Small cell lung cancer (SCLC) is a special kind of lung cancers, lymph or blood metastasis of SCLC usually occurs in early stage. Studies in breast and colon cancer showed over expression of SATB1 could promote tumor cell growth and inhibit apoptosis. Therefore, we studied the expression of SATB1 in SCLC.
The level of SATB1 was analyzed in SCLC tissues, metastatic lymphoid nodes and adjacent normal lung tissues by immunohistochemistry. Meanwhile, small interfering SATB1-targeting RNA was constructed and transfected into human SCLC cell line NCI-H446 to evaluate the effects of SATB1-siRNA on cell proliferation, invasion and apoptosis.
SATB1 protein was overexpressed in SCLC tissues and metastasis lymphoid nodes compared with adjacent normal lung tissues. Compared with control group, SATB1-siRNA inhibits the proliferation and invasion of SCLC cells and induces SCLC cells apoptosis statistically (P<0.05) in vitro.
Our results suggest that SATB1 plays an important role in the metastasis of human SCLC cell.
SATB1; Small cell lung cancer; siRNA interfering; Apoptosis
Background & Aim
A key feature in the pathogenesis of liver fibrosis is fibrillar collagen-I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSC), could drive fibrogenesis by modulating the HSC profibrogenic phenotype and collagen-I expression.
rOPN up-regulated collagen-I protein in primary HSC in a TGFβ-independent fashion whereas it down-regulated matrix metalloprotease-13 (MMP13) thus favoring scarring. rOPN activated primary HSC -confirmed by increased α-smooth muscle actin (α-SMA) expression- and enhanced their invasive and wound-healing potential. HSC isolated from wild type (WT) mice were more profibrogenic than those from Opn-/- mice and infection of primary HSC with an Ad-OPN increased collagen-I, indicating correlation between both proteins. The OPN induction of collagen-I occurred via integrin αvβ3 engagement and activation of the PI3K-pAkt-NFκB signaling pathway, while CD44-binding and mTOR-p70S6K were not involved. Neutralization of integrin αvβ3 prevented the OPN-mediated activation of the PI3K-pAkt-NFκB signaling cascade and collagen-I up-regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN-mediated collagen-I increase. HCV-cirrhotic patients showed co-induction of collagen-I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by carbon tetrachloride (CCl4)-injection or thioacetamide (TAA)-treatment elevated OPN expression. Reactive oxygen species up-regulated OPN in vitro and in vivo and antioxidants prevented this effect. OpnHEP Tg mice developed spontaneous liver fibrosis compared to WT mice. Lastly, chronic CCl4-injection and TAA-treatment caused more liver fibrosis to WT than to Opn-/- mice and the reverse occurred in OpnHEP Tg mice.
OPN emerges as a cytokine within the ECM protein network driving the increase in collagen-I protein contributing to scarring and liver fibrosis.
Extracellular matrix; fibrosis; hepatic stellate cells; oxidant stress
Planetary gearboxes exhibit complicated dynamic responses which are more difficult to detect in vibration signals than fixed-axis gear trains because of the special gear transmission structures. Diverse advanced methods have been developed for this challenging task to reduce or avoid unscheduled breakdown and catastrophic accidents. It is feasible to make fault features distinct by using multiwavelet denoising which depends on the feature separation and the threshold denoising. However, standard and fixed multiwavelets are not suitable for accurate fault feature detections because they are usually independent of the measured signals. To overcome this drawback, a method to construct customized multiwavelets based on the redundant symmetric lifting scheme is proposed in this paper. A novel indicator which combines kurtosis and entropy is applied to select the optimal multiwavelets, because kurtosis is sensitive to sharp impulses and entropy is effective for periodic impulses. The improved neighboring coefficients method is introduced into multiwavelet denoising. The vibration signals of a planetary gearbox from a satellite communication antenna on a measurement ship are captured under various motor speeds. The results show the proposed method could accurately detect the incipient pitting faults on two neighboring teeth in the planetary gearbox.
