External validation of a pharmacokinetic model for target-controlled infusion of cefazolin as a prophylactic antibiotic.

AIMS: This study aimed to evaluate the predictive performance of previously constructed cefazolin pharmacokinetic models and determine whether cefazolin administration via the target-controlled infusion (TCI) method may be possible in clinical practice. METHODS: Twenty-five gastrectomy patients receiving cefazolin as a prophylactic antibiotic were enrolled. Two grams of cefazolin was dissolved in 50 mL of normal saline to give a concentration of 40 mg mL-1 . Before skin incision, cefazolin was administered using a TCI syringe pump, and its administration continued until the end of surgery. The target total plasma concentration was set to 100 µg mL-1 . Total and unbound plasma concentrations of cefazolin were measured in three arterial blood samples collected at 30, 60 and 120 min after the start of cefazolin administration. The predictive performance of the TCI system was evaluated using four measures: inaccuracy, divergence, bias and wobble. RESULTS: Total (n = 75) and unbound (n = 75) plasma concentration measurements from 25 patients were included in the analysis. The pooled median (95% confidence interval) biases and inaccuracies were 6.3 (4.0-8.5) and 10.5 (8.6-12.4) for the total concentration model and -10.3 (-16.8 to -3.7) and 22.4 (18.2-26.7) for the unbound concentration model, respectively. All unbound concentrations were above 10 µg mL-1 . CONCLUSION: Administration of cefazolin by the TCI method showed a clinically acceptable performance. Applying the TCI method by setting the total concentration as the target concentration rather than the unbound concentration is effective in maintaining a constant target concentration of cefazolin.

Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds.

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T0 generation at editing frequencies of 0.1-84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1-9 nucleotides and insertions of 1 nucleotide in several soybean lines (T0). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T1 plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T2 seeds but did not show any defects in plant growth and seed development.

Syringic Acid Alleviates Cesium-Induced Growth Defect in Arabidopsis.

Syringic acid, a phenolic compound, serves a variety of beneficial functions in cells. Syringic acid increases in plants in response to cesium, and exogenous application of syringic acid resulted in a significant attenuation of cesium-induced growth defects in Arabidopsis. In addition, cesium or syringic acid application to plants also resulted in increased lignin deposition in interfascicular fibers. To better understand the role of lignin and syringic acid in attenuating cesium-induced growth defects, two mutants for Arabidopsis REDUCED EPIDERMAL FLUORESCENE 4 (REF4) and fourteen laccase mutants, some of which have lower levels of lignin, were evaluated for their response to cesium. These mutants responded differently to cesium stress, compared to control plants, and the application of syringic acid alleviated cesium-induced growth defects in the laccase mutants but not in the ref4 mutants. These findings imply that lignin plays a role in cesium signaling but the attenuation of cesium stress defects by syringic acid is mediated by regulatory components of lignin biosynthesis and not lignin biosynthesis itself. In contrast, syringic acid did not alleviate any low potassium-induced growth defects. Collectively, our findings provide the first established link between lignin and cesium stress via syringic acid in plants.

Clinical and biochemical relevance of monounsaturated fatty acid metabolism targeting strategy for cancer stem cell elimination in colon cancer.

Lipid metabolism is associated with colon cancer prognosis and incidence. Stearoyl-CoA desaturase 1 (SCD1), which converts fully saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs), has been suggested as a vulnerable target for selective elimination of cancer stem cells (CSCs). However, the clinical significance and physiological role of SCD1 in CSCs has not been well demonstrated. Here, we showed the clinical and biochemical relevance of blocking SCD1 to target CSCs by analyzing human colon cancer data from TCGA and through lipidomic profiling of CSCs with or without SCD1 inhibition using mass spectrometry. Positive associations between SCD1 expression and colorectal cancer patient clinical status and the expression of CSC-related genes (WNT and NOTCH signaling) were found based on TCGA data analysis. Lipidomic profiling of CSCs and bulk cancer cells (BCCs) using mass spectrometry revealed that colon CSCs contained a distinctive lipid profile, with higher free MUFA and lower free SFA levels than in BCCs, suggesting that enhanced SCD1 activity generates MUFAs that may support WNT signaling in CSCs. In addition, all identified phosphatidyl-ethanolamine-containing MUFAs were found at higher levels in CSCs. Interestingly, we observed lower phosphatidyl-serine (18:1/18:0), phosphatidyl-choline (PC; p-18:0/18:1)), and sphingomyelin (SM; d18:1/20:0 or d16:1/22:0) levels in CSCs than in BCCs. Of those, SCD1 inhibition, which efficiently diminished free MUFA levels, increased those specific PC and SM and MUFAs in CSCs promptly. These results suggest that these specific lipid composition is critical for CSC stem cell maintenance. In addition, not only free MUFAs, which are known to be required for WNT signaling, but also other phospholipids, such as SM, which are important for lipid raft formation, may mediate other cell signaling pathways that support CSC maintenance. Comparison of the lipidomic profiles of colon cancer cells with those of previously reported for glioma cells further demonstrated the tissue specific characteristics of lipid metabolism in CSCs.

