Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line.

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called "oncogene-induced senescence (OIS)", prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called "HBEC3KT"), immortalized with hTERT ("T") and Cdk4 ("K"), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated ß-galactosidase staining (SA-ßG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

The Effect of Co-Ingestion of Carbohydrate with Milk after Exercise in Healthy Women: Study Considering the Menstrual Cycle.

This study aimed to assess the effects of co-ingestion of carbohydrate with milk (MILK) and isocaloric carbohydrate beverage (CHO) on post-exercise recovery and subsequent exercise capacity, considering the menstrual cycle. This study included 12 women with regular menstrual cycles who completed four test days, which started with glycogen-depleting exercise using a cycle ergometer in the early follicular phase (EF) and late follicular phase (LF), followed by 240 min of recovery from the ingestion of 200 mL of CHO or MILK every 30 min immediately after the exercise (POST0) until 210 min post-exercise. After 240 min, participants performed an exercise capacity test. Blood samples and breathing gas samples were collected before the exercise (PRE), POST0, and 120 (POST120) and 240 min after the end of exercise (POST240) to determine the concentrations of estradiol, progesterone, blood glucose, blood lactate, free fatty acid (FFA), and insulin and the respiratory exchange ratio, fat oxidation, and carbohydrate oxidation. The exercise time at exercise capacity test was not significantly different in terms of menstrual cycle phases and recovery beverages ingested. However, there was a significant positive correlation between the exercise capacity test and area under the curve (AUC) of FFA concentrations from POST0 to POST240 in each group (EF + CHO, p < 0.05; LF + CHO, p < 0.05; EF + MILK, p < 0.01; and LF + MILK, p < 0.05). The AUC of FFA from POST120 to POST240 showed no difference between EF (CHO and MILK) and LF (CHO and MILK). However, the AUC of FFA concentrations from POST120 to POST240 was significantly greater in MILK (EF and LF) than that in CHO (EF and LF) (p < 0.05). In active women, circulating substrates and hormone concentrations during short recovery post-exercise are not affected by the menstrual cycle. However, MILK may affect circulating substrates during recovery and the exercise capacity after recovery.

Relationship between weight management and menstrual status in female athletes: a cross-sectional survey.

The aim of this study was to investigate the effects of weight management on menstrual status in female athletes. A total of 225 collegiate athletes and 27 para-athletes who belonged to teams affiliated with the Japanese Paralympic Committee were included in this cross-sectional survey. A self-reported questionnaire (containing information on the demographic characteristics, medical history, lifestyle habits, weight management, menstruation status, physical symptoms related to menstrual cycle, and the influence of physical symptoms experienced during the luteal phase of menstruation during training or competition.) was used to assess the results. In the collegiate athletes, the rate of regular menstrual cycle was significantly lower in those with weight loss than in those without (56.7% vs. 75.0%, P < .05). Furthermore, stress fractures were found significantly more often in those with weight loss than those without (36.1% vs. 20.3%, P < .05). In the para-athletes, 46.2% of experience in weight loss had irregular menstruations (P < .01), and all of them had physical symptoms that negatively affected their training or competition (P < .05). To prevent menstrual dysfunction related to energy deficiency in female athletes with weight management, menstrual status must be considered.

Effects of the Menstrual Cycle on Serum Carnitine and Endurance Performance of Women.

This study aimed to investigate the effect of the menstrual cycle on serum carnitine and the endurance performance of healthy women. Fifteen eumenorrheic women underwent cycle ergometer exercise at 60% maximal oxygen uptake (V̇ O2max) for 45 min, followed by exercise at an intensity that was increased to 80% V̇ O 2max until exhaustion, during two menstrual cycle phases, including the early follicular phase (FP) and the midluteal phase (LP). The blood levels of estradiol, progesterone, total carnitine, free carnitine, and acylcarnitine were assessed. Compared with the FP, the LP had significantly lower serum total carnitine (p<0.05) and free carnitine (p<0.01). Moreover, the group with decreased endurance performance in the LP than in the FP showed a significantly higher change in serum free carnitine compared with the group that showed improved endurance performance in the LP than in the FP (p<0.05). The results of this study suggested that the changes in serum free carnitine during the menstrual cycle might influence endurance performance.

