RNA m6A detection using raw current signals and basecalling errors from Nanopore direct RNA sequencing reads.

MOTIVATION: Nanopore direct RNA sequencing (DRS) enables the detection of RNA N6-methyladenosine (m6A) without extra laboratory techniques. A number of supervised or comparative approaches have been developed to identify m6A from Nanopore DRS reads. However, existing methods typically utilize either statistical features of the current signals or basecalling-error features, ignoring the richer information of the raw signals of DRS reads. RESULTS: Here, we propose RedNano, a deep-learning method designed to detect m6A from Nanopore DRS reads by utilizing both raw signals and basecalling errors. RedNano processes the raw-signal feature and basecalling-error feature through residual networks. We validated the effectiveness of RedNano using synthesized, Arabidopsis, and human DRS data. The results demonstrate that RedNano surpasses existing methods by achieving higher area under the ROC curve (AUC) and area under the precision-recall curve (AUPRs) in all three datasets. Furthermore, RedNano performs better in cross-species validation, demonstrating its robustness. Additionally, when detecting m6A from an independent dataset of Populus trichocarpa, RedNano achieves the highest AUC and AUPR, which are 3.8%-9.9% and 5.5%-13.8% higher than other methods, respectively. AVAILABILITY AND IMPLEMENTATION: The source code of RedNano is freely available at https://github.com/Derryxu/RedNano.

Gate-controlled silicon controlled rectifier with adjustable clamping voltage using a photoelectric mechanism.

In order to flexibly control the voltage-clamping capability of silicon controlled rectifiers (SCRs), this paper proposes a photoelectric gate-controlled SCR (PGCSCR). Equivalent circuits and technology computer aided design (TCAD) simulations are used to analyze how the device works. The device has been validated by a standard 0.18 µm Bipolar CMOS DMOS (BCD) process. The ES620-50 Transmission Line Pulse (TLP) test system was used to verify the impact of the photoelectric effect on the electro-static discharge (ESD) characteristics of the device. The test result shows that the clamping voltage at the holding point of the PGCSCR under the light-free condition is 4.308 V. When the optical power is 5 W/µm2 and the 450 nm wavelength spot is irradiated on the surface of the device, the clamping voltage at the holding point of the PGCSCR is reduced to 3.655 V. And by changing the wavelength of the incident light spot (600 nm), the clamping voltage (3.409 V) of the device changes. Finally, based on the avalanche multiplication effect and the photoelectric effect, the change in the clamping voltage of the device can be further explained. PGCSCR can flexibly adjust the clamping voltage of the device according to the ESD window requirements of the target chip without changing the structure and size, and is expected to be applied in the fields of integrated optical circuits, opto-coupling, and optical communication.

Predicting Antitumor Effect of Deoxypodophyllotoxin in NCI-H460 Tumor-Bearing Mice on the Basis of In Vitro Pharmacodynamics and a Physiologically Based Pharmacokinetic-Pharmacodynamic Model.

Antitumor evaluation in tumor-bearing mouse is time- and energy-consuming. We aimed to investigate whether in vivo antitumor efficacy could be predicted on the basis of in vitro pharmacodynamics using deoxypodophyllotoxin (DPT), an antitumor candidate in development, as a model compound. Proliferation kinetics of monolayer-cultivated NCI-H460 cells under various DPT concentrations were quantitatively investigated and expressed as calibration curves. Koch two-phase natural growth model combined with sigmoid Emax model, i.e., dM/dt = 2λ0λ1M/(λ1 + 2λ0M) - Emax C γ /(EC50γ + C γ )·M, was introduced to describe cell proliferation (M) against time under DPT treatment (C). Estimated in vitro pharmacodynamic parameters were: EC50, 8.97 nM; Emax , 0.820 day-1, and γ, 7.13. A physiologically based pharmacokinetic model including tumor compartment was introduced to predict DPT disposition in plasma, tumor tissue, and main normal tissues of NCI-H460 tumor-bearing mice following a single dose. The in vivo pharmacodynamic model and parameters were assumed the same as the in vitro ones, and linked with simulated tumor pharmacokinetic profiles by a physiologically based pharmacokinetic (PBPK) model to build a PBPK-pharmacodynamic (PBPK-PD) model. After natural growth parameters (λ0 and λ1) were estimated, the objective in this study was to predict with the PBPK-PD model the tumor growth in NCI-H460 tumor-bearing mice during multidose DPT treatment, a use of the model similar to what others have reported. In our work, the model was successfully applied to predict tumor growth in SGC-7901 tumor-bearing mice. The resulting data indicated that in vivo antitumor efficacy might be predicted on the basis of in vitro cytotoxic assays via a PBPK-PD model approach. We demonstrated that the approach is reasonable and applicable and may facilitate and accelerate anticancer candidate screening and dose regimen design in the drug discovery process.