Arabidopsis CNGC Family Members Contribute to Heavy Metal Ion Uptake in Plants.

Heavy metal ions, including toxic concentrations of essential ions, negatively affect diverse metabolic and cellular processes. Heavy metal ions are known to enter cells in a non-selective manner; however, few studies have examined the regulation of heavy metal ion transport. Plant cyclic nucleotide-gated channels (CNGCs), a type of Ca2+-permeable-channel, have been suggested to be involved in the uptake of both essential and toxic cations. To determine the candidates responsible for heavy metal ion transport, a series of Arabidopsis CNGC mutants were examined for their response to Pb2+ and Cd2+ ions. The primary focus was on root growth and the analysis of the concentration of heavy metals in plants. Results, based on the analysis of primary root length, indicated that AtCNGC1, AtCNGC10, AtCNGC13 and AtCNGC19 play roles in Pb2+ toxicity, while AtCNGC11, AtCNGC13, AtCNGC16 and AtCNGC20 function in Cd2+ toxicity in Arabidopsis. Ion content analysis verified that the mutations of AtCNGC1 and AtCNGC13 resulted in reduced Pb2+ accumulation, while the mutations of AtCNGC11, AtCNGC15 and AtCNGC19 resulted in less Pb2+ and Cd2+ accumulation in plants. These findings provide functional evidence which support the roles of these AtCNGCs in the uptake and transport of Pb2+ or Cd2+ ion in plants.

Silencing of an α-dioxygenase gene, Ca-DOX, retards growth and suppresses basal disease resistance responses in Capsicum annum.

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the ß-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.

Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma.

PURPOSE: The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor-stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs. METHODS: Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC-MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry. RESULTS: CAFs were demonstrated to increase expressions and activities of 17ßHSD2, 17ßHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17ßHSD2, 17ßHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17ßHSD2 and 17ßHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17ßHSD2 and 17ßHSD5 status in TNBC tissues, especially AR-positive cases. CONCLUSIONS: Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.

Multiple UDP-Glucuronosyltransferase and Sulfotransferase Enzymes are Responsible for the Metabolism of Verproside in Human Liver Preparations.

Verproside, an active iridoid glycoside component of Veronica species, such as Pseudolysimachion rotundum var. subintegrum and Veronica anagallis-aquatica, possesses anti-asthma, anti-inflammatory, anti-nociceptive, antioxidant, and cytostatic activities. Verproside is metabolized into nine metabolites in human hepatocytes: verproside glucuronides (M1, M2) via glucuronidation, verproside sulfate (M3) via sulfation, picroside II (M4) and isovanilloylcatalpol (M5) via O-methylation, M4 glucuronide (M6) and M4 sulfate (M8) via further glucuronidation and sulfation of M4, and M5 glucuronide (M7) and M5 sulfate (M9) via further glucuronidation and sulfation of M5. Drug-metabolizing enzymes responsible for verproside metabolism, including sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT), were characterized. The formation of verproside glucuronides (M1, M2), isovanilloylcatalpol glucuronide (M7), and picroside II glucuronide (M6) was catalyzed by commonly expressed UGT1A1 and UGT1A9 and gastrointestinal-specific UGT1A7, UGT1A8, and UGT1A10, consistent with the higher intrinsic clearance values for the formation of M1, M2, M6, and M7 in human intestinal microsomes compared with those in liver microsomes. The formation of verproside sulfate (M3) and M5 sulfate (M9) from verproside and isovanilloylcatalpol (M5), respectively, was catalyzed by SULT1A1. Metabolism of picroside II (M4) into M4 sulfate (M8) was catalyzed by SULT1A1, SULT1E1, SULT1A2, SULT1A3, and SULT1C4. Based on these results, the pharmacokinetics of verproside may be affected by the co-administration of relevant UGT and SULT inhibitors or inducers.