planetary gearbox; fault detection; vibration sensor signals; customized multiwavelets; redundant symmetric lifting schemes; improved neighboring coefficients
Motivation: Genome-wide fitness is an emerging type of high-throughput
biological data generated for individual organisms by creating libraries of knockouts,
subjecting them to broad ranges of environmental conditions, and measuring the resulting
clone-specific fitnesses. Since fitness is an organism-scale measure of gene regulatory
network behaviour, it may offer certain advantages when insights into such phenotypical
and functional features are of primary interest over individual gene expression. Previous
works have shown that genome-wide fitness data can be used to uncover novel gene
regulatory interactions, when compared with results of more conventional gene expression
analysis. Yet, to date, few algorithms have been proposed for systematically using
genome-wide mutant fitness data for gene regulatory network inference.
Results: In this article, we describe a model and propose an inference
algorithm for using fitness data from knockout libraries to identify underlying gene
regulatory networks. Unlike most prior methods, the presented approach captures not only
structural, but also dynamical and non-linear nature of biomolecular systems involved. A
state–space model with non-linear basis is used for dynamically describing gene
regulatory networks. Network structure is then elucidated by estimating unknown model
parameters. Unscented Kalman filter is used to cope with the non-linearities introduced in
the model, which also enables the algorithm to run in on-line mode for practical use.
Here, we demonstrate that the algorithm provides satisfying results for both synthetic
data as well as empirical measurements of GAL network in yeast
Saccharomyces cerevisiae and TyrR–LiuR network
in bacteria Shewanella oneidensis.
Availability: MATLAB code and datasets are available to download at
http://www.duke.edu/∼lw174/Fitness.zip and http://genomics.lbl.gov/supplemental/fitness-bioinf/
firstname.lastname@example.org or email@example.com
Supplementary data are available at Bioinformatics
Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are inhibited by the second mitocondrial activator of caspases (SMAC). Previously, a small subset of TNFα-expressing non-small cell lung cancers (NSCLCs) was found to be sensitive to SMAC mimetics alone. In this study we determined if a SMAC mimetic (JP1201) could sensitize non-responsive NSCLC cell lines to standard chemotherapy. We found that JP1201 sensitized NSCLCs to doxorubicin, erlotinib, gemcitabine, paclitaxel, vinorelbine, and the combination of carboplatin with paclitaxel in a synergistic manner at clinically achievable drug concentrations. Sensitization did not occur with platinum alone. Furthermore, sensitization was specific for tumor compared to normal lung epithelial cells, increased in NSCLCs harvested after chemotherapy treatment, and did not induce TNFα secretion. Sensitization also was enhanced in vivo with increased tumor inhibition and increased survival of mice carrying xenografts. These effects were accompanied by caspase 3, 4, and 9 activation, indicating that both mitochondrial and ER stress-induced apoptotic pathways are activated by the combination of vinorelbine and JP1201. Chemotherapies that induce cell death through the mitochondrial pathway required only inhibition of XIAP for sensitization, while chemotherapies that induce cell death through multiple apoptotic pathways required inhibition of cIAP1, cIAP2, and XIAP. Therefore, the data suggest that IAP-targeted therapy using a SMAC mimetic provides a new therapeutic strategy for synergistic sensitization of NSCLCs to standard chemotherapy agents, which appears to occur independently of TNFα secretion.
non-small cell lung cancer; smac mimetic; vinorelbine; gemcitabine; IAPs
Cultured neural stem cells (NSCs) provide a powerful means for investigating central nervous system disease, neuron development, differentiation, and regeneration. To obtain sufficient neurospheres, subculturing is essential following establishment of the primary NSC culture. Passaging the primary neurospheres is a key issue that is often ignored. We evaluated the influence of different passaging schedules on primary cultured NSCs. Passaging was performed on day 5, 7 or 9. We observed more neurospheres with diameters of 200–250 μm on day 7 than on day 5 or 9. Prolonging the time of primary culture reduced the cell metabolic activity by the MTT assay and cell proliferation by colony-forming assay and the differentiation to neurons from cells at P2 and later decreased. Additionally, more cells were in G0/G1 phase, and higher expression of p16INK4a and lower expression of cyclin D1 was found when the time of primary culture was prolonged to 9 days compared to 7-days cultures. Thus, in this study, we established that the optimal time for subculturing aggregated NSCs was on day 7 based on the primary culture.
