Impaired neuronal activity and differential gene expression in STXBP1 encephalopathy patient iPSC-derived GABAergic neurons.

Syntaxin-binding protein 1 (STXBP1; also called MUNC18-1), encoded by STXBP1, is an essential component of the molecular machinery that controls synaptic vesicle docking and fusion. De novo pathogenic variants of STXBP1 cause a complex set of neurological disturbances, namely STXBP1 encephalopathy (STXBP1-E) that includes epilepsy, neurodevelopmental disorders and neurodegeneration. Several animal studies have suggested the contribution of GABAergic dysfunction in STXBP1-E pathogenesis. However, the pathophysiological changes in GABAergic neurons of these patients are still poorly understood. Here, we exclusively generated GABAergic neurons from STXBP1-E patient-derived induced pluripotent stem cells (iPSCs) by transient expression of the transcription factors ASCL1 and DLX2. We also generated CRISPR/Cas9-edited isogenic iPSC-derived GABAergic (iPSC GABA) neurons as controls. We demonstrated that the reduction in STXBP1 protein levels in patient-derived iPSC GABA neurons was slight (approximately 20%) compared to the control neurons, despite a 50% reduction in STXBP1 mRNA levels. Using a microelectrode array-based assay, we found that patient-derived iPSC GABA neurons exhibited dysfunctional maturation with reduced numbers of spontaneous spikes and bursts. These findings reinforce the idea that GABAergic dysfunction is a crucial contributor to STXBP1-E pathogenesis. Moreover, gene expression analysis revealed specific dysregulation of genes previously implicated in epilepsy, neurodevelopment and neurodegeneration in patient-derived iPSC GABA neurons, namely KCNH1, KCNH5, CNN3, RASGRF1, SEMA3A, SIAH3 and INPP5F. Thus, our study provides new insights for understanding the biological processes underlying the widespread neuropathological features of STXBP1-E.

Craniofacial Characteristics and Orthodontic Treatment of Diamond Blackfan Syndrome: Two Case Reports.

Diamond Blackfan anemia (DBA) is a chronic congenital form of erythrocytic hypoplasia in which erythroid precursor cell levels are low. DBA reflects ribosomal dysfunction and is accompanied by hematopoietic cell apoptosis, anemia, and various somatic symptoms. We report the characteristic symptoms of the craniofacial region and the orthodontic treatments of two DBA cases. Case 1 was a 12-year-old female. The typical physical and facial characteristics of DBA were lacking. On initial examination, she exhibited a skeletal Class II jaw and end to end molar relationships and a large overjet. An edgewise appliance was placed after extraction of the first maxillary premolars. After 3 years and 11 months, an appropriate overjet and overbite, rigid intercuspation, and an acceptable profile were evident without any clinical adverse effects. Case 2 was a 13-year-old female. She exhibited a skeletal Class I jaw relationship, a spaced dental arch, the maxillofacial dysplasia characteristic of Binder syndrome, hypoplasia of the right mandibular condyle, and labial protrusions of the maxillary and mandibular incisors. We placed an edgewise appliance and after 1 year and 7 months, the occlusion was optimal in the absence of any adverse effects. Our two DBA cases exhibited a broad spectrum of physical and dentofacial symptoms. Patients with DBA are often prescribed combined steroid/bisphosphonate therapies. Both agents are likely to affect alveolar bone remodeling after tooth extraction and orthodontic tooth movement. Careful consideration of medication with reference to various dentofacial characteristics is necessary.

Evaluation of indices for predicting recovery of exercise tolerance in patients surviving allogenic hematopoietic stem cell transplantation.

