors indicating an extensive network of stress response factors in transcriptional regulation of target genes. Further analysis of effects of ATF3 knockdown on these targets revealed that stress-induced ATF3 regulates genes in metabolic pathways, cell cycle, apoptosis, cell adhesion, and signalling including insulin, p53, Wnt, and VEGF pathways. Cancer-associated ATF3 is involved in regulation of distinct sets of genes in processes such as calcium signalling, Wnt, p53 and diabetes pathways. Notably, stress-induced ATF3 binds to 40% of p53 targets and activates pro-apoptotic genes such as TNFRSF10B/DR5 and BBC3/PUMA. Cancer-associated ATF3, by contrast, represses these pro-apoptotic genes in addition to CDKN1A/p21. Taken together, our data reveal an extensive network of stress-inducible transcription factors and demonstrate that ATF3 has opposing, cell context-dependent effects on p53 target genes in DNA damage response and cancer development.

Hox genes play important roles in haematopoietic development in mammals. ASH1 is a member of the trithorax group (trxG) that is required for proper expression of Hox genes and is preferentially expressed in haematopoietic stem cells. We have recently reported that ASH1 methylates histone H3 at lysine 36 (K36) but its biological function has remained elusive. Here we show that ASH1 regulates Hox gene expression positively and negatively in a leukemic cell line K562 and is required for myelomonocytic differentiation of murine haematopoietic stem cells. ASH1 binds to endogenous Hox loci in K562 cells and its knockdown causes reduced expression of Hox genes. In addition, ASH1 and MLL1 induce more than 100-fold activation of Hox promoters in HeLa cells if expressed simultaneously but not individually. Notably, ASH1 harbouring a point mutation that kills methyltransferase activity is more efficient than wild type ASH1 in Hox gene activation, indicating that K36 methylation is not a prerequisite for Hox gene expression. Moreover, tethering wild type or catalytically inactive methyltransferase domain of ASH1 to a heterologous promoter causes downregulation or upregulation, respectively, of transcription, supporting a hypothesis that K36 methylation imparts repression. Knockdown of ASH1 in K562 cells in vitro causes increased expression of ε-globin gene and reduced expression of myelomonocytic markers GPIIb and GPIIIa, whereas knockdown of ASH1 in murine haematopoietic stem cells in vivo results in decreased number of macrophages and granulocytes, a phenotype similar to that induced by loss of mll1 function. Taken together, our data suggest that ASH1 and MLL1 synergize in activation of Hox genes and thereby regulate development of myelomonocytic lineages from haematopoietic stem cells.

The ERas gene promotes the proliferation of and formation of teratomas by mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues as assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi- scid , IL-2Rγ null (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.

Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/?cnull (NOG) mice, teratomas developed in all the animals transplanted with 1 × 105 or more cells, but were not observed in any mouse transplanted with 1 × 103 cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratomas to form in all mice increased to 5 × 105. When the clump cells were injected subcutaneously (instead of intramuscularly), the number also increased to 5 × 105. When cynomolgus ES cell-derived progenitor cells (1 × 106), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.