Arabidopsis ROOT PHOTOTROPISM2 Contributes to the Adaptation to High-Intensity Light in Phototropic Responses.

Living organisms adapt to changing light environments via mechanisms that enhance photosensitivity under darkness and attenuate photosensitivity under bright light conditions. In hypocotyl phototropism, phototropin1 (phot1) blue light photoreceptors mediate both the pulse light-induced, first positive phototropism and the continuous light-induced, second positive phototropism, suggesting the existence of a mechanism that alters their photosensitivity. Here, we show that light induction of ROOT PHOTOTROPISM2 (RPT2) underlies photosensory adaptation in hypocotyl phototropism of Arabidopsis thaliana. rpt2 loss-of-function mutants exhibited increased photosensitivity to very low fluence blue light but were insensitive to low fluence blue light. Expression of RPT2 prior to phototropic stimulation in etiolated seedlings reduced photosensitivity during first positive phototropism and accelerated second positive phototropism. Our microscopy and biochemical analyses indicated that blue light irradiation causes dephosphorylation of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) proteins and mediates their release from the plasma membrane. These phenomena correlate closely with the desensitization of phot1 signaling during the transition period from first positive phototropism to second positive phototropism. RPT2 modulated the phosphorylation of NPH3 and promoted reconstruction of the phot1-NPH3 complex on the plasma membrane. We conclude that photosensitivity is increased in the absence of RPT2 and that this results in the desensitization of phot1. Light-mediated induction of RPT2 then reduces the photosensitivity of phot1, which is required for second positive phototropism under bright light conditions.

Synthesis, antitumor activity, and cytotoxicity of 4-substituted 1-benzyl-5-diphenylstibano-1H-1,2,3-triazoles.

Trisubstituted 5-organostibano-1H-1,2,3-triazoles (3a-f) were synthesized by the Cu-catalyzed azide-alkyne cycloaddition of various ethynylstibanes (1) with benzylazide (2) in the presence of CuBr (5 mol%) under aerobic conditions. The reaction of 5-stibanotriazoles with HCl afforded C5-unsubstituted 1,2,3-triazoles (4a-f). The antitumor activity of trisubstituted 5-organostibano-1H-1,2,3-triazoles (3a-f) and their 5-unsubstituted 1,2,3-triazoles (4a-f) were evaluated in several tumor cell lines. All 5-stibanotriazoles (3a-f) exerted an excellent antitumor activity. On the contrary, 5-unsubstituted 1,2,3-triazoles (4a-f) without a diphenylantimony group in the molecule exhibited very low antitumor activity compared with 5-stibanotriazoles (3a-f). In compounds of both the series, the substituted 4-butyl group appeared to decrease antitumor activity. However, results suggested that organometal (antimony) in the molecule was required for greater antitumor activity. In addition, all 5-stibanotriazoles (3a-f), but not all 5-unsubstituted 1,2,3-triazoles (4a-f), exhibited cytotoxicity in normal vascular endothelial cells derived from bovine aorta. Among the compounds (3b-e) that exhibited excellent antitumor activity, those with 4-methylphenyl (3b) and 1-cyclohexenyl (3e) showed relatively low cytotoxicity to vascular endothelial cells. Together, these results suggest that trisubstituted 5-organostibano-1H-1,2,3-triazoles, including compounds 3b and 3e, may serve as potential anticancer therapeutic drugs in the future.

Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington's model mice.

Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine (polyQ) tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here, we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer life spans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyQ length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions.

Synthesis and photophysical properties of novel benzophospholo[3,2-b]indole derivatives.

The parent benzophospholo[3,2-b]indole was prepared by the reaction of dichlorophenylphosphine with a dilithium intermediate, which was prepared in two steps from 2-ethynyl-N,N-dimethylaniline. Using the obtained benzophosphole-fused indole as a common starting material, simple modifications were carried out at the phosphorus center of the phosphole, synthesizing various functionalized analogs. The X-ray structure analysis of trivalent phosphole and phosphine oxide showed that the fused tetracyclic moieties are planar. The benzophosphole-fused indoles, such as phosphine oxide, phospholium salt, and borane complex, exhibited strong photoluminescence in dichloromethane (Φ = 67-75%).

Copper-catalyzed [3 + 2] cycloaddition of (phenylethynyl)di-p-tolylstibane with organic azides.

