Hepatic gene expression profiles during fed-fasted-refed state in mice.

Background: Regulation of nutrient status during fasting and refeeding plays an important role in maintaining metabolic homeostasis in the liver. Thus, we investigated the impact of the physiological Fed-Fast-Refed cycle on hepatic gene expression in nutrient-sensitive mice. Methods: We performed transcriptomic analysis of liver samples in fed, fasted and refed groups of mice. Through mRNA-sequencing (RNA-Seq) and miRNA-Seq, we compared fasted and fed states (fasted versus fed cohort) as well as refed and fasted states (refed versus fasted cohort) to detect dynamic alterations of hepatic mRNA-miRNA expression during the fed-fasted-refed cycle. Results: We found dozens of dysregulated mRNAs-miRNAs in the transition from fed to fasted and from fasted to refed states. Gene set enrichment analysis showed that gene expression of the two cohorts shared common pathways of regulation, especially for lipid and protein metabolism. We identified eight significant mRNA and three miRNA clusters that were up-downregulated or down-upregulated during the Fed-Fast-Refed cycle. A protein-protein interaction network of dysregulated mRNAs was constructed and clustered into 22 key modules. The regulation between miRNAs and target mRNAs was presented in a network. Up to 42 miRNA-mRNA-pathway pairs were identified to be involved in metabolism. In lipid metabolism, there were significant correlations between mmu-miR-296-5p and Cyp2u1 and between mmu-miR-novel-chr19_16777 and Acsl3. Conclusion: Collectively, our data provide a valuable resource for the molecular characterization of the physiological Fed-Fast-Refed cycle in the liver.

Invasive mucinous adenocarcinoma misdiagnosed as pneumonia: A case report.

Common imaging findings of invasive mucinous adenocarcinoma (IMA) include consolidation of the lung parenchyma, nodules, and ground-glass changes. However, the IMA imaging findings in the present case included diffuse, patchy and blurry density shadows through both lungs. To the best of the authors' knowledge, this image pattern has rarely been reported. The patient provided his consent and authorized the publication of photographs featuring his likeness. The present study reported a patient was diagnosed with IMA via pathologic and genetic analyses. Following antibiotic treatment, the lesions in both sides became larger. Further examinations were completed and IMA was confirmed by biopsy pathohistological examination. Pathological specimens were negative for almost all driver genes mutations, except KRAS. The patients and family refused further treatment, including chemotherapy, radiotherapy and interventional chemotherapy and the patient was discharged from The First Affiliated Hospital of Chengdu Medical College. The present case report emphasized that IMA should be suspected when imaging studies show diffuse lesions throughout both lungs. When a patient does not respond to treatment, clinicians should consider alternative diagnoses.

Comprehensive RNA expression profile analysis of γδ T cells from peripheral blood and decidual tissues in normal pregnancy (NP) donors and patients with recurrent pregnancy loss (RPL).

Recurrent pregnancy loss (RPL) is a significant adverse pregnancy complication. The loss of immune tolerance has been proposed in the pathogenesis of RPL, however, the role of γδ T cells in RPL is still controversial. In this study, the gene expression patterns of circulated and decidual tissue-resident γδ T cells from normal pregnancy donors and patients with RPL were analyzed by SMART-seq. We demonstrate that the transcriptional expression profile of different subsets of γδ T cells in peripheral blood and decidual tissue is strikingly different. Vδ2 γδ T cells, as the major cytotoxic subset, are found to be enriched considerably, and the potential cytotoxicity of this subset is further enhanced in the decidua of RPL patients may be due to detrimental ROS reduction, enhanced metabolic activity, downregulation of immunosuppressive molecules expression in resident γδ T cells. Time-series Expression Miner (STEM) analysis of transcriptome indicates complex changes in gene expression in decidual γδ T cells over time from NP and RPL patients. Taken together, our work identifies high heterogeneity of gene signature in γδ T cells from NP and RPL patients between peripheral blood and decidua, which will be a useful resource for further studies of the critical roles of γδ T cells in RPL.