PURPOSE: Decline in physical function in the early stage after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a major challenge. Exercise tolerance tests, such as the 6-min walk test, are useful markers for predicting exercise tolerance and various other traits, including cardiometabolic risk and non-relapse mortality. This retrospective cohort study aimed to investigate and identify predictors of recovery of exercise tolerance in the early stage after allo-HSCT. METHODS: Ninety-eight patients were classified into recovery and non-recovery groups according to the median 6-min walk distance (6MWD) at discharge. RESULTS: Logistic regression analysis revealed that pre-post change in knee extensor strength (ΔKES) and hematopoietic cell transplantation comorbidity index were useful predictors of recovery of exercise tolerance at discharge and moderate predictors of 6MWD recovery in the early post-transplant period. Receiver operating characteristic analysis showed that pre-transplant ΔKES was an accurate predictor of 6MWD recovery in the early post-transplant period. The cutoff point for ΔKES calculated using the Youden index was - 1.17 Nm/kg. CONCLUSIONS: The results of this study emphasize the importance of the need for programs designed to prevent muscle weakness in the early period after allo-HSCT. The results from markers of recovery of exercise tolerance are promising and can be used for patient education in rehabilitation programs after allo-HSCT.

School-based cognitive behavioural intervention programme for addressing anxiety in 10- to 11-year-olds using short classroom activities in Japan: a quasi-experimental study.

BACKGROUND: Although several school-based cognitive behavioural intervention programmes have been developed in Japan to prevent and improve children's anxiety disorders, the substantial time required for their completion remains a problem. METHODS: A brief version of the cognitive behavioural programme called 'Journey of the Brave', developed for Japanese children was conducted among 90 children aged 10‒11 years using 20-min short classroom activities, and its effectiveness was examined. The children were divided into two groups: the intervention (n = 31) and control groups (n = 59). The control group did not attend any programme sessions and followed regular school curriculum. We conducted 14 weekly programme sessions and assessed children at pre-intervention, post-intervention, and 2-month follow-up (6 months after the beginning). The primary and secondary outcome measures were the Spence Children's Anxiety Scale (SCAS) to assess children's anxiety symptoms and the Strengths and Difficulties Questionnaire (SDQ) to measure behaviour problems, respectively. RESULTS: A statistically significant reduction in the SCAS score in the intervention group was found at 2-month follow-up compared with the control group. A significant reduction was also observed in the SDQ score. CONCLUSIONS: Our findings suggested that the 'Journey of the Brave' programme, which requires only 5 h of short classroom activities, demonstrates promising results compared with previous programmes. A larger randomised control trial would be desirable. TRIAL REGISTRATION: UMIN, UMIN000009021, Registered 10 March 2012.

Ten-m/Odz3 regulates migration and differentiation of chondrogenic ATDC5 cells via RhoA-mediated actin reorganization.

Tenascin-like molecule major (Ten-m)/odd Oz (Odz), a type II transmembrane molecule, is well known to modulate neural development. We have reported that Ten-m/Odz3 is expressed in cartilaginous tissues and cells. Actin cytoskeleton and its regulator ras homolog gene family member A (RhoA) are closely associated with chondrogenesis. The present study aimed to evaluate the function and molecular mechanism of Ten-m/Odz3 during chondrogenesis, focusing on RhoA and the actin cytoskeleton. Ten-m/Odz3 was expressed in precartilaginous condensing mesenchyme in mouse limb buds. Ten-m/Odz3 knockdown in ATDC5 induced actin cytoskeleton reorganization and change of cell shape through modulation of RhoA activity and FGF2 expression. Ten-m/Odz3 knockdown suppressed ATDC5 migration and expression of genes associated with chondrogenesis, such as Sox9 and type II collagen, via RhoA. On the other hand, Ten-m/Odz3 knockdown inhibited proliferation of ATDC5 in a RhoA-independent manner. These findings suggest that Ten-m/Odz3 plays an important role in early chondrogenesis regulating RhoA-mediated actin reorganization.

Analysis of factors associated with patient-reported physical functioning scores at discharge of allogeneic hematopoietic stem cell transplantation patients: a cross-sectional study.

