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Abstract The one-leaf plant Monophyllaea glabra exhibits a unique developmental manner in which only one cotyledon continues growing without producing new vegetative organs. This morphology is formed by specific meristems, the groove meristem (GM) and the basal meristem (BM), which are thought to be modified shoot apical meristem (SAM) and leaf meristem. In this study, we analysed the expression of the organ boundary gene CUP-SHAPED COTYLEDON (CUC) and the SAM maintenance gene SHOOT MERISTEMLESS (STM) orthologs by whole-mount in situ hybridisation. We found that CUCs did not show clear border patterns around GM and BM during the vegetative phase. Furthermore, double-colour detection analysis at the cellular level revealed that CUC and STM expression overlapped in the GM region during the vegetative phase. We also found that this overlap is dissolved in the reproductive phase when normal shoot organogenesis is observed. Since co-expression of these genes occurs during SAM initiation under embryogenesis in Arabidopsis, our results demonstrate that GM is a prolonged stage of pre-mature SAM. Therefore, we propose that neotenic meristems could be a novel plant trait acquired by one-leaf plants.

Abstract In seed plants, growth of shoots and roots is indeterminate, while leaves are typically determinate organs that cease to grow after a certain developmental stage. This is due to the characteristics of the leaf meristem, where cell proliferation activity is retained only for a limited period. However, several plants exhibit indeterminacy in their leaves, exemplified by the pinnate compound leaves of Guarea and Chisocheton genera in the Meliaceae family. In these plants, the leaf meristem at the tip of the leaf retains meristematic activity and produces leaflets over years, resulting in a single leaf that resembles a twig. The molecular mechanism underlying the indeterminate leaf meristem of these plants has not been examined. In this research, we used Guarea glabra as a model to investigate the development of indeterminate pinnate leaves. Transcriptome analyses revealed that the gene expression profile in leaf apex tissue differed from that in the shoot apex. However, a class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) gene which is lost in Brassicaceae was highly expressed in both tissues. We established an in situ hybridisation system for this species using Technovit 9100 to analyse the spatial expression patterns of genes. We revealed that the leaf meristematic region of G. glabra expresses KNOX1, LEAFY and ANGUSTIFORIA3 simultaneously, suggesting the involvement of these genes in the indeterminacy of the leaf meristem.

Abstract Bile acids have received increasing attention as a marker of the long-term prognosis and a potential therapeutic target in patients with biliary atresia, which is a progressive disease of the hepatobiliary system. A detailed analysis of serum and urinary bile acid compositions was conducted to assess the characteristics of bile acid profiles and the correlation between bile acid profiles and liver fibrosis markers in adult patients with biliary atresia who achieved bilirubin normalization. Serum total bile acids and glucuronide-conjugated (glyco- and tauro-) cholic acids (GCA and TCA) and chenodeoxycholic acids (GCDCA and TCDCA) were significantly higher in patients with biliary atresia than in healthy controls, whereas unconjugated CA and CDCA showed no significant difference. There were no significant differences in CA to CDCA ratios and glycine-to-taurine-conjugated ratios. Urinary glycocholic acid 3-sulfate (GCA-3S) was significantly higher in patients with biliary atresia. Serum GCDCA showed a strong positive correlation with Mac-2 binding protein glycosylation isomer (M2BPGi). These results demonstrate that bile acid congestion persists into adulthood in patients with biliary atresia, even after cholestasis has completely improved after Kasai portoenterostomy. These fundamental data on bile acid profiles also suggest the potential value of investigating bile acid profiles in patients with biliary atresia.

The cosmopolitan genus Callitriche (Plantaginaceae) is a clade of small herbaceous plants that encompasses terrestrial and aquatic species. In Japan, six Callitriche species have been identified: four native and two naturalised species. Callitriche terrestris, a naturalised terrestrial species, was first reported in 1984 in Kanagawa Prefecture and it is thriving today.We report the presence of a new naturalised terrestrial species, Callitriche deflexa, which has been previously misidentified as C. terrestris because of its similar morphology. Callitriche deflexa can be distinguished from C. terrestris through genetic differences and distinct morphological traits, such as longer pedicels. Re-examination of herbarium specimens in the Kanagawa Prefectural Museum of Natural History confirmed that most of the specimens labelled as C. terrestris, including voucher specimens from the original report, were indeed C. terrestris, but a few were C. deflexa. We also noted that the plants referred to as “C. terrestris” in our previous developmental studies should be corrected to C. deflexa.

