Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

<div><p>Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. <i>In-vitro</i> BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy. </p> </div

Changes in the percentage of EpCAM-positive cells by using the target activation array scan protocol (A), and CYP3A4 and Bmi mRNA levels (B) in HAK1-B cells cultured in RPMI1640 containing 10% FBS only, or with 1, 2, or 4 mM BCAA added for 72 h.

<div><p>Dunnett's test, *p < 0.05, **p < 0.01 n = 6, mean ± SE.</p> <p>The percentage of Annexin V-positive cells (C) and relative viable cell number (D) by array scan in HAK-1B cells cultured in RPMI1640 containing 10% FBS with or without 2 mM BCAA in the presence (1 or 2 µg/mL) or absence of 5-FU for 72 h by using target activation protocol of array scan. Student <i>t</i>-test, *p < 0.05, **p < 0.01, n = 7, mean ± SE.</p></div

The rate change of EpCAM positive cell in 5000 cells in the presence of 100 nM rapamycin treatment for 1 h or 72 h (A).

<div><p>Dunnett's test, *p < 0.05, ***p <0.001 vs. control, n = 6, mean ± SE.</p> <p>The relative expressions of EpCAM, c-myc, and FOXO3a mRNA upon Raptor and Rictor knockdown (B-D).</p> <p>Dunnett's test, **p < 0.01, ***p < 0.001 vs. control n = 8, mean ± SE.</p></div

Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan.

<div><p>The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C).</p> <p>The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). </p> <p>Tukey’s test **p < 0.01 vs. control, $$$p < 0.001 vs. BCAA, n = 8, mean ± SE (A).</p> <p>Student t-test *p < 0.05, ****p < 0.0001, n = 8, mean ± SE (B,C).</p></div

Tumor volume change over 14 days (A) and tumor weight (B) in the HAK-1B xenograft mouse model on the 14^th day after administration of BCAA and 5-FU injection.

<p>The relative expression of mRNA of various molecules of each tumor was associated with CSC properties (C, D). Control: 10% DMSO/saline/tumor injection + 3% casein containing diet, 5-FU: 250 µg/tumor injection + 3% casein containing diet, BCAA diet: 10% DMSO/saline/tumor injection + 3% BCAA containing, 5-FU+BCAA diet: 250 µg/tumor injection + 3% BCAA containing diet for 14 days. Tukey’s test: *p < 0.05, **p < 0.01, ***p < 0.001 vs. control, #p < 0.05 vs. 5-FU, $$p < 0.01 vs. BCAA, n = 6, mean ± SE.</p

Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min.

<div><p>Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4^th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B).</p> <p>Dunnett's test, *p<0.05, ***p < 0.001 vs. N.C. n = 5, mean ± SE.</p></div

Effects of Five Amino Acids (Serine, Alanine, Glutamate, Aspartate, and Tyrosine) on Mental Health in Healthy Office Workers: A Randomized, Double-Blind, Placebo-Controlled Exploratory Trial

Background: The importance of maintaining good mental health with overall well-being has recently drawn attention from various spheres of academics and the working population. Amino acid intake has been reported to reduce depression symptoms and other mental health problems. However, the effectiveness of amino acid intake (i.e., single or combined) remains unknown. In this study, we assessed a combination of five amino acids (serine, alanine, glutamate, aspartate, and tyrosine; SAGAT) reported to regulate mental health. Methods: A randomized, double-blind, placebo-controlled exploratory trial was conducted. Participants, aged between 20 and 65 years with fatigue sensation, were randomized to receive either SAGAT or the placebo and ingested them for four weeks. A transient mental work was loaded at day 0 and after four weeks of intervention. As the primary outcomes, the fatigue sensation was assessed. The mood status, cognitive function, work efficiency, and blood marker were also measured as secondary outcomes. Results: The number of participants analyzed for the efficacy evaluation were 20 in SAGAT and 22 in the placebo. There were no significant differences in the primary outcomes. However, as the secondary outcomes, the SAGAT group showed a significant improvement in motivation and cognitive function in the recovery period after mental work loaded in a four-week intervention compared to the placebo. Conclusion: The current findings suggest that SAGAT contributes to maintaining proper motivation and cognitive function. Clinical Trial Registration: University Hospital Medical Information Network Clinical Trial Registry (ID: UMIN 000041221)

Development of Microwave Antenna for Operation of (5mm) ^3 Volume Solid State Spins

5立方ミリメートルの固体スピン（NVセンター）を駆動するマイクロ波アンテナの開発14th International Conference on New Diamond and Nano Carbons 2020/202

A compact quantum sensor head with side excitation of CVD diamond

The study on A compact quantum sensor head with side excitation of CVD diamondThe 4th International Forum on Quantum Metrology and Sensin

Simultaneous thermometry and magnetometry using a fiber-coupled quantum diamond sensor

Energy conservation and battery life extension are key challenges for the next-generation hybrid electric vehicles. In particular, the temperature and electric currents in a storage battery need to be monitored simultaneously with 1 kHz signal bandwidth for optimum battery usage. Here we introduce a centimeter-scale portable quantum sensor head, consisting of a diamond substrate hosting an ensemble of nitrogen-vacancy (NV) color centers with a density of 3 x 1017 cm3. One diamond surface is attached to a multi-mode fiber for simultaneous optical excitation and readout of the NV centers, while the other diamond surface is attached to a coplanar microwave guide for NV spin ground-state mixing. Signal bandwidth of 1 kHz was realized through time-domain multiplexing of the two-tone microwave frequency modulation at 20 kHz. Two microwave frequencies were locked to the two resonance points that were determined from the optically detected magnetic resonance spectrum. From the mean and the difference of the deviation from the two locked frequencies, the temperature and magnetic field were obtained simultaneously and independently, with sensitivities of 3.5 nT/Hz1/2 and 1.3 mK/Hz1/2, respectively. We also showed that our sensor reached a minimum detectable magnetic field of 5 pT by accumulating signals for over 10 000 s