PURPOSE: The purpose of this study was to clarify the independent factors related to patient-reported physical functioning (PF) scores at discharge of patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: A total of 103 patients who underwent allo-HSCT were included in this cross-sectional study. As a screening method, a single regression analysis was conducted with the PF domain in the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 at discharge as the dependent variable, and body mass index, adverse events related to HSCT, and objective physical functions as independent variables. Multiple regression analysis was performed with PF as the dependent variable and variables that passed the screening by single regression analysis and confounders as independent variables. RESULTS: The mean PF score at discharge of the patients was 76.5 (standard deviation: 15.2). Based on the results of screening by the single regression analysis, length of stay, infections (+ / -), acute graft-versus-host disease grade, brief fatigue inventory score (BFI), knee extensor strength, and 6-min walk distance (6MWD) were included in the multiple regression analysis. BFI (B = - 11.94, p < 0.001) and 6MWD (per 10 m) (B = 0.56, p = 0.001) were extracted as significant independent variables governing the PF at discharge in the multiple regression model (adjusted R2 = 0.59). CONCLUSION: Higher exercise tolerance and lower fatigue in patients who underwent allo-HSCT were associated independently with patient-reported better PF scores at discharge.

Effect of the severity of acute graft-versus-host disease on physical function after allogeneic hematopoietic stem cell transplantation.

PURPOSE: The purpose of this study was to retrospectively investigate the effect of the severity of acute graft-versus-host disease (GVHD) on physical function after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: 76 patients were included as subjects of this study. Severity of acute GVHD was classified according to the method defined by Grucksberg. To evaluate physical function, the knee extensor strength and six-minute walk distance (6MWD) were performed. RESULTS: Among these patients, 54% developed acute GVHD; of these, 32%, 54%, and 15% of patients had grade I, grade II, and grades III-IV GVHD, respectively. In the grade I-II groups, mild acute GVHD following allo-HSCT resulted in a gradual decline in physical function, which improved at discharge. However, in cases of severe acute GVHD, physical function deteriorated, implementation of rehabilitation became difficult, and the decline in physical function persisted even at discharge. CONCLUSION: These results indicate that severe acute GVHD negatively affects physical function leading to longer hospital days because of inadequate rehabilitation interventions.

Establishment of isogenic iPSCs from an individual with SCN1A mutation mosaicism as a model for investigating neurocognitive impairment in Dravet syndrome.

Dravet syndrome (DS) is a severe childhood epilepsy typically caused by de novo dominant mutations in SCN1A. Although patients with DS frequently have neurocognitive abnormalities, the precise neural mechanisms responsible for their expression have not been elucidated. There are wide phenotypic differences among individuals with SCN1A mutations, suggesting that factors other than the SCN1A mutation modify the phenotype. Therefore, a well-controlled cellular model system is required to improve our understanding of the mechanisms underlying DS. Here we generated induced pluripotent stem cell (iPSC) lines from an individual with SCN1A mutation mosaicism, and separately cloned iPSC lines both with and without the SCN1A mutation. These clones theoretically have the same genetic backgrounds, except for the SCN1A gene, and should serve as an ideal pair for investigating the pathophysiology caused by SCN1A mutations. Quantitative reverse transcription-PCR and western blot analysis revealed higher tyrosine hydroxylase mRNA and protein expression levels in mutant neurons than in wild-type neurons. Moreover, dopamine concentrations in media collected from mutant neural cultures were higher than those from wild-type neural cultures. Our findings suggest that SCN1A mutation leads to changes in the dopamine system that may contribute to the behavioral abnormalities in DS.

Mislocalization of syntaxin-1 and impaired neurite growth observed in a human iPSC model for STXBP1-related epileptic encephalopathy.

