Effect of valproic acid on the formation and migration of cranial neural crest cells at the early developmental stages in rat embryos.

Cranial neural crest cells (NCCs) are critical for craniofacial development. The administration of valproic acid (VPA) to pregnant females causes craniofacial malformations in offspring. However, the in vivo influence of VPA on mammalian cranial NCCs remains unclear. In this study, we aimed to elucidate the developmental stage-specific effect of VPA on cranial NCCs through the administration of a single dose of VPA to pregnant rat females immediately prior to the formation of the cranial neural crest (NC). We performed whole-mount immunohistochemistry or in situ hybridization to examine localization changes of gene transcripts associated with the epithelial-mesenchymal transition of the cranial NC (i.e., cranial NCC formation) and cranial NCC migration. The results showed that Hoxa2 mRNA was abnormally detected and Sox9 mRNA expression was decreased in the midbrain-rhombomere (R) 1/2 NC, which forms cranial NCCs that migrate to the frontonasal mass (FNM) and branchial arch (BA) 1, through VPA administration, thus reducing the formation of SNAI2-positive NCCs. Hoxa2-positive NCCs were detected normally in BA2 and abnormally in FNM and BA1, which are normally Hox-free, implying VPA-induced abnormal cranial NCC migration. In vitro verification experiments using the whole embryo culture system revealed that midbrain-R4 NCC migration was abnormal. These results indicate that VPA reduces the formation/delamination of the midbrain-R1/2 NCCs in a developmental stage-specific manner and subsequently causes the abnormal migration of R4 NCCs, which suggests that the abnormal formation and migration of cranial NCCs contribute to the inhibition of axonal elongation in the trigeminal nerve and a reduction in head size.

HDAC Inhibition Induces CD26 Expression on Multiple Myeloma Cells via the c-Myc/Sp1-mediated Promoter Activation.

CD26 is ubiquitously and intensely expressed in osteoclasts in patients with multiple myeloma, whereas its expression in plasma cells of patients with multiple myeloma is heterogeneous because of its cellular diversity, immune escape, and disease progression. Decreased expression levels of CD26 in myeloma cells constitute one of the mechanisms underlying resistance to humanized anti-CD26 mAb therapy in multiple myeloma. In the current study, we show that histone deacetylase inhibition (HDACi) with broad or class-specific inhibitors involves the induction of CD26 expression on CD26neg myeloma cells both transcriptionally and translationally. Furthermore, dipeptidyl peptidase Ⅳ (DPPⅣ) enzymatic activity was concomitantly enhanced in myeloma cells. Combined treatment with HDACi plus CD26mAb synergistically facilitated lysis of CD26neg myeloma cells not only by antibody-dependent cellular cytotoxicity but also by the direct effects of mAb. Of note, its combination readily augmented lysis of CD26neg cell populations, refractory to CD26mAb or HDACi alone. Chromatin immunoprecipitation assay revealed that HDACi increased acetylation of histone 3 lysine 27 at the CD26 promoter of myeloma cells. Moreover, in the absence of HDACi, c-Myc was attached to the CD26 promoter via Sp1 on the proximal G-C box of myeloma cells, whereas, in the presence of HDACi, c-Myc was detached from Sp1 with increased acetylation of c-Myc on the promoter, leading to activation of the CD26 promoter and initiation of transcription in myeloma cells. Collectively, these results confirm that HDACi plays crucial roles not only through its anti-myeloma activity but by sensitizing CD26neg myeloma cells to CD26mAb via c-Myc/Sp1-mediated CD26 induction, thereby augmenting its cytotoxicity. SIGNIFICANCE: There is a desire to induce and sustain CD26 expression on multiple myeloma cells to elicit superior anti-myeloma response by humanized anti-CD26 mAb therapy. HDACi upregulates the expression levels of CD26 on myeloma cells via the increased acetylation of c-MycK323 on the CD26 promoter, leading to initiation of CD26 transcription, thereby synergistically augments the efficacy of CD26mAb against CD26neg myeloma cells.

The effect of guardians' health literacy on the child's spending time at home: A cross-sectional study among Japanese schoolchildren.