Differential effects of pravastatin on the pharmacokinetics of paroxetine in normal and diabetic rats.

1. Diabetes is often accompanied with depression and hypercholesterolemia. It is possible that paroxetine and pravastatin are co-administered to diabetic patients. The aim of this study was to research the differential effect of pravastatin on plasma exposure of paroxetine in normal and diabetic rats. 2. Pharmacokinetics of paroxetine was investigated following oral administration of paroxetine with and without pravastatin in normal and diabetic rats. Effects of pravastatin on metabolism, intestinal absorption and hepatic uptake of paroxetine were investigated. Activity and expression of hepatic Oatp1 and Oatp2 were also assessed. 3. Pravastatin decreased plasma exposure of paroxetine in normal rats, but increased exposure of paroxetine in diabetic rats. Pravastatin neither affected metabolism nor intestinal absorption of paroxetine. Data from hepatocytes demonstrated that hepatic uptake of paroxetine were involved in Oatp1 and Oatp2. Diabetes suppressed Oatp1 activity and expression, but enhanced Oatp2 activity and expression. Pravastatin stimulated Oatp1 but inhibited Oatp2 activity. 4. We concluded that differential effects of pravastatin on plasma exposure of paroxetine in normal and diabetic rats was partly due to the fact that diabetes suppressed Oatp1 activity and expression but enhanced Oatp2 activity and expression as well as that pravastatin stimulated Oatp1 activity but inhibited Oatp2 activity.

Acute liver failure impairs function and expression of breast cancer-resistant protein (BCRP) at rat blood-brain barrier partly via ammonia-ROS-ERK1/2 activation.

We once reported that P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) were oppositely regulated at the blood-brain barrier (BBB) of thioacetamide-induced acute liver failure (ALF) rats. This study aimed to investigate whether ALF affected function and expression of breast cancer-resistant protein (BCRP) at the BBB of rats and the role of ammonia in the regulation. ALF rats were developed by intraperitoneal (i.p.) injection of thioacetamide (300 mg/kg) for 2 days. Hyperammonemic rats were developed by NH4 Ac (i.p. 4.5 mmol/kg). BCRP function and expression were measured by brain distribution of specific substrates (prazosin and methotrexate) and western blot, respectively. MDCK-BCRP cells and primarily cultured rat brain microvessel endothelial cells (rBMECs) were employed to investigate possible mechanisms through which ammonia regulated BCRP function and expression. The results showed that both ALF and hyperammonemia significantly weakened function and expression of BCRP in the brain of rats. The function and expression of BCRP in MDCK-BCRP cells and rBMECs were strikingly decreased after exposure to NH4 Cl and H2 O2 , accompanied by remarkable increases in the levels of phosphorylated ERK1/2 and reactive oxygen species (ROS). The altered BCRP expression and function by ammonia and H2 O2 were restored by ROS scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. Markedly increased levels of ERK1/2 phosphorylation and ROS were found in the brains of ALF rats and hyperammonemic rats. All above results indicated ALF down-regulated expression and function of BCRP at BBB of rats partly via hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia impaired BCRP expression and function at the BBB. The present study showed that the expression and function of breast cancer resistant protein (BCRP) at blood-brain barrier (BBB) of thioacetamide-induced ALF rats were down-regulated which partly attribute to hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia suppressed BCRP expression and function. Impaired BCRP at BBB might enhanced pharmacological/toxic effects of corresponding substrates on CNS.