Syntaxin-binding protein 1 (STXBP1) is essential for synaptic vesicle exocytosis. Mutations of its encoding gene, STXBP1, are among the most frequent genetic causes of epileptic encephalopathies. However, the precise pathophysiology of STXBP1 haploinsufficiency has not been elucidated. Using patient-derived induced pluripotent stem cells (iPSCs), we aimed to establish a neuronal model for STXBP1 haploinsufficiency and determine the pathophysiologic basis for STXBP1 encephalopathy. We generated iPSC lines from a patient with Ohtahara syndrome (OS) harboring a heterozygous nonsense mutation of STXBP1 (c.1099C>T; p.R367X) and performed neuronal differentiation. Both STXBP1 messenger RNA (mRNA) and STXBP1 protein expression levels of OS-derived neurons were approximately 50% lower than that of control-derived neurons, suggesting that OS-derived neurons are a suitable model for elucidating the pathophysiology of STXBP1 haploinsufficiency. Through Western blot and immunocytochemistry assays, we found that OS-derived neurons show reduced levels and mislocalization of syntaxin-1, a component of soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. In addition, OS-derived neurons have impaired neurite outgrowth. In conclusion, this model enables us to investigate the neurobiology of STXBP1 encephalopathy throughout the stages of neurodevelopment. Reduced expression of STXBP1 leads to changes in the expression and localization of syntaxin-1 that may contribute to the devastating phenotype of STXBP1 encephalopathy.

Contributions of the light environment and co-sleeping to sleep consolidation into nighttime in early infants: A pilot study.

BACKGROUND: Sleep consolidation into nighttime is considered the primary goal of sleep development in early infants. However, factors contributing to sleep consolidation into nighttime remain unclear. AIM: To clarify the influences of the light environment and nighttime co-sleeping on sleep consolidation into nighttime in early infants. STUDY DESIGN: Cross-sectional study. SUBJECTS AND METHODS: Sleep-wake time and light stimulation were measured in infants for 4 consecutive days using actigraphy. The infants' mothers were asked to complete a sleep events diary and a questionnaire about childcare, including "co-sleeping", defined as when the infant and mother slept on the same surface throughout the night. OUTCOME MEASURES: The data were analyzed with a focus on daytime and nighttime sleep parameters. RESULTS: Daytime light stimulation reduced daytime "active sleep", tended to reduce daytime sleep, and increased daytime waking. Nighttime light stimulation reduced nighttime "quiet sleep" and nighttime sleep and increased nighttime waking. Co-sleeping reduced nighttime waking, and, as a result, nighttime sleep time and sleep efficiency increased. Co-sleeping reduced daytime sleep and tended to increase daytime waking. Consequently, co-sleeping tended to increase the ratio of nighttime sleep to daytime sleep. CONCLUSIONS: The present findings suggest that an appropriate light environment promotes daytime waking and nighttime sleep in early infants, but it does not contribute to sleep consolidation into nighttime by itself. On the other hand, co-sleeping may promote sleep consolidation into nighttime. Therefore, further methods for safe co-sleeping need to be established while avoiding risk factors for sudden unexpected death in infancy/sudden infant death syndrome.

Influence of feeding a soft diet on proteoglycan expression in rat temporomandibular joint discs.

OBJECTIVES: Extracellular matrix components play a significant role in maintaining tissue integrity and pathological processes of the temporomandibular joint (TMJ). This study aimed to evaluate the influence of a soft diet on the mRNA expression of proteoglycans and glycosaminoglycans (GAGs) linked to proteoglycan core proteins in rat TMJ discs. METHODS: Thirty 4-week-old male Wistar rats were assigned to one of two groups: a control group fed a regular pellet diet and a soft diet group fed a powdered diet for 4 weeks. The mRNA expression levels of 12 proteoglycans in TMJ discs were evaluated using real-time polymerase chain reaction (PCR). In addition, histomorphometric and biochemical analyses were performed to evaluate the thickness and deoxyribonucleic acid (DNA), GAG, and water content of the TMJ discs. RESULTS: The TMJ disc thickness in the anterior, intermediate, and posterior bands decreased significantly in the soft diet group. The GAG content decreased significantly in the soft-diet group, whereas no significant differences in DNA content or water content ratio were observed between the groups. Real-time PCR indicated that the expression levels of aggrecan, versican, biglycan, decorin, fibromodulin, lumican, and chondroadherin decreased in the soft diet group. The expression levels of all versican isoforms decreased in the soft diet group. CONCLUSIONS: These results indicate that the biomechanical environment of the TMJ caused by a soft diet is closely related to the expression of proteoglycans in TMJ discs, which may eventually increase the fragility of the TMJ discs.