Trisubstituted 5-stibano-1H-1,2,3-triazoles were synthesized in moderate to excellent yields by the Cu-catalyzed [3 + 2] cycloaddition of a ethynylstibane with organic azides in the presence of CuBr (5 mol %) under aerobic conditions. The reaction of 5-stibanotriazole with HCl, I2, and NOBF4 afforded 1-benzyl-4-phenyltriazole, 1-benzyl-5-iodo-4-phenyltriazole, and a pentavalent organoantimony compound, respectively.

Intracellular localization and splicing regulation of FUS/TLS are variably affected by amyotrophic lateral sclerosis-linked mutations.

TLS (translocated in liposarcoma), also known as FUS (fused in sarcoma), is an RNA/DNA-binding protein that plays regulatory roles in transcription, pre-mRNA splicing and mRNA transport. Mutations in TLS are responsible for familial amyotrophic lateral sclerosis (ALS) type 6. Furthermore, TLS-containing intracellular inclusions are found in polyglutamine diseases, sporadic ALS, non-SOD1 familial ALS and a subset of frontotemporal lobar degeneration, indicating a pathological significance of TLS in a wide variety of neurodegenerative diseases. Here, we identified TLS domains that determine intracellular localization of the murine TLS. Among them, PY-NLS located in the C-terminus is a strong determinant of intracellular localization as well as splicing regulation of an E1A-derived minigene. Disruption of PY-NLS promoted the formation of cytoplasmic granules that were partially overlapped with stress granules and P-bodies. Some of the ALS-linked mutations altered both intracellular localization and splicing regulation of TLS, while most mutations alone did not affect splicing regulation. However, phospho-mimetic substitution of Ser505 (or Ser513 in human) could enhance the effects of ALS mutations, highlighting interplay between post-translational modification and ALS-linked mutations. These results demonstrate that ALS-linked mutations can variably cause loss of nuclear functions of TLS depending on the degree of impairment in nuclear localization.

Recent Advances in Localized Immunomodulation Technology: Application of NIR-PIT toward Clinical Control of the Local Immune System.

Current immunotherapies aim to modulate the balance among different immune cell populations, thereby controlling immune reactions. However, they often cause immune overactivation or over-suppression, which makes them difficult to control. Thus, it would be ideal to manipulate immune cells at a local site without disturbing homeostasis elsewhere in the body. Recent technological developments have enabled the selective targeting of cells and tissues in the body. Photo-targeted specific cell therapy has recently emerged among these. Near-infrared photoimmunotherapy (NIR-PIT) has surfaced as a new modality for cancer treatment, which combines antibodies and a photoabsorber, IR700DX. NIR-PIT is in testing as an international phase III clinical trial for locoregional recurrent head and neck squamous cell carcinoma (HNSCC) patients (LUZERA-301, NCT03769506), with a fast-track designation by the United States Food and Drug Administration (US-FDA). In Japan, NIR-PIT for patients with recurrent head and neck cancer was conditionally approved in 2020. Although NIR-PIT is commonly used for cancer therapy, it could also be exploited to locally eliminate certain immune cells with antibodies for a specific immune cell marker. This strategy can be utilized for anti-allergic therapy. Herein, we discuss the recent technological advances in local immunomodulation technology. We introduce immunomodulation technology with NIR-PIT and demonstrate an example of the knockdown of regulatory T cells (Tregs) to enhance local anti-tumor immune reactions.

Effects of the menstrual cycle on EPOC and fat oxidation after low-volume high-intensity interval training.

