Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17β-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11β-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.

Although widespread cortical thinning centered on the fronto-temporal regions in schizophrenia has been reported, the findings in at-risk mental state (ARMS) patients have been inconsistent. In addition, it remains unclear whether abnormalities of cortical thickness (CT) in ARMS individuals, if present, are related to their functional decline irrespective of future psychosis onset. In this multicenter study in Japan, T1-weighted magnetic resonance imaging was performed at baseline in 107 individuals with ARMS, who were subdivided into resilient (77, good functional outcome) and non-resilient (13, poor functional outcome) groups based on the change in Global Assessment of Functioning scores during 1-year follow-up, and 104 age- and sex-matched healthy controls recruited at four scanning sites. We measured the CT of the entire cortex and performed group comparisons using FreeSurfer software. The relationship between the CT and cognitive functioning was examined in an ARMS subsample (n = 70). ARMS individuals as a whole relative to healthy controls exhibited a significantly reduced CT, predominantly in the fronto-temporal regions, which was partly associated with cognitive impairments, and an increased CT in the left parietal and right occipital regions. Compared with resilient ARMS individuals, non-resilient ARMS individuals exhibited a significantly reduced CT of the right paracentral lobule. These findings suggest that ARMS individuals partly share CT abnormalities with patients with overt schizophrenia, potentially representing general vulnerability to psychopathology, and also support the role of cortical thinning in the paracentral lobule as a predictive biomarker for short-term functional decline in the ARMS population.

Low body weight at birth has been shown to be a risk factor for future metabolic disorders, as well as stress response abnormalities and depression. We showed that low-birthweight rats had prolonged high blood corticosterone levels after stress exposure, and that an increase in Gas5 lncRNA, a decoy receptor for glucocorticoid receptors (GRs), reduces glucocorticoid responsiveness. Thus, we concluded that dampened pituitary glucocorticoid responsiveness disturbed the glucocorticoid feedback loop in low-birthweight rats. However, it remains unclear whether such glucocorticoid responsiveness is suppressed solely in the pituitary or systemically. The expression of Gas5 lncRNA increased only in the pituitary, and the intact induction of expression of the GR co-chaperone factor Fkbp5 against dexamethasone was seen in the liver, muscle, and adipose tissue. Intervention with a methyl-modulator diet (folate, VB12, choline, betaine, and zinc) immediately before or one week after delivery reversed the expression level of Gas5 lncRNA in the pituitary of the offspring. Consequently, it partially normalized the blood corticosterone levels after restraint stress exposure. In conclusion, the mode of glucocorticoid response in low-birthweight rats is impaired solely in the pituitary, and intervention with methyl-modulators ameliorates the impairment, but with a narrow therapeutic time window.

The lack of robust neuroanatomical markers of psychosis risk has been traditionally attributed to heterogeneity. A complementary hypothesis is that variation in neuroanatomical measures in the majority of individuals at psychosis risk may be nested within the range observed in healthy individuals.

In schizophrenia, social cognitive impairment is considered one of the greatest obstacles to social participation. Although numerous measures have been developed to assess social cognition, only a limited number of them have become available in Japan. We are therefore planning this evaluation study for social cognition measures in Japan (ESCoM) to confirm their psychometric characteristics and to promote research focused on social cognition. Participants in the cross-sectional observational study will be 140 patients with schizophrenia recruited from three Japanese facilities and 70 healthy individuals. In our primary analysis, we will calculate several psychometric indicators with a focus on whether they can independently predict social functioning. In secondary analyses, we will assess the reliability and validity of the Japanese translations of each measure and conduct an exploratory investigation of patient background, psychiatric symptoms, defeatist performance belief, and gut microbiota as determinants of social cognition. The protocol for this study is registered in UMIN-CTR, unique ID UMIN000043777.

We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52-57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.

Smoking during pregnancy is one of the causes of low birthweight. Ingestion of nicotine during pregnancy has various metabolic impacts on the fetus and offspring. According to the developmental origins of health and disease theory, low birthweight is a risk factor for developing various non-communicable diseases, including diabetes. We hypothesized that when nicotine-induced low-birthweight rats, when exposed to a high-fat diet (HFD) after growth, are predisposed to glucose intolerance as a result of a mismatch between the eutrophic environment and small body size. Therefore, we investigated whether hyperinsulinemia was caused by exposure of nicotine-induced low-birthweight rats to HFD, including whether this phenomenon exhibited possible sex differences. The average birthweight and body weight at weaning day of offspring from nicotine-administered dams was lower than those of controls. The offspring from nicotine-administered dams did not show rapid fat accumulation after exposure to HFD, and weight and body fat ratio of these animals did not differ from those of the controls. Blood glucose levels did not differ between the groups, but insulin levels increased only in male HFD-exposed offspring from nicotine-administered dams. Similarly, only in HFD-exposed male from nicotine-administered dams showed decreases in the insulin receptor expression in the liver. We conclude that male rats subjected to prenatal nicotine exposure develop hyperinsulinemia when exposed to HFD after growth. Our results suggest that decreased expression of insulin receptors in the liver may be involved in the mechanism underlying hyperinsulinemia in low-birthweight offspring, a phenomenon that appeared to exhibit a sex-specific bias.

