Background & Aims
Integrity of the intestinal epithelium is required for nutrition absorption and defense against pathogens. Claudins are cell adhesion molecules that localize at tight junctions (TJs); many are expressed in the intestinal tract, but little is known about their functions. Claudin-7 is unique in that it has a stronger basolateral membrane distribution than other claudins, which localize primarily to apical TJs in the intestinal epithelium. We investigated the basolateral functions of claudin-7 and assessed the effects of disruption of Cldn7 in intestines of mice.
We generated Cldn7−/− mice and examined their intestines by histology, molecular and cellular biology, and biochemistry approaches. We carried out gene silencing experiments in epithelial cell lines using small interfering (si)RNAs.
The Cldn7−/− mice had severe intestinal defects that included mucosal ulcerations, epithelial cell sloughing, and inflammation. Intestines of Cldn7−/− mice produced significantly higher levels of cytokines, the NF-κB p65 subunit, and COX-2; they also upregulated expression of matrix metalloproteinases (MMPs)-3 and -7. siRNA in epithelial cell lines demonstrated that the increased expression of MMP-3 resulted directly from claudin-7 depletion, whereas that of MMP-7 resulted from inflammation. Electron microscopy analysis showed that intestines of Cldn7−/− mice had intercellular gaps below TJs and cell-matrix loosening. Deletion of Cldn7 reduced expression and altered localization of the integrin α2 subunit; disrupted formation of complexes of claudin-7, integrin α2, and claudin-1 that normally form in epithelial basolateral compartments of intestines.
In mice, claudin-7 has non-TJ functions, including maintenance of epithelial cell–matrix interactions and intestinal homeostasis.
Mucosal integrity; epithelial barrier; mouse model; permeability; IBD
Tight junctions are the most apical component of the junctional complex critical for epithelial cell barrier and polarity functions. Although its disruption is well documented during cancer progression such as epithelial-mesenchymal transition, molecular mechanisms by which tight junction integral membrane protein claudins affect this process remain largely unknown. In this report, we found that claudin-7 was normally expressed in bronchial epithelial cells of human lungs but was either downregulated or disrupted in its distribution pattern in lung cancer. To investigate the function of claudin-7 in lung cancer cells, we transfected claudin-7 cDNA into NCI-H1299, a human lung carcinoma cell line that has no detectable claudin-7