During leaf development, the timing of transition from cell proliferation to expansion is an important factor in determining the final organ size. However, the regulatory system involved in this transition remains less understood. To get an insight into this system, we investigated the compensation phenomenon, in which the cell number decreases while the cell size increases in organs with determinate growth. Compensation is observed in several plant species suggesting coordination between cell proliferation and expansion. In this study, we examined an Arabidopsis mutant of ANGUSTIFOLIA 3 (AN3)/GRF-INTERACTING FACTOR 1, a positive regulator of cell proliferation, which exhibits the compensation. Though the AN3 role has been extensively investigated, the mechanism underlying excess cell expansion in the an3 mutant remains unknown. Focusing on the early stage of leaf development, we performed kinematic, cytological, biochemical, and transcriptome analyses, and found that the cell size had already increased during the proliferation phase, with active cell proliferation in the an3 mutant. Moreover, at this stage, chloroplasts, vacuoles, and xylem cells developed earlier than in the wild-type cells. Transcriptome data showed that photosynthetic activity and secondary cell wall biosynthesis were activated in an3 proliferating cells. These results indicated that precocious cell differentiation occurs in an3 cells. Therefore, we suggest a novel AN3 role in the suppression of cell expansion/differentiation during the cell proliferation phase.

Abstract Background Dysregulation of the terminal differentiation of bladder urothelium is associated with the pathogenesis of urinary tract disorders. Fibroblast growth factor (Fgf)7 and Fgf10 stimulate urothelial proliferation; however, their roles in cellular differentiation remain unclear. In this study, we used an organoid system to investigate the roles of these Fgfs in regulating bladder urothelium differentiation and identify their distribution patterns in the mouse bladder. Methods Adult bladder epithelia (AdBE) isolated from adult mouse bladder tissues (AdBTs) were used to culture adult bladder organoids (AdBOs) in the presence of Fgf7 and Fgf10. The differentiation status of the cells in AdBTs, AdBEs, AdBOs, and neonatal bladder tissues (NeoBTs) was analyzed via quantitative real-time-PCR for the presence of undifferentiated cell markers (Krt5, Trp63, and Krt14) and differentiated cell markers (Krt20, Upk1a, Upk2, and Upk3a). Organoid cell proliferation was assessed by counting cell numbers using the trypan blue method. The effects of Fgf7 and Fgf10 on organoid differentiation were assessed using different doses of Fgfs, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) signaling in these processes was tested by introducing a PPARγ agonist (Rosiglitazone) and antagonist (T0070907) to the culture. The expression patterns of Fgf7 and Fgf10 were examined via in situ hybridization of AdBTs. Results AdBOs showed higher expression of undifferentiated cell markers and lower expression of differentiated cell markers than AdBTs, NeoBTs, and AdBEs, indicating the relatively immature state of AdBOs. Differentiation of AdBOs was enhanced by Rosiglitazone and Fgf7, suggesting an interplay of intracellular signals between Fgf7 and PPARγ. Co-addition of T0070907 suppressed Fgf7-mediated differentiation, demonstrating that PPARγ is activated downstream of Fgf7 to promote cellular differentiation into umbrella cells. Furthermore, we found that Fgf7 is predominantly expressed in the umbrella cells of the urothelium, whereas Fgf10 is predominantly expressed in the urothelium and stroma of AdBTs. Conclusions We demonstrated that unlike Fgf10, Fgf7 induces cellular differentiation via PPARγ activity and has a unique tissue distribution pattern in the adult bladder. Further studies on the Fgf7-PPARγ signaling axis would provide insights into the differentiation mechanisms toward functional umbrella cells and the pathogenesis of several urinary tract diseases.

As master transcription factors of stomatal development, SPEECHLESS, MUTE, and FAMA, collectively termed SMFs, are primary targets of molecular genetic analyses in the model plant Arabidopsis thaliana. Studies in other model systems identified SMF orthologs as key players in evolutionary developmental biology studies on stomata. However, recent studies on the astomatous liverwort Marchantia polymorpha revealed that the functions of these genes are not limited to the stomatal development, but extend to other types of tissues, namely sporophytic setal and gametophytic epidermal tissues. These studies provide insightful examples of gene-regulatory network co-opting, and highlight SMFs and related transcription factors as general toolkits for novel trait evolution in land plant lineages. Here, we critically review recent literature on the SMF-like gene in M. polymorpha and discuss their implications for plant evolutionary biology.