Neurosphere; Neural stem cells; Primary culture; Cell passage; Optimal time
This paper proposes a method to select a set of genes from a large number of genes with the ability of classifying types of diseases. The proposed gene selection method is designed according to correlation analysis and the concept of 95% reference range. The method is very simple and uses the information of all genes. We have used the method in leukemia patients and achieved good classification results.
CYP2E1 induction and TNF-α production are key risk factors in alcoholic liver injury. Increased oxidative stress from CYP2E1 induction by pyrazole in vivo sensitizes the liver to TNF-α-induced hepatotoxicity by a mechanism involving activation of c-jun N-terminal kinase (JNK) and mitochondrial damage. The goal of this study was to evaluate whether JNK1 or JNK2 plays a role in this potentiated hepatotoxicity. Wild type (wt), jnk1−/− and jnk2−/− mice were used to identify changes of hepatotoxicity, damage to mitochondria and production of oxidative stress following pyrazole plus TNF-α treatment. Increased serum ALT, inflammatory infiltration and central necrosis were observed in the jnk2−/− and wt mice treated with pyrazole plus TNF-α, but not in the jnk1−/− mice. Pyrazole elevated the activity and protein level of CYP2E1 in all mice. There was a significant increase of malondialdehyde, 4-hydroxynonenal adducts, 3-nitrotyrosine and inducible nitric oxide synthase in the jnk2−/− and wt mice compared to the jnk1−/− mice upon pyrazole plus TNF-α treatment, or compared to mice treated with either pyrazole alone or TNF-α alone. The antioxidants catalase, GPx-4, thioredoxin and glutathione were lowered and cytochrome c was released from the mitochondria in the jnk2−/− and wt mice. Mitochondrial production of superoxide was increased in the jnk2−/− and wt mice compared to the jnk1−/− mice upon pyrazole plus TNF-α treatment. Electron microscopy showed altered mitochondrial structure in the jnk2−/− and wt but not the jnk1−/− mice.
JNK1 plays a role in the hepatotoxicity, mitochondrial dysfunction and oxidative stress mediated by pyrazole plus TNF-α treatment. These findings raise the question as to the potential mechanisms of JNK1 activation related to alcoholic liver injury.
Cytochrome P450 2e1; c-Jun N-terminal kinase; Oxidative stress; Liver injury; Mitochondrial damage
Resting-state fMRI is a novel approach to measure spontaneous brain activity in patients with major depressive disorder (MDD). Although most resting-state fMRI studies have focused on the examination of temporal correlations between low-frequency oscillations (LFOs), few studies have explored the amplitude of these LFOs in MDD. In this study, we applied the approaches of amplitude of low-frequency fluctuation (ALFF) and fractional ALFF to examine the amplitude of LFOs in MDD.
A total of 36 subjects, 18 first-episode, treatment-naive patients with MDD matched with 18 healthy controls (HCs) completed the fMRI scans. Compared with HCs, MDD patients showed increased ALFF in the right fusiform gyrus and the right anterior and posterior lobes of the cerebellum but decreased ALFF in the left inferior temporal gyrus, bilateral inferior parietal lobule, and right lingual gyrus. The fALFF in patients was significantly increased in the right precentral gyrus, right inferior temporal gyrus, bilateral fusiform gyrus, and bilateral anterior and posterior lobes of the cerebellum but was decreased in the left dorsolateral prefrontal cortex, bilateral medial orbitofrontal cortex, bilateral middle temporal gyrus, left inferior temporal gyrus, and right inferior parietal lobule. After taking gray matter (GM) volume as a covariate, the results still remained.