High-temperature GC-MS-based serum cholesterol signatures may reveal sex differences in vasospastic angina.

Alterations of cholesterol metabolism are responsible for vasospastic angina and atherosclerosis. To comprehensively evaluate cholesterol metabolism, 18 sterols, including cholesterol, 6 cholesteryl esters (CEs), 3 cholesterol precursors, and 8 hydroxycholesterols (OHCs), were simultaneously analyzed using hybrid solid-phase extraction (SPE) purification coupled to high-temperature gas chromatography-mass spectrometry (HTGC-MS). Methanol-based hybrid SPE increased the selective extraction, and HTGC resulted in a good chromatographic resolution for the separation of lipophilic compounds. The limits of quantification of cholesterol and CEs ranged from 0.2 to 10.0 µg/ml, while OHCs and cholesterol precursors ranged from 0.01 to 0.10 µg/ml. Linearity as the correlation coefficient was higher than 0.99 with the exception of cholesteryl laurate, myristate, oleate, and linoleate (r² > 0.98). The precision (% coefficient of variation) and accuracy (% bias) ranged from 1.1 to 9.8% and from 75.9 to 125.1%, respectively. The overall recoveries of CEs ranged from 26.1 to 64.0%, and the recoveries of other sterols ranged from 83.8 to 129.3%. The cholesterol signatures showed sex differences in patients with vasospastic angina and may associate with 24-reductases. This technique can be useful for making clinical diagnoses and for an increased understanding of the pathophysiology of vasospastic angina.

Global metabolomics and targeted steroid profiling reveal that rifampin, a strong human PXR activator, alters endogenous urinary steroid markers.

Activation of the pregnane X receptor (PXR) alters the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. To identify endogenous biomarkers of PXR activation in humans, rifampin, a strong PXR activator, was administered to 12 healthy male subjects, and their urine was analyzed before and after rifampin administration. Ultraperformance liquid chromatography time-of-flight mass spectrometry (UPLC/QTOF-MS)-based global metabolomics and gas chromatography-mass spectrometry (GC-MS)-based profiling of 75 steroids were used to screen the urine samples. Global metabolomics revealed that hydroxytestosterone sulfate and glycochenodeoxycholate sulfate levels were significantly increased and that androsterone sulfate, dehydroepiandrosterone (DHEA) sulfate, and p-cresol sulfate levels were significantly decreased following rifampin administration compared with controls. Urinary steroid profiling showed that 16α-OH-androstenedione (16α-OH-A-dione), 16α-OH-DHEA, 7α-DHEA, 7ß-DHEA, and 11ß-OH-A-dione levels were increased, whereas DHEA, androsterone, etiocholanolone, estrone, ß-cortolone, and allo-tetrahydrocortisone levels were decreased in the rifampin group. The analysis of the metabolic pathway and the metabolic ratio of steroids enabled the estimation of the induction of CYP1A/3A/7B/11B/2C and the inhibition of CYP17A/19A in response to PXR activation. These human urinary biomarkers may be useful for predicting the extent of PXR activation, monitoring the activity of DMEs, and anticipating drug-drug interactions in patients administered PXR-activating drugs.

GC-MS-based quantitative signatures of cytochrome P450-mediated steroid oxidation induced by rifampicin.