Background: The contents of children's daily activities and the amount of time spent on them has been directly linked to their health and development. Parental health behavior has also been considered a key factor, and the aim of this study was to determine the relationship between parent/guardian health literacy (HL) and their child's time spent at home by behavioral types. The study was conducted in elementary schools in Japan. Method: The target subjects for this study were elementary schoolchildren (all grades, aged 6 to 12 years) and their parents/guardians, and almost 3000 schoolchildren and their parents/guardians in the Northern and Southern districts in Japan participated. The questionnaire for parents/guardians included amount of time spent per day on the seven major behavioral contents of their child's time at home, on weekdays and weekends, respectively, and a shortened five-item health literacy (HL) scale. Parent/guardian HL results were categorized into two groups (low HL group and high HL group), and we analyzed the association between the HL and child's time spent at home by behavioral contents. Results: Children in the high HL parent/guardian group spent significantly less time watching TV and playing games than those in the low HL group, both on weekdays and weekends. Time spent playing outside on weekdays and on hobbies on weekdays and weekends was significantly longer for children in the high HL parent/guardian group than in the low HL group. Results of logistic regression analyses adjusted for confounders showed that higher parental/guardian HL reduced children's spending more than 30 minutes watching TV or playing games and increased children's spending more than 30 minutes on outside playing and doing hobbies. Conclusions: Parental/guardian HL affected the child's time spent at home. The results could suggest that increasing parental/guardian HL has strong potential to improve children's major lifestyle behaviors．.

The effect of aromatase inhibitor on controlled ovarian stimulation for oocyte cryopreservation in adolescent and young cancer patients.

AIM: To determine the impact of aromatase inhibitor (AI) use in oocyte cryopreservation among Japanese adolescent and young adult (AYA) cancer patients for fertility preservation, we evaluated the oocyte cryopreservation outcomes following AI therapy in combination with the follicular phase start (FPS) and random start (RS) protocols. METHODS: This retrospective study included 81 cycles of controlled ovarian stimulation (COS) among 73 AYA patients with cancer who underwent oocyte cryopreservation to maintain fertility. The outcome measures were the total number of matured oocytes that were retrieved and cryopreserved, as well as their maturation rates. The AI (+) and AI (-) groups were compared using the RS and FPS protocols. RESULTS: Our results showed that the combined use of AI and COS decreases serum E2 levels and maintains the number of retrieved and cryopreserved mature oocytes. We also confirmed the efficacy of the RS protocol, which was found to have comparable outcomes to that of the FPS protocol in both AI (+) and AI (-) groups. CONCLUSION: The combined use of AI and COS is beneficial for oocyte cryopreservation in patients with estrogen-sensitive cancer, regardless of the menstrual cycle phase of COS initiation.

Hippocampus as a sorter and reverberatory integrator of sensory inputs.

The hippocampus must be capable of sorting and integrating multiple sensory inputs separately but simultaneously. However, it remains to be elucidated how the hippocampus executes these processes simultaneously during learning. Here we found that synchrony between conditioned stimulus (CS)-, unconditioned stimulus (US)- and future retrieval-responsible cells occurs in the CA1 during the reverberatory phase that emerges after sensory inputs have ceased, but not during CS and US inputs. Mutant mice lacking N-methyl-D-aspartate receptors (NRs) in CA3 showed a cued-fear memory impairment and a decrease in synchronized reverberatory activities between CS- and US-responsive CA1 cells. Optogenetic CA3 silencing at the reverberatory phase during learning impaired cued-fear memory. Thus, the hippocampus uses reverberatory activity to link CS and US inputs, and avoid crosstalk during sensory inputs.

Eating behaviour characteristics and dietary intake among Japanese junior high school students: A cross-sectional study.

BACKGROUND: Psychological characteristics of eating behaviour may be related to dietary habits. AIM: We investigated the association between eating behaviour characteristics and nutrition and food intake adequacy in Japanese adolescents. METHODS: This cross-sectional survey was conducted among 136 junior high school students (boys: 90, girls: 46) at a junior high school in Tokyo, Japan. Eating behaviour was categorised into three types (emotional, external, and restrained) using scores on the Japanese version of the Dutch Eating Behaviour Questionnaire. Dietary intake was assessed using a validated, brief self-administered diet history questionnaire. Inadequate nutrient intake was determined by counting the number of nutrients not meeting the dietary reference intake (DRI) for the Japanese population. The statistical analyses included Wilcoxon signed-rank tests, Spearman's rank correlation coefficient, and multiple regression analysis using JMP ver.14 (SAS Institute Inc., Cary, NC, USA). All reported p values are two-tailed, and p < 0.05 was regarded as statistically significant. RESULTS: Multiple regression analysis showed that restrained eating score was inversely associated with the number of nutrients not meeting the DRI (ß = - 0.28; p = 0.0027) and with total weight of snack intake (ß = - 0.30; p = 0.0010). Neither emotional nor external eating was significantly associated with the number of nutrients not meeting the DRI and with total weight of snack intake. CONCLUSIONS: These results suggest that adolescents with low restrained eating scores may have less self-control over their eating behaviour and may therefore have inadequate dietary intake.