These findings indicate that MDD patients have altered LFO amplitude in a number of regions distributed over the frontal, temporal, parietal, and occipital cortices and the cerebellum. These aberrant regions may be related to the disturbances of multiple emotion- and cognition-related networks observed in MDD and the apparent heterogeneity in depressive symptom domains. Such brain functional alteration of MDD may contribute to further understanding of MDD-related network imbalances demonstrated in previous fMRI studies.
Typically, the first phase of a genome wide association study (GWAS) includes genotyping across hundreds of individuals and validation of the most significant SNPs. Allelotyping of pooled genomic DNA is a common approach to reduce the overall cost of the study. Knowledge of haplotype structure can provide additional information to single locus analyses. Several methods have been proposed for estimating haplotype frequencies in a population from pooled DNA data.
We introduce a technique for haplotype frequency estimation in a population from pooled DNA samples focusing on datasets containing a small number of individuals per pool (2 or 3 individuals) and a large number of markers. We compare our method with the publicly available state-of-the-art algorithms HIPPO and HAPLOPOOL on datasets of varying number of pools and marker sizes. We demonstrate that our algorithm provides improvements in terms of accuracy and computational time over competing methods for large number of markers while demonstrating comparable performance for smaller marker sizes. Our method is implemented in the "Tree-Based Deterministic Sampling Pool" (TDSPool) package which is available for download at http://www.ee.columbia.edu/~anastas/tdspool.
Using a tree-based determinstic sampling technique we present an algorithm for haplotype frequency estimation from pooled data. Our method demonstrates superior performance in datasets with large number of markers and could be the method of choice for haplotype frequency estimation in such datasets.
A small-molecule mimetic of Smac/Diablo that specifically counters the apoptosis-inhibiting activity of IAP proteins has been shown to enhance apoptosis induced by cell surface death receptors as well as chemotherapeutic drugs. Survey of a panel of 50 human non-small-cell lung cancer cell lines has revealed, surprisingly, that roughly one-quarter of these lines are sensitive to the treatment of Smac mimetic alone, suggesting that an apoptotic signal has been turned on in these cells and is held in check by IAP proteins. This signal has now been identified as the autocrine-secreted cytokine tumor necrosis factor alpha (TNFα). In response to autocrine TNFα signaling, the Smac mimetic promotes formation of a RIPK1-dependent caspase-8-activating complex, leading to apoptosis.
The goal of this study was to determine to what extent mechanical stability affects vascular repair during fracture healing.
Stabilized and non-stabilized tibia fractures were created in adult mice. Fracture tissues were collected at multiple time points during early fracture healing. Vasculature in fractured limbs was visualized by immunohistochemistry with an anti-PECAM-1 antibody on tissue sections and then quantified with stereology. Oxygen tension, vascular endothelial growth factor (VEGF) expression, and lactate accumulation at the fracture site were measured. Gene expression was compared between stabilized and non-stabilized fractures by micro-array analysis.
We found that new blood vessel formation was robust by 3 days after fracture. Quantitative analysis showed that non-stabilized fractures had higher length density and surface density than stabilized fractures at 3 days after injury, suggesting that non-stabilized fractures were more vascularized. Oximetry analysis did not detect significant difference in oxygen tension at the fracture site between stabilized and non-stabilized fractures during the first 3 days after injury. Further micro-array analysis was performed to determine the effects of mechanical stability on the expression of angiogenic factors. No significant difference in the expression of VEGFs and other angiogenic factors was detected between stabilized and non-stabilized fractures.
Mechanical instability promotes angiogenesis during early fracture healing and further research is required to determine the underlying mechanisms.
mechanical stability; fracture; angiogenesis; microarray; VEGF
IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.
Genetic manipulation reveals that Mule is vital for B cell development, proliferation, and homeostasis as a result of its ability to regulate p53 and ATM.
Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell–specific Mule knockout (BMKO) mice were generated using the Cre–LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 transactivation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho–ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apoptosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule-deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM–p53 axis to maintain B cell homeostasis under both steady-state and stress conditions.
Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1 induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40–60% in E47 but not C34 cells with 80–90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity by either a glutathione synthesis inhibitor or TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 hrs of siRNA treatment. JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1 induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.
thioredoxin; CYP2E1; HepG2 cells; oxidative stress; cell toxicity
Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.