Chronic administration of caderofloxacin, a new fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats.

AIM: Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs. METHODS: Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a "cocktail" of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively. RESULTS: Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results. CONCLUSION: Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

AIM: Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. METHODS: Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). RESULTS: BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 µmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. CONCLUSION: UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Ciprofloxacin blocked enterohepatic circulation of diclofenac and alleviated NSAID-induced enteropathy in rats partly by inhibiting intestinal ß-glucuronidase activity.

AIM: Diclofenac is a non-steroidal anti-inflammatory drug (NSAID), which may cause serious intestinal adverse reactions (enteropathy). In this study we investigated whether co-administration of ciprofloxacin affected the pharmacokinetics of diclofenac and diclofenac-induced enteropathy in rats. METHODS: The pharmacokinetics of diclofenac was assessed in rats after receiving diclofenac (10 mg/kg, ig, or 5 mg/kg, iv), with or without ciprofloxacin (20 mg/kg, ig) co-administered. After receiving 6 oral doses or 15 intravenous doses of diclofenac, the rats were sacrificed, and small intestine was removed to examine diclofenac-induced enteropathy. ß-Glucuronidase activity in intestinal content, bovine liver and E coli was evaluated. RESULTS: Following oral or intravenous administration, the pharmacokinetic profile of diclofenac displayed typical enterohepatic circulation, and co-administration of ciprofloxacin abolished the enterohepatic circulation, resulted in significant reduction in the plasma content of diclofenac. In control rats, ß-glucuronidase activity in small intestinal content was region-dependent: proximal intestine

Decreased exposure of atorvastatin in diabetic rats partly due to induction of hepatic Cyp3a and Oatp2.

1. Atorvastatin is frequently prescribed for lowering blood cholesterol and for prevention of events associated with cardiovascular disease. The aim of this study was to investigate the pharmacokinetics of atorvastatin in diabetic rats. 2. Diabetes was induced in rats by combination of high-fat diet and low-dose streptozotocin (35 mg/kg). Plasma concentrations of atorvastatin following oral (10 mg/kg) and intravenous (2 mg/kg) administrations to rats were measured by LC-MS. Metabolism and uptake of atorvastatin in primary hepatocytes of experimental rats were assessed. Protein expressions and activities of hepatic Cyp3a and Oatp2 were further investigated. 3. Clearances of atorvastatin in diabetic rats following oral and intravenous administrations were remarkably increased, leading to marked decreases in area-under-the-plasma concentration-time curve (AUC). The estimated oral and systematic clearances of atorvastatin in diabetic rats were 4.5-fold and 2.0-fold of control rats, respectively. Metabolism and uptake of atorvastatin in primary hepatocytes isolated from diabetic rats were significantly increased, which were consistent with the up-regulated protein expressions and activities of hepatic Cyp3a and Oatp2. 4. All these results demonstrated that the plasma exposure of atorvastatin was significantly decreased in diabetic rats, which was partly due to the up-regulated activities and expressions of both hepatic Cyp3a and Oatp2.

Sensitive determination of glucose in Dulbecco's modified Eagle medium by high-performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone derivatization: application to gluconeogenesis studies.

A new pre-column derivative high-performance liquid chromatography (HPLC) method for determination of d-glucose with 3-O-methyl-d-glucose (3-OMG) as the internal standard was developed and validated in order to study the gluconeogenesis in HepG2 cells. Samples were derivatized with 1-phenyl-3-methy-5-pyrazolone at 70°C for 50 min. Glucose and 3-OMG were extracted by liquid-liquid extraction and separated on a YMC-Triart C18 column, with a gradient mobile phase composed of acetonitrile and 20 mm ammonium acetate solution containing 0.09% tri-ethylamine at a flow rate of 1.0 mL/min. The eluate were detected using a UV detector at 250 nm. The assay was linear over the range 0.39-25 µm (R(2) = 0.9997, n = 5) and the lower limit of quantitation was 0.39 µm (0.070 mg/mL). Intra- and inter-day precision and accuracy were <15% and within ±3%, respectively. After validation, the HPLC method was applied to investigate the gluconeogenesis in Dulbecco's modified Eagle medium (DMEM) cultured HepG2 cells. Glucose concentration was determined to be about 1-2.5 µm in this gluconeogenesis assay. In conclusion, this method has been shown to determine small amounts of glucose in DMEM successfully, with lower limit of quantitation and better sensitivity when compared with common commercial glucose assay kits.