Selective engram coreactivation in idling brain inspires implicit learning.

Passive priming of prior knowledge to assimilate ongoing experiences underlies advanced cognitive processing. However, the necessary neural dynamics of memory assimilation remains elusive. Uninstructed brain could also show boosted creativity, particularly after idling states, yet it remains unclear whether the idling brain can spontaneously spark relevant knowledge assimilations. We established a paradigm that links/separates context-dependent memories according to geometrical similarities. Mice exploring one of four contexts 1 d before undergoing contextual fear conditioning in a square context showed a gradual fear transfer to preexposed geometrically relevant contexts the next day, but not after 15 min. Anterior cingulate cortex neurons representing relevant, rather than distinct, memories were significantly coreactivated during postconditioning sleep only, before their selective integration the next day during testing. Disrupting sleep coreactivations prevented assimilation while preserving recent memory consolidation. Thus, assimilating pertinent memories during sleep through coreactivation of their respective engrams represents the neural underpinnings of sleep-triggered implicit cortical learning.

Ultrasonographic findings of placental abruption observed on superb microvascular imaging.

OBJECTIVE: To demonstrate ultrasound pathological findings of placental abruption (PA) detected using a new Doppler method: superb microvascular imaging (SMI). CASE REPORT: The patient was a pregnant woman with dark brown vaginal discharge at 32 + 4 weeks of gestation. Conventional ultrasound revealed an exophytic heterogeneous area measuring 3 cm, between the placenta and myometrium. SMI showed no minor blood flow inside the area. A diagnosis of marginal sub-chorionic hematoma was made. On the seventh day of hospitalization, SMI showed pulsation of blood flow in the inter-villous space and fetal blood flow in the villous trees. Due to an increase in the frequency of uterine contractions, an emergency cesarean section was performed. Histopathological examination showed hematomas beneath the decidual tissue, and the decidual layer was undamaged. The inter-villous space was preserved. CONCLUSION: SMI can contribute to a more accurate PA diagnosis that may lead to timely administration of obstetric intervention.

Novel method utilizing bisulfite conversion with dual amplification-refractory mutation system polymerase chain reaction to detect circulating pancreatic ß-cell cfDNA.

AIMS/INTRODUCTION: Several research groups have reported methods for quantifying pancreatic beta cell (ß-cell) injury by measuring ß-cell-specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next-generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment. MATERIALS AND METHODS: We established a novel method for detecting four CpG unmethylations of the insulin gene using two-step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults. RESULTS: The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose ß-cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA-positive type 1 diabetes. CONCLUSIONS: We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real-time PCR system. This method would be useful for analyzing dynamic profiles of ß-cells in human disease such as type 1 diabetes.

A Dome-Shaped Aerosol Box for Protection During a Pandemic.

With the rise of COVID-19, the use of aerosol boxes when interacting with COVID-19 patients has increased. However, their use has been controversial. We have been involved in the development of a dome-shaped aerosol containment device with negative pressure (DAWN), an aerosol box that can maintain negative pressure inside at all times. There are two types of DAWN: one is mounted on a bed (bed type) and the other is mounted on a stretcher (stretcher type). Each device has its own characteristics and can be selected depending on the situation. The bed type has enough space inside to allow procedures to be performed easily. The stretcher type can be attached to a stretcher and can maintain negative pressure when the patient is being moved. Due to the negative pressure structure and easy change of nonwoven fabric adopted in both types of DAWN, it is expected to prevent the scattering of aerosol when it is removed, which is a problem of conventional aerosol boxes. DAWN will contribute to reducing the enormous psychological stress of medical personnel who treat infections, and will contribute to reducing aerosol dispersion.