BACKGROUND: Drug-induced cytochrome P450 (CYP) activity affects endocrine function and drug clearance rates, leading to the development of unpredictable pathologic and toxicologic risks. METHODS: Urinary steroid profiling based on gas chromatography-mass spectrometry (GC-MS) was used for simultaneous quantification of CYP-mediated regioselective hydroxysteroids and their substrates, including 26 androgens, 9 estrogens, 5 progestins, and 7 corticoids. The quantitative data were visualized using a hierarchically clustered heat map to allow identification of CYP-mediated steroid signatures. Twelve healthy subjects were orally administered 600 mg of rifampicin a day for 7 days, and their CYP enzyme activity was evaluated. RESULTS: Using GC-MS, all 47 steroids were well separated with good peak shapes. This assay had good linearity (r > 0.994) in a dynamic range, and the interassay imprecision (% CV) and inaccuracy (% bias) were 3.0%-15.6% and 98.0%-109.2%, respectively. Administration of the CYP3A4 inducer rifampicin produced distinct differences in CYP3A4 and CYP11B1, CYP19A1, HSD11B, and HSD17B, which were indicated by their heat map-visualized steroid signatures. CONCLUSIONS: This CYP-mediated steroid signature profile allows simultaneous assessment of CYP1A, CYP1B, CYP2C, CYP3A, CYP11B, CYP17A, CYP19A, and CYP21A in urine samples. This method could therefore be a useful tool for assessing drug efficacy.

Comparison of metabolic ratios of urinary estrogens between benign and malignant thyroid tumors in postmenopausal women.

BACKGROUND: Estrogen metabolism may be associated with the pathophysiological development of papillary thyroid carcinoma (PTC). METHODS: To evaluate the differential estrogen metabolism between benign and malignant PTCs, estrogen profiling by gas chromatography-mass spectrometry was applied to urine samples from postmenopausal patients with 9 benign tumors and 18 malignant stage I and III/IV PTCs. RESULTS: The urinary concentration of 2-methoxyestradiol was significantly lower in the stage I malignant patients (3.5-fold; P < 0.025) than in the benign group. The metabolic ratios of 16α-OH-estrone/estrone and estriol/estradiol, which are responsible for 16α-hydroxylase activity, were increased more than 2.5-fold in the advanced-stage malignant PTC (P < 0.02 each). The more than 6.2-fold decrease in the urinary 2-/16α-hydroxylase ratio in stage III/IV malignant PTC was consistent with the ratio in postmenopausal patients with endocrine gland cancers. In addition, reductive 17ß-hydroxysteroid dehydrogenase (17ß-HSD; estradiol/estrone or estriol/16α-OH-estrone) was present at significantly higher levels in subjects with stage III/IV malignant PTCs than in benign subjects (>3.5-fold difference; P < 0.002). In particular, the estriol/16α-OH-estrone ratio differentiated between the benign and early-stage malignant patients (P < 0.01). CONCLUSIONS: Increased 16α-hydroxylation and/or a decreased 2-/16α-ratio, as well increased reductive 17ß-HSD, with regard to estrogen metabolism could provide potential biomarkers. The devised profiles could be useful for differentiating malignant thyroid carcinomas from benign adenomas in postmenopausal women.

Isolation of novel chemical components and their plant target proteins under selenium stress.

Selenium is recognized as a beneficial nutrient in living organisms. Excessive amounts of selenium, however, can have a significant negative impact on organisms. Screening of novel chemical compounds that regulate and/or moderate selenium in plants was conducted. The present chapter discusses (1) the design of a chemical screening strategy, (2) methods used to identify and select candidate chemicals, and (3) the identification of chemical-binding target proteins. We identified a novel chemical compound, C9H8N2OS2, in our screening program that enhances selenate accumulation and stress tolerance. The target protein, beta-glucosidase 23, in Arabidopsis was found to regulate selenium accumulation, as well as plant response to selenate stress.

A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis.