The important role of glycerophospholipid metabolism in the protective effects of polyphenol-enriched Tartary buckwheat extract against alcoholic liver disease.

Alcoholic liver disease (ALD) is a mounting public health problem with significant medical, economic and social burdens. Tartary buckwheat (F. tataricum (L.) Gaertn, bitter buckwheat) is a kind of healthy and nutritious food, which has been demonstrated to protect against ALD, but the underlying mechanism has not been fully studied. Herein, we aimed to elucidate the beneficial effects of Tartary buckwheat extract (mainly composed of polyphenols including rutin, quercetin, kaempferol and kaempferol-3-O-rutinoside) in terms of lipid metabolism with the aid of lipidomic analysis. In our study, we employed C57BL/6J mice and a Lieber-DeCarli alcohol liquid diet to construct an ALD model and found that Tartary buckwheat extract was able to prevent ALD-induced histopathological lesions, liver injury and abnormal plasma lipid levels. These beneficial effects might be attributed to the regulation of energy metabolism-related genes (SIRT1, LKB1 and AMPK), lipid synthesis-related genes (ACC, SREBP1c and HMGR) and lipid oxidation-related genes (PPARα, CPT1 and CPT2). In addition, lipidomic profiling and KEGG pathway analysis showed that glycerophospholipid metabolism contributed the most to elucidating the regulatory mechanism of Tartary buckwheat extract. In specific, chronic ethanol intake reduced the level of phosphatidylcholines (PC) and increased the level of phosphatidylethanolamines (PE) in the liver, resulting in a decrease in the PC/PE ratio, which could be all significantly restored by Tartary buckwheat extract intervention, indicating that the Tartary buckwheat extract might regulate PC/PE homeostasis to exert its lipid-lowering effect. Overall, we demonstrated that Tartary buckwheat extract could prevent ALD by modulating hepatic glycerophospholipid metabolism, providing the theoretical basis for its further exploitation as a medical plant or nutritional food.

Genistein promotes M1 macrophage apoptosis and reduces inflammatory response by disrupting miR-21/TIPE2 pathway.

Cardiovascular diseases are a major cause of mortality, and vascular injury, a common pathological basis of cardiovascular disease, is deeply correlated with macrophage apoptosis and inflammatory response. Genistein, a type of phytoestrogen, exerts cardiovascular protective activities, but the underlying mechanism has not been fully elucidated. In this study, RAW264.7 cells were treated with genistein, lipopolysaccharide (LPS), nuclear factor-kappa B (NF-κB) inhibitor, and/or protein kinase B (AKT) agonist to determine the role of genistein in apoptosis and inflammation in LPS-stimulated cells. Simultaneously, high fat diet-fed C57BL/6 mice were administered genistein to evaluate the function of genistein on LPS-induced cardiovascular injury mouse model. Here, we demonstrated that LPS obviously increased apoptosis resistance and inflammatory response of macrophages by promoting miR-21 expression, and miR-21 downregulated tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) expression by targeting the coding region. Genistein reduced miR-21 expression by inhibiting NF-κB, then blocked toll-like receptor 4 (TLR4) pathway and AKT phosphorylation dependent on TIPE2, resulting in inhibition of LPS. Our research suggests that miR-21/TIPE2 pathway is involved in M1 macrophage apoptosis and inflammatory response, and genistein inhibits the progression of LPS-induced cardiovascular injury at the epigenetic level via regulating the promoter region of Vmp1 by NF-κB.

Comprehensive Transcriptome Profiling of NAFLD- and NASH-Induced Skeletal Muscle Dysfunction.