Background Following on from an earlier study published in 2008 about left pulmonary artery (LPA) flow measured on serial echocardiography being strongly prognostic in left-sided congenital diaphragmatic hernia (CDH) and the ratio of LPA to right pulmonary artery (RPA) diameters being a simple and reliable indicator for commencing nitric oxide (NO) therapy, the ratio of LPA:RPA diameters (PA ratio or PAR) was hypothesized to possibly reflect cardiopulmonary stresses accompanying CDH better.Methods Subjects with isolated left-sided CDH treated between 2007 and 2020 at a single pediatric surgical center were recruited and classified according to survival. Data obtained retrospectively for subject demographics, clinical course, LPA/RPA diameters, and PAR were compared between survivors and non-survivors. The value of PAR for optimizing the prognostic value of PA diameter data in CDH were analyzed with receiver operating characteristic (ROC) curve analysis.Results Of 65 subjects, there were 54 survivors (82.3%) and 11 non-survivors (17.7%); 7 of 11 non-survivors died before surgical repair could be performed. Mean PAR for survivors (0.851±0.152) was significantly higher than for non-survivors (0.672±0.108) (p=0.0003). Mean PAR for non-survivors was not affected by surgical repair. Characteristics of survivors were: LPA ≥2 mm (n=52 of 54; mean PAR=0.866±0.146) and RPA ≥3 mm (n=46 of 54; mean PAR=0.857±0.152). Non-survivors with similar LPA and RPA diameters to survivors had significantly lower mean PAR. ROC curve cut-off for PAR was 0.762. Subjects with high PAR (≥0.762) required high-frequency oscillatory ventilation/NO less than subjects with low PAR (

Background: : Sufficient knowledge of anatomy including normal patterns and aberrant variations is crucial in thoracoscopic lobectomy for congenital lung malformations (CLMs). This study aimed to investigate the prevalence of anatomical variations and their correlations with surgical outcome. Methods: : Video-recordings of thoracoscopic lobectomy performed for CLMs between 2009 and 2021 were reviewed. The incidences of surgically relevant variations, including abnormal branches of pulmonary arteries and veins, aberrant systemic arteries, and incomplete fissures (as classified by Craig) were assessed in comparison with the previously published reports. Moreover, multiple univariate analyses were performed to seek correlations between surgical finding and prolonged operative times and postoperative drainage. Results: : Fifty-six lobectomies were reviewed. Variations of pulmonary arteries were more commonly seen in the left lung (29.6%) compared to right (10.7%). The prevalence of atypical pulmonary venous branches was similar between the right (13.8%) and left (18.5%). The right upper oblique fissure was frequently associated with Grade 2 incomplete fissure (44.8%). Grade 3 incomplete fissure was rare (3.4%) in the right lower oblique fissure but relatively common (14.3 – 18.5%) in other locations. Grade 4 incomplete fissure was limited to the right horizontal fissure (1.7%). Multiple univariate analyses demonstrated a significant correlation between any degree of incomplete fissure and prolonged operative time (p < 0.05), whereas no factor was identified to affect need for postoperative drainage. Conclusions: : The incidence of anatomical variations in CLM patients was similar to that reported in adults. Review of intra-operative video footage was useful to closely evaluate and understand anatomical variations.

Pancreatic neuroendocrine tumors (PNETs) are relatively rare, especially in the pediatric age group. This report describes a pediatric case of acute pancreatitis secondary to stenosis of the main pancreatic duct due to a PNET. The patient was a boy, thirteen and a half years old, who presented with persistent low-grade fever, nausea, and abdominal pain. He was diagnosed with acute pancreatitis based on the elevation of serum pancreatic enzyme levels and abdominal ultrasonography findings of enlargement of the pancreas and dilatation of the main pancreatic duct. Abdominal contrast-enhanced computed tomography (CT) showed a 5.5 mm, contrast-enhanced mass in the head of the pancreas. His symptoms resolved with conservative treatment, although the pancreatic tumor grew slowly. At fifteen years and four months, since the tumor had enlarged to 8.0 mm, the patient underwent pancreaticoduodenectomy for therapeutic and diagnostic purposes. Based on the pathological evaluation, he was diagnosed with PNET (grade: G1). The patient has been free of tumor recurrence for 10 years and requires no additional therapy. In this report, the clinical characteristics of PNETs are also discussed, comparing the clinical features of adult-onset and pediatric-onset cases that initially present as acute pancreatitis.