Three-Dimensional Evaluation of Treatment Effects and Post-Treatment Stability of Maxillary Molar Intrusion Using Temporary Anchorage Devices in Open Bite Malocclusion.

Background: We investigated treatment outcomes and post-treatment stability in 10 patients with an anterior open bite and nonsurgical orthodontics. Methods: The patients underwent maxillary molar intrusion using temporary anchorage devices (TADs) to deepen the overbite due to mandibular autorotation. Lateral cephalograms and dental cast models were obtained before treatment (T0), immediately after it (T1), and >1 year after it (T2). Skeletal and dental cephalometric changes and three-dimensional movements of the maxillary dentitions were evaluated. Results: At T0, cephalometric analysis indicated that patients had skeletal class I with tendencies for a class II jaw relationship and a skeletal open bite. During active treatment (T0 to T1), the maxillary first molar intruded by 1.6 mm, the mandibular first molar extruded by 0.3 mm, the Frankfort-mandibular plane angle decreased by 1.1°, and the overbite increased by 4.1 mm. Statistically significant changes were observed in the amount of vertical movement of the maxillary first molar, Frankfort-mandibular plane angle, and overbite. Three-dimensional (3D) dental cast analysis revealed that the maxillary first and second molars intruded, whereas the anterior teeth extruded, with the second premolar as an infection point. In addition, the maxillary molar was tipped distally by 2.9° and rotated distally by 0.91°. Statistically significant changes were observed in the amount of vertical movement of the central incisor, lateral incisor, canine and first molar, and molar angulation. From T1 to T2, no significant changes in cephalometric measurements or the 3D position of the maxillary dentition were observed. The maxillary and mandibular dentitions did not significantly change during post-treatment follow-up. Conclusions: Maxillary molar intrusion using mini-screws is an effective treatment for open bite correction, with the achieved occlusion demonstrating 3D stability at least 1 year after treatment.

[Modulation of transcriptional regulation during bone and cartilage development and their disease].

Genetic and biochemical studies have identified transcription factors critical and specific for bone and cartilage development. More recent studies revealed the molecular mechanisms how these transcription factors regulate bone and cartilage development. Especially, we appreciate recent advances in molecular function of the complex assembled by these transcription factors and epigenetic regulation of them. Aging, inflammation, biological stress, and disorder of endocrine system induce several bone and/or cartilage diseases by affecting the transcriptional and epigenetic regulation. In this review, we would like to describe the transcriptional and epigenetic regulation during developmental and pathological stages. In addition, we discuss possible application of these information in regeneration of bone and cartilage.

SMN promotes mitochondrial metabolic maturation during myogenesis by regulating the MYOD-miRNA axis.

Spinal muscular atrophy (SMA) is a congenital neuromuscular disease caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although the primary cause of progressive muscle atrophy in SMA has classically been considered the degeneration of motor neurons, recent studies have indicated a skeletal muscle-specific pathological phenotype such as impaired mitochondrial function and enhanced cell death. Here, we found that the down-regulation of SMN causes mitochondrial dysfunction and subsequent cell death in in vitro models of skeletal myogenesis with both a murine C2C12 cell line and human induced pluripotent stem cells. During myogenesis, SMN binds to the upstream genomic regions of MYOD1 and microRNA (miR)-1 and miR-206. Accordingly, the loss of SMN down-regulates these miRs, whereas supplementation of the miRs recovers the mitochondrial function, cell survival, and myotube formation of SMN-deficient C2C12, indicating the SMN-miR axis is essential for myogenic metabolic maturation. In addition, the introduction of the miRs into ex vivo muscle stem cells derived from Δ7-SMA mice caused myotube formation and muscle contraction. In conclusion, our data revealed novel transcriptional roles of SMN during myogenesis, providing an alternative muscle-oriented therapeutic strategy for SMA patients.

