Structural alterations in the amygdala and impaired social incentive learning in a mouse model of a genetic variant associated with neurodevelopmental disorders.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and their dimensions and changes in brain structures and behavior. However, as CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how the genes in the 22q11.2 region individually contribute to structural alterations and associated mental illnesses and their dimensions. Our previous studies have identified Tbx1, a T-box family transcription factor encoded in 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes in congenic Tbx1 heterozygous mice. Our data show that the volumes of anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were reduced in Tbx1 heterozygous mice. Moreover, we examined the behavioral consequences of an altered volume of the amygdala. Tbx1 heterozygous mice were impaired for their ability to detect the incentive value of a social partner in a task that depends on the amygdala. Our findings identify the structural basis for a specific social dimension associated with loss-of-function variants of TBX1 and 22q11.2 CNV.

Structural alterations in the amygdala and impaired social incentive learning in a mouse model of a genetic variant associated with neurodevelopmental disorders.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and their dimensions and changes in brain structures and behavior. However, as CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how the genes in the 22q11.2 region individually contribute to structural alterations and associated mental illnesses and their dimensions. Our previous studies have identified Tbx1, a T-box family transcription factor encoded in 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes in congenic Tbx1 heterozygous mice. Our data show that the volumes of anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were reduced in Tbx1 heterozygous mice. Moreover, we examined the behavioral consequences of an altered volume of the amygdala. Tbx1 heterozygous mice were impaired for their ability to detect the incentive value of a social partner in a task that depends on the amygdala. Our findings identify the structural basis for a specific social dimension associated with loss-of-function variants of TBX1 and 22q11.2 CNV.

Cranial Shape Measurements Obtained Using a Caliper and Elastic Bands Are Useful for Brachycephaly and Deformational Plagiocephaly Screening.

We assessed a method for screening the cranial shape of 1-month-old infants using a simple measuring instrument instead of a three-dimensional scanner. The Mimos craniometer was used to measure cranial length, cranial width, and two diagonal lengths to calculate the cranial index (CI) and cranial asymmetry (CA). We defined a CI > 90% as brachycephaly and CA > 5 mm as deformational plagiocephaly (DP). Intra- and inter-examiner accuracy analyses were performed on a dummy doll and 1-month-old infants. The measurements of healthy 1-month-old infants were compared with previously reported three-dimensional scanner measurements. Intra- and inter-rater measurements showed good accuracy; diagnostic accuracy comparisons of brachycephaly and DP using a three-dimensional scanner showed kappa values of 1.0 and 0.8, respectively. Comparisons were made among 113 infants matched for day-age at the date of measurement; there were no significant differences in the CI (85.0% vs. 85.2%, p = 0.98) and CA (5.9 mm vs. 6.0 mm, p = 0.48) between the scanner and caliper measurements, nor in the prevalence of brachycephaly (12.4% vs. 17.7%, p = 0.35) or DP (58.4% vs. 56.6%, p = 0.89). This simple measurement method using calipers and bands was useful in screening for brachycephaly and DP in 1-month-old infants.

Denominator of Cranial Vault Asymmetry Index: Choosing Between Longer and Shorter Diagonal Lengths.

Since it was proposed in this journal in 2001, the cranial vault asymmetry index (CVAI) has been an important parameter for assessing cranial shape. However, different publications currently use different variables in the denominator of the CVAI formula. We thus investigated the use of long and short diagonal lengths as variables in the denominator of the CVAI formula. We searched the databases of PubMed, Google Scholar, and Scopus for articles published between 2016 and 2022 that cited the original work article of CVAI. Articles were included if they were written in English and if the denominator of the CVAI formula was specified. For multiple articles by the same author, only the most recent article was included. In total, 30 articles were included; 10 articles used the longer diagonal length as the denominator and 20 articles used the shorter diagonal length. No uniform trend was observed by a country or journal of publication. Application of the CVAI formula using different denominators yielded interchangeable results, and the resulting values had only negligible differences clinically. However, it would be necessary to create a standard formula for using the CVAI as a parameter for reporting cranial shape assessments consistently.

Evaluating Cranial Growth in Japanese Infants Using a Three-dimensional Scanner: Relationship between Growth-related Parameters and Deformational Plagiocephaly.