Aim: To present the use of an additional trocar (AT) in the lower thorax during thoracoscopic pulmonary lobectomy (TPL) in children with congenital pulmonary airway malformation.Methods: For a lower lobe TPL (LL), an AT is inserted in the 10th intercostal space (IS) in the posterior axillary line after trocars for a 5-mm 30° scope, and the surgeon's left and right hands are inserted conventionally in the 6th, 4th, and 8th IS in the anterior axillary line, respectively. For an upper lobe TPL (UL), the AT is inserted in the 9th IS, and trocars are inserted in the 5th, 3rd, and 7th IS, respectively. By switching between trocars (6th↔8th for the scope, 4th↔6th for the left hand, and 8th↔10th for the right hand during LL and 5th↔7th, 3rd↔5th, and 7th↔9th during UL, respectively), vital anatomic landmarks (pulmonary veins, bronchi, and feeding arteries) can be viewed posteriorly. The value of AT was assessed from blood loss, operative time, duration of chest tube insertion, requirement for post-operative analgesia, and incidence of perioperative complications.Results: On comparing AT+ (n = 28) and AT– (n = 27), mean intraoperative blood loss (5.6 vs. 13.0 ml), operative time (3.9 vs. 5.1 h), and duration of chest tube insertion (2.2 vs. 3.4 days) were significantly decreased with AT (p < 0.05, respectively). Differences in post-operative analgesia were not significant. There were three complications requiring conversion to open/mini-thoracotomy: AT– (n = 2; bleeding), AT+: (n = 1; erroneous stapling).Conclusions: An AT and switching facilitated posterior dissection during TPL in children with congenital pulmonary airway malformation enhancing safety and efficiency.

A 10-year-old Japanese boy presented the day after a bicycle handlebar injury with mild abdominal pain. Computed tomography (CT) confirmed pancreatic transection and possible main pancreatic duct (MPD) injury because of a fluid collection around the pancreas. Conservative management (fasting, antibiotics, total parental nutrition, protease inhibitor) was initially successful, but an oral challenge with plain water on day 23 caused a relapse. CT identified a pancreatic pseudocyst. Endoscopic trans-papillary drainage was attempted but failed because the MPD was completely obstructed. Endoscopic ultrasound (EUS)-guided drainage of the pseudocyst with a lumen apposing metal stent (LAMS) was performed on day 29 via the stomach. On day 36, EUS-guided pancreatic drainage (EUS-PD) with a 5Fr plastic stent was performed via the stomach with rapid improvement in symptoms and elevated enzyme levels. Repeat oral challenge on day 39 was successful, the LAMS was removed on day 59 without recurrence. He was discharged well on day 69 and remains asymptomatic 20 months after treatment.Our case highlights the potential of minimally invasive endoscopic procedures as options for the non-surgical management of complete MPD disruption in a child. However, further research to develop dedicated pediatric endoscopic devices is required.

Purpose: We compared laparoscopic redo fundoplications performed for failed laparoscopic Toupet fundoplication (LTF) and failed laparoscopic Nissen fundoplications (LNFs). Methods: Redo LTF (R-LTF; n = 4) and redo LNF (R-LNF; n = 6) performed between 2007 and 2014 were assessed retrospectively for severity of intraperitoneal adhesions on a scale of 0–3, identification/preservation of the anterior/posterior/hepatic branches of the vagus nerve (VN), complications, and outcome. Results: Redos were performed after a mean of 34 months in R-LTF and 32 months in R-LNF (P = ns) indicated for sliding hernia (n = 3; 2 with partial wrap dehiscence) and partial wrap dehiscence (n = 1) in R-LTF and sliding hernia (n = 6; 4 with partial wrap dehiscence) in R-LNF. The mean adhesion severity score was 1.5 in R-LTF and 2.5 in R-LNF (P < 0.05). The mean number of VN branches identified/preserved was 2.0 in R-LTF and 0.8 in R-LNF (P < 0.05). Mean operative times and mean blood loss were similar. Intraoperative complications were accidental local trauma (n = 1 in R-LTF and n = 3 in R-LNF, one requiring conversion to open repair) (P = ns). Gastric outlet obstruction developed in two R-LNF cases; both were managed conservatively. There have been no further recurrences to date. Conclusion: Although our series is small, adhesions were less, and identification/preservation of VN was easier during R-LTF.