Nonalcoholic fatty liver disease (NAFLD), characterized by extensive triglyceride accumulation in hepatocytes, may progress to nonalcoholic steatohepatitis (NASH) with liver fibrosis and inflammation and increase the risk of cirrhosis, cancer, and death. It has been reported that physical exercise is effective in ameliorating NAFLD and NASH, while skeletal muscle dysfunctions, including lipid deposition and weakness, are accompanied with NAFLD and NASH. However, the molecular characteristics and alterations in skeletal muscle in the progress of NAFLD and NASH remain unclear. In the present study, we provide a comprehensive analysis on the similarity and heterogeneity of quadriceps muscle in NAFLD and NASH mice models by RNA sequencing. Importantly, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway functional enrichment analysis revealed that NAFLD and NASH led to impaired glucose and lipid metabolism and deteriorated functionality in skeletal muscle. Besides this, we identified that myokines possibly mediate the crosstalk between muscles and other metabolic organs in pathological conditions. Overall, our analysis revealed a comprehensive understanding of the molecular signature of skeletal muscles in NAFLD and NASH, thus providing a basis for physical exercise as an intervention against liver diseases.

Preparation and Bolometric Responses of MoS2 Nanoflowers and Multi-Walled Carbon Nanotube Composite Network.

Due to their broadband optical absorption ability and fast response times, carbon nanotube (CNT)-based materials are considered promising alternatives to the toxic compounds used in commercial infrared sensors. However, the direct use of pure CNT networks as infrared sensors for simple resistance read-outs results in low sensitivity values. In this work, MoS2 nanoflowers are composited with CNT networks via a facile hydrothermal process to increase the bolometric performance. The thermal diffusivity (α) against temperature (T) is measured using the transient electro-thermal (TET) technique in the range of 320 K to 296 K. The α-T curve demonstrates that the composite containing MoS2 nanoflowers provides significant phonon scattering and affects the intertube interfaces, decreasing the α value by 51%. As the temperature increases from 296 K to 320 K, the relative temperature coefficient of resistance (TCR) increases from 0.04%/K to 0.25%/K. Combined with the enhanced light absorption and strong anisotropic structure, this CNT-MoS2 composite network exhibits a more than 5-fold greater surface temperature increase under the same laser irradiation. It shows up to 18-fold enhancements in resistive responsivity ((Ron - Roff)/Roff) compared with the pure CNT network for a 1550 nm laser at room temperature (RT).

Comparison of hepatic gene expression profiles between three mouse models of Nonalcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disorder worldwide. Murine models of NAFLD have been widely used to explore its pathogenesis. In this study, we performed a systematic evaluation of hepatic genome-wide mRNA expression by RNA-Sequencing using three mouse models of NAFLD: leptin receptor deficient db/db mice, high-fat high-sugar diet (HSHF)-induced obese mice, and dexamethasone (DEX)-induced NAFLD mice. As a result, we found both distinct and common pathways in the regulation of lipid metabolism from transcriptomes of three mouse models. Moreover, only a total of 12 differentially expressed genes (DEGs) were commonly detected among all three mouse groups, indicating very little overlap among all three models. Therefore, our results suggest that NAFLD is a heterogeneous disease with highly variable molecular mechanisms.

Nutritional-status dependent effects of microplastics on activity and expression of alkaline phosphatase and alpha-amylase in Brachionus rotundiformis.

Tissue-nonspecific alkaline phosphatase (ALPL) and alpha-amylase (AMY) are essential in the immune and digestive systems, respectively. Microplastics (MPs) pose a risk to zooplankton which may be in a state of feeding, starvation, or subsequent refeeding. However, molecular characterization of both enzymes and the regulated mechanisms affected by nutritional statuses and MPs remain unclear in zooplankton. In the present study, four full-length genes encoding ALPL and two genes encoding AMY were cloned and characterized from an isolated marine rotifer, Brachionus rotundiformis, including alplA, alplB, alplC, alplD, amy2a, and amy2al. AMY activity and expression of amy2a and amy2al were reduced by starvation and recovered after refeeding compared with feeding. ALPL activity remained unchanged among different statuses, while alplA, alplB and alplD were down-regulated by starvation and refeeding compared with feeding. ALPL activity was not affected by exposure to 10, 100 and 1000 µg/L MPs in rotifers subjected to feeding, starvation and refeeding, whereas AMY activity was significantly enhanced by 1000 µg/L MPs in rotifers subjected to refeeding. Gene expression of the tested genes, except amy2a, was significantly responsive to MPs, especially in the feeding rotifers, depending on MPs concentrations and nutritional statuses. Two-way ANOVA confirmed that these changes were strongly associated with the interaction between MPs concentrations and nutritional statuses. The present study is the first to demonstrate a nutritional status-dependent impact of MPs on immune and digestive responses, and provides more sensitive molecular biomarkers for assessing MPs toxicity using the species as model animals.