A Cross-Sectional Study on the Characteristics of Physical Activity in Pre-Frail Older Adults.

This cross-sectional study aimed to characterize the physical activity (PA) of older adults with pre-frail status by examining sedentary behavior (SB) and PA using triaxial accelerometer data, with non-frail older adults as the control group. In this study, we divided the study participants into older adults who regularly participated in self-initiated citizen group exercise activities and those who did not. Data were collected between September and December 2017. We analyzed data from 256 older adults (women: 86.3%) aged ≥65 years. The interaction effect of participation status (participation and non-participation group) and frailty status (pre-frail and non-frail group) for moderate-to-vigorous PA (F = 9.178, p = 0.003) and daily mean number of steps (F = 9.351, p = 0.002) was significant. For the participation group, there was no difference between pre-frail older adults and non-frail older adults regarding length of SB and PA time, indicating that PA level was maintained in the participating pre-frail older adults. In contrast, moderate-to-vigorous PA and daily mean number of steps were low in pre-frail older adults who did not participate in the activities. The opportunity to participate in self-initiated group exercise activities and other PAs in the community may help pre-frail older adults maintain their PA.

Spatially restricted substrate-binding site of cortisol-synthesizing CYP11B1 limits multiple hydroxylations and hinders aldosterone synthesis.

Human cytochromes P45011ß (CYP11B1) and P450aldo (CYP11B2) are monooxygenases that synthesize cortisol through steroid 11ß-hydroxylation and aldosterone through a three-step process comprising 11ß-hydroxylation and two 18-hydroxylations, respectively. CYP11B1 also catalyzes 18-monohydroxylation and 11ß,18-dihydroxylation. To study the molecular basis of such catalytic divergence of the two enzymes, we examined a CYP11B1 mutant (Mt-CYP11B1) with amino acid replacements on the distal surface by determining the catalytic activities and crystal structure in the metyrapone-bound form at 1.4-Å resolution. Mt-CY11B1 retained both 11ß-hydroxylase and 18-hydroxylase activities of the wild type (Wt-CYP11B1) but lacked 11ß,18-dihydroxylase activity. Comparisons of the crystal structure of Mt-CYP11B1 to those of Wt-CYP11B1 and CYP11B2 that were already reported show that the mutation reduced the innermost space putatively surrounding the C3 side of substrate 11-deoxycorticosterone (DOC) bound to Wt-CYP11B1, while the corresponding space in CYP11B2 is enlarged markedly and accessible to bulk water through a channel. Molecular dynamics simulations of their DOC-bound forms supported the above findings and revealed that the enlarged space of CYP11B2 had a hydrogen bonding network involving water molecules that position DOC. Thus, upon positioning 11ß-hydroxysteroid for 18-hydroxylation in their substrate-binding sites, steric hindrance could occur more strongly in Mt-CYP11B1 than in Wt-CYP11B1 but less in CYP11B2. Our investigation employing Mt-CYP11B1 sheds light on the divergence in structure and function between CYP11B1 and CYP11B2 and suggests that CYP11B1 with spatially-restricted substrate-binding site serves as 11ß-hydroxylase, while CYP11B2 with spatially-extended substrate-binding site successively processes additional 18-hydroxylations to produce aldosterone.

Primary malignant melanoma of uterine cervix treated with pembrolizumab as adjuvant immunotherapy.

This is the case report of primary malignant melanoma (MM) of uterine cervix treated by immune checkpoint inhibitor: the Pembrolizumab. Despite the merge of the novel drugs that has been strikingly improving prognosis of MM, we still struggle treatment of MM of uterine cervix that has aggressive characteristics with unknown etiology. We present our case to contribute its rarity of the disease case report, the primary MM of the uterine cervix that had poor response to pembrolizumab and had OS of 6 months. The treatment ineffectiveness is mainly considered for mucosal MM of low tumor mutation burden and its unusual type of pathology. Accumulation of retrospective studies exclusively on cervical melanoma needs to be proceeded to investigate on characteristics between poor and long survival to establish standardized treatment.

Examining the Beneficial Aspects of Nutritional Guidance Using Estimated Daily Salt Intake in Cancer Patients with Ischemic Heart Disease.