Combined Contribution of Increased Intestinal Permeability and Inhibited Deglycosylation of Ginsenoside Rb1 in the Intestinal Tract to the Enhancement of Ginsenoside Rb1 Exposure in Diabetic Rats after Oral Administration.

Panax ginseng is becoming a promising antidiabetic herbal medication. As the main active constituents of Panax ginseng, ginsenosides are well known, poorly absorbed chemicals. However, the pharmacokinetic behavior of ginsenosides under diabetic conditions is not fully understood. This study aimed to explore the alterations and potential mechanisms of pharmacokinetic behavior of ginsenoside Rb1 in diabetic rats compared with normal rats and rats fed a high-fat diet. Systemic exposure (area under the concentration-time curve extrapolated from zero to infinity) was significantly increased in diabetic rats after oral administration of Rb1. Oral bioavailability of Rb1 was significantly higher in diabetic rats (2.25%) compared with normal rats (0.90%) and rats fed a high-fat diet (0.78%). Further studies revealed that increased Rb1 exposure in diabetic rats may be mainly attributed to increased Rb1 absorption via the intestine and inhibited Rb1 deglycosylation by the intestinal microflora. Neither metabolic enzymes nor drug transporters displayed appreciable effects on Rb1 disposition. The transport of paracellular markers (fluorescein sodium and fluorescein isothiocyanate-dextran of 4 kDa) as well as Rb1 itself across the Caco-2 monolayer cultured with diabetic serum was promoted, demonstrating that increased paracellular permeability of the Caco-2 monolayer may benefit intestinal Rb1 absorption. In addition, Rb1 exposure was decreased in diabetic rats after Rb1 intravenous administration, which may result from increased Rb1 urinary excretion. In conclusion, Rb1 oral exposure was significantly increased under diabetic conditions, which is of positive significance to clinical treatment. The potential mechanism may be associated with the combined contribution of increased gut permeability and inhibited deglycosylation of ginsenoside Rb1 by intestinal microflora.

High-fat diet enhanced retinal dehydrogenase activity, but suppressed retinol dehydrogenase activity in liver of rats.

Evidence has shown that hyperlipidemia is associated with retinoid dyshomeostasis. In liver, retinol is mainly oxidized to retinal by retinol dehydrogenases (RDHs) and alcohol dehydrogenases (ADHs), further converted to retinoic acid by retinal dehydrogenases (RALDHs). The aim of this study was to investigate whether high-fat diet (HFD) induced hyperlipidemia affected activity and expression of hepatic ADHs/RDHs and RALDHs in rats. Results showed that retinol levels in liver, kidney and adipose tissue of HFD rats were significantly increased, while plasma retinol and hepatic retinal levels were markedly decreased. HFD rats exhibited significantly downregulated hepatic ADHs/RDHs activity and Adh1, Rdh10 and Dhrs9 expression. Oppositely, hepatic RALDHs activity and Raldh1 expression were upregulated in HFD rats. In HepG2 cells, treatment of HFD rat serum inhibited ADHs/RDHs activity and induced RALDHs activity. Among the tested abnormally altered components in HFD rat serum, cholesterol reduced ADHs/RDHs activity and RDH10 expression, while induced RALDHs activity and RALDH1 expression in HepG2 cells. Contrary to the effect of cholesterol, cholesterol-lowering agent pravastatin upregulated ADHs/RDHs activity and RDH10 expression, while suppressed RALDHs activity and RALDH1 expression. In conclusion, hyperlipidemia oppositely altered activity and expression of hepatic ADHs/RDHs and RALDHs, which is partially due to the elevated cholesterol levels.

Co-administration of paroxetine increased the systemic exposure of pravastatin in diabetic rats due to the decrease in liver distribution.