Members of the genus Monophyllaea are unique in that they produce no new organ during the vegetative phase in the shoot; instead, one of the cotyledons grows indeterminately. The mechanism of this unique trait is unclear, in part because of the lack of suitable assessment techniques. We therefore established a whole-mount in situ hybridization technique, a powerful means of examining spatial patterns in gene expression, for Monophyllaea glabra. By using this, we examined the expression pattern of a SHOOT MERISTEMLESS (STM) ortholog, which is indispensable for the formation and maintenance of the shoot apical meristem (SAM) in typical angiosperms. Expression was confined to the groove meristem (GM), which corresponds to the SAM. We also assessed the expression pattern of ANGUSTIFOLIA3 (AN3), a key promoter for cell division in the leaf meristem. It was expressed not only in the basal meristem (BM) tissue with active cell division in the basal part of the growing cotyledon but also in the GM. The findings suggest that the unusual gene expression pattern of the GM underpins the fuzzy morphogenesis of Monophyllaea.

Background We assessed the impact of anorectal malformation (ARM) on the kidneys of children with dilating vesicoureteral reflux (D-VUR) ≥grade III using a simple dimercaptosuccinic acid (DMSA) scintigraphy scan based renal dysfunction score (RDS).Methods The medical records of 121 patients with D-VUR treated between 2000 and 2014 were reviewed retrospectively. After excluding patients with secondary D-VUR (n=18), presence of ARM was used to create two groups: ARM+ (n=12 cases; 15 ureters) and ARM– (n=91 cases; 131 ureters). Types of ARM, grades of D-VUR, bladder and bowel dysfunction (BBD) and RDS were compared.Results Patient demographics, mean follow-up, grades of D-VUR and history of urinary tract infections were not significantly different. BBD was significantly higher in ARM+ (41.7% versus 7.7%, p=0.0006). RDS was significantly higher in ARM+ (p=0.036). Grades of D-VUR were significantly lower in ARM– with low RDS (p=0.008). During follow-up, changes in DMSA uptake over time were not observed in ARM+.Conclusions While renal cortical lesions were correlated with grade of D-VUR in ARM– and RDS was significantly higher in ARM+, BBD did not appear to contribute to progressive renal dysfunction as is commonly believed. In fact, no progression in renal cortical lesions was observed in ARM+ based on RDS data. Renal cortical lesions may possibly be a feature of ARM, a topic that warrants further study.

Heterophylly, or phenotypic plasticity in leaf form, is a remarkable feature of amphibious plants. When the shoots of these plants grow underwater, they often develop surprisingly different leaves from those that emerge in air. Among aquatic plants, it is typical for two or more distinct leaf development processes to be observed in the same individual exposed to different environments. Here, we analyze the developmental processes of heterophylly in the amphibious plant Callitriche palustris L. (Plantaginaceae). First, we reliably cultured this species under laboratory conditions and established a laboratory strain. We also established a framework for molecular-based developmental analyses, such as whole-mount in situ hybridization. We observed several developmental features of aerial and submerged leaves, including changes in form, stomata and vein formation, and transition of the meristematic zone. Then we defined developmental stages for C. palustris leaves. We found that in early stages, aerial and submerged leaf primordia had similar forms, but became discriminable through cell divisions with differential direction, and later became highly distinct via extensive cell elongation in submerged leaf primordia.

There is a risk for high-output congestive heart failure to be associated with an extra-lobar pulmonary sequestration (ELS) when there is left-to-left shunting caused by a large systemic arterial supply to a sequestration and venous drainage via the pulmonary veins into the left atrium. We present a 2-year-old boy who underwent thoracoscopic resection of an unusually hypervascular right ELS with a high output left-to-left shunt between the aorta and the left atrium via a pulmonary vein. This case is of particular interest because computed tomographic angiography identified a hypervascular nidus with indistinct borders between arterial and venous vessels suggestive of an arteriovenous malformation.

Umbilical hernia (UH) is one of the most common conditions in infants and young children and usually closes spontaneously during the early years of life. Complications, such as incarceration, are rare. To the best of our knowledge, there have been only 35 cases of incarcerated UHs reported in children in the English literature. We present three cases of incarcerated UH in infants below the age of 1 year. A survey of the literature suggests that incarceration and strangulation tend to occur in infants younger than 6 months old with medium-sized (0.5–1.5 cm) UH rings, and that the mechanism may be the same as Richter's hernia. We conclude that guardians of infants with UH should be aware of the signs of incarceration.