A case of immunoglobulin G4-related retroperitoneal fibrosis and hypophysitis with antecedent respiratory disease followed by spontaneous remission and recurrence.

A 65-year-old man presented with apparent bronchopneumonia. After treatment with antibiotics, he showed eosinophilia. Computed tomography (CT) imaging revealed bilateral consolidation, ground-glass opacities with nodular consolidations, and pleural effusion. Lung biopsy showed organising pneumonia with lymphoplasmacytic infiltration in the alveolar septa and in the thickened pleura and interlobular septa. All pulmonary abnormalities spontaneously went into remission within 12 months. At 73 years old, a follow-up CT scan revealed small nodules in both lungs and the review of the head CT scan showed thickening of the pituitary stalk in studying prolonged headache. Two years later, he visited the hospital complaining of severe oedema on the lower extremities with high serum immunoglobulin (Ig)G4 186 mg/dl. A whole-body CT scan showed retroperitoneal mass surrounding aortic bifurcation and compressing inferior vena cava, pituitary stalk thickening and gland swelling, and enlarged pulmonary nodules. Anterior pituitary stimulation tests showed central hypothyroidism, central hypogonadism, and adult growth hormone deficiency with partial primary hypoadrenocorticism. Retroperitoneal mass biopsy showed storiform fibrosis and obliterative phlebitis with marked lymphoplasmacytic infiltration with moderate IgG4-positivity. Immunostaining of the former lung specimen revealed dense interstitial infiltration of IgG4-positive cells. These findings indicated metachronous development of IgG4-related disease in lung, hypophysis, and retroperitoneum, according to the recent comprehensive diagnostic criteria of IgG4-related disease. Glucocorticoid therapy ameliorated oedema, on the other hand, unmasked partial diabetes insipidus at the initial dose of the treatment. Hypothyroidism and retroperitoneal mass regressed at 6 months of the treatment. This case warns us that long-term follow-up from prodromal to remission is necessary for the treatment of IgG4-related disease.

[Role and function regulation of transcription factors in endochondral ossification].

Endochondral ossification plays critical roles in skeletal development and tissue patterning. Disruption of endochondral ossification and cartilage homeostasis results in skeletal dysplasia and cartilage diseases such as osteoarthritis. Several transcription factors regulate chondrocyte differentiation and expression of chondrogenic matrices, and consequently maintains order of endochondral ossification. In this review article, we would like to introduce recent advancement in understanding of role and functional regulation of transcription factors in endochondral ossification.

Bacterial meningitis due to the Streptococcus mitis group in children with cerebrospinal fluid leak.

The Streptococcus mitis group constitutes a part of the oral flora in humans and has been reported to cause infective endocarditis, brain abscesses, sepsis, pneumonia, and peritonitis. However, the S. mitis group rarely causes meningitis in children. We experienced a case of bacterial meningitis due to the S. mitis group in a 14-year-old girl with Gorham-Stout disease undergoing treatment with sirolimus for skull base osteolysis and cerebrospinal fluid (CSF) leak. Antibiotic treatment was initiated with linezolid and levofloxacin due to allergies against ß-lactam antibiotics. On the third treatment day, antibiotics were switched to penicillin G according to CSF culture results, which were positive for penicillin-susceptible S. mitis group. Antibiotic therapy was successfully completed after 14 days without any neurological sequelae. There have apparently been no reports of S. mitis meningitis in pediatric patients with skull base osteolysis and CSF leak as in our case. Our findings suggest that clinicians should be aware of the possibility of S. mitis meningitis for patients with skull base osteolysis and/or CSF leakage.