BACKGROUND: Low-volume high-intensity interval training (HIIT) for weight loss has become prevalent in recent years, with increased excess post-exercise oxygen consumption (EPOC) as the mechanism. However, the influence of the menstrual cycle on EPOC and fat oxidation following low-volume HIIT is unclear. This study aimed to investigate the effect of the menstrual cycle on the increase in EPOC and fat oxidation after low-volume HIIT. METHODS: Twelve eumenorrheic women participated during their early follicular and luteal phases. On each experimental day, they performed low-volume HIIT comprising fifteen repeated 8 s sprint cycling tests with 12 s rests, for 5 min. Expired gas samples were collected before and every 60 min until 180 min post-exercise. EPOC was defined as the increase in oxygen consumption from the resting state, and the total EPOC and fat oxidation were calculated from the total time of each measurement. Blood samples for serum estradiol, progesterone, free fatty acids, blood glucose, lactate, and plasma noradrenaline were collected and assessed before immediately after, and at 180 min post-exercise and were assessed. RESULTS: Serum estradiol and progesterone were significantly higher in the luteal phase than the follicular phase (P<0.01 for both). No significant differences in total EPOC and fat oxidation were found between the menstrual phases. Serum free fatty acid, blood glucose, lactate, and plasma noradrenaline concentrations were not affected by the menstrual cycle. CONCLUSIONS: These results suggest that the menstrual cycle does not affect the increase in EPOC or fat oxidation after low-volume HIIT.

Contrast-enhanced ultrasound imaging for monitoring the efficacy of near-infrared photoimmunotherapy.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy combining NIR-light irradiation with an antibody and IR700DX, a light-sensitive substance, to destroy tumours. However, homogeneous irradiation is difficult because the light varies depending on the distance and tissue environment. Therefore, markers that indicate sufficient irradiation are necessary. Nanoparticles sized 10∼200 nm show enhanced permeation and retention within tumours, which is further enhanced via NIR-PIT (super enhanced permeability and retention, SUPR). We aimed to monitor the effectiveness of NIR-PIT by measuring SUPR. METHODS: A xenograft mouse tumour model was established by inoculating human cancer cells in both buttocks of Balb/C-nu/nu mice, and NIR-PIT was performed on only one side. To evaluate SUPR, fluorescent signal examination was performed using QD800-fluorescent nanoparticles and NIR-fluorescent poly (d,l-lactide-co-glycolic acid) (NIR-PLGA) microparticles. Harmonic signals were evaluated using micro-bubbles of the contrast agent Sonazoid and contrast-enhanced ultrasound (CEUS) imaging. The correlation between SUPR immediately after treatment and NIR-PIT effectiveness on the day after treatment was evaluated. FINDINGS: QD800 fluorescent signals persisted only in the treated tumours, and the intensity of remaining signals showed high positive correlation with the therapeutic effect. NIR-PLGA fluorescent signals and Sonazoid-derived harmonic signals remained for a longer time in the treated tumours than in the controls, and the kE value of the two-compartment model correlated with NIR-PIT effectiveness. INTERPRETATION: SUPR measurement using Sonazoid and CEUS imaging could be easily adapted for clinical use as a therapeutic image-based biomarker for monitoring and confirming of NIR-PIT efficacy. FUNDING: This research was supported by ARIM JAPAN of MEXT, the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, 21K07217) (JSPS), CREST (JPMJCR19H2, JST), and FOREST-Souhatsu (JST). Mochida Memorial Foundation for Medical and Pharmaceutical Research; Takeda Science Foundation; The Japan Health Foundation; and Princess Takamatsu Cancer Research Fund. Funders only provided financial support and had no role in the study design, data collection, data analysis, interpretation, and writing of the report.

Silencing of GRHL2 induces epithelial­to­mesenchymal transition in lung cancer cell lines with different effects on proliferation and clonogenic growth.

Grainyhead-like 2 (GRHL2) is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT). It has been previously shown that GRHL2 can confer both oncogenic and tumor-suppressive roles in human cancers, including breast, pancreatic and colorectal cancers. However, its role in lung cancer remains elusive. In the present study, a meta-analysis of multiple gene expression datasets with clinical data revealed that GRHL2 expression was increased in lung cancer compared with that in the normal tissues. Copy number analysis of GRHL2, performed using datasets of whole exome sequencing involving 151 lung cancer cell lines, revealed frequent amplifications, suggesting that the increased GRHL2 expression may have resulted from gene amplification. A survival meta-analysis of GRHL2 using The Cancer Genome Atlas (TCGA) dataset showed no association of GRHL2 expression with overall survival. GRHL2 expression was found to be associated with EMT status in lung cancer in TCGA dataset and lung cancer cell lines. GRHL2 knockdown induced partial EMT in the hTERT/Cdk4-immortalized normal lung epithelial cell line HBEC4KT without affecting proliferation measured by CCK-8 assays. In addition, GRHL2 silencing caused three lung cancer cell lines, H1975, H2009 and H441, to undergo partial EMT. However, the proliferative effects differed significantly. GRHL2 silencing promoted proliferation but not colony formation in H1975 cells whilst suppressing colony formation without affecting proliferation in H2009 cells, but it did not affect proliferation in H441 cells. These results suggest cell type-dependent effects of GRHL2 knockdown. Downstream, GRHL2 silencing enhanced the phosphorylation of AKT and ERK, assessed by western blotting with phospho-specific antibodies, in HBEC4KT, H1975 and H2009 cell lines but not in the H441 cell line. By contrast, transient GRHL2 overexpression did not affect A549 cell proliferation, which lack detectable endogenous expression of the GRHL2 protein. However, GRHL2 overexpression did suppress E-cadherin expression in A549 cells. These results suggested that GRHL2 does not only function as a tumor suppressor of EMT but can also behave as an oncogene depending on the lung cancer cell-type context.