A boy with myelomeningocele who had sigmoidocolocystoplasty and ureteric reimplantation when 2-years old and normal annual cystoscopies, developed hematuria and abdominal pain with liver dysfunction 14 years postoperatively. Computed tomography showed a tumor on the left side of the augmented bladder, a large lymph node, and large multiple probable metastases in the liver. Cystoscopy 3 months earlier had been normal, but when repeated showed a tumor originating from the augmented sigmoid colon. Biopsy showed undifferentiated sarcoma. Despite chemotherapy, he died 3 months later. The diagnosis at autopsy was undifferentiated sarcoma originating from the sigmoid colon. We report the first case of undifferentiated sarcoma developing 14 years after sigmoidocolocystoplasty for meningomyelocele, and also review the 55 cases of post-bladder augmentation malignancy in the literature.

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.

Although thoracoscopic lobectomy for severe bronchiectasis has been reported in children, this is the first report of double lobectomy of the right middle and lower lobes performed using the video-assisted thoracoscopy in a 9-year-old girl. The post-operative course was uneventful and she is currently well after 18 months′ follow-up.

Abstract Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody–drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4–MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.

Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1-, TDP2-, and TDP1/TDP2-deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1/TDP2-deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1/TDP2-deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.

To perform basic Research and Development for future Accelerator-driven Systems (ADSs), Japan Proton Accelerator Research Complex (J-PARC) will construct an ADS target test facility. A Lead⁻Bismuth Eutectic (LBE) spallation target will be installed in the target test facility and bombarded by pulsed proton beams (250 kW, 400 MeV, 25 Hz, and 0.5 ms pulse duration). To realize the LBE spallation target, cavitation damage due to pressure changes in the liquid metal should be determined, preliminarily, because such damage is considered to be very critical, from the viewpoint of target safety and lifetime. In this study, cavitation damage due to pressure waves caused by pulsed proton beam injection and turbulent liquid metal flow, were studied, numerically, from the viewpoint of single cavitation bubble dynamics. Specifically, the threshold of cavitation and effects of flow speed fluctuation on cavitation bubble dynamics, in an orifice structure, were investigated in the present work. The results showed that the LBE spallation target did not undergo cavitation damage, under normal nominal operation conditions, mainly because of the long pulse duration of the pulsed proton beam and the low liquid metal flow velocity. Nevertheless, the possibility of cavitation damage in the orifice structure, under certain extreme transient LBE flow conditions, cannot be neglected.

At the Japan Proton Accelerator Research Complex (J-PARC), a pulsed spallation neutron source provides neutrons with high intensity and narrow pulse width pulse to promote researches on a variety of science in the Materials and Life Science Experimental Facility (MLF). It was designed to be driven by a proton beam with an energy of 3 GeV, a power of 1 MW at a repetition rate of 25 Hz, that is world’s highest power level. It is still on the way towards the goal to accomplish the operation with a 1 MW proton beam. In this review, distinctive features of the target-moderator-reflector system of the pulsed spallation neutron source are presented.

Basal cell adhesion molecule (BCAM), known to be a splicing variant of Lutheran glycoprotein (LU), is an immunoglobulin superfamily membrane protein that acts as a laminin α5 receptor. The high affinity of BCAM/LU for laminin α5 is thought to contribute to the pathogenesis of sickle red blood cells and to various developmental processes. However, the function of BCAM in carcinogenesis is poorly understood. Based on microarray expression analysis, we found that BCAM was one of the target genes of the oncogenic 14-3-3β-FBI1/Akirin2 complex, which acts as a transcriptional repressor and suppresses MAPK phosphatase-1 gene expression. To elucidate the detailed function of BCAM in malignant tumors, we established BCAM-expressing hepatoma K2 cells. These cells lost the malignant characteristics of parental cells, such as anchorage-independent growth, migration, invasion, and tumorigenicity. Moreover, luciferase reporter assays and chromatin immunoprecipitation analysis revealed that the 14-3-3β-FBI1/Akirin2 complex bound to the BCAM promoter and repressed transcription. Thus, these data indicate that BCAM is a suppressive oncoprotein, and that FBI1/Akirin2 is involved in tumorigenicity and metastasis of hepatoma through the downregulation of suppressive oncogenes.