In Vivo Metabolite Profiles of an N-Acetylgalactosamine-Conjugated Antisense Oligonucleotide AZD8233 Using Liquid Chromatography High-Resolution Mass Spectrometry: A Cross-Species Comparison in Animals and Humans.

AZD8233, a liver-targeting antisense oligonucleotide (ASO), inhibits subtilisin/kexin type 9 protein synthesis. It is a phosphorothioated 3-10-3 gapmer with a central DNA sequence flanked by constrained 2'-O-ethyl 2',4'-bridged nucleic acid (cEt-BNA) wings and conjugated to a triantennary N-acetylgalactosamine (GalNAc) ligand at the 5'-end. Herein we report the biotransformation of AZD8233, as given by liver, kidney, plasma and urine samples, after repeated subcutaneous administration to humans, mice, rats, rabbits, and monkeys. Metabolite profiles were characterized using liquid chromatography high-resolution mass spectrometry. Metabolite formation was consistent across species, mainly comprising hydrolysis of GalNAc sugars, phosphodiester-linker hydrolysis releasing the full-length ASO, and endonuclease-mediated hydrolysis within the central DNA gap followed by exonuclease-mediated 5'- or 3'-degradation. All metabolites contained the 5'- or 3'-cEt-BNA terminus. Most shortmer metabolites had the free terminal alcohol at 5'- and 3'-positions of ribose, although six were found retaining the terminal 5'-phosphorothioate group. GalNAc conjugated shortmer metabolites were also observed in urine. Synthesized metabolite standards were applied for (semi)quantitative metabolite assessment. Intact AZD8233 was the major component in plasma, whereas the unconjugated full-length ASO was predominant in tissues. In plasma, most metabolites were shortmers retaining the 3'-cEt-BNA terminus, whereas metabolites containing the 5'- or 3'-cEt-BNA terminus were detected in both tissues and urine. All metabolites in human plasma were also detected in all nonclinical species, and all human urine metabolites were detected in monkey urine. In general, metabolite profiles in animal species were qualitatively similar and quantitatively exceeded the exposures of the circulating metabolites in humans at the doses studied. SIGNIFICANCE STATEMENT: This study presents metabolite identification and profiling of AZD8233, an N-acetylgalactosamine-conjugated antisense oligonucleotide (ASO), across species. A biotransformation strategy for ASOs was established by utilizing biologic samples collected from toxicology and/or clinical studies and liquid chromatography high-resolution mass spectrometry analysis without conducting bespoke radiolabeled absorption, distribution, metabolism, and excretion studies. The generated biotransformation package was considered adequate by health authorities to progress AZD8233 into a phase 3 program, proving its applicability to future metabolism studies of ASOs in drug development.

Biotransformation of the Novel Myeloperoxidase Inhibitor AZD4831 in Preclinical Species and Humans.

We report herein an in-depth analysis of the metabolism of the novel myeloperoxidase inhibitor AZD4831 ((R)-1-(2-(1-aminoethyl)-4-chlorobenzyl)-2-thioxo-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one) in animals and human. Quantitative and qualitative metabolite profiling were performed on samples collected from mass balance studies in rats and humans. Exposure of circulating human metabolites with comparable levels in animal species used in safety assessment were also included. Structural characterization of 20 metabolites was performed by liquid chromatography high-resolution mass spectrometry, and quantification was performed by either 14C analysis using solid phase scintillation counting or accelerator mass spectrometry and, where available, authentication with synthesized metabolite standards. A complete mass balance study in rats is presented, while data from dogs and human are limited to metabolite profiling and characterization. The metabolism of AZD4831 is mainly comprised of reactions at the primary amine nitrogen and the thiourea sulfur, resulting in several conjugated metabolites with or without desulfurization. A carbamoyl glucuronide metabolite of AZD4831 (M7) was the most abundant plasma metabolite in both human healthy volunteers and heart failure patients after single and repeated dose administration of AZD4831, accounting for 75%-80% of the total drug-related exposure. Exposures to M7 and other human circulating metabolites were covered in rats and/or dogs, the two models most frequently used in the toxicology studies, and were also highly abundant in the mouse, the second model other than rat used in carcinogenicity studies. The carbamoyl glucuronide M7 was the main metabolite in rat bile, while a desulfurized and cyclized metabolite (M5) was abundant in rat plasma and excreta. SIGNIFICANCE STATEMENT: The biotransformation of AZD4831, a novel myeloperoxidase inhibitor inhibiting xanthine derivative bearing thiourea and primary aliphatic amine functions, is described. Twenty characterized metabolites demonstrate the involvement of carbamoylation with glucuronidation, desulfurization, and cyclization as main biotransformation reactions. The carbamoyl glucuronide was the main metabolite in human plasma, likely governed by a significant species difference in plasma protein binding for this metabolite, but this and other human plasma metabolites were covered in animals used in the toxicity studies.