Mer inhibitors; acute lymphoblastic leukemia; pyrazolopyrimidines; chemosensitizer
Small GTP-binding proteins function as regulators of specific intercellular fundamental biological processes. In this study, a small GTP-binding protein Rab7 gene, designated as TaRab7, was identified and characterized from a cDNA library of wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst) the wheat stripe rust pathogen. The gene was predicted to encode a protein of 206 amino acids, with a molecular mass of 23.13 KDa and an isoeletric point (pI) of 5.13. Further analysis revealed the presence of a conserved signature that is characteristic of Rab7, and phylogenetic analysis demonstrated that TaRab7 has the highest similarity to a small GTP binding protein gene (BdRab7-like) from Brachypodium distachyon. Quantitative real-time PCR assays revealed that the expression of TaRab7 was higher in the early stage of the incompatible interactions between wheat and Pst than in the compatible interaction, and the transcription level of TaRab7 was also highly induced by environmental stress stimuli. Furthermore, knocking down TaRab7 expression by virus induced gene silencing enhanced the susceptibility of wheat cv. Suwon 11 to an avirulent race CYR23. These results imply that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance.
Cerebral palsy is currently one of the major diseases that cause severe paralysis of the nervous system in children; approximately 9–30% of cerebral palsy patients are also visually impaired, for which no effective treatment is available. Bone marrow mesenchymal stem cells (BMSCs) have very strong self-renewal, proliferation, and pluripotent differentiation potentials. Therefore, autologous BMSC transplantation has become a novel method for treating cerebral palsy.
An 11-year-old boy had a clear history of dystocia and asphyxia after birth; at the age of 6 months, the family members observed that his gaze roamed and noted that he displayed a lack of attention. A brain MRI examination at the age of 7 years showed that the child had cerebral palsy with visual impairment (i.e., posterior visual pathway injury). The patient was hospitalized for 20 days and was given four infusions of intravenous autologous BMSCs. Before transplantation and 1, 6, and 12 months after transplantation, a visual evoked potential test, an electrocardiogram, routine blood tests, and liver and kidney function tests were performed.
The patient did not have any adverse reactions during hospitalization or postoperative follow-up. After discharge, the patient could walk more smoothly than he could before transplantation; furthermore, his vision significantly improved 6 months after transplantation, which was also supported by the electrophysiological examinations.
The clinical application of BMSCs is effective for improving vision in a patient with cerebral palsy combined with visual impairment.
Curation of information from bioscience literature into biological knowledge databases is a crucial way of capturing experimental information in a computable form. During the biocuration process, a critical first step is to identify from all published literature the papers that contain results for a specific data type the curator is interested in annotating. This step normally requires curators to manually examine many papers to ascertain which few contain information of interest and thus, is usually time consuming. We developed an automatic method for identifying papers containing these curation data types among a large pool of published scientific papers based on the machine learning method Support Vector Machine (SVM). This classification system is completely automatic and can be readily applied to diverse experimental data types. It has been in use in production for automatic categorization of 10 different experimental datatypes in the biocuration process at WormBase for the past two years and it is in the process of being adopted in the biocuration process at FlyBase and the Saccharomyces Genome Database (SGD). We anticipate that this method can be readily adopted by various databases in the biocuration community and thereby greatly reducing time spent on an otherwise laborious and demanding task. We also developed a simple, readily automated procedure to utilize training papers of similar data types from different bodies of literature such as C. elegans and D. melanogaster to identify papers with any of these data types for a single database. This approach has great significance because for some data types, especially those of low occurrence, a single corpus often does not have enough training papers to achieve satisfactory performance.
We successfully tested the method on ten data types from WormBase, fifteen data types from FlyBase and three data types from Mouse Genomics Informatics (MGI). It is being used in the curation work flow at WormBase for automatic association of newly published papers with ten data types including RNAi, antibody, phenotype, gene regulation, mutant allele sequence, gene expression, gene product interaction, overexpression phenotype, gene interaction, and gene structure correction.