In this study, we aimed to evaluate the longitudinal changes in the cranial shape of healthy Japanese infants using a three-dimensional scanner and construct a normal values database for the growth process. Preterm infants (gestational age < 37 weeks), infants with neonatal asphyxia (5-minute Apgar score of <7), and patients who started helmet therapy for deformational plagiocephaly were excluded from this study. The first scan was performed at approximately 1 month of age, followed by two scans conducted at 3 and 6 months of age. The parameters considered were as follows: cranial length, width, height, circumference, volume, cranial vault asymmetry index, and cephalic index. A cranial vault asymmetry index >5% was defined as deformational plagiocephaly. Changes in each parameter were examined using repeated-measures analysis of variance classified by sex and deformational plagiocephaly status. The rate of increase in each parameter was also examined. In total, 88 infants (45 boys and 43 girls) were included in this study. All growth-related parameters were noted to increase linearly with time. Sex differences were observed in all parameters except cranial length. Deformational plagiocephaly was found to have no effect on growth-related parameters. Cranial volume increased by 60% from 1 to 6 months of age. The growth almost uniformly influenced the rate of increase in volume in each coordinate axis direction. Overall, the mean trends in three-dimensional parameters in infants up to 6 months of age were obtained using a three-dimensional scanner. These trends could be used as a guide by medical professionals involved in cranioplasty.

Cranial shapes of Japanese preterm infants at one month of age using a three-dimensional scanner.

BACKGROUND: Recently, cranial shape measurements of preterm infants have been performed using handheld three-dimensional (3D) scanners and can now be objectively quantified. AIMS: To measure the cranial shapes of Japanese preterm infants at one month of age using a 3D scanner, compare these values with those of healthy term infants, and examine the risk factors for dolichocephaly. STUDY DESIGN: A multicenter, retrospective cohort study. SUBJECTS: Preterm infants born at <37 weeks of gestation and staying in the neonatal intensive care unit or visiting an outpatient clinic for a one-month checkup between April 2020 and March 2022. OUTCOME MEASURES: A 3D scanner was used to quantify cranial shape. Comparison was made with full-term, one-month-old infants. RESULTS: Ninety-four preterm infants (42 boys) and 165 full-term infants were enrolled. Preterm infants had a significantly lower cephalic index (77.9% and 85.0%, p < 0.01) and a higher incidence of dolichocephaly (54.3% and 13.3%, p < 0.01) compared to term infants. No significant difference in incidence of deformational plagiocephaly was found between the groups (41.5% vs. 47.3%, p = 0.44). The risk of dolichocephaly was significantly higher for female sex (odds ratio [OR], 3.32; 95% confidence interval, 1.30-8.50), cesarean section (OR, 4.07; 95% confidence interval, 1.23-13.5), and use of mechanical ventilation (OR, 4.66; 95% confidence interval, 1.09-20.0). CONCLUSIONS: Japanese preterm infants at the first month of life had longer heads than full-term infants; the risk factors identified were female sex, cesarean section, and use of mechanical ventilation.

Three-Dimensional versus Two-Dimensional Evaluations of Cranial Asymmetry in Deformational Plagiocephaly Using a Three-Dimensional Scanner.

This study aimed to assess the measurement precision of a three-dimensional (3D) scanner that detects the geometric shape as surface data and to investigate the differences between two-dimensional (2D) and 3D evaluations in infants with deformational plagiocephaly. Using the 3D scanner that can perform both 2D and 3D evaluations, we calculated cranial asymmetry (CA) for the 2D evaluation, and the anterior symmetry ratio (ASR) and posterior symmetry ratio (PSR) for the 3D evaluation. Intra- and inter-examiner precision analyses revealed that the coefficients of the variation measurements were extremely low (<1%) for all variables, except CA (5%). In 530 infants, the coincidence rate of CA severity by the 2D evaluation and the 3D evaluation was 83.4%. A disagreement on severity was found between 2D and 3D evaluations in 88 infants (16.6%): 68 infants (12.8%) were assessed as severe by 2D evaluation and mild by the 3D evaluation, while 20 infants (3.8%) were evaluated as mild by 2D and severe by 3D evaluation. Overall, the 2D evaluation identified more infants as severe than the 3D evaluation. The 3D evaluation proved more precise than the 2D evaluation. We found that approximately one in six infants differed in severity between 2D and 3D evaluations.

Cranial Shape in Infants Aged One Month Can Predict the Severity of Deformational Plagiocephaly at the Age of Six Months.