Mass Balance and Absorption, Distribution, Metabolism, and Excretion Properties of Balcinrenone following Oral Administration in Combination with Intravenous Microtracer in Healthy Subjects.

An absorption, distribution, metabolism, and excretion study was performed to determine the basic pharmacokinetic parameters, mass balance, and metabolite profiles of balcinrenone, a mineralocorticoid receptor modulator, in humans. This open-label, single-center, nonrandomized study had a two-period design. In period 1, eight healthy male subjects were dosed with a microtracer intravenous infusion of [14C]balcinrenone shortly after receiving an oral dose of unlabeled balcinrenone in a capsule. Following a 7-day washout, the same group of subjects subsequently received an oral dose of [14C]balcinrenone as a suspension in period 2. Clearance and absolute bioavailability of balcinrenone were determined to be 14.2 l/h and 52%, respectively. Renal clearance was determined to be 5.4 l/h (>fu • glomerular filtration rate), indicating elimination via active tubular secretion, which was potentially mediated by P-glycoprotein 1 and/or organic anion transporter 3, according to in vitro transporter data. In total, 94.1% of the oral dose was recovered: 45.2% in the urine and 48.9% in the feces. Balcinrenone was primarily metabolized via oxidation, and in vitro data suggest that cytochrome P450 3A4 was the main enzyme responsible. Intact [14C]balcinrenone accounted for 55% of drug-related material in the plasma; four metabolites were identified, each representing <6% of the total plasma radioactivity. In conclusion, this two-period study has determined the basic pharmacokinetic parameters of balcinrenone in humans, including absolute bioavailability and disposition. No metabolites warranted further evaluation on account of their low representation, and any contribution to the pharmacodynamic response or potential drug-drug interactions was deemed negligible. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the pharmacokinetics, disposition, and metabolism of balcinrenone following oral and microtracer intravenous administration in humans. In vitro phenotyping and transporter data granted mechanistic insights into the absorption, distribution, metabolism, and excretion properties of balcinrenone. This knowledge will guide future nonclinical and clinical studies evaluating drug-drug interactions, organ dysfunction, and safety of metabolites.

Ameliorative effects of silybin against avermectin-triggered carp spleen mitochondrial dysfunction and apoptosis through inhibition of PERK-ATF4-CHOP signaling pathway.

The aim of this study was to investigate the splenic tissue damage of environmental biological drug avermectin to freshwater cultured carp and to evaluate the effect of silybin on the splenic tissue damage of carp induced by avermectin. A total of 60 carp were divided into 4 groups with 15 carp in each group, including the control group fed with basic diet, experimental group fed with basal diet and exposed to avermectin (avermectin group), experimental group fed with basal diet supplement silybin (silybin group), and experimental group fed with basal diet supplement silybin and exposed to avermectin (silybin + avermectin group). The whole test period lasted for 30 days, and spleen tissue was collected for analysis. In this study, H&E staining, mitochondrial purification and membrane potential detection, ATP detection, DHE staining, biochemical tests, qPCR, immunohistochemistry, and apoptosis staining were used to evaluate the biological processes of spleen tissue injury, mitochondrial function, oxidative stress, apoptosis, and endoplasmic reticulum stress. The results show that silybin protected carp splenic tissue damage caused by chronic avermectin exposure, decreased mitochondrial membrane potential, decreased ATP content, ROS accumulation, oxidative stress, apoptosis, and endoplasmic reticulum stress. Silybin may ameliorate the splenic tissue damage of cultured freshwater carp caused by environmental biopesticide avermectin by alleviating mitochondrial dysfunction and inhibiting PERK-ATF4-CHOP-driven mitochondrial apoptosis. Adding silybin into the diet becomes a feasible strategy to resist the pollution of avermectin and provides a theoretical basis for creating a good living environment for freshwater carp.

