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The hypocholesterolemic peptide, LPYPR, was successfully introduced into the VR-1, VR-2, and VR-5 regions of the mung bean 8Sα globulin. The mutant protein (MP) has 96.69% structural homology and 97% sequence homology compared to the wild type (WT). Expression of the mutant protein in E. coli HMS174(DE3) was 40.66%, which was 144.42% higher than that of the WT. The WT protein and MP had MWs of about 48.4 and 48.7 kDa, respectively. These were purified using HIC and digested with trypsin. UPLC analysis of the tryptic digests of the MP revealed the successful release of the LPYPR peptide. Unlike the WT protein, cholesterol-binding capacity (mg/g sample) of the MP increased over time of tryptic digestion (average growth rate of 9.5% for crude MP and 12.5% for HIC-purified MP) for its undigested form (crude: 220.96 ± 8.65, purified: 214.71 ± 11.91), with maximum values of 380.76 ± 6.61 and 434.44 ± 10.88 were obtained for the 24-h digests of the crude and purified proteins, respectively. Similarly, the sodium taurocholate binding capacity (%) was also found to increase over time of tryptic digestion (average growth rate of 4% for crude MP and 5.67% for HIC-purified MP) for the tryptic digests of the MP. Minimum values for % bound sodium taurocholate was obtained with the undigested samples (crude: 46.71 ± 0.42, purified: 44.49 ± 0.13), while maximum values thereof were obtained with the 24-h digest samples (crude: 59.75 ± 0.30, purified 61.95 ± 0.51). [ABSTRACT FROM AUTHOR]

Bioactive peptides from okra seeds were successfully isolated, purified, and characterized for its antihypertensive properties. Okra seeds were decorticated, defatted, and suspended in 35 mM phosphate buffer (pH 7.6, with 0.4 M NaCl and 0.02% NaN3) to isolate the okra seed crude protein extract (OSCPE). The protein content of OSCPE was estimated to be 13.8 ± 0.6 mg/mL. Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) revealed six major polypeptide bands with molecular weights of 11, 13, 16, 18, 25, and 49 kDa. Bioactive peptides from OSCPE were released through separate enzymatic hydrolysis, using pepsin and α-chymotrypsin, from 0 min to 24 h of incubation. OSCPE was more resistant to enzymatic hydrolysis with α-chymotrypsin than pepsin. The 12-h hydrolysate from both enzymes exhibited the highest angiotensin I-converting enzyme (ACE) inhibitory activity. Both hydrolysates were fractionated through anion-exchange chromatography (IEX) using a DEAE-Sephacel column (1.6 cm × 5.0 cm). Fractions from prominent peaks were subjected to ACE inhibition assay. The 17th peptic IEX fraction and the 15th chymotryptic IEX fraction showed the highest ACE inhibitory activities of 68.68 ± 2.91% and 71.89 ± 7.77%, with IC50 values of 8.62 ± 1.17 μg/mL and 2.44 ± 0.87 μg/mL, respectively. Aside from sustaining an individual's daily protein requirements, okra seeds also have the potential to further improve health by releasing bioactive peptides that can alleviate hypertension. [ABSTRACT FROM AUTHOR]