In this study, we aimed to monitor changes in cranial shape using three-dimensional (3D) scanning to determine whether the severity of deformational plagiocephaly (DP) at the age of 6 months could be predicted at the age of 1 month. The cranial shape was measured at the ages of 1, 3, and 6 months (T1, T2, and T3, respectively) in 92 infants. We excluded those who received helmet treatment before T3. The cranial vault asymmetry index (CVAI) using 3D scanning was evaluated in all infants. DP was defined as a CVAI > 5.0% with mild (CVAI ≤ 6.25%) or moderate/severe severity (CVAI > 6.25%). The CVAI cut-off value at T1 for severe DP at T3 was determined using receiver operating characteristic (ROC) curves. At T1, T2, and T3, the respective CVAI median values were 5.0%, 5.8%, and 4.7% and the DP incidence was 50.0%, 56.8%, and 43.2%, respectively. The DP severity temporarily worsened from T1 to T2 but then improved at T3. Among the infants, 73.9% had a similar DP severity at T1 and T3 (p = 1.0). A ROC curve analysis revealed a CVAI cut-off value of 7.8% at T1 predicted severe DP. It was concluded that later DP severity could be predicted using 3D scanning at T1 with properly defined cut-off values.

Reference Values for Cranial Morphology Based on Three-dimensional Scan Analysis in 1-month-old Healthy Infants in Japan.

Currently, molded helmet therapy is used to treat infants with deformational plagiocephaly. However, the indices of normal cranial shape remain unclear, and thus, the prevalence of deformational plagiocephaly is unknown, particularly in Japan. We investigated the reference values for cranial morphological characteristics in 1-month-old Japanese infants using a three-dimensional scanner, to determine the prevalence of deformational plagiocephaly. One hundred fifty-three healthy infants who visited three hospitals (from April 2020 to March 2021) were enrolled. Cranial shape was measured using a three-dimensional scanner and was analyzed using image analysis software. Outcome measures were cranial volume, length, width, length-width ratio, circumference, asymmetry, and vault asymmetry index; cephalic index; and anterior, posterior, and overall symmetry ratios. The cranial vault asymmetry index >3.5% or ≥10% were diagnosed as deformational or severe deformational plagiocephaly, respectively. The mean age at measurement was 35.7 days. The mean cranial volume was 559 mL; cranial length, 129 mm; cranial width, 110 mm; length-width ratio, 118%; cephalic index, 85.2%; cranial circumference, 377 mm, cranial asymmetry, 6.4 mm; cranial vault asymmetry index, 5.0%; and anterior, posterior, and overall asymmetry ratios, 93.1%, 91.3%, and 96.4%, respectively. The prevalence of deformational and severe deformational plagiocephaly was 64.7% and 6.6%, respectively. Sex-based differences were observed for cranial volume and width. The results obtained in this study can be considered standard values that can facilitate the differentiation of abnormal infant cranial morphological characteristics for Japanese medical practitioners.

Correction: Orita et al. Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse ß-Oxidation Pathway in the Presence of Lanthanide. Microorganisms 2022, 10, 184.

The authors wish to make the following corrections to this paper [...].

Methotrexate mediates the integrity of intestinal stem cells partly through nitric oxide-dependent Wnt/ß-catenin signaling in methotrexate-induced rat ileal mucositis.

This study aimed to elucidate the role of nitric oxide (NO) in intestinal stem cells in methotrexate-induced ileal mucositis in rats. Methotrexate induced the mRNA expressions of the Wnt/ß-catenin target genes Wnt3a, Sox9, and Lgr5 and the Wnt-antagonist gene sFRP-1 and the protein expressions of Lgr5 and sFRP-1. Methotrexate also induced Lgr5+ cells and lysozyme+ cells. A non-selective NO inhibitor inhibited the methotrexate induction of Wnt/ß-catenin target genes and Lgr5+ cells but enhanced that of sFRP-1 expression. Thus, methotrexate mediates the integrity of intestinal stem cells partly through NO-dependent Wnt/ß-catenin signaling and may enhance tolerability to methotrexate-induced injury.

Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse ß-Oxidation Pathway in the Presence of Lanthanide.

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse ß-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Natural-Course Evaluation of Infants with Positional Severe Plagiocephaly Using a Three-Dimensional Scanner in Japan: Comparison with Those Who Received Cranial Helmet Therapy.

This study aimed to clarify the natural course of positional plagiocephaly using a three-dimensional (3D) scanner and investigate the effectiveness of cranial helmet therapy (CHT). One hundred infants with severe plagiocephaly who visited our institutions between April 2020 and March 2021 were included. Cranial shape was measured using an Artec Eva 3D scanner. A cranial asymmetry (CA) >12 mm was diagnosed as severe plagiocephaly. An infant whose CA subsided to <12 mm was considered to have improved naturally or by CHT. The difference in CA between the second and initial scans was defined as the improvement value (median scan interval was two months). In the natural-course group comprising 56 infants with severe plagiocephaly, 37 (66%) with a median CA of 15.6 mm exhibited no improvement after two months. In the scan age- and evaluation interval-matched case-control study, the CA value in the CHT group improved by three times that in the natural-course group (-4.6 mm [n = 33] vs. -1.55 mm [n = 24], p < 0.001). Severe plagiocephaly did not improve naturally in 66% of the cases. Therefore, CHT should be considered if the CA is >12 mm on the initial evaluation.