RAE1 is a prognostic biomarker and is correlated with clinicopathological characteristics of patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a primary malignant tumor that accounts for approximately 90% of all cases of primary liver cancer worldwide. Microtubule alterations may contribute to the broad spectrum of resistance to chemotherapy, tumor development, and cell survival. This study aimed to assess the value of ribonucleic acid export 1 (RAE1), as a regulator of microtubules, in the diagnosis and prognosis of HCC, and to analyze its correlation with genetic mutations and pathways in HCC. RESULTS: The mRNA and protein levels of RAE1 were significantly elevated in HCC tissues compared with those in normal tissues. The high expression level of RAE1 was correlated with T stage, pathologic stage, tumor status, histologic grade, and alpha-fetoprotein level. HCC patients with a higher expression level of RAE1 had a poorer prognosis, and the expression level of RAE1 showed the ability to accurately distinguish tumor tissues from normal tissues (area under the curve (AUC) = 0.951). The AUC values of 1-, 3-, and 5-year survival rates were all above 0.6. The multivariate Cox regression analysis showed that RAE1 expression level was an independent prognostic factor for a shorter overall survival of HCC patients. The rate of RAE1 genetic alterations was 1.1% in HCC samples. Gene ontology and kyoto encyclopedia of genes and genomes pathway enrichment analyses indicated the co-expressed genes of RAE1 were mainly related to chromosome segregation, DNA replication, and cell cycle checkpoint. Protein-protein interaction analysis showed that RAE1 was closely correlated with NUP205, NUP155, NUP214, NUP54, and NXF1, all playing important roles in cell division and mitotic checkpoint. CONCLUSION: RAE1 can be a potential diagnostic and prognostic biomarker associated with microtubules and a therapeutic target for HCC.

IL-35 promotes EMT through STAT3 activation and induces MET by promoting M2 macrophage polarization in HCC.

The tumor microenvironment and interplay with cancer cells could promote tumor growth and metastasis. Here we report that polarization state of macrophages could affect epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). IL-35 level secreted by M1 macrophage was significantly higher than M2 macrophage and it facilitated EMT process through activation of STAT3 in hepatocellular carcinoma cells. Interestingly, IL-35 could not directly promote MET, but it could indirectly induce MET of HCC cells through M2 macrophage polarization. These results indicated the level of IL-35 in tumor microenvironment may fluctuate at different stages of oncogenesis to regulate epithelial plasticity of HCC and provide potential therapeutic targets for tumor metastasis.

Metabolism of Strained Rings: Glutathione S-transferase-Catalyzed Formation of a Glutathione-Conjugated Spiro-azetidine without Prior Bioactivation.

AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] is a melanin-concentrating hormone receptor 1 antagonist designed for the treatment of obesity. In this study, metabolite profiles of AZD1979 in human hepatocytes revealed a series of glutathione-related metabolites, including the glutathionyl, cysteinyl, cysteinylglycinyl, and mercapturic acid conjugates. The formation of these metabolites was not inhibited by coincubation with the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole. In efforts to identify the mechanistic features of this pathway, investigations were performed to characterize the structure of the glutathionyl conjugate M12 of AZD1979 and to identify the enzyme system catalyzing its formation. Studies with various human liver subcellular fractions established that the formation of M12 was NAD(P)H-independent and proceeded in cytosol and S9 fractions but not in microsomal or mitochondrial fractions. The formation of M12 was inhibited by ethacrynic acid, an inhibitor of glutathione S-transferases (GSTs). Several human recombinant GSTs, including GSTA1, A2-2, M1a, M2-2, T1-1, and GST from human placenta, were incubated with AZD1979. All GSTs tested catalyzed the formation of M12, with GSTA2-2 being the most efficient. Metabolite M12 was purified from rat liver S9 incubations and its structure elucidated by NMR. These results establish that M12 is the product of the GST-catalyzed glutathione attack on the carbon atom α to the nitrogen atom of the strained spiro-azetidinyl moiety to give, after ring opening, the corresponding amino-thioether conjugate product, a direct conjugation pathway that occurs without the prior substrate bioactivation by P450. SIGNIFICANCE STATEMENT: The investigated compound, AZD1979, contains a 6-substituted-2-oxa-6-azaspiro[3.3]heptanyl derivative that is an example of strained heterocycles, including spiro-fused ring systems, that are widely used in synthetic organic chemistry. An unusual azetidinyl ring-opening reaction involving a nucleophilic attack by glutathione, which does not involve prior cytochrome P450-catalyzed bioactivation of the substrate and which is catalyzed by glutathione transferases, is reported. We propose a mechanism involving the protonated cyclic aminyl intermediate that undergoes nucleophilic attack by glutathione thiolate anion in this reaction, catalyzed by glutathione transferases.

The effect of ketoconazole on praziquantel pharmacokinetics and the role of CYP3A4 in the formation of X-OH-praziquantel and not 4-OH-praziquantel.

AIM: The study sought to determine the effect of ketoconazole (KTZ) on the pharmacokinetics of praziquantel (PZQ) and on the formation of its major hydroxylated metabolites, cis- and trans-4-OH-PZQ, and X-OH-PZQ in healthy subjects. METHODS: Two treatments were evaluated by single-dose PK studies; the reference treatment was a 20 mg/kg dose of praziquantel given alone. The test treatment was a 20 mg/kg dose of praziquantel given in combination with 200 mg of ketoconazole. The study had a balanced and randomised cross-over design. Serial blood samples were collected between 0 and 12 h after each drug administration. PZQ, and cis- and trans-4-OH-PZQ and X-OH-PZQ concentrations in plasma were determined by LC-MS. A non-compartmental approach was used for pharmacokinetic analysis. Data were analysed using ANOVA and assessment of the 90% confidence interval of the geometric means of the log-transformed PK parameters obtained for each treatment. RESULTS: The pharmacokinetics of PZQ following the two treatments, PZQ alone and PZQ + KTZ, were not equivalent based on the assessment of the 90% CI of the geometric mean ratios of the AUC and Cmax (α = 0.05). The geometric mean ratios of the AUC and Cmax were found to be 176.8% and 227% respectively. The 90% CI of the AUC and Cmax were found to be 129.8%-239.8% and 151.4%-341.4% respectively. The AUC of PZQ was increased by 75% with KTZ co-administration (3516 vs 6172 ng h/ml) (p < 0.01). Meanwhile, the mean AUC of trans-4-OH-PZQ increased by 67% (61,749 ng h/ml vs 103,105 ng h/ml) (p < 0.01). X-OH-PZQ levels were reduced by about 57% (semi-quantified as 7311 ng h/ml vs 3109 ng h/ml by using trans-4-OH as standards) (p < 0.01) with KTZ co-administration. CONCLUSIONS: The relative bioavailability of praziquantel was increased by concomitant KTZ administration. KTZ preferentially inhibited the formation of X-OH-PZQ rather than 4-OH-PZQ, confirming in vitro data which implicates CYP3A4 in the formation of X-OH-PZQ rather than 4-OH-PZQ. The 4-hydroxylation of PZQ was shown to be the major metabolic pathway of PZQ, as evidenced by larger quantities of 4-OH-PZQ produced, thus explaining the modest albeit significant effect of ketoconazole on PZQ pharmacokinetics.

Two new secolignans from the roots of Urtica fissa.

Two new secolignans, 3,4-trans-3-hydroxymethyl-4-[bis(4-hydroxy-3- methoxyphenyl)methyl]butyrolactone (1) and 3,4-trans-3-hydroxymethyl-4- [bis(3,4-dimethoxyphenyl)methyl]butyrolactone (2) have been isolated from the roots of Urtica fissa E.Pritz. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS. The inhibitory effects on N1 and N2, two subtypes of neuraminidases (NAs), of these two compounds were assayed.