Introduction: NGLY1-associated congenital disorder of deglycosylation (CDDG1: OMIM #615273) is a rare autosomal recessive disorder caused by a functional impairment of endoplasmic reticulum in degradation of glycoproteins. Neurocognitive dysfunctions have been documented in patients with CDDG1; however, deteriorating phenotypes of affected individuals remain elusive., Case Presentation: A Japanese boy with delayed psychomotor development showed ataxic movements from age 5 years and myoclonic seizures from age 12 years. Appetite loss, motor and cognitive decline became evident at age 12 years. Electrophysiological studies identified paroxysmal discharges on myoclonic seizure and a giant somatosensory evoked potential. Perampanel was effective for controlling myoclonic seizures. Exome sequencing revealed that the patient carried compound heterozygous variants in NGLY1, NM_018297.4: c.857G > A and c.-17_12del, which were inherited from mother and father, respectively. A literature review confirmed that myoclonic seizures were observed in 28.5% of patients with epilepsy. No other patients had progressive myoclonic epilepsy or cognitive decline in association with loss-of-function variations in NGLY1., Conclusion: Our data provides evidence that a group of patients with CDDG1 manifest slowly progressive myoclonic epilepsy and cognitive decline during the long-term clinical course., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest concerning the present study., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction: Open-lip-type schizencephaly is characterized by trans-cerebral clefts filled with cerebrospinal fluid (CSF) between the subarachnoid space at the hemisphere surface and the lateral ventricles. Disorders related to CSF retention, including hydrocephalus and arachnoid cysts, have reportedly been associated with open-lip schizencephaly and have induced intracranial hypertension in some cases. However, detailed neuroimaging and surgical treatment findings have rarely been described., Case Presentation: We report 2 cases of open-lip schizencephaly with an expanding CSF-filled cavity overlying the ipsilateral cerebral hemisphere that manifested as signs of intracranial hypertension. Detailed three-dimensional heavily T2-weighted imaging revealed thin borders between the CSF-filled cavity and the subarachnoid space, but no separating structures between the cavity and the lateral ventricle, suggesting that the cavity was directly connected to the lateral ventricle through the schizencephalic cleft but not to the subarachnoid space. Neuroendoscopic observation in case 1 confirmed this finding. Endoscopic fenestration of the cavity to the prepontine cistern was ineffective in case 1. Shunting between the lateral ventricle (case 1) or CSF-filled cavity (case 2) and the peritoneal cavity slightly decreased the size of the CSF-filled cavity., Discussion: We speculate that the thin borders along the margin of the CSF-filled cavity are membranes that previously covered the schizencephalic cleft and are now pushed peripherally. In addition, we believe that the cavity is a ventricular diverticulum protruding through the cleft and that shunting operation is effective against such expanding cavity. Detailed magnetic resonance imaging can be useful for evaluating patients with schizencephaly associated with CSF retention disorders., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Competing Interests: Declaration of Competing Interest None.

Mutations in the gene encoding the transient receptor potential vanilloid member 4 (TRPV4), a Ca 2+ permeable nonselective cation channel, cause TRPV4-related disorders. TRPV4 is widely expressed in the brain; however, the pathogenesis underlying TRPV4-mediated Ca 2+ deregulation in neurodevelopment remains unresolved and an effective therapeutic strategy remains to be established. These issues were addressed by isolating mutant dental pulp stem cells from a tooth donated by a child diagnosed with metatropic dysplasia with neurodevelopmental comorbidities caused by a gain-of-function TRPV4 mutation, c.1855C > T (p.L619F). The mutation was repaired using CRISPR/Cas9 to generate corrected isogenic stem cells. These stem cells were differentiated into dopaminergic neurons and the pharmacological effects of folic acid were examined. In mutant neurons, constitutively elevated cytosolic Ca 2+ augmented AKT-mediated α-synuclein (α-syn) induction, resulting in mitochondrial Ca 2+ accumulation and dysfunction. The TRPV4 antagonist, AKT inhibitor, or α-syn knockdown, normalizes the mitochondrial Ca 2+ levels in mutant neurons, suggesting the importance of mutant TRPV4/Ca 2+ /AKT-induced α-syn in mitochondrial Ca 2+ accumulation. Folic acid was effective in normalizing mitochondrial Ca 2+ levels via the transcriptional repression of α-syn and improving mitochondrial reactive oxygen species levels, adenosine triphosphate synthesis, and neurite outgrowth of mutant neurons. This study provides new insights into the neuropathological mechanisms underlying TRPV4-related disorders and related therapeutic strategies., (© 2023 The Authors. FASEB BioAdvances published by Wiley Periodicals LLC on behalf of The Federation of American Societies for Experimental Biology.)

