The purpose of this study was to observe the effect and feasibility of hyperthermia and the influence of heat on surrounding organs in a VX2 rabbit liver model exposed to an alternating magnetic field after embolization with ferromagnetic nanoparticles.
Forty rabbits containing implanted hepatic VX2 carcinomas were divided into four groups, each containing ten rabbits. Fourteen days after tumor transplantation, we opened the abdomen to observe the size and shape of the tumor. A transfemoral retrograde approach was then used for hepatic arterial catheterization in groups B, C, and D to perform angiography and embolization. The next day, three rabbits in group B and all rabbits in group D were exposed to an alternating magnetic field, and the temperature was recorded simultaneously in the center of the tumor, at the edge of the tumor, and in the normal liver parenchyma. On day 28, all animals was euthanized to observe changes in the implanted liver tumor and the condition of the abdomen. A pathologic examination was also done.
Before surgery, there was no significant difference in tumor volume between the four groups. Three different temperature points (cen ter of the tumor, edge of the tumor, and in the normal liver parenchyma) of group B under an alternating magnetic field were 37.2°C ± 1.1°C, 36.8°C ± 1.2°C, and 36.9°C ± 2.1°C, none of which were significantly different from pretreatment values. Three points basal temperature in group D showed no significant difference (F = 1.038, P = 0.413). Seven to 26 minutes after hyperthermia, the temperature at the center of the tumor and at the edge of the tumor in group D was significantly different from the corresponding points in group B and from normal liver tissue in group D (FB–D center = 5.431, PB–D center = 0.041, FB–D edge = 9.744, PB–D edge = 0.011; FD = 8.379, PD = 0.002). The highest temperature recorded at the rim of the tumor was 46°C in group D. Fourteen days later, the tumor volume in the four groups was group A 31.4 ± 20.6 cm3, group B 26.7 ± 18.2 cm3, group C 28.7 ± 9.1 cm3, and group D 25.8 ± 13.9 cm3, with no significant difference found between the groups (F = 0.218, P = 0.883). The increase in tumor volume was greatest in group A and least in group D, while that in groups B and D was similar.
It is feasible to treat a VX2 tumor in an alternating magnetic field after embolization with magnetic nanoparticles without a significant effect on the surrounding normal liver parenchyma.
hyperthermia; ferromagnetic nanoparticles; Lipiodol®; hepatocellular carcinoma; animal model
The goal of the current study was to evaluate whether CYP2E1 plays a role in binge-ethanol induced steatosis and if autophagy impacts CYP2E1-mediated hepatotoxicity, oxidative stress and fatty liver formation produced by ethanol. Wild type (WT), CYP2E1 knockin (KI) and CYP2E1 knockout (KO) mice were gavaged with 3g/kg body wt ethanol twice a day for four days. This treatment caused fatty liver, elevation of CYP2E1 and oxidative stress in WT and KI mice but not KO mice. Autophagy was impaired in ethanol-treated KI mice compared to KO mice as reflected by a decline in the LC3-II/LC3-I ratio and lower total LC-3 and Beclin-1 levels coupled to increases in P62, pAKT/AKT and mTOR. Inhibition of macroautophagy by administration of 3-methyladenine enhanced the binge ethanol hepatotoxicity, steatosis and oxidant stress in CYP2E1 KI, but not CYP2E1 KO mice. Stimulation of autophagy by rapamycin blunted the elevated steatosis produced by binge ethanol. Treatment of HepG2 E47 cells which express CYP2E1 with 100 mM ethanol for 8 days increased fat accumulation and oxidant stress but decreased autophagy. Ethanol had no effect on these reactions in HepG2 C34 cells which do not express CYP2E1. Inhibition of autophagy elevated ethanol toxicity, lipid accumulation and oxidant stress in the E47, but not C34 cells. The antioxidant N-acetylcysteine, and CYP2E1 inhibitor chlormethiazole blunted these effects of ethanol. These results indicate that CYP2E1 plays an important role in binge ethanol-induced fatty liver. We propose that CYP2E1-derived reactive oxygen species inhibit autophagy, which subsequently causes accumulation of lipid droplets. Inhibition of autophagy promotes binge ethanol induced hepatotoxicity, steatosis and oxidant stress via CYP2E1.