If sufficient nutrition is not obtained during pregnancy, the fetus changes its endocrine system and metabolism to protect the brain, resulting in a loss of body size. The detailed mechanisms that determine the success or failure of growth catch-up are still unknown. Therefore, we investigated the mechanism by which catch-up growth failure occurs. The body weights of rat pups at birth from dams whose calorie intake during pregnancy was reduced by 40% were significantly lower than those of controls, and some offspring failed to catch up. Short-body-length and low-bodyweight rats showed blood IGF-1 levels and mRNA expression levels of IGF-1 and growth hormone receptor (GHR) in the liver that were lower than those in controls. The next generation offspring from low-bodyweight non-catch-up (LBW-NCG) rats had high expression of miR-322 and low expression of GHR and IGF-1. The expression of miR-322 showed a significant negative correlation with GHR expression and body length, and overexpression of miR-322 suppressed GHR expression. We found that insufficient intake of calories during pregnancy causes catch-up growth failure due to increased expression of miR-322 and decreased expression of GHR in the livers of offspring, and this effect is inherited by the next generation.

In schizophrenia spectrum disorders (SSD), social cognition mediates the relationship between neurocognition and social functioning. Although people with major depressive disorder (MDD) also exhibit cognitive impairments, which are often prolonged, little is known about the role of social cognition in MDD.

Forward genetic screens of mutated variants are a versatile strategy for protein engineering and investigation, which has been successfully applied to various studies like directed evolution (DE) and deep mutational scanning (DMS). While next-generation sequencing can track millions of variants during the screening rounds, the vast and noisy nature of the sequencing data impedes the estimation of the performance of individual variants. Here, we propose ACIDES that combines statistical inference and in-silico simulations to improve performance estimation in the library selection process by attributing accurate statistical scores to individual variants. We tested ACIDES first on a random-peptide-insertion experiment and then on multiple public datasets from DE and DMS studies. ACIDES allows experimentalists to reliably estimate variant performance on the fly and can aid protein engineering and research pipelines in a range of applications, including gene therapy.

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyze the reduction of 17-ketosteroids and the oxidation of 17β-hydroxysteroids to regulate the production of androgens and estrogens. Among them, 17β-HSD type 3 (HSD17B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of 17β-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of 17β-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD17B1 and HSD17B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B3. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins.

Being delivered as a low birthweight (LBW) infant is a risk factor for elevated blood pressure and future problems with cardiovascular and cerebellar diseases. Although premature babies are reported to have low numbers of nephrons, some unclear questions remain about the mechanisms underlying elevated blood pressure in full-term LBW infants. We previously reported that glucocorticoids increased miR-449a expression, and increased miR-449a expression suppressed Crhr1 expression and caused negative glucocorticoid feedback. Therefore, we conducted this study to clarify the involvement of pituitary miR-449a in the increase in blood pressure caused by higher glucocorticoids in LBW rats. We generated a fetal low-carbohydrate and calorie-restricted model rat (60% of standard chow), and some individuals showed postnatal growth failure caused by growth hormone receptor expression. Using this model, we examined how a high-fat diet (lard-based 45kcal% fat)-induced mismatch between prenatal and postnatal environments could elevate blood pressure after growth. Although LBW rats fed standard chow had slightly higher blood pressure than control rats, their blood pressure was significantly higher than controls when exposed to a high-fat diet. Observation of glomeruli subjected to periodic acid methenamine silver (PAM) staining showed no difference in number or size. Aortic and cardiac angiotensin II receptor expression was altered with compensatory responses. Blood aldosterone levels were not different between control and LBW rats, but blood corticosterone levels were significantly higher in the latter with high-fat diet exposure. Administration of metyrapone, a steroid synthesis inhibitor, reduced blood pressure to levels comparable to controls. We showed that high-fat diet exposure causes impairment of the pituitary glucocorticoid negative feedback via miR-449a. These results clarify that LBW rats have increased blood pressure due to high glucocorticoid levels when they are exposed to a high-fat diet. These findings suggest a new therapeutic target for hypertension of LBW individuals.