Genome-wide identification of seven superoxide dismutase genes in the marine rotifer Brachionus rotundiformis and modulated expression and enzymatic activity in response to microplastics and nutritional status.

Microplastics (MPs) pollution has attracted worldwide attention. Superoxide dismutase (SOD) is a sensitive indicator for assessing the toxic effects of MPs in aquatic organisms. However, few studies have been performed to identify all genes encoding SOD in aquatic invertebrates. Especially, effects of MPs on SOD activity and expression in aquatic organisms under starvation or a subsequent refeeding status are unclear. In the present study, all full-length genes encoding SOD were cloned and characterized from the marine rotifer Brachionus rotundiformis, including CuZnSOD1, CuZnSOD2, CuZnSOD3, CuZnSOD4, CuZnSOD5, MnSOD1, and MnSOD2. The CuZnSOD1, CuZnSOD2 and MnSOD2 are homologous to SODs from vertebrates and the other SOD proteins are rotifer-specific according to the results from the phylogenetic tree. The conserved signature sequences and binding sites of Cu2+, Zn2+and Mn2+ were also identified in the seven SOD proteins. Compared with feeding, starvation down-regulated SOD activity and mRNA expression of CuZnSOD2, CuZnSOD4, CuZnSOD5, MnSOD1 and MnSOD2 while refeeding maintained SOD activity comparable to the feeding level and up-regulated CuZnSOD5 and MnSOD2. Intake of MPs by B. rotundiformis was observed by examining fluorescence signals from the fluorescently-labeled microplastics under different nutritional status. Exposure to MPs reduced rotifer density and increased malondialdehyde (MDA) content and SOD activity in the rotifers under the refeeding condition, but did not affect these indicators under the feeding and starvation conditions. However, mRNA expression of some tested genes was responsive to MPs in the fed, starved and refed rotifers. The present study for the first time demonstrated a nutritional status-dependent effect of MPs on oxidative stress response, and provided more sensitive molecular biomarkers for assessing the toxicity of MPs using B. rotundiformis as a model animal.

Analysis of N6-Methyladenosine Methylation Modification in Fructose-Induced Non-Alcoholic Fatty Liver Disease.

Improvements in living standards have led to non-alcoholic fatty liver disease (NAFLD), one of the most common chronic liver diseases worldwide. Recent studies have shown that N6-methyladenosine (m6A), a type of RNA modification, is strongly associated with many important biological processes. However, the relationship between m6A methylation modifications and NAFLD remains poorly understood. In the present study, through methylated RNA immunoprecipitation sequencing and RNA transcriptome sequencing in high fructose diet-induced NAFLD mice, we found that hypermethylation-encoding genes were mainly enriched in lipid metabolism processes. We identified 266 overlapping and differentially expressed genes (DEGs) that changed at both the mRNA expression level and m6A modification level. Among them, 193 genes displayed increased expression and m6A modification, indicating that m6A RNA modifications tend to be positively correlated with NAFLD. We further compared the high fructose diet-induced NAFLD mouse model with leptin receptor-deficient mice and found that DEGs enriched in the lipid metabolism pathway were up-regulated in both groups. In contrast, DEGs associated with the immune inflammatory response were up-regulated in the high fructose diet group, but down-regulated in leptin receptor-deficient mice. Taken together, our results demonstrate that m6A methylation modifications may play an important role in the development of NAFLD.

A novel immune-related ceRNA network that predicts prognosis and immunotherapy response in lung adenocarcinoma.