Binge ethanol; Autophagy; Steatosis; hepatotoxicity; CYP2E1; Oxidative Stress
Binge alcohol drinking induces hepatic steatosis. Recent studies showed that chronic ethanol-induced fatty liver was, at least in part, CYP2E1 dependent. The mechanism of acute alcohol induced steatosis and whether CYP2E1 plays any role is still unclear. Increasing oxidative stress by alcohol can activate the JNK MAP kinase signaling pathway, suggesting that JNK might be a target for prevention of alcohol induced steatosis. We used CYP2E1 knockout (KO) mice, a JNK inhibitor, and JNK1 or JNK2 knockout mice to test the role of CYP2E1, JNK, and the individual role of JNK1 and JNK2 in acute alcohol-induced steatosis. In wild type (WT) mice, acute alcohol activates CYP2E1 and increases oxidative stress, which reciprocally increases activation of the JNK signaling pathway. Acute alcohol–induced fatty liver and oxidative stress was blunted in CYP2E1 KO mice and by the JNK inhibitor in WT mice. The antioxidant N-acetylcysteine decreased the acute alcohol induced oxidative stress, activation of JNK and the steatosis but not the activation of CYP2E1. Acute alcohol decreased autophagy and increased expression of SREBP, effects blocked by the JNK inhibitor. Acute alcohol–induced fatty liver was the same in either JNK1 or JNK2 KO mice as WT mice, thus either JNK1 or JNK2 per se is sufficient in induction of steatosis by acute alcohol.
acute alcohol elevation of CYP2E1, oxidative stress and activation of JNK interact to lower autophagy and increase lipogenic SREBP resulting in fatty liver.
oxidative stress; CYP2E1; JNK; autophagy; liver steatosis
A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death.
In all multicellular plants and animals, cells are continuously dying and being replaced. There are a number of different types of cell death, but two of the best studied are apoptosis and necrosis. Apoptosis, sometimes referred to as ‘cell suicide’, is a form of programmed cell death that is generally beneficial to the organism. Necrosis, however, occurs whenever cells are damaged—for example, due to a lack of oxygen—and can trigger harmful inflammation in surrounding tissue. Although the processes leading up to apoptosis and necrosis are very different, they both involve regulated changes in mitochondria—the organelles that supply cells with chemical energy.
Mitochondria have a distinctive appearance, being enclosed by two membranes, the innermost of which is highly folded. During apoptosis, large pores form in the outer membranes of mitochondria. These pores are generated by two proteins—Bax and Bak—and they enable the mitochondrion to release proteins that activate processes involved in apoptosis. Pores also form in the mitochondrial membrane during necrosis. However, these mitochondrial permeability transition pores (MPTPs) occur simultaneously in both the inner and outer membranes and are thought to lead to swelling and rupture of mitochondria.
Now, Karch et al. have shown that Bax and Bak are also involved in the formation of these permeability pores that underlie necrosis. When mouse cells that had been genetically modified to lack Bak and Bax were grown in cell culture, they were found to be resistant to substances that normally induce necrosis. Instead, their mitochondria continued to function normally, suggesting that MPTPs cannot form in the absence of Bak and Bax.
Karch et al. then generated mice with heart cells that lack Bax and Bak, and deprived their hearts of oxygen to simulate a heart attack. Compared to normal mice, the genetically modified animals experienced less damage to their heart muscle, suggesting that the absence of Bax and Bak prevents cell death due to necrosis. If Bax and Bak are involved in both apoptosis and necrosis, inhibiting them could be a powerful therapeutic approach for preventing all forms of cell death during heart attacks or in certain degenerative diseases.
cell death; mitochondria; necrosis; Mouse
The C-terminal domain (CTD) of eukaryotic RNA polymerase II is an essential regulator for RNA polymerase II-mediated transcription. It is composed of multiple repeats of a consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. Ser2 and Ser5 are the major phosphorylation sites in vivo while Pro3 and Pro6 can adopt either cis or trans conformations. CTD regulation of transcription is mediated both by phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases.
Experimental evidence of cross-talk between these two regulatory mechanisms has been elusive. Pin1 is a highly conserved phosphorylation-specific peptidyl-prolyl isomerase (PPIase) that recognizes the phospho-Ser/Thr (pSer/Thr)-Pro motif with CTD as one of its primary substrates in vivo. In the present study, we provide structural snapshots and kinetic evidence that support the concept of cross-talk between prolyl isomerization and phosphorylation. We determined the structures of Pin1 bound with two substrate isosteres that mimic peptides containing pSer/Thr-Pro motifs in cis or trans conformations. The results unequivocally demonstrate the utility of both cis- and trans-locked alkene isosteres as close geometric mimics of peptides bound to a protein target. Building on this result, we identified a specific case in which Pin1 differentially affects the rate of dephosphorylation catalyzed by two phosphatases (Scp1 and Ssu72) that target the same serine residue in the CTD heptad repeat but that have different preferences for the isomerization state of the adjacent proline residue. These data exemplify for the first time how modulation of proline isomerization can kinetically impact signal transduction in transcription regulation.