Effects of acute aerobic exercise on arterial stiffness in transgender men.

Testosterone replacement therapy (TRT) in transgender men (TM) results in side effects such as elevated triglycerides and increased arterial stiffness. Exercise may be useful to ameliorate such effects, but no studies have examined the effects of acute aerobic exercise in TM. This study aimed to investigate the effects of acute aerobic exercise on arterial stiffness in TM. Thirty-six participants were included, comprising 12 TM (duration of TRT: 57.4 ± 30.3 months), 12 males and 12 females. All participants performed acute aerobic exercise on a treadmill at 50% heart rate reserve for 30 min. Arterial stiffness as measured by brachial-ankle pulse wave velocity (baPWV) was measured before exercise (Pre), 30 min after exercise (Post30), and 60 min after exercise (Post60). Serum sex hormone levels, and serum lipid profile were determined only before exercise. Serum low-density lipoprotein cholesterol (LDL-C) levels before exercise were significantly higher in TM than in males or females (males: p < 0.01; females: p < 0.05). At all points, baPWV in TM was significantly higher than in females (p < 0.05) and significantly lower than in males (p < 0.05). However, when comparing changes in baPWV over time in each group, significant decreases in Post30 and Post60 were seen in males compared to Pre (both p < 0.05), but no significant change after aerobic exercise was seen in TM or females. These results suggest that acute aerobic exercise yield different effects in TM than in males, but is unlikely to reduce arterial stiffness in TM receiving TRT.

Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction.

In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

Near-Infrared Photoimmunotherapy for Thoracic Cancers: A Translational Perspective.

The conventional treatment of thoracic tumors includes surgery, anticancer drugs, radiation, and cancer immunotherapy. Light therapy for thoracic tumors has long been used as an alternative; conventional light therapy also called photodynamic therapy (PDT) has been used mainly for early-stage lung cancer. Recently, near-infrared photoimmunotherapy (NIR-PIT), which is a completely different concept from conventional PDT, has been developed and approved in Japan for the treatment of recurrent and previously treated head and neck cancer because of its specificity and effectiveness. NIR-PIT can apply to any target by changing to different antigens. In recent years, it has become clear that various specific and promising targets are highly expressed in thoracic tumors. In combination with these various specific targets, NIR-PIT is expected to be an ideal therapeutic approach for thoracic tumors. Additionally, techniques are being developed to further develop NIR-PIT for clinical practice. In this review, NIR-PIT is introduced, and its potential therapeutic applications for thoracic cancers are described.

Expression of the auxin biosynthetic genes YUCCA1 and YUCCA4 is dependent on the boundary regulators CUP-SHAPED COTYLEDON genes in the Arabidopsis thaliana embryo.

During embryogenesis of eudicots, the apical region of the embryo develops two cotyledon primordia and the shoot meristem. In Arabidopsis thaliana, this process is dependent on the functionally redundant activities of the CUP-SHAPED COTYLEDON (CUC) transcription factors, namely CUC1, CUC2, and CUC3, as well as the phytohormone auxin. However, the relationship between the CUC proteins and auxin has yet to be fully elucidated. In the present study, we examined whether the expression of auxin biosynthetic genes is dependent on CUC gene activities. Comprehensive quantitative RT-PCR analysis of the main auxin biosynthetic gene families of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATED and YUCCA (YUC) showed that YUC1 and YUC4 expression levels were lower in cuc double mutant embryos than the expression levels of these genes in wild type embryos. Reporter analysis also revealed that the expression of YUC1 and YUC4 in the cotyledon boundary region was reduced in cuc double mutant embryos. In contrast, the loss of function mutation in the SHOOT MERISTEMLESS gene, a shoot stem cell regulator that acts downstream of the CUC genes, did not markedly affect YUC1 expression levels. These results demonstrate that CUC genes play an important role in the regulation of auxin biosynthetic gene expression during embryogenesis; furthermore, they raise the possibility that the auxin produced by this regulation contributes to cotyledon boundary development.