BACKGROUND: The tumor microenvironment plays an important role in the progression and malignancy of lung adenocarcinoma and affects the immunotherapy response. There is increasing evidence that long non-coding RNAs (lncRNAs) as competing endogenous RNAs (ceRNAs) have significant functions in the development and treatment response of various kinds of cancer. This study aimed to explore the association between immune-related lncRNA-microRNA (miRNA)-messenger RNA (mRNA)-ceRNA networks, and the prognosis of and immunotherapy response in lung adenocarcinoma. METHODS: RNA-sequencing (RNA-seq) and miRNA-seq data from The Cancer Genome Atlas (TCGA) were used to evaluate the infiltration of immune cells in lung adenocarcinoma samples by undertaking a single-sample gene set enrichment analysis (ssGSEA) to divide the cells into high and low immune cell infiltration groups. The differentially expressed mRNA (DEmRNA) was further analyzed by a weighted gene co-expression network analysis (WGCNA), search tool for recurring instances of neighboring genes (STRING), and Cytoscape to select hub genes. The ceRNA network was constructed using Cytoscape. Additionally, survival analyses were conducted to screen out prognostic candidate genes. RESULTS: Seven thousand five hundred and thirty-eight mRNAs, 540 lncRNAs, and 138 miRNAs were found to be differentially expressed between the high and low immune cell infiltration groups. The two DEmRNA modules most significantly associated with immune cell infiltration were further analyzed, and four clusters, including 179 DEmRNAs, were selected based on Molecular Complex Detection (MCODE) scores. The selected DEmRNAs in the four clusters were mainly enriched in pathways involved in regulating the immune response. Ultimately, a ceRNA network of SNHG6-hsa-miR-30e-5p-CYSLTR1 was identified as being associated with the prognosis of and immunotherapy response in lung adenocarcinoma. CONCLUSIONS: The present study extends understandings of immune-related lncRNA-miRNA-mRNA-ceRNA networks and identifies novel targets and a regulatory pathway for anti-tumor immunotherapy.

Sparcl1 promotes nonalcoholic steatohepatitis progression in mice through upregulation of CCL2.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of chronic liver disease ranging from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms of NASH progression remain incompletely understood. White adipose tissue (WAT) has emerged as an important endocrine organ and contributes not only to the initial stage of NAFLD, but also to its severity. In the current study, through transcriptomic analysis we identified increased expression of Sparcl1, a secreted glycoprotein, in the WAT from NASH mice. Plasma Sparcl1 levels were similarly elevated and positively correlated with hepatic pathological features in NASH patients. Functional studies showed that both chronic injection of recombinant Sparcl1 protein and overexpression of Sparcl1 exaggerated hepatic inflammation and liver injury in mice. In contrast, genetic ablation of Sparcl1, knockdown of Sparcl1 in WAT, and treatment with a Sparcl1-neutralizing antibody dramatically alleviated diet-induced NASH pathogenesis. Mechanistically, Sparcl1 promoted the expression of C-C motif chemokine ligand 2 (CCL2) in hepatocytes through binding to Toll-like receptor 4 (TLR4) and activation of the NF-κB/p65 signaling pathway. Genetically or pharmacologically blocking the CCL2/CCR2 pathway attenuated the hepatic inflammatory response evoked by Sparcl1. Thus, our results demonstrated an important role for Sparcl1 in NASH progression, suggesting a potential target for therapeutic intervention.

Transferrin receptor 1 ablation in satellite cells impedes skeletal muscle regeneration through activation of ferroptosis.

BACKGROUND: Satellite cells (SCs) are critical to skeletal muscle regeneration. Inactivation of SCs is linked to skeletal muscle loss. Transferrin receptor 1 (Tfr1) is associated with muscular dysfunction as muscle-specific deletion of Tfr1 results in growth retardation, metabolic disorder, and lethality, shedding light on the importance of Tfr1 in muscle physiology. However, its physiological function regarding skeletal muscle ageing and regeneration remains unexplored. METHODS: RNA sequencing is applied to skeletal muscles of different ages to identify Tfr1 associated to skeletal muscle ageing. Mice with conditional SC ablation of Tfr1 were generated. Between Tfr1SC/WT and Tfr1SC/KO (n = 6-8 mice per group), cardiotoxin was intramuscularly injected, and transverse abdominal muscle was dissected, weighted, and cryosectioned, followed by immunostaining, haematoxylin and eosin staining, and Masson staining. These phenotypical analyses were followed with functional analysis such as flow cytometry, tread mill, Prussian blue staining, and transmission electron microscopy to identify pathological pathways that contribute to regeneration defects. RESULTS: By comparing gene expression between young (2 weeks old, n = 3) and aged (80 weeks old, n = 3) mice among four types of muscles, we identified that Tfr1 expression is declined in muscles of aged mice (~80% reduction, P < 0.005), so as to its protein level in SCs of aged mice. From in vivo and ex vivo experiments, Tfr1 deletion in SCs results in an irreversible depletion of SCs (~60% reduction, P < 0.005) and cell-autonomous defect in SC proliferation and differentiation, leading to skeletal muscle regeneration impairment, followed by labile iron accumulation, lipogenesis, and decreased Gpx4 and Nrf2 protein levels leading to reactive oxygen species scavenger defects. These abnormal phenomena including iron accumulation, activation of unsaturated fatty acid biosynthesis, and lipid peroxidation are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis further exacerbates SC proliferation and skeletal muscle regeneration. Ferrostatin-1, a ferroptosis inhibitor, could not rescue ferroptosis. However, intramuscular administration of lentivirus-expressing Tfr1 could partially reduce labile iron accumulation, decrease lipogenesis, and promote skeletal muscle regeneration. Most importantly, declined Tfr1 but increased Slc39a14 protein level on cellular membrane contributes to labile iron accumulation in skeletal muscle of aged rodents (~80 weeks old), leading to activation of ferroptosis in aged skeletal muscle. This is inhibited by ferrostatin-1 to improve running time (P = 0.0257) and distance (P = 0.0248). CONCLUSIONS: Satellite cell-specific deletion of Tfr1 impairs skeletal muscle regeneration with activation of ferroptosis. This phenomenon is recapitulated in skeletal muscle of aged rodents and human sarcopenia. Our study provides mechanistic information for developing novel therapeutic strategies against muscular ageing and diseases.

7-Difluoromethoxy-5,4'-dimethoxy-genistein attenuates macrophages apoptosis to promote plaque stability via TIPE2/TLR4 axis in high fat diet-fed ApoE-/- mice.

Promoting plaque stability is of great significance for prevention and treatment of cardiovascular diseases. 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) is a novel active compound synthesized using genistein, which exerts anti-atherosclerotic effect. In this study, we evaluated effects of DFMG on plaque stability in ApoE-/- mice fed with high fat diet (HFD), and explored the molecular mechanism by using ApoE-/-TLR4-/- mice and RAW264.7 cells. Here, we found that DFMG significantly reduced plaque areas, macrophages infiltration and apoptosis, and TLR4 expression in HFD-fed ApoE-/- mice. Meanwhile, DFMG increased collagen fibers, smooth muscle cells and TIPE2 expression in plaques and media. Besides, TLR4 knockout promoted the protective effects of DFMG on plaques. In vitro, DFMG decreased lysophosphatidylcholine (LPC)-induced macrophages apoptosis and TLR4, while upregulated TIPE2. Moreover, TIPE2 reduced TLR4, MyD88, p-NF-κB p65Ser276, cleaved Caspase-3 overproduction, and enhanced effects of DFMG on LPC-induced macrophages. Overall, our study demonstrates that DFMG can promote plaque stability by reducing macrophage apoptosis through TIPE2/TLR4 signaling pathway, which suggests DFMG should be used to develop food additives or drugs for preventing atherosclerosis.

Resistin increases cisplatin-induced cytotoxicity in lung adenocarcinoma A549 cells via a mitochondria-mediated pathway.

Lung cancer is the most commonly diagnosed cancer with a high mortality rate. Cisplatin is one of the most important chemotherapeutic agents for the treatment of lung cancer patients, especially in advanced stages. Recent studies show that cisplatin may interact with mitochondria to induce apoptosis, which may partly account for its cytotoxicity. In the study, we explored the effect of resistin on cisplatin-induced cytotoxicity in A549 cells and assessed whether mitochondria-dependent apoptosis was involved. Our results found that 25 ng/ml resistin could significantly increase cisplatin-induced apoptosis and G2/M phase arrest, enhance reactive oxygen species generation, exacerbate the collapse of mitochondrial membrane potential, promote the distribution of cytochrome C in the cytoplasm from mitochondria, and activate caspase 3. Therefore, the results suggested that resistin might increase cisplatin-induced cytotoxicity via a mitochondria-mediated pathway in A549 cells. However, the precise mechanism remains to be explored in the future.

Genistein alleviates chronic vascular inflammatory response via the miR­21/NF­κB p65 axis in lipopolysaccharide­treated mice.

Chronic vascular inflammatory response is an important pathological basis of cardiovascular disease. Genistein (GEN), a natural compound, exhibits anti­inflammatory effects. The aim of the present study was to investigate the effects of GEN on lipopolysaccharide (LPS)­induced chronic vascular inflammatory response in mice and explore the underlying anti­inflammatory mechanisms. C57BL/6 mice were fed with a high­fat diet combined with intraperitoneal injection of LPS to induce chronic vascular inflammation. The expression levels of TNF­α, IL­6 and microRNA (miR)­21 in the vasculature were detected via reverse transcription­quantitative (RT­q)PCR. The protein levels of inducible nitric oxide synthase (iNOS) and NF­κB p65 were detected via western blotting. NF­κB p65 was also analyzed via immunohistochemistry and immunofluorescence (IF). In addition, after transfection with miR­21 mimic or inhibitor for 24 h, vascular endothelial cells (VECs) were treated with GEN and LPS. RT­qPCR and western blot analyses were performed to detect the expression of TNF­α, IL­6, miR­21 and iNOS, and the protein levels of iNOS and NF­κB p65, respectively. IF was used to measure NF­κB p65 nuclear translocation. The results revealed that GEN significantly decreased the expression of inflammation­associated vascular factors in LPS­treated C57BL/6 mice, including TNF­α, IL­6, iNOS, NF­κB p65 and miR­21. Furthermore, miR­21 antagomir enhanced the anti­inflammatory effects of GEN. In LPS­induced VECs, miR­21 mimic increased inflammation­associated factor expression and attenuated the anti­inflammatory effects of GEN, whereas miR­21 inhibitor induced opposing effects. Therefore, the results of the present study suggested that GEN inhibited chronic vascular inflammatory response in mice, which may be associated with the inhibition of VEC inflammatory injury via the miR­21/NF­κB p65 pathway.

Application of Population Pharmacokinetic Analysis to Characterize CYP2C19 Mediated Metabolic Mechanism of Voriconazole and Support Dose Optimization.

Purpose: The aims of this study were to establish a joint population pharmacokinetic model for voriconazole and its N-oxide metabolite in immunocompromised patients, to determine the extent to which the CYP2C19 genetic polymorphisms influenced the pharmacokinetic parameters, and to evaluate and optimize the dosing regimens using a simulating approach. Methods: A population pharmacokinetic analysis was conducted using the Phoenix NLME software based on 427 plasma concentrations from 78 patients receiving multiple oral doses of voriconazole (200 mg twice daily). The final model was assessed by goodness of fit plots, non-parametric bootstrap method, and visual predictive check. Monte Carlo simulations were carried out to evaluate and optimize the dosing regimens. Results: A one-compartment model with first-order absorption and mixed linear and concentration-dependent-nonlinear elimination fitted well to concentration-time profile of voriconazole, while one-compartment model with first-order elimination well described the disposition of voriconazole N-oxide. Covariate analysis indicated that voriconazole pharmacokinetics was substantially influenced by the CYP2C19 genetic variations. Simulations showed that the recommended maintenance dose regimen would lead to subtherapeutic levels in patients with different CYP2C19 genotypes, and elevated daily doses of voriconazole might be required to attain the therapeutic range. Conclusions: The joint population pharmacokinetic model successfully characterized the pharmacokinetics of voriconazole and its N-oxide metabolite in immunocompromised patients. The proposed maintenance dose regimens could provide a rationale for dosage individualization to improve clinical outcomes and minimize drug-related toxicities.

The Nuclear Orphan Receptor NR2F6 Promotes Hepatic Steatosis through Upregulation of Fatty Acid Transporter CD36.

Nuclear receptors (NRs) are a superfamily of transcription factors which sense hormonal signals or nutrients to regulate various biological events, including development, reproduction, and metabolism. Here, this study identifies nuclear receptor subfamily 2, group F, member 6 (NR2F6), as an important regulator of hepatic triglyceride (TG) homeostasis and causal factor in the development of non-alcoholic fatty liver disease (NAFLD). Adeno-associated virus (AAV)-mediated overexpression of NR2F6 in the liver promotes TG accumulation in lean mice, while hepatic-specific suppression of NR2F6 improves obesity-associated hepatosteatosis, insulin resistance, and methionine and choline-deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH). Mechanistically, the fatty acid translocase CD36 is identified as a transcriptional target of NR2F6 to mediate its steatotic role. NR2F6 is able to bind directly onto the CD36 promoter region in hepatocytes and increases the enrichment of nuclear receptor coactivator 1 (SRC-1) and histone acetylation at its promoter. Of pathophysiological significance, NR2F6 is significantly upregulated in the livers of obese mice and NAFLD patients. Moreover, treatment with metformin decreases NR2F6 expression in obese mice, resulting in suppression of CD36 and reduced hepatic TG contents. Therefore, these results provide evidence for an unpredicted role of NR2F6 that contributes to liver steatosis and suggest that NR2F6 antagonists may present a therapeutic strategy for reversing or treating NAFLD/NASH pathogenesis.

Chronic Periodontitis and Alzheimer Disease: A Putative Link of Serum Proteins Identification by 2D-DIGE Proteomics.

Increasing evidence indicates Chronic Periodontitis (CP) is a comorbidity of Alzheimer's disease (AD), which is the most common form of age-related dementia, and for the latter, effective diagnostic and treatment strategies are lacking. Although inflammation is present in both diseases, the exact mechanisms and cross-links between CP and AD are poorly understood; and a direct association between the two has not been reported. This study aimed to identify a direct serum proteins link between AD and CP. Two-dimensional differential in-gel electrophoresis was employed to analyze serum samples from 12 CP patients and 12 age-matched controls. Furthermore, to determine the molecular link between CP and AD, neuroblastoma SK-N-SH APPwt cells were treated with 1 µg/ml of lipopolysaccharide from Porphyromonas gingivalis (P.g-LPS). Ten differentially expressed proteins were identified in CP patients. Among them, nine proteins were up-regulated, and one protein was down-regulated. Of the 10 differentially expressed proteins, five proteins were reportedly involved in the pathology of AD: Cofilin-2, Cathepsin B, Clusterin, Triosephosphate isomerase, and inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4). Western blotting indicated significantly higher expression of Cofilin-2, Cathepsin B, and Clusterin and lower expression of ITI-H4 in the CP group than in the Control group. The serum concentration of Cathepsin B has a good correlation with MMSE scores. Moreover, the protein level of Cathepsin B (but not that of ADAM10 and BACE1) increased significantly along with a prominent increase in Aß1-40 and Aß1-42 in the cell lysates of P.g-LPS-treated SK-N-SH APPwt cells. Cathepsin B inhibition resulted in a sharp decrease in Aß1-40 and Aß1-42 in the cell lysates. Furthermore, TNF-α was one of the most important inflammatory cytokines for the P.g-LPS-induced Cathepsin B upregulation in SK-N-SH APPwt cells. These results show that CP and AD share an association, while Cathepsin B could be a key link between the two diseases. The discovery of the identical serum proteins provides a potential mechanism underlying the increased risk of AD in CP patients, which could be critical for elucidating the pathophysiology of AD.