Estrogen metabolites play important roles in the development of female-related disorders and homeostasis of the bone. To improve detectability, a validated gas chromatography-mass spectrometry (GC-MS) method was conducted with two-phase extractive ethoxycarbonlyation (EOC) and subsequent pentafluoropropionyl (PFP) derivatization was introduced. The resulting samples were separated through a high-temperature MXT-1 column within an 8 min run and were detected in the selected ion monitoring (SIM) mode. The optimized analytical conditions led to good separation with a symmetric peak shape for 19 estrogens as their EOC-PFP derivatives. The limit of quantification (LOQ) was from 0.02 to ∼0.1 ng/ml for most estrogens analyzed, except for 2-hydroxyestriol (0.5 ng/ml). The devised method was found to be linear (r² > 0.995) in the range from the LOQ to 40 ng/ml, whereas the precision (% CV) and accuracy (% bias) ranged from 1.4 to 10.5% and from 91.4 to 108.5%, respectively. The good sensitivity and selectivity of this method even allowed quantification of the estrogen metabolites in urine samples obtained from the postmenopausal female patients with osteoporosis. The present technique can be useful for clinical diagnosis as well as to better understand the pathogenesis of estrogen-related disorders in low-level quantification.

Metabolic alteration of urinary steroids in pre- and post-menopausal women, and men with papillary thyroid carcinoma.

BACKGROUND: To evaluate the metabolic changes in urinary steroids in pre- and post-menopausal women and men with papillary thyroid carcinoma (PTC). METHODS: Quantitative steroid profiling combined with gas chromatography-mass spectrometry was used to measure the urinary concentrations of 84 steroids in both pre- (n = 21, age: 36.95 ± 7.19 yr) and post-menopausal female (n = 19, age: 52.79 ± 7.66 yr), and male (n = 16, age: 41.88 ± 8.48 yr) patients with PTC. After comparing the quantitative data of the patients with their corresponding controls (pre-menopause women: n = 24, age: 33.21 ± 10.48 yr, post-menopause women: n = 16, age: 49.67 ± 8.94 yr, male: n = 20, age: 42.75 ± 4.22 yr), the levels of steroids in the patients were normalized to the mean concentration of the controls to exclude gender and menopausal variations. RESULTS: Many urinary steroids were up-regulated in all PTC patients compared to the controls. Among them, the levels of three active androgens, androstenedione, androstenediol and 16α-hydroxy DHEA, were significantly higher in the pre-menopausal women and men with PTC. The corticoid levels were increased slightly in the PTC men, while progestins were not altered in the post-menopausal PTC women. Estrogens were up-regulated in all PTC patients but 2-hydroxyestrone and 2-hydroxy-17ß-estradiol were remarkably changed in both pre-menopausal women and men with PTC. For both menopausal and gender differences, the 2-hydroxylation, 4-hydroxylation, 2-methoxylation, and 4-methoxylation of estrogens and 16α-hydroxylation of DHEA were differentiated between pre- and post-menopausal PTC women (P < 0.001). In particular, the metabolic ratio of 2-hydroxyestrone to 2-hydroxy-17ß-estradiol, which could reveal the enzyme activity of 17ß-hydroxysteroid dehydrogenase, showed gender differences in PTC patients (P < 1 × 10-7). CONCLUSIONS: These results are expected be helpful for better understanding the pathogenic differences in PTC according to gender and menopausal conditions.

Cesium tolerance is enhanced by a chemical which binds to BETA-GLUCOSIDASE 23 in Arabidopsis thaliana.

Cesium (Cs) is found at low levels in nature but does not confer any known benefit to plants. Cs and K compete in cells due to the chemical similarity of Cs to potassium (K), and can induce K deficiency in cells. In previous studies, we identified chemicals that increase Cs tolerance in plants. Among them, a small chemical compound (C17H19F3N2O2), named CsToAcE1, was confirmed to enhance Cs tolerance while increasing Cs accumulation in plants. Treatment of plants with CsToAcE1 resulted in greater Cs and K accumulation and also alleviated Cs-induced growth retardation in Arabidopsis. In the present study, potential target proteins of CsToAcE1 were isolated from Arabidopsis to determine the mechanism by which CsToAcE1 alleviates Cs stress, while enhancing Cs accumulation. Our analysis identified one of the interacting target proteins of CsToAcE1 to be BETA-GLUCOSIDASE 23 (AtßGLU23). Interestingly, Arabidopsis atßglu23 mutants exhibited enhanced tolerance to Cs stress but did not respond to the application of CsToAcE1. Notably, application of CsToAcE1 resulted in a reduction of Cs-induced AtßGLU23 expression in wild-type plants, while this was not observed in a high affinity transporter mutant, athak5. Our data indicate that AtßGLU23 regulates plant response to Cs stress and that CsToAcE1 enhances Cs tolerance by repressing AtßGLU23. In addition, AtHAK5 also appears to be involved in this response.