[Research progress of platelet activating factor(PAF) receptor antagonist].

Platelet activating factor(PAF), an endogenous synthesized phospholipid transmitter, has widely biological activities. It has "signal transmission" effect in various life processes, but abnormality of concentration in vivo will promote or aggravate the diseases, such as, cerebral ischemia, myocardial injury, multi-organ failure, asthma, injury of liver and kidney, severely affecting the normal life activities of body. In recent years, with the development of medical science and technology, more and more attention has been paid to the research of platelet activating factor receptor antagonist. Components of animals, plants, microbial fermentation, and synthetic composition all can reflect such activity. Ginkgolide B and cytopone are the most representative herbal compositions at present. This paper referred to the research status of platelet activating factor receptor antagonist in recent years, made a summary of the researches on biological effect of platelet activating factor and platelet receptor antagonist of different sources, so as to provide a reference for the exploration of effective and safe platelet activating factor receptor antagonists.

Oxetane Substrates of Human Microsomal Epoxide Hydrolase.

Oxetanyl building blocks are increasingly used in drug discovery because of the improved drug-like properties they confer on drug candidates, yet little is currently known about their biotransformation. A series of oxetane-containing analogs was studied and we provide the first direct evidence of oxetane hydrolysis by human recombinant microsomal epoxide hydrolase (mEH). Incubations with human liver fractions and hepatocytes were performed with and without inhibitors of cytochrome P450 (P450), mEH and soluble epoxide hydrolase (sEH). Reaction dependence on NADPH was investigated in subcellular fractions. A full kinetic characterization of oxetane hydrolysis is presented, in both human liver microsomes and human recombinant mEH. In human liver fractions and hepatocytes, hydrolysis by mEH was the only oxetane ring-opening metabolic route, with no contribution from sEH or from cytochrome P450-catalyzed oxidation. Minimally altering the structural elements in the immediate vicinity of the oxetane can greatly modulate the efficiency of hydrolytic ring cleavage. In particular, higher pKa in the vicinity of the oxetane and an increased distance between the oxetane ring and the benzylic nitrogen improve reaction rate, which is further enhanced by the presence of methyl groups near or on the oxetane. This work defines oxetanes as the first nonepoxide class of substrates for human mEH, which was previously known to catalyze the hydrolytic ring opening of electrophilic and potentially toxic epoxide-containing drugs, drug metabolites, and exogenous organochemicals. These findings will be of value for the development of biologically active oxetanes and may be exploited for the biocatalytic generation of enantiomerically pure oxetanes and diols.

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane.

Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.

CYP3A Specifically Catalyzes 1ß-Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity.

The endogenous bile acid metabolite 1ß-hydroxy-deoxycholic acid (1ß-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1ß-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [(2)H4]-labeled analog of 1ß-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1ß-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1ß-OH-DCA formation. This indicated that 1ß-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1ß-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1ß-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1ß-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1ß-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1ß-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies.

Application of liquid chromatographic/tandem mass spectrometric method to a urinary excretion study of subutinib and active metabolite in human urine.

A novel and selective liquid chromatographic-mass spectrometric method (LC-MS/MS) has been established and validated for simultaneous determination of subutinib and active metabolite in human urine. Urine samples were extracted by liquid-liquid extraction with ethyl acetate and separated on a Wondasil C18 (150 × 2.1 mm, 3.5 µm), with methanol-0.2% formic acid solution (73:27, v/v) as mobile phase at flow rate of 0.2 mL/min. The linear range was 0.5000-200.0 ng/mL for subutinib and active metabolite, with a lower limit of quantitation of 0.5000 ng/mL. Intra- and inter-run precisions were all <11.8 and 14.3%, and the accuracies were all <4.5 and 5.4%, with the extraction recoveries 88.8-97.5 and 93.8-99.4% for the two analytes, respectively. The carryover values were all <15% for the two anayltes. The method was successfully applied to study urinary excretion of subutinib and active metabolite in human after oral administration of subutinib maleate capsules in fed and fasting states.

Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes.

Human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep) hold great potential as an unlimited cell source for toxicity testing in drug discovery research. However, little is known about mechanisms of compound toxicity in hiPSC-Hep. In this study, modified mRNA was used to reprogram foreskin fibroblasts into hiPSC that were differentiated into hiPSC-Hep. The hiPSC-Hep expressed characteristic hepatic proteins and exhibited cytochrome P450 (CYP) enzyme activities. Next, the hiPSC-Hep, primary cryopreserved human hepatocytes (cryo-hHep) and the hepatic cell lines HepaRG and Huh7 were treated with staurosporine and acetaminophen, and the toxic responses were compared. In addition, the expression of genes regulating and executing apoptosis was analyzed in the different cell types. Staurosporine, an inducer of apoptosis, decreased ATP levels and activated caspases 3 and 7 in all cell types, but to less extent in Huh7. Furthermore, a hierarchical clustering and a principal component analysis (PCA) of the expression of apoptosis-associated genes separated cryo-hHep from the other cell types, while an enrichment analysis of apoptotic pathways identified hiPSC-Hep as more similar to cryo-hHep than the hepatic cell lines. Finally, acetaminophen induced apoptosis in hiPSC-Hep, HepaRG and Huh7, while the compound initiated a direct necrotic response in cryo-hHep. Our results indicate that for studying compounds initiating apoptosis directly hiPSC-Hep may be a good alternative to cryo-hHep. Furthermore, for compounds with more complex mechanisms of toxicity involving metabolic activation, such as acetaminophen, our data suggest that the cause of cell death depends on a balance between factors controlling death signals and the drug-metabolizing capacity.

Evidence for kinetic nucleation in helical nanofiber formation directed by chiral solvent for a perylene bisimide organogelator.

The self-assembly behavior of an achiral perylene bisimide (PBI) organogelator that bears two 3,4,5-tridodecyloxybenzoylaminoethyl substituents at the imide positions has been investigated in chiral solvents (R)- and (S)-limonene in great detail by circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). CD spectroscopic studies on dilute solutions revealed a preferential population of one-handed helical assemblies in chiral solvent with an enantiomeric excess close to 100 %, whereas AFM images of more than 100 nanofibers of the organogel obtained from more concentrated solutions were found to consist of both handed helices with an enantiomeric excess of only 20 %. This discrepancy is attributed to the fast gelation process at high dye concentration that evidently proceeds through non-equilibrated nuclei in a kinetic rather than thermodynamic self-assembly process. Under these conditions the chiral induction from the homochiral solvent may not be adequate in effectively populating only one-handed helices.

EZH2-mediated H3K27me3 promotes autoimmune hepatitis progression by regulating macrophage polarization.

Autoimmune hepatitis (AIH) is a chronic progressive liver disease related to abnormal immune stimulation, leading to liver cirrhosis, liver cancer and liver failure. There is an urgent need to find novel biomarkers and potential drug targets for effective treatment of the disease. Although previous studies have shown that EZH2, as a histone methyltransferase, plays critical roles in tumor and autoimmune diseases, its role in autoimmune hepatitis remains largely unknown. In this study, we reported that the EZH2 and H3K27me3 expression level was significantly upregulated in liver tissues during the progression of AIH. High expression of EZH2 enhanced autoimmune hepatitis, immune response and liver fibrosis through H3K27me3. EZH2 inhibition induced the phenotype of hepatic macrophages to switch from M1 to M2 in the development of AIH. These findings indicated that EZH2-mediated H3K27me3 promoted autoimmune hepatitis by regulating the polarization of hepatic macrophages. EZH2 may be a promising therapeutic target for the prevention or treatment of autoimmune hepatitis.

Photophysical and quantum chemical study on a J-aggregate forming perylene bisimide monomer.