Coexistence of Hereditary Spastic Paraplegia Type 4 and Narcolepsy: A Case Report.

Spastic paraplegia type 4 (SPG4) is the most common type of hereditary spastic paraplegia (HSP) caused by the mutations in the SPAST gene, which encodes a microtubule-severing protein named spastin. Spastin regulates the number and mobility of microtubules and is essential for axonal outgrowth and neuronal morphogenesis. Herein, we report a patient with SPG4 harboring a novel donor splice site mutation in the SPAST gene (c.1616+1dupG). Although SPG4 usually manifests itself as a pure form of HSP, this patient exhibited a slow progressive cognitive decline and also developed narcolepsy type 2 (narcolepsy without cataplexy) prior to the onset of SPG4. Recently, cognitive decline has attracted attention as a main non-motor symptom of SPG4. However, this is the first reported case of a patient developing both SPG4 and narcolepsy, although it remains unclear whether the manifestation of the two diseases is a coincidence or an association. In this report, we describe the clinical symptoms and genetic background of the patient.

Maternal approach behaviors toward neonatal calls are impaired by mother's experiences of raising pups with a risk gene variant for autism.

How the intrinsic sequence structure of neonatal mouse pup ultrasonic vocalization (USV) and maternal experiences determine maternal behaviors in mice is poorly understood. Our previous work showed that pups with a Tbx1 heterozygous (HT) mutation, a genetic risk for autism spectrum disorder (ASD), emit altered call sequences that do not induce maternal approach behaviors in C57BL6/J mothers. Here, we tested how maternal approach behaviors induced by wild-type and HT USVs are influenced by the mother's experience in raising pups of these two genotypes. The results showed that wild-type USVs were effective in inducing maternal approach behaviors when mothers raised wild-type but not HT pups. The USVs of HT pups were ineffective regardless of whether mothers raised HT or wild-type pups. However, the sequence structure of pup USVs had no effect on the general, non-directional incentive motivation of maternal behaviors. Our data show how the mother's experience with a pup with a genetic risk for ASD alters the intrinsic incentive values of USV sequences in maternal approach behaviors.

Accumulative scFv-Fc antibody gene integration into the hprt chromosomal locus of Chinese hamster ovary cells.

We have previously developed an accumulative site-specific gene integration system (AGIS) using Cre-recombinase and mutated loxP sites. AGIS enables repeated transgene integration into a predetermined chromosomal site in mammalian cells. However, the process of establishing cells with multiple integrated copies of the transgene is still time-consuming. In the present study, we describe an improved version of AGIS that facilitates and accelerates the establishment of high-producer Chinese hamster ovary (CHO) cells. Two donor vectors were simultaneously introduced into the cells in a single transfection. Cells with successfully targeted transgene integration were screened based on a change in the color of the reporter fluorescent protein that they express. Repeated rounds of integration allowed the transgene copy number to be increased. As a model, an scFv-Fc antibody gene was integrated into the hprt locus of the CHO cell genome. After three rounds of integration, a high-producer CHO cell clone with six copies of the scFv-Fc gene was successfully established. scFv-Fc productivity was approximately four-fold greater than a control cell line harboring a single copy of the transgene. This newly designed AGIS procedure should facilitate the development of producer cells suitable for biopharmaceutical protein production.

The heavy analogue of CpLi: lithium 1,2-disila-3-germacyclopentadienide, a 6pi-electron aromatic system.

Lithium 1,2-disila-3-germacyclopentadienide 2-.Li+ was synthesized by the reduction of 1,2-disila-3-germacyclopenta-2,4-diene 1 with potassium graphite followed by treatment with an excess amount of LiBr. The X-ray analysis of 2-.[Li+(THF)] revealed a delocalized aromatic cyclopentadienide-type structure with the diagnostic eta5-coordination of the Li+ cation to the five-membered ring. Aromaticity of this compound was verified and confirmed by theoretical calculations. The solution behavior of the 2-.Li+ is different in nonpolar and polar solvents; in nonpolar toluene, 2-.Li+ maintained the properties of a delocalized aromatic compound with the characteristically shielded 7Li NMR resonance at -5.4 ppm, whereas in polar THF, 2-.Li+ exhibited the properties of a localized nonaromatic compound with the negative charge situated on the Ge atom.