The hypothalamic-pituitary-gonadal axis controls muscle stem cell senescence through autophagosome clearance.

BACKGROUND: With organismal aging, the hypothalamic-pituitary-gonadal (HPG) activity gradually decreases, resulting in the systemic functional declines of the target tissues including skeletal muscles. Although the HPG axis plays an important role in health span, how the HPG axis systemically prevents functional aging is largely unknown. METHODS: We generated muscle stem cell (MuSC)-specific androgen receptor (Ar) and oestrogen receptor 2 (Esr2) double knockout (dKO) mice and pharmacologically inhibited (Antide) the HPG axis to mimic decreased serum levels of sex steroid hormones in aged mice. After short-term and long-term sex hormone signalling ablation, the MuSCs were functionally analysed, and their aging phenotypes were compared with those of geriatric mice (30-month-old). To investigate pathways associated with sex hormone signalling disruption, RNA sequencing and bioinformatic analyses were performed. RESULTS: Disrupting the HPG axis results in impaired muscle regeneration [wild-type (WT) vs. dKO, P < 0.0001; Veh vs. Antide, P = 0.004]. The expression of DNA damage marker (in WT = 7.0 ± 1.6%, dKO = 32.5 ± 2.6%, P < 0.01; in Veh = 13.4 ± 4.5%, Antide = 29.7 ± 5.5%, P = 0.028) and senescence-associated ß-galactosidase activity (in WT = 3.8 ± 1.2%, dKO = 10.3 ± 1.6%, P < 0.01; in Veh = 2.1 ± 0.4%, Antide = 9.6 ± 0.8%, P = 0.005), as well as the expression levels of senescence-associated genes, p16Ink4a and p21Cip1 , was significantly increased in the MuSCs, indicating that genetic and pharmacological inhibition of the HPG axis recapitulates the progressive aging process of MuSCs. Mechanistically, the ablation of sex hormone signalling reduced the expression of transcription factor EB (Tfeb) and Tfeb target gene in MuSCs, suggesting that sex hormones directly induce the expression of Tfeb, a master regulator of the autophagy-lysosome pathway, and consequently autophagosome clearance. Transduction of the Tfeb in naturally aged MuSCs increased muscle mass [control geriatric MuSC transplanted tibialis anterior (TA) muscle = 34.3 ± 2.9 mg, Tfeb-transducing geriatric MuSC transplanted TA muscle = 44.7 ± 6.7 mg, P = 0.015] and regenerating myofibre size [eMyHC+ tdTomato+ myofibre cross-section area (CSA) in control vs. Tfeb, P = 0.002] after muscle injury. CONCLUSIONS: Our data show that the HPG axis systemically controls autophagosome clearance in MuSCs through Tfeb and prevents MuSCs from senescence, suggesting that sustained HPG activity throughout life regulates autophagosome clearance to maintain the quiescence of MuSCs by preventing senescence until advanced age.

Increased biosynthesis and accumulation of cholesterol in maternal plasma, but not amniotic fluid in pre-eclampsia.