Study of Orally Disintegrating Tablets Using Erythritol as an Excipient Produced by Moisture-Activated Dry Granulation (MADG).

Moisture-activated dry granulation (MADG) is an eco-friendly granulation method that uses a small amount of water and insoluble excipients to absorb moisture. MADG is expected to improve productivity and reduce costs. Erythritol, an excipient used for preparing orally disintegrating tablets (ODTs), has poor tabletability and is difficult to form into tablets by conventional methods, such as high-shear granulation (HSG) and direct compression. In this study, we optimized the manufacturing conditions for ODTs to improve the tabletability of erythritol using MADG. The disintegration time of tablets made using the MADG method was approximately one-tenth that of those made using the HSG method, and the hardness was approximately 1.4 times higher. Moreover, MADG could delay disintegration and improve tabletability. We further attempted to optimize the manufacturing conditions using MADG, particularly in terms of the amount of water used. The disintegration time increased as the amount of added water increased. Moreover, water absorption tests revealed that capillary wetting decreased as the amount of water added increased, but the initial wetting did not change. These results suggested that the disintegration time was prolonged because of the increase in granule density and decrease in capillary wetting with the increase in the amount of added water. The hardness of the tablets increased because of the easy deformation of the granules after the addition of up to 3% water; however, when more than 3% water was added, the hardness decreased because of the aggregation of the granules with the excess water. Finally, two-dimensional maps of the effect of the amount of added water and water activity indicated that tablets with a hardness of ≥80 N and a disintegration time of ≤15 s could be produced by adjusting the amount of added water to within the range of 2.2-3.3% and water activity to 0.3-0.53. These results indicate that MADG can improve the tabletability of erythritol and be used for the granulation of ODTs. Tablets with appropriate hardness and disintegration properties can be produced by adjusting the water content to approximately 2.7% and the water activity to approximately 0.4 when producing ODTs with MADG.

Influence of menstrual cycle on muscle glycogen utilization during high-intensity intermittent exercise until exhaustion in healthy women.

The present study investigated the effects of the menstrual cycle on muscle glycogen and circulating substrates during high-intensity intermittent exercise until exhaustion in healthy women who habitually exercised. In total, 11 women with regular menstrual cycles completed three tests, which comprised the early follicular phase (E-FP), late follicular phase (L-FP), and luteal phase (LP) of the menstrual cycle. High-intensity intermittent exercise until exhaustion was performed on each test day. Evaluation of muscle glycogen concentration by 13C-magnetic resonance spectroscopy and measurement of estradiol, progesterone, blood glucose, lactate, free fatty acids (FFA), and insulin concentrations were conducted before exercise (Pre) and immediately after exercise (Post). Muscle glycogen concentrations from thigh muscles at Pre and Post were not significantly different between menstrual cycle phases (P = 0.57). Muscle glycogen decreases by exercise were significantly greater in L-FP (59.0 ± 12.4 mM) than in E-FP (48.3 ± 14.4 mM, P < 0.05). Nonetheless, blood glucose, blood lactate, serum FFA, serum insulin concentrations, and exercise time until exhaustion in E-FP, L-FP, and LP were similar. The study results suggest that although exercise time does not change according to the menstrual cycle, the menstrual cycle influences muscle glycogen utilization during high-intensity intermittent exercise until exhaustion in women with habitual exercise activity. Novelty: This study compared changes in muscle glycogen concentration across the menstrual cycle during high-intensity intermittent exercise until exhaustion using 13C-magnetic resonance spectroscopy. Our results highlight the influence of the menstrual cycle on muscle glycogen during high-intensity intermittent exercise in healthy women.