BACKGROUND The outcomes associated with nutritional guidance for patients with ischemic heart disease undergoing cancer treatment have not been explored. We examined the effects of nutritional guidance using estimated daily salt intake in cancer patients with ischemic heart disease. MATERIAL AND METHODS We examined the data from physical examinations and laboratory assessments of 27 patients with suspected excessive salt intake who underwent coronary angiography for the first time and received nutritional guidance on their next visit to the Department of Cardiology of Shizuoka Cancer Center between May 2018 and March 2020. Salinity measurement was not used in the nutritional guidance method, but the patients were instructed to reduce consumption of salt-containing foods. We compared the frequency of the estimated daily salt intake with the frequency of categories requiring salt control (food, cooking, and table salts). RESULTS The median age of the participants was 74 (range, 63-86) years. The estimated daily salt intake and the rate of change in the triglyceride level were negatively correlated (r=-0.61, P<0.01). The estimated daily salt intake was reduced in 16 cases; there was a relative decrease in the frequency of food intake among categories requiring salt control compared with that in the nonimproved cases (P<0.01). No difference was found between the cancer stage and the affected site of the digestive system in either group (P=0.64, P=0.39). CONCLUSIONS Nutritional guidance on dietary habits without using salinity measurement was beneficial in preventing ischemic heart disease and food intake reduction in cancer patients.

Energy metabolism profile of the effects of amino acid treatment on hepatocytes: Phenylalanine and phenylpyruvate inhibit glycolysis of hepatocytes.

OBJECTIVES: Amino acids are not only the building blocks of proteins, but also can be metabolized to energy substances or function as signaling molecules. The aim of this study was to profile whether amino acid treatment (essential amino acids and alanine) affects the energy metabolism (glycolysis, mitochondrial respiration) of cultured hepatocytes. METHODS: AML12 hepatocytes were treated with 5 mM of each amino acid for 1 h and the energy metabolism was then measured by using an extracellular flux analyzer. RESULTS: The results showed that phenylalanine and lysine decreased the extracellular acidification rate (ECAR), an indirect indicator of glycolysis, whereas isoleucine and histidine increased the ECAR. Amino acids did not affect the oxygen consumption rate, an indirect indicator of mitochondrial respiration. The glycolysis stress test revealed that treatment of the hepatocytes with phenylalanine inhibited glycolysis when the concentration of the substrate for glycolysis was sufficient in cultured media. We also investigated the effect of metabolites derived from conversion of phenylalanine on glycolysis in hepatocytes and found that phenylpyruvate inhibited glycolysis, whereas tyrosine and phenylethylamine did not affect glycolysis. CONCLUSIONS: The findings from the present study complement basic knowledge of the effects of amino acid treatment on energy metabolism in cultured hepatocytes and indicate that phenylalanine and phenylpyruvate inhibit glycolysis.

Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes.

OBJECTIVES: Amino acids are not only components of proteins, but also can be metabolized to energy substances or be used as signaling molecules. However, basic knowledge of the relationship between amino acid treatment and energy metabolism is still insufficient. The aims of this study was to profile the effects of essential amino acid and alanine treatment on the energy metabolism of both myoblasts and myotubes and to contribute to the understanding of the basic relationship between amino acid treatment and energy metabolism of skeletal muscle cell. METHODS: We profiled whether amino acid (essential amino acids and alanine) treatment can affect the energy metabolism (glycolysis, mitochondrial respiration) of cultured skeletal muscle cells. C2C12 myoblasts and differentiated myotubes were treated with 5 mM each amino acid for 1 h, then the energy metabolism was measured by using extracellular flux analyzer. RESULTS: Although not all of the amino acid treatments could affect the energy metabolism of C2C12 myoblasts, leucine, isoleucine, lysine, phenylalanine, and histidine decreased the extracellular acidification rate, an indirect indicator of glycolysis, in differentiated myotubes without alteration of oxygen consumption rate, an indirect indicator of mitochondrial respiration. By glycolysis stress test, we found that leucine treatment inhibited glycolysis of myotubes when the substrate of glycolysis is sufficient in cultured media. The inhibitory effect of glycolysis by leucine was not canceled by rapamycin (an inhibitor for mTOR). But, 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (an inhibitor for branched-chain α ketoacid dehydrogenase complex kinase) increased branched-chain amino acid catabolism, which decreased the glycolysis of myotubes. CONCLUSION: Findings from the present study complemented the basic knowledge of amino acid treatment on the energy metabolism of cultured skeletal muscle cells and suggested the inhibitory effects of glycolysis by branched-chain amino acid catabolism.