Vitamin deficiencies are an emerging concern in the management of children with autism spectrum disorder (ASD). Particular attention is required for recognizing the variable signs caused by unbalanced food intakes. We herein report two patients with multiple vitamin deficiencies who needed critical care showing different prognoses. Patient 1 with 'Shoshin' beriberi presenting with cardiac arrest had thiamine deficiency developed severe neurological sequelae despite rapid vitamin supplementation. Patient 2, who had leg pain and a limping gait, showed a rapid recovery with intravenous infusion and tube feeding after being diagnosed with scurvy. A literature search revealed several children with ASD with critically ill thiamine deficiency, but few reports documented a life-threatening condition in the form of cardiac arrest at the onset. Considering the high observation rate of food selectivity in children with ASD, early intervention is required to prevent the exacerbation of vitamin deficiencies to severe neurological disabilities., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: To determine the neurodevelopmental outcomes of very-low-birth-weight infants (VLBWIs, birth weight <1,500 g) after 9 years of follow-up., Methods: This study prospectively recruited 224 VLBWIs born from 2003 to 2009 in Kyushu University Hospital, Japan. Comorbidities of neurocognitive impairment, epilepsy, and autism spectrum disorder or attention-deficit hyperactivity disorder (ASD/ADHD) were assessed at age 3, 6, and 9 years., Results: Neurodevelopmental profiles were obtained from 185 (83%), 150 (67%), and 119 (53%) participants at age 3, 6, and 9 years, respectively. At age 9 years, 25 (21%) VLBWIs showed intelligence quotient (IQ) <70, 11 (9%) developed epilepsy, and 14 (12%) had a diagnosis of ASD/ADHD. The prevalence of epilepsy was higher in children with an IQ <70 at age 9 years than in those with an IQ ≥70 (44% vs 0%). In contrast, ASD/ADHD appeared at similar frequencies in children with an IQ <70 (16%) and ≥70 (11%). Perinatal complications and severe brain lesions on MRI were considered common perinatal risks for developmental delay and epilepsy but not for ASD/ADHD. Male sex was identified as a unique risk factor for ASD/ADHD., Conclusion: These data suggest that VLBWIs showed a higher prevalence of developmental delay, epilepsy, and ASD/ADHD at age 9 years than the general population. Distinct mechanisms might be involved in the pathogenic process of ASD/ADHD from those of developmental delay and epilepsy., (© 2021 American Academy of Neurology.)

Background: Malnutrition and vitamin deficiency are growing concerns in the clinical management of children with autism spectrum disorder (ASD). This case report presents a boy with ASD who developed vitamin A deficiency during follow-up., Case Report: A 7-y-old boy had been diagnosed with ASD and developmental delay at age 18 mo. He developed convulsions associated with hypocalcemia and vitamin D deficiency at 3 y of age. Although vitamin D supplementation was continued, he was only able to eat rice, green tea, and fried potatoes from 3 y of age to age 7 y. He had started rubbing his eyes and had refused to open his eyes 9 mo before. An ophthalmologic examination showed bilateral corneal ulcers and right corneal perforation. Vitamin A was immediately supplemented with a nasogastric tube; however, his right eye was surgically enucleated against the persistent infection., Literature Review: A search of the relevant literature from 1993 to 2020 identified 11 cases of patients with ASD (5-17 y of age) who developed vitamin A deficiency owing to malnutrition. Only 4 cases (36%) had a full recovery in visual acuity., Conclusion: Vitamin A deficiency frequently causes irreversible visual impairment in children with ASD. Vigilant monitoring of vitamin levels prevents unfavorable outcomes in children with ASD and difficulty in food intake., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: To investigate the association between breast feeding and infant development during the first year of life using sibling comparison., Design: Nationwide prospective birth cohort study with sibling pair analysis., Setting: 15 regional centres that participated in the Japan Environment and Children's Study., Participants: This study included 77 119 children (singleton, term birth and no malformation/severe diseases) whose mothers were registered between January 2011 and March 2014, including 3521 duos or trios of siblings., Primary Outcome Measures: The primary outcome was developmental delay at 6 and 12 months of age, assessed using the Japanese translation of the Ages and Stages Questionnaires, third edition. Multivariable regression analyses adjusted for confounders were performed to estimate the risk ratios of delay associated with any or exclusive breast feeding. Pairs of siblings discordant for statuses were selected, and conditional regression analyses were conducted with a matched cohort design., Results: Developmental delay was identified in 6162 (8.4%) and 10 442 (14.6%) children at 6 and 12 months of age, respectively. Any breast feeding continued until 6 months or 12 months old was associated with reduced developmental delay at 12 months of age (adjusted risk ratio (95% CI): 0.81 (0.77 to 0.85) and 0.81 (0.78 to 0.84), respectively). Furthermore, exclusive breast feeding until 3 months was associated with reduced developmental delay at 12 months of age (adjusted risk ratio, 0.86 (95% CI 0.83 to 0.90)). In sibling pair analysis, the association between any breast feeding until 12 months and reduced developmental delay at 12 months of age persisted (adjusted risk ratio, 0.64 (95% CI 0.43 to 0.93))., Conclusions: The present study demonstrated the association of continuous breast feeding with reduced developmental delay at 1 year of age using sibling pair analysis, in which unmeasured confounding factors are still present but less included. This may provide an argument to promote breastfeeding continuation., Competing Interests: Competing interests: Dr MSa and Professor SO received a grant and director’s fees, respectively, from Morinaga Houshikai outside of the submitted work. The other authors have no conflicts of interest relevant to this article to disclose., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Absence epilepsy shows age-related clinical features, as is observed in childhood and juvenile absence epilepsy. Electroencephalogram (EEG) is characterized by bursts of 3 Hz spike-and-wave complex (SWC). We noticed a morphological variation of the slow-wave component of SWCs between patients. This study investigated whether the waveform of SWC might be associated with the child's age of this epilepsy., Methods: Digitally-recorded EEGs under medication-free conditions were collected from 25 children who received the diagnosis of childhood or juvenile absence epilepsy. The morphology of slow wave in SWC in the frontal midline region was quantitatively compared between younger and older children using a cluster-based permutation test., Results: At <7 years of age (2.9-6.5 years of age, n = 6), the electrical potential of the descending slope in the slow wave was positively correlated with age whereas this correlation was not observed in patients of ≥7 years of age (7.1-12.9 years, n = 19). A cluster-based permutation test confirmed the results-among the entire slow wave period (0-285 msec), the period of the descending slope (195-260 msec) showed significantly lower potential in patients of <7 years of age in comparison to patients of ≥7 years of age (sum of t-values: 46.57, p-value: 0.011)., Conclusions: The current study demonstrated an age-dependent morphological difference in the slow-wave components of SWCs in EEGs of patients with pediatric absence epilepsy. This finding may provide a clue to understanding the age-related clinical manifestations of this epilepsy., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Transient receptor potential vanilloid member 4 (TRPV4) is a Ca 2+ permeable nonselective cation channel, and mutations in the TRPV4 gene cause congenital skeletal dysplasias and peripheral neuropathies. Although TRPV4 is widely expressed in the brain, few studies have assessed the pathogenesis of TRPV4 mutations in the brain. We aimed to elucidate the pathological associations between a specific TRPV4 mutation and neurodevelopmental defects using dopaminergic neurons (DNs) differentiated from dental pulp stem cells (DPSCs). DPSCs were isolated from a patient with metatropic dysplasia and multiple neuropsychiatric symptoms caused by a gain-of-function TRPV4 mutation, c.1855C>T (p.L619F). The mutation was corrected by CRISPR/Cas9 to obtain isogenic control DPSCs. Mutant DPSCs differentiated into DNs without undergoing apoptosis; however, neurite development was significantly impaired in mutant vs. control DNs. Mutant DNs also showed accumulation of mitochondrial Ca 2+ and reactive oxygen species, low adenosine triphosphate levels despite a high mitochondrial membrane potential, and lower peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial content. These results suggested that the persistent Ca 2+ entry through the constitutively activated TRPV4 might perturb the adaptive coordination of multiple mitochondrial functions, including oxidative phosphorylation, redox control, and biogenesis, required for dopaminergic circuit development in the brain. Thus, certain mutations in TRPV4 that are associated with skeletal dysplasia might have pathogenic effects on brain development, and mitochondria might be a potential therapeutic target to alleviate the neuropsychiatric symptoms of TRPV4-related diseases., Competing Interests: All authors declare no conflicts of interest., (© 2021 The Authors. Published by Elsevier B.V.)