Preeclampsia is one of the most serious complications during pregnancy, defined as development of hypertension during late pregnancy affecting other organ systems (proteinuria, thrombocytopenia, renal insufficiency, liver involvement, cerebral symptoms or pulmonary edema). Preeclampsia is known to be associated with significant dyslipidemia, but the cause or mechanism of this metabolic aberration is not clear. Quantitative analysis of cholesterol precursors and metabolites can reveal metabolic signatures of cholesterol, and provide insight into cholesterol biosynthetic and degradation pathways. We undertook this study to compare the metabolic signatures of cholesterol in serum and amniotic fluid collected from women who delivered in the late preterm period. Matching serum and amniotic fluid samples were collected from women who delivered in the late preterm period (34-0/7-36-6/7 weeks), had undergone amniocentesis within 3 days of delivery, had no evidence of rupture of membranes or intra-amniotic infection/inflammation, and who had not received antenatal corticosteroid prior to amniocentesis. Patients were classified into 3 groups according to the etiology of their preterm birth: Group 1, preeclampsia; Group 2, spontaneous preterm labor; Group 3, other maternal medical indications for iatrogenic preterm birth. Quantitative metabolite profiling of cholesterols was performed using gas chromatography-mass spectrometry. A total of 39 women were included in the analysis (n = 14 in Group 1, n = 16 in Group 2, n = 9 in Group 3). In maternal blood, patients in Group 1 had significantly higher ratios of cholesterol/desmosterol and cholesterol/7-dehydrocholesterol (which represent 24- and 7-reductase enzyme activity, respectively) than those in Group 3 (p < 0.05 for each), which suggests increased cholesterol biosynthesis. In contrast, patients in Group 1 had significantly decreased ratios of individual cholesterol esters/cholesterol and total cholesterol esters/cholesterol than those in Groups 3 (p < 0.01 for each), suggesting increased reverse cholesterol transport. No differences in cholesterol ratios were found in amniotic fluid among the 3 groups. In conclusion, the metabolic signatures of cholesterol suggest increased cholesterol biosynthesis and accumulation in the maternal blood (but not amniotic fluid) of women with preeclampsia.

Effects of alternate day fasting and exercise on cholesterol metabolism in overweight or obese adults: A pilot randomized controlled trial.

BACKGROUND AND PURPOSE: The objective of this pilot randomized controlled trial was to investigate the effect of alternate day fasting (ADF) and exercise on serum sterol signatures, which are surrogate markers of cholesterol absorption and biosynthesis. METHODS: We randomly assigned 112 overweight or obese participants to four groups: 1) ADF and exercise (E-ADF); 2) ADF; 3) exercise; and 4) control. We studied 31 completers in this exploratory analysis and measured their serum sterol signatures using gas chromatography-mass spectrometry. RESULTS: After intervention, most serum sterol signatures that correspond to cholesterol metabolism were significantly different between groups (p < 0.05 by analysis of covariance [ANCOVA]). We found no differences in plant sterols, which are markers of cholesterol absorption. In the exercise group, desmosterol, cholesteryl esters, and oxysterols decreased significantly. Furthermore, only changes in physical activity levels negatively correlated with changes in the metabolic ratios of desmosterol and 7-dehydrocholesterol to cholesterol, which reflect cholesterol biosynthesis (r = -0.411; p = 0.030, and r = -0.540; p = 0.003, respectively). CONCLUSION: These findings suggest that exercise with or without ADF improves cholesterol metabolism as measured by serum sterol signatures, and increased physical activity has a greater effect on cholesterol biosynthesis than weight reduction or calorie restriction.

Validated gas chromatographic-mass spectrometric analysis of urinary cannabinoids purified with a calcium-hardened beta-cyclodextrin polymer.

A comprehensive solid-phase extraction (SPE) technique based on the formation of an inclusion complex between beta-cyclodextrin (betaCD) and cannabinoids including Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) was developed in gas chromatographic-mass spectrometric (GC-MS) analysis. A betaCD/epichlorohydrin copolymer was prepared and then 'hardened' in aqueous solution with 0.3M CaCl(2) to yield a stable particulate copolymer, which was used as a novel SPE sorbent. An internal standard THC-COOH-d(9) was added to urine samples containing 3 cannabinoids and then purified with the hardened betaCD polymer. The cannabinoids were extracted from the hardened betaCD using tetrahydrofuran. Resulting extracts were evaporated and derivatized with MSTFA/NH(4)I/dithioerythritol (500:4:2, v/w/w) and analyzed by GC-MS in selected-ion monitoring (SIM) mode. Overall recoveries ranged from 85% to 102%, with a detection limit of 0.2 microg L(-1) for the three cannabinoids tested. The precision (% CV) and accuracy (% bias) of the assay were 1.2-5.1% and 93-111% in 0.2-50 microg L(-1) calibration range, respectively (r(2)>0.9997). Twenty actual samples positive by fluorescence polarization immunoassay were also quantitatively analyzed. The devised technique based on the calcium-hardened betaCD sorption of cannabinoids and subsequent GC-SIM/MS resulted in better selectivity and extraction efficiency than is possible using the conventional hydrophobicity-based SPE methods.