Our methods are applicable to a variety of data types with training set containing several hundreds to a few thousand documents. It is completely automatic and, thus can be readily incorporated to different workflow at different literature-based databases. We believe that the work presented here can contribute greatly to the tremendous task of automating the important yet labor-intensive biocuration effort.
A major pathway for chronic ethanol-induced liver injury is ethanol-induced oxidant stress. Several pathways contribute to mechanisms by which ethanol induces oxidant stress. While some studies support a role for cytochrome P450 2E1 (CYP2E1), others do not. Most previous studies were conducted in the intragastric infusion model of ethanol administration. There is a need to develop oral models of significant liver injury and to evaluate the possible role of CYP2E1 in ethanol actions in such models. We evaluated chronic ethanol-induced liver injury, steatosis and oxidant stress in wild type (WT) mice, CYP2E1 knockout (KO) mice and in humanized CYP2E1 knockin (KI) mice, where the human 2E1 was added back to mice deficient in the mouse 2E1. WT mice and the CYP2E1 KO and KI mice (both provided by Dr F. Gonzalez, NCI) were fed a high fat Lieber-DeCarli ethanol liquid diet for 3 weeks; pair-fed controls received dextrose. Ethanol produced fatty liver and oxidant stress in WT mice but liver injury (transaminases, histopathology) was minimal. Ethanol-induced steatosis and oxidant stress was blunted in the KO mice (no liver injury) but restored in the KI mice. Signicant liver injury was produced in the ethanol-fed KI mice with elevated transaminases, necrosis, and increased levels of collagen type 1 and smooth muscle actin. This liver injury in the KI mice was associated with elevated oxidant stress and elevated levels of the human CYP2E1 compared to levels of the mouse 2E1 in WT mice. Activation of JNK and decreased levels of Bcl-2 and Bcl-XL were observed in the ethanol-fed KI mice compared to the other groups. Fatty liver in the WT and the KI mice was associated with lower levels of PPAR alpha and acyl CoA oxidase. No such changes were found in the ethanol-fed KO mice. These results show that CYP2E1 plays a major role in ethanol-induced fatty liver and oxidant stress. It is the absence of CYP2E1 in the KO mice responsible for the blunting of steatosis and oxidant stress since restoring the CYP2E1 restores the fatty liver and oxidant stress. Moreover, it is the human CYP2E1 which restores these effects of ethanol which suggests that results on fatty liver and oxidant stress from rodent models of ethanol intake and mouse CYP2E1 can be extrapolated to human models of ethanol intake and to human CYP2E1.
Chronic Alcohol; CYP2E1; Fatty Liver; Oxidative Stress; Hepatotoxcity
Since its release in 2000, WormBase (http://www.wormbase.org) has grown from a small resource focusing on a single species and serving a dedicated research community, to one now spanning 15 species essential to the broader biomedical and agricultural research fields. To enhance the rate of curation, we have automated the identification of key data in the scientific literature and use similar methodology for data extraction. To ease access to the data, we are collaborating with journals to link entities in research publications to their report pages at WormBase. To facilitate discovery, we have added new views of the data, integrated large-scale datasets and expanded descriptions of models for human disease. Finally, we have introduced a dramatic overhaul of the WormBase website for public beta testing. Designed to balance complexity and usability, the new site is species-agnostic, highly customizable, and interactive. Casual users and developers alike will be able to leverage the public RESTful application programming interface (API) to generate custom data mining solutions and extensions to the site. We report on the growth of our database and on our work in keeping pace with the growing demand for data, efforts to anticipate the requirements of users and new collaborations with the larger science community.