1. Liver distribution and systemic exposure of pravastatin were the determinant factors of efficacy and toxicity of pravastatin. Aim of the present study was to investigate the effect of paroxetine on the liver distribution and systemic exposure of pravastatin in diabetic rats induced by combining high fat diet (HFD) and low-dose streptozotocin (STZ). 2. Plasma concentrations and liver distribution of pravastatin were measured in the presence of paroxetine. Effect of paroxetine on pravastatin excretion via bile, intestine, feces and urine, as well as pravastatin absorption via intestine was documented. Freshly isolated hepatocytes and Caco-2 cells were used to investigate the effect of paroxetine on pravastatin transport. 3. Paroxetine increased the systemic exposure of pravastatin and decreased hepatic distribution of pravastatin in diabetic rats. In vitro, paroxetine inhibited the hepatic uptake of pravastatin and promoted the efflux of pravastatin in freshly isolated hepatocytes, which may partly explain the decreased hepatic distribution of pravastatin by paroxetine. It was also observed that paroxetine promoted the absorption of pravastatin via jejunum and the uptake of pravastatin in Caco-2 cells. 4. We concluded that paroxetine increased the systemic exposure of pravastatin partly via promoting absorption via jejunum and inhibiting hepatic uptake of pravastatin.

Aggravation of clozapine-induced hepatotoxicity by glycyrrhetinic acid in rats.

Clozapine (CLZ) was reported to be associated with hepatotoxicity. Glycyrrhetinic acid (GA) has a liver protective effect. Our preliminary experiments showed that GA aggravated rather than attenuated CLZ-induced hepatotoxicity in primary cultured rat hepatocytes. The study aimed to describe the enhancing effect of GA on CLZ-induced hepatotoxicity in vivo and in vitro. Data from primary cultured rat hepatocytes showed the decreased formation of metabolites demethylclozapine (nor-CLZ) and clozapine N-oxide (CLZ N-oxide). The results in vivo showed that 7-day CLZ treatment led to marked accumulation of triglyceride (TG) and increase in γ-glutamyl transpeptidase (γ-GT) activity, liver weight, and serum AST in rats. Co-administration of GA enhanced the increases in hepatic TG, γ-GT, liver weight, and serum total cholesterol induced by CLZ. GA decreased plasma concentrations of nor-CLZ and CLZ N-oxide. Compared with control rats, hepatic microsomes of GA rats exhibited the decreased formations of nor-CLZ and CLZ N-oxide, accompanied by decreases in activities of CYP2C11 and CYP2C19 and increased activity of CYP1A2. QT-PCR analysis demonstrated that GA enhanced expression of CYP1A2, but suppressed expression of CYP2C11 and CYP2C13. All these results support the conclusion that GA aggravated CLZ-induced hepatotoxicity, which was partly via inhibiting CYP2C11 and CYP2C13 or inducing CYP1A2.

Co-administration of paroxetine and pravastatin causes deregulation of glucose homeostasis in diabetic rats via enhanced paroxetine exposure.

AIM: Clinical evidence shows that co-administration of pravastatin and paroxetine deregulates glucose homeostasis in diabetic patients. The aim of this study was to verify this phenomenon in diabetic rats and to elucidate the underlying mechanisms. METHODS: Diabetes mellitus was induced in male SD rats by a high-fat diet combined with a low-dose streptozotocin injection. The rats were orally administered paroxetine (10 mg/kg) and pravastatin (10 mg/d) or both the drugs daily for 28 d. The pharmacokinetics of paroxetine and pravastatin were examined on d 1 and d 28. Biochemical parameters including serum insulin, glucose and lipids were monitored during the treatments. An insulin-secreting cell line (INS-1) was used for measuring insulin secretion. RESULTS: In diabetic rats, co-administration of paroxetine and pravastatin markedly increased the concentrations of both the drugs compared with administration of each drug alone. Furthermore, co-administration severely impaired glucose homeostasis in diabetic rats, as demonstrated by significantly increased serum glucose level, decreased serum and pancreatic insulin levels, and decreased pancreatic Insulin-2 mRNA and tryptophan hydroxylase-1 (Tph-1) mRNA levels. Treatment of INS-1 cells with paroxetine (5 and 10 µmol/L) significantly inhibited insulin secretion, decreased the intracellular insulin, 5-HT, Insulin-2 mRNA and Tph-1 mRNA levels. Treatment of the cells with pravastatin (10 µmol/L) significantly stimulated insulin secretion, which was weakened by co-treatment with paroxetine. CONCLUSION: Paroxetine inhibits insulin secretion at least via decreasing intracellular 5-HT and insulin biosynthesis. The deregulation of glucose homeostasis by co-administration of paroxetine and pravastatin in diabetic rats can be attributed to enhanced paroxetine exposure.