Perylene bisimides (PBIs) are excellent dyes and versatile building blocks for supramolecular structures. Only recently have PBIs been shown to depict absorption characteristics of J-aggregates. We apply electronic structure calculations and femtosecond pump-probe spectroscopy to the monomeric, bay-substituted building-block of a PBI aggregate in dichloromethane to investigate its electronically excited states in order to provide the ingredients for the description of excitons in the aggregates and their annihilation processes. The PBI S(1)←S(0) absorption spectrum and the S(1)→S(0) emission spectrum have been assigned based on time-dependent Density Functional Theory calculations for the geometry-optimized electronic ground state and excited state structures in the gas phase. The monomeric absorption spectrum contains a strong transition at 580 nm and a broad shoulder between 575-500 nm, both features are attributed to a vibrational progression with an effective vibrational mode of 1415 cm(-1) whose major contributing vibrational normal modes are breathing modes of the perylene body. The effective vibrational mode of the emission spectrum is characterized by a frequency of 1369 cm(-1), whose major contributing vibrational normal modes are characterized by perylene and phenol (bay-substituent) CH bendings. The S(n)←S(1) excited state absorption spectrum is assigned based on Multi-Reference Configuration Interaction methodology. Here, we identify three transitions which give rise to two broad experimental features, one being located between 500 and 600 nm and the other one ranging from 650 to 750 nm.

One-dimensional exciton diffusion in perylene bisimide aggregates.

The dynamics and mobility of excitons in J-aggregates of perylene bisimides are investigated by transient absorption spectroscopy with a time resolution of 50 fs. The transient spectra are compatible with an exciton delocalization length of two monomers and indicate that vibrational and configurational relaxation processes are not relevant for the spectroscopic properties of the aggregates. Increasing the pump pulse energy and in that way the initial exciton density results in an accelerated signal decay and pronounced exciton-exciton annihilation dynamics. Modeling the data by assuming a diffusive exciton motion reveals that the excitons cannot migrate freely in all three directions of space but their mobility is restricted to one dimension. The observed anisotropy supports this picture and points against direct Förster-transfer-mediated annihilation between the excitons. A diffusion constant of 1.29 nm(2)/ps is deduced from the fitting procedure that corresponds to a maximal exciton diffusion length of 96 nm for the measured exciton lifetime of 3.6 ns. The findings indicate that J-aggregates of perylene bisimides are promising building blocks to facilitate directed energy transport in optoelectronic organic devices or artificial light-harvesting systems.

Effect of modified radical mastectomy combined with latissimus dorsi musculocutaneous flap breast reconstruction on patients' psychology and quality of life.

BACKGROUND: Breast carcinoma (BC) is a commonly seen malignancy in women. Although traditional radical mastectomy can improve the survival of patients, it can cause breast loss and chest wall deformities, which seriously affects the daily life of patients and causes anxiety and depression. The purpose of this research project is to investigate the effect of breast reconstruction with latissimus dorsi myocutaneous flap (LDMF) after nipple- and areola-sparing modified radical mastectomy (MRM) on the psychological mood and quality of life (QoL) of patients with stage I BC. METHODS: A total of 102 patients with BC (research group, RG) treated in the Shanghai Fifth People's Hospital, Fudan University from January 2018 to December 2020 were selected for phase I breast reconstruction with LDMF after nipple- and areola-sparing MRM. Concurrently, 50 BC patients (control group, CG) who underwent traditional total mastectomy in our hospital were collected. The activities of daily living (ADL), self-rating anxiety scale (SAS) and self-rating depression scale (SDS) scores were observed before and 1 month after treatment. The intraoperative indicators, postoperative complications, postoperative satisfaction rate and overall survival rate were compared. RESULTS: The Functional Assessment of Cancer Therapy-Breast Cancer (FACT-B) score was higher after treatment, while SAS and SDS scores were lower in RG than in CG (P<0.05). No statistical difference was observed in intraoperative blood loss, wound drainage time, operation time, postoperative complications and overall survival rate between the two cohorts (P>0.05). RG showed higher satisfaction degree and overall satisfaction rate, as well as better QoL than CG (P<0.05). CONCLUSIONS: Breast reconstruction with LDMF after nipple- and areola-sparing MRM can alleviate adverse emotions of patients with stage I BC and improve their QoL.