Selective targeting of mRNA and the following protein synthesis of CaMKIIα at the long-term potentiation-induced site.

Late-phase long-term potentiation (L-LTP) in hippocampus, thought to be the cellular basis of long-term memory, requires new protein synthesis. Neural activity enhances local protein synthesis in dendrites, which in turn mediates long-lasting synaptic plasticity. Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is a locally synthesized protein crucial for this plasticity, as L-LTP is impaired when its local synthesis is eliminated. However, the distribution of Camk2a mRNA during L-LTP induction remains unclear. In this study, we investigated the dendritic targeting of Camk2a mRNA after high-frequency stimulation, which induces L-LTP in synapses of perforant path and granule cells in the dentate gyrus in vivoIn situ hybridization studies revealed that Camk2a mRNA was immediately but transiently targeted to the site receiving high-frequency stimulation. This was associated with an increase in de novo protein synthesis of CaMKIIα. These results suggest that dendritic translation of CaMKIIα is locally mediated where L-LTP is induced. This phenomenon may be one of the essential processes for memory establishment.

Orchestrated ensemble activities constitute a hippocampal memory engram.

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory.

The association of urinary estrogen levels with urinary isoflavone levels: Difference between premenopausal women and postmenopausal women.

Results of studies on the associations of soy food intake with urinary estrogen levels in premenopausal women and in postmenopausal women have been inconsistent. We examined the associations of urinary isoflavone levels as well as soy food intake with estrone (E1) and estradiol (E2) in pre- and postmenopausal women. In addition, we compared the levels of isoflavones, E1 and E2 across current hormone users such as those receiving hormone replacement therapy and those using oral contraceptives and non-users among both pre- and postmenopausal women. Urinary levels of isoflavones, E1 and E2 in 498 women (36 hormone users and 462 non-users) were analyzed. Premenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, but there were no significant associations between E1 and E2 levels and urinary isoflavone levels. Levels of E1 and E2 in hormone users were significantly lower than those in hormone non-users among premenopausal women, but levels of E1 and E2 in hormone users were significantly higher than those in hormone non-users among postmenopausal women. Postmenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, and postmenopausal women with high urinary isoflavone levels had significantly higher E1 and E2 levels. In conclusion, the associations of urinary isoflavone levels with urinary estrogen levels differed with menopausal status. Urinary levels of E1 and E2 were high in postmenopausal women with high urinary isoflavone levels but not in premenopausal women with high urinary isoflavone levels.

Leucyl-tRNA synthetase is required for the myogenic differentiation of C2C12 myoblasts, but not for hypertrophy or metabolic alteration of myotubes.

Mammalian target of rapamycin (mTOR) signaling controls skeletal muscle cell differentiation, growth, and metabolism by sensing the intracellular energy status and nutrients. Recently, leucyl-tRNA synthetase (Lars) was identified as an intracellular sensor of leucine involved in the activation of mTOR signaling. However, there is still no evidence for the activation of mTOR signaling by Lars and its physiological roles in skeletal muscle cells. In this study, we determined the potential roles of Lars for the activation of mTOR signaling, skeletal muscle cell differentiation, hypertrophy, and metabolism using small interfering (si)-RNA knockdown. siRNA-mediated knockdown of Lars decreased phosphorylated p70 S6 kinase and inhibited the differentiation of C2C12 mouse myoblasts into myotubes, as evidenced by a decreased fusion index and decreased mRNA and protein expression levels of myogenic markers. Importantly, si-Lars decreased the level of Insulin-like growth factor 2 (Igf2) mRNA expression from the early stages of differentiation, indicating the possibility of an association between the mTOR-IGF2 axis and Lars. However, Lars knockdown did not decrease phosphorylated mTOR in differentiated myotubes, nor did it affect the hypertrophy of myotubes as evidenced by measuring their diameters and detecting the mRNA and protein expression of hypertrophy markers. Similarly, an extracellular flux analyzer showed that Lars knockdown did not affect the metabolism (glycolysis and mitochondrial respiration) of myotubes. These results demonstrate that Lars is required for skeletal muscle differentiation through the activation of mTOR signaling, but not for hypertrophy or metabolic alteration of myotubes.