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of neurotransmitters and trace amines. MAO has two subtypes (MAO-A and MAO-B) that are encoded by different genes. In the brain, MAO-B is highly expressed in the paraventricular thalamic nucleus (PVT); however, its substrate in PVT remains unclear. To identify the MAO-B substrate in PVT, we generated Maob knockout (KO) mice and measured five candidate substrates (i.e., noradrenaline, dopamine, 3-methoxytyramine, serotonin, and phenethylamine [PEA]) by liquid chromatography tandem mass spectrometry. We showed that only PEA levels were markedly elevated in the PVT of Maob KO mice. To exclude the influence of peripheral MAO-B deficiency, we developed brain-specific Maob KO mice, finding that PEA in the PVT was increased in brain-specific Maob KO mice, whereas the extent of PEA increase was less than that in global Maob KO mice. Given that plasma PEA levels were elevated in global KO mice, but not in brain-specific KO mice, and that PEA passes across the blood-brain barrier, the substantial accumulation of PEA in the PVT of Maob KO mice was likely due to the increase in plasma PEA. These data suggest that PEA is a substrate of MAO-B in the PVT as well as other tissues.

Low birth weight (LBW) is related to increased incidence of common cardiovascular and metabolic disorders, and psychopathologies later in life. Recent studies have suggested that maternal malnutrition affects fetal hypothalamic-pituitary-adrenal (HPA) axis programing although the mechanism is unknown. We demonstrated that LBW offspring delivered from malnourished dams showed prolonged elevated plasma corticosterone concentrations when compared with those of normal-birth-weight (NBW) offspring and impaired downregulation of corticotropin-releasing factor receptor type 1 (CRF-R1, Crhr1) in the anterior pituitary in restraint. Restraint increased expression of miR449a, which we had previously demonstrated to be involved in Crhr1 downregulation, in the anterior pituitary and serum exosomal miR449a contents through glucocorticoids in NBW offspring, but not in LBW offspring. Although plasma corticosterone concentrations were higher at 2000 h than at 0800 h in both LBW and NBW offspring, they were significantly higher in LBW offspring than in NBW offspring at 2000 and 0200 h. There were no significant diurnal changes in miR449a expression levels in the anterior pituitary of either NBW or LBW offspring, but the expression was significantly lower in LBW offspring than in NBW offspring at 1400, 2000, and 0200 h. The expression levels of GAS5, which inhibits glucocorticoid receptor (GR) binding to glucocorticoid-responsive element, in the anterior pituitary of LBW offspring were elevated when compared with those of NBW offspring. The downregulation of GR found in NBW offspring did not occur in restrained LBW offspring. These results indicate that impaired miR449a expression, probably induced by increased GAS5 expression, causes dysregulation of Crhr1 expression in the anterior pituitary, resulting in prolonged HPA axis activation in restrained LBW offspring.

Individuals at Clinical High Risk for Psychosis (CHR-P) demonstrate heterogeneity in clinical profiles and outcome features. However, the extent of neuroanatomical heterogeneity in the CHR-P state is largely undetermined. We aimed to quantify the neuroanatomical heterogeneity in structural magnetic resonance imaging measures of cortical surface area (SA), cortical thickness (CT), subcortical volume (SV), and intracranial volume (ICV) in CHR-P individuals compared with healthy controls (HC), and in relation to subsequent transition to a first episode of psychosis. The ENIGMA CHR-P consortium applied a harmonised analysis to neuroimaging data across 29 international sites, including 1579 CHR-P individuals and 1243 HC, offering the largest pooled CHR-P neuroimaging dataset to date. Regional heterogeneity was indexed with the Variability Ratio (VR) and Coefficient of Variation (CV) ratio applied at the group level. Personalised estimates of heterogeneity of SA, CT and SV brain profiles were indexed with the novel Person-Based Similarity Index (PBSI), with two complementary applications. First, to assess the extent of within-diagnosis similarity or divergence of neuroanatomical profiles between individuals. Second, using a normative modelling approach, to assess the 'normativeness' of neuroanatomical profiles in individuals at CHR-P. CHR-P individuals demonstrated no greater regional heterogeneity after applying FDR corrections. However, PBSI scores indicated significantly greater neuroanatomical divergence in global SA, CT and SV profiles in CHR-P individuals compared with HC. Normative PBSI analysis identified 11 CHR-P individuals (0.70%) with marked deviation (>1.5 SD) in SA, 118 (7.47%) in CT and 161 (10.20%) in SV. Psychosis transition was not significantly associated with any measure of heterogeneity. Overall, our examination of neuroanatomical heterogeneity within the CHR-P state indicated greater divergence in neuroanatomical profiles at an individual level, irrespective of psychosis conversion. Further large-scale investigations are required of those who demonstrate marked deviation.

The lack of robust neuroanatomical markers of psychosis risk has been traditionally attributed to heterogeneity. A complementary hypothesis is that variation in neuroanatomical measures in individuals at psychosis risk may be nested within the range observed in healthy individuals.