Decreased exposure of simvastatin and simvastatin acid in a rat model of type 2 diabetes.

AIM: Simvastatin is frequently administered to diabetic patients with hypercholesterolemia. The aim of the study was to investigate the pharmacokinetics of simvastatin and its hydrolysate simvastatin acid in a rat model of type 2 diabetes. METHODS: Diabetes was induced in 4-week-old rats by a treatment of high-fat diet combined with streptozotocin. After the rats received a single dose of simvastatin (20 mg/kg, po, or 2 mg/kg, iv), the plasma concentrations of simvastatin and simvastatin acid were determined. Simvastatin metabolism and cytochrome P4503A (Cyp3a) activity were assessed in hepatic microsomes, and its uptake was studied in freshly isolated hepatocytes. The expression of Cyp3a1, organic anion transporting polypeptide 2 (Oatp2), multidrug resistance-associated protein 2 (Mrp2) and breast cancer resistance protein (Bcrp) in livers was measured using qRT-PCR. RESULTS: After oral or intravenous administration, the plasma concentrations and areas under concentrations of simvastatin and simvastatin acid were markedly decreased in diabetic rats. Both simvastatin metabolism and Cyp3a activity were markedly increased in hepatocytes of diabetic rats, accompanied by increased expression of hepatic Cyp3a1 mRNA. Furthermore, the uptake of simvastatin by hepatocytes of diabetic rats was markedly increased, which was associated with increased expression of the influx transporter Oatp2, and decreased expression of the efflux transporters Mrp2 and Bcrp. CONCLUSION: Diabetes enhances the metabolism of simvastatin and simvastatin acid in rats via up-regulating hepatic Cyp3a activity and expression and increasing hepatic uptake.

DNA 5-methylcytosine detection and methylation phasing using PacBio circular consensus sequencing.

Long single-molecular sequencing technologies, such as PacBio circular consensus sequencing (CCS) and nanopore sequencing, are advantageous in detecting DNA 5-methylcytosine in CpGs (5mCpGs), especially in repetitive genomic regions. However, existing methods for detecting 5mCpGs using PacBio CCS are less accurate and robust. Here, we present ccsmeth, a deep-learning method to detect DNA 5mCpGs using CCS reads. We sequence polymerase-chain-reaction treated and M.SssI-methyltransferase treated DNA of one human sample using PacBio CCS for training ccsmeth. Using long (≥10 Kb) CCS reads, ccsmeth achieves 0.90 accuracy and 0.97 Area Under the Curve on 5mCpG detection at single-molecule resolution. At the genome-wide site level, ccsmeth achieves >0.90 correlations with bisulfite sequencing and nanopore sequencing using only 10× reads. Furthermore, we develop a Nextflow pipeline, ccsmethphase, to detect haplotype-aware methylation using CCS reads, and then sequence a Chinese family trio to validate it. ccsmeth and ccsmethphase can be robust and accurate tools for detecting DNA 5-methylcytosines.

Combined contributions of impaired hepatic CYP2C11 and intestinal breast cancer resistance protein activities and expression to increased oral glibenclamide exposure in rats with streptozotocin-induced diabetes mellitus.