This case series aimed to characterize the clinical features, management, and outcomes of apnea in infants with trisomy 18. Participants in this study were infants with trisomy 18 who were born alive and admitted to the neonatal intensive care unit in Kyushu University Hospital from 2000 to 2018. Retrospective analysis was performed on clinical data recorded in our department. Twenty-seven infants with trisomy 18 were admitted to our hospital during the study period, of which 25 (nine males, 16 females) were enrolled as eligible participants in this study. Among them, 14 started presenting with apnea from median 3.5 days of age (range 0-47d). In these infants with apnea, eight received respiratory support of positive pressure ventilation (PPV). The 1-year survival rate of infants in the PPV group was higher than that of non-PPV-supported infants (5 out of 8 vs 0 out of 6 infants). Five PPV-supported infants received a diagnosis of epilepsy, which was controlled by antiepileptic drugs. Postnatal respiratory intervention provides better prognosis in infants with trisomy 18. Improved survival leads to accurate diagnosis and treatment of apneic events in association with epilepsy. WHAT THIS PAPER ADDS: Respiratory support is effective against apnea in infants with trisomy 18. Intervention with ventilation provides a higher chance of prolonged survival. Improved survival leads to the accurate diagnosis and treatment of epilepsy-associated apnea., (© 2019 Mac Keith Press.)