In this study, the effect of ordered rod-like FA coatings of metal discs on adipose-derived stem cell (ASC)’s growth, differentiation and mineralization was studied in vitro; and their mineral inductive effects in vivo. After 3 and 7 days, the cell number on the metal surfaces was significantly higher than those on the ordered and disordered FA surfaces. However, after 4 weeks much greater amounts of mineral formation was induced on the two FA surfaces with and even without osteogenesis induction. The osteogenic profiles showed the up regulation of a set of pro-osteogenic transcripts and bone mineralization phenotypic markers when the ASCs were grown on FA surfaces compared to metal surfaces at 7 and 21 days. In addition to BMP and TGFβ signaling pathways, EGF and FGF pathways also appeared to be involved in ASC differentiation and mineralization. In vivo studies showed accelerated and enhanced mineralized tissue formation integrated within ordered FA coatings. After 5 weeks, over 80 % of the ordered FA coating was integrated with a mineralized tissue layer covering the implants. Both the intrinsic properties of the FA crystals and the topography of the FA coating appeared to dominate the cell differentiation and mineralization process.
Fluorapatite; differentiation; mineralization; gene profile; stem cells
In this work, a novel combined system by Fe-Ag or Fe-Ni nanoparticles and microwave (MW) radiation were used for the debromination of tetrabromobisphenol A (TBBPA) in aqueous solutions. Core-shell structure bimetallic nanoparticles were prepared by replacement reaction in liquid phase and then characterized by X-ray diffraction, transmission electron microscopy, and X-ray photoelectron spectroscopy techniques. MW radiation can enhance the degradation of TBBPA by Fe-Ag or Fe-Ni observably. The rate of reduction reactions for bimetallic nanoparticles under MW were first compared with those under conventional heating conditions. Compared with nano-iron, the deposition of Ag or Ni also accelerated the debromination, and Fe-Ag was more reactive than Fe-Ni toward TBBPA reduction. Removal efficiencies increased with increasing Fe-Ag dosage and MW energy level. Major reduction products of TBBPA identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were tri-BBPA, di-BBPA, mono-BBPA, and BPA, which indicated a stepwise debromination process. It provides an effective technology for TBBPA laden wastewater treatment.
debromination; TBBPA; bimetallic; nanoparticles; microwave radiation
Double-side replication of polymer substrates is beneficial to the design and the fabrication of 3-demensional devices. The ultrasonic embossing method is a promising, high efficiency and low cost replication method for thermoplastic substrates. It is convenient to apply silicon molds in ultrasonic embossing, because microstructures can be easily fabricated on silicon wafers with etching techniques. To reduce the risk of damaging to silicon molds and to improve the replication uniformity on both sides of the polymer substrates, thermal assisted ultrasonic embossing method was proposed and tested. The processing parameters for the replication of polymethyl methacrylate (PMMA), including ultrasonic amplitude, ultrasonic force, ultrasonic time, and thermal assisted temperature were studied using orthogonal array experiments. The influences of the substrate thickness, pattern style and density were also investigated. The experiment results show that the principal parameters for the upper and lower surface replication are ultrasonic amplitude and thermal assisted temperature, respectively. As to the replication uniformity on both sides, the ultrasonic force has the maximal influence. Using the optimized parameters, the replication rate reached 97.5% on both sides of the PMMA substrate, and the cycle time was less than 50 s.
Clustering is an important data processing tool for interpreting microarray data and genomic network inference. In this article, we propose a clustering algorithm based on the hierarchical Dirichlet processes (HDP). The HDP clustering introduces a hierarchical structure in the statistical model which captures the hierarchical features prevalent in biological data such as the gene express data. We develop a Gibbs sampling algorithm based on the Chinese restaurant metaphor for the HDP clustering. We apply the proposed HDP algorithm to both regulatory network segmentation and gene expression clustering. The HDP algorithm is shown to outperform several popular clustering algorithms by revealing the underlying hierarchical structure of the data. For the yeast cell cycle data, we compare the HDP result to the standard result and show that the HDP algorithm provides more information and reduces the unnecessary clustering fragments.
The aim of this study is to explore signal transducer and activator of transcription 3 (STAT3) expression in breast cancer and to analyze the detailed mechanism that STAT3 contributes to the progression of breast cancer.
We retrospectively analyzed the clinicopathologic characteristics and overall survival (OS) of 140 breast cancer patients after curative surgery, and detected STAT3 expression, phosphorylated STAT3 (pSTAT3) expression, Ki-67 expression, vascular endothelial growth factor (VEGF)-C and -D expression in breast cancer tissues, and adjacent nontumor tissues. Survival analysis and relationship analysis were adopted for demonstrated the important mechanism of STAT3 contribution to progression of breast cancer.
STAT3 expression, pSTAT3 expression, Ki-67 expression, VEGF-C expression, and VEGF-D expression in breast cancer tissues were significantly higher than those in adjacent nontumor tissues, respectively. With survival analysis, only number of lymph node metastasis (N stage) was identified as the independent predictors of the OS of breast cancer patients. Besides, we demonstrated there was the most prominent correlation between STAT3 expression and lymph node metastasis in breast cancer tissues by using the multinominal regression method.
STAT3, a poor survival biomarker potential association with lymph node metastasis, was suitable for predication the OS of breast cancer patients after curative resection.
Breast neoplasms; Neoplasm metastasis; Prognosis; STAT3 transcription factor
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