The purpose of this study was to evaluate the contributions of impaired cytochrome P450 and breast cancer resistance protein (BCRP) activity and expression to drug pharmacokinetics under diabetic conditions. Diabetes was induced in rats with the intraperitoneal administration of streptozocin. Glibenclamide (GLB), a substrate of BCRP, served as a model drug. The pharmacokinetics of orally administered GLB (10 mg/kg) were studied. The results showed that diabetes mellitus significantly increased exposure (area under the curve and peak concentration) to GLB after oral administration. Data from hepatic microsomes suggested impairment of GLB metabolism in diabetic rats. GLB metabolism in hepatic microsomes was significantly inhibited by a selective inhibitor (sulfaphenazole) of CYP2C11 and an anti-CYP2C11 antibody. Western blotting further indicated the contribution of impaired CYP2C11 expression to the impairment of GLB metabolism. Excretion data showed that ∼72% of the orally administered dose was excreted in the feces of normal rats, which indicates an important role for intestinal BCRP. Diabetes significantly decreased the recovery from feces, which was only 40% of the orally administered dose. Results from in situ, single-pass, intestinal perfusion experiments revealed that diabetes significantly increased the apparent effective permeability and decreased the efflux of GLB through the intestine; this suggests impairment of intestinal BCRP function, which may play a role in the increased exposure to orally administered GLB in diabetic rats. Insulin treatment partly or completely reversed the changes in diabetic rats. All results yielded the conclusion that impaired hepatic CYP2C11 and intestinal BCRP expression and activity induced by diabetes contributed to the increased exposure of orally administered GLB.

Characteristic analysis of volatile avalanche diode threshold switching for bionic nerve synapse applications.

The combination of biological neurology and memristive theory has greatly promoted the development of neuromorphic computing. To build a large-scale artificial intelligence alert system, the exploration of bionic synapses compatible with standard processes has become an urgent problem to be solved in the next step. In response to the above application requirements, this paper proposes a volatile avalanche diode threshold switching (VADTS) that is fully compatible with standard semiconductor technology to simulate the various functions of the synapse. Technology computer-aided design device-level simulation can verify the bionic principle of VADTS. The function of VADTS's bionic synapse was verified by the experimental test platform. The results show that under the action of the excitation signal (11.25 V), the device can continuously change from a high-resistance state to a low-resistance state. When the excitation signal is lower than the threshold, VADTS presents a "no adaptation" state of nerve synapses. When the excitation signal is higher than the threshold and changes continuously, the current changes along with the amplitude of the excitation signal, similar to the "sensitization" state of the nerve synapse.

Atorvastatin impaired glucose metabolism in C2C12 cells partly via inhibiting cholesterol-dependent glucose transporter 4 translocation.

Skeletal muscle accounts for approximately 75% of glucose disposal in body and statins impair glucose metabolism. We aimed to investigate the effect of atorvastatin on glucose metabolism in C2C12 cells. Glucose metabolism and expression of glucose transporter 4 (GLUT4) and hexokinase II (HXKII) were measured following incubation with atorvastatin or pravastatin. Roles of cholesterol in atorvastatin-induced glucose metabolism impairment were investigated via adding cholesterol or mevalonic acid and confirmed by cholesterol depletion with methyl-ß-cyclodextrin. Hypercholesterolemia mice induced by high fat diet (HFD) feeding, orally received atorvastatin (6 and 12 mg/kg) or pravastatin (12 mg/kg) for 22 days. Results showed that atorvastatin not pravastatin concentration-dependently impaired glucose consumption, glucose uptake and GLUT4 membrane translocation in C2C12 cells without affecting expression of HXKII or total GLUT4 protein. The atorvastatin-induced alterations were reversed by cholesterol or mevalonic acid. Cholesterol depletion exerted similar impact to atorvastatin, which could be alleviated by cholesterol supplement. Glucose consumption or GLUT4 translocation was positively associated with cellular cholesterol levels. In HFD mice, atorvastatin not pravastatin significantly increased blood glucose levels following glucose or insulin dose and decreased expression of membrane not total GLUT4 protein in muscle. Glucose exposure following glucose or insulin dose was negatively correlated to muscular free cholesterol concentration. Expression of membrane GLUT4 protein was positively related to free cholesterol in muscle. In conclusion, atorvastatin impaired glucose utilization in muscle cells partly via inhibiting GLUT4 membrane translocation due to inhibition of cholesterol synthesis by atorvastatin, at least, partly contributing to glucose intolerance in HFD mice.