Background: Pierson syndrome (PS) is a rare autosomal recessive disorder, characterized by congenital nephrotic syndrome and microcoria. Advances in renal replacement therapies have extended the lifespan of patients, whereas the full clinical spectrum of PS in infancy and beyond remains elusive., Case Presentation: We present the case of a 12-month-old boy with PS, manifesting as the bilateral microcoria and congenital nephrotic syndrome. He was born without asphyxia, and was neurologically intact from birth through the neonatal period. Generalized muscle weakness and hypotonia were recognized from 3 months of age. The infant showed recurrent vomiting at age 5 months of age, and was diagnosed with gastroesophageal reflux and intestinal malrotation. Despite the successful surgical treatment, vomiting persisted and led to severely impaired growth. Tulobuterol treatment was effective in reducing the frequency of vomiting. Targeted sequencing confirmed that he had a compound heterozygous mutation in LAMB2 (NM&#95;002292.3: p.Arg550X and p.Glu1507X). A search of the relevant literature identified 19 patients with severe neuro-muscular phenotypes. Among these, only 8 survived the first 12 months of life, and one had feeding difficulty with similar gastrointestinal problems., Conclusions: This report demonstrated that severe neurological deficits and gastrointestinal dysfunction may emerge in PS patients after the first few months of life.

Background: Hypokalemic periodic paralysis (HypoPP) is an autosomal dominant disease characterized by the episodic weakness of skeletal muscles and hypokalemia. More than half patients with HypoPP carry mutations in CACNA1S, encoding alpha-1 subunit of calcium channel. Few reports have documented the non-neuromuscular phenotypes of HypoPP., Methods: The proband is a Japanese woman who developed HypoPP at 6 years of age. An excessive insulin secretion with the oral glucose tolerance test rationalized that she had experienced frequent attacks of paralysis on high-carbohydrate diets., Results: Voglibose and acetazolamide effectively controlled her paralytic episodes. Her 8-year-old son and 2-year-old daughter started showing the paralytic symptoms from 4 and 2 years of age, respectively. Laboratory tests revealed high concentrations of creatinine kinase in serum and elevated renin activities in plasma of these children. The targeted sequencing confirmed that these three patients had an identical heterozygous mutation (p.V876E) in CACNA1S., Conclusion: Our data indicate that the p.V876E mutation in CACNA1S contributes to the early onset of neuromuscular symptoms and unusual clinical phenotypes of HypoPP., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Objective: To determine clinical features of very low birth weight infants (VLBWIs) who had developed epilepsy by age 3 years., Study Design: Multicenter cohort study using the Neonatal Research Network of Japan database. We analyzed clinical variables of 8431 VLBWIs who had recorded data of neurological sequelae at age 3 years. Logistic regression identified the association between variables and development of epilepsy., Result: One hundred and forty-three (1.7%) infants developed epilepsy, 683 (8.1%) showed cerebral palsy (CP), and 1114 (13.2%) had psychomotor delay. Epilepsy was associated with history of sepsis [adjusted odds ratio (AOR) 3.23], severe intraventricular hemorrhage (IVH; AOR 5.13), and cystic periventricular leukomalacia (PVL; AOR 12.7). Severe IVH and cystic PVL were also frequently associated with CP and psychomotor delay., Conclusion: Severe IVH and cystic PVL are strongly associated with development of epilepsy, as well as other neurological sequelae, and are potential critical therapeutic targets.

Objective: To delineate the critical decision-making processes that paediatricians apply when treating children with life-threatening conditions and the psychosocial experience of paediatricians involved in such care., Design: We conducted semistructured, individual face-to-face interviews for each participant from 2014 to 2015. The content of each interview was subjected to a comprehensive qualitative analysis. The categories of dilemma were extracted from a second-round content analysis., Participants: Participants were board-certified paediatricians with sufficient experience in making decisions in relation to children with severe illnesses or disabilities. We repeated purposive sampling and analyses until we reached saturation of the category data., Results: We performed interviews with 15 paediatricians. They each reported both unique and overlapping categories of dilemmas that they encountered when making critical decisions. The dilemmas included five types of causal elements: (1) paediatricians' convictions; (2) the quest for the best interests of patients; (3) the quest for medically appropriate plans; (4) confronting parents and families and (5) socioenvironmental issues. Dilemmas occurred and developed as conflicting interactions among these five elements. We further categorised these five elements into three principal domains: the decision-maker (decider); consensus making among families, colleagues and society (process) and the consequential output of the decision (consequence)., Conclusions: This is the first qualitative study to demonstrate the framework of paediatricians' decision-making processes and the complex structures of dilemmas they face. Our data indicate the necessity of establishing and implementing an effective support system for paediatricians, such as structured professional education and arguments for creating social consensus that assist them to reach the best plan for the management of severely ill children., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)