The regulation and function of autophagy and lipid metabolism have recently been reported to be reciprocally related. Macroautophagy mediates the breakdown of lipids stored in lipid droplets. An inhibition of autophagy leads to the development of a fatty liver. We evaluated the ability of CYP2E1 to modulate the effects of ethanol on lipid accumulation and autophagy in vitro. The E47 HepG2 cell which expresses CYP2E1 was treated with ethanol at 50, 100 and 150 mM for 4 or 5 days. Ethanol induced lipid accumulation and an increase of triglycerides (TG) in E47 cells to a greater extent than in control C34 cells which do not express CYP2E1. In contrast, autophagy (LC3 II/LC3 I ratio) was significantly induced by ethanol in C34 cells to a greater extent than in E47 cells. P62 was significantly increased in E47 cells after ethanol treatment. Thus, there is a reciprocal relationship between the effects of ethanol on lipid accumulation and autophagy in the CYP2E1-expressing cells. Inhibition of autophagy by 3-methyladenine (3MA), increased lipid accumulation and TG levels in C34 cells which display elevated autophagy, but enhanced lipid accumulation and TG level to a lesser extent in E47 cells which displayed lower autophagy. Ethanol induced CYP2E1 activity and oxidative stress in E47 cells compared with C34 cells. These experiments suggest that the expression of CYP2E1 may impair autophagy formation which contributes to lipid accumulation in the liver. We hypothesize that CYP2E1-induced oxidative stress promotes the accumulation of lipid droplets by ethanol and this may be responsible for the suppression of autophagy in the liver.
Ethanol; CYP2E1; ROS; HepG2 E47 cells; steatosis; autophagy
Protein complexes are a cornerstone of many biological processes and together they form various types of molecular machinery. A broad understanding of these protein complexes is crucial for revealing and building models of protein function and regulation. Pancreatic cancer is a highly lethal disease which is difficult to diagnose at early stage and even more difficult to cure. In this study, we applied a gradient clear native gel system combined with subsequent second-dimensional SDS-PAGE to separate protein complexes from cell lysates of SW1990 and PANC-1 pancreatic cancer cell lines with different degrees of differentiation. Ten heat-shock-protein- (HSP-) associated protein complexes were separated and identified, and the differentially expressed proteins related to cancers were also found, such as HSP60, protein disulfide-isomerase A4 (ERp72), and transitional endoplasmic reticulum ATPase (TER ATPase).
The mechanisms by which alcohol causes cell injury are not clear. A major mechanism is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other; however, interactions between them, have not been extensively evaluated. Increased oxidative stress from induction of CYP2E1 sensitizes hepatocytes to LPS and TNFα toxicity and oxidants, activation of inducible nitric oxide synthase and p38 and JNK MAP kinases, and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNFα-potentiated hepatotoxicity. This paper will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved including activation of the mitogen-activated kinase kinase kinase ASK-1. Decreasing either cytosolic or mitochondrial thioredoxin in HepG2 cells expressing CYP2E1 causes loss of cell viability and elevated oxidative stress via an ASK-1/JNK-dependent mechanism. We hypothesize that similar interactions occur as a result of ethanol induction of CYP2E1 and TNFα.
Finding conserved motifs in genomic sequences represents one of essential bioinformatic problems. However, achieving high discovery performance without imposing substantial auxiliary constraints on possible motif features remains a key algorithmic challenge. This work describes BAMBI—a sequential Monte Carlo motif-identification algorithm, which is based on a position weight matrix model that does not require additional constraints and is able to estimate such motif properties as length, logo, number of instances and their locations solely on the basis of primary nucleotide sequence data. Furthermore, should biologically meaningful information about motif attributes be available, BAMBI takes advantage of this knowledge to further refine the discovery results. In practical applications, we show that the proposed approach can be used to find sites of such diverse DNA-binding molecules as the cAMP receptor protein (CRP) and Din-family site-specific serine recombinases. Results obtained by BAMBI in these and other settings demonstrate better statistical performance than any of the four widely-used profile-based motif discovery methods: MEME, BioProspector with BioOptimizer, SeSiMCMC and Motif Sampler as measured by the nucleotide-level correlation coefficient. Additionally, in the case of Din-family recombinase target site discovery, the BAMBI-inferred motif is found to be the only one functionally accurate from the underlying biochemical mechanism standpoint. C++ and Matlab code is available at http://www.ee.columbia.edu/~guido/BAMBI or http://genomics.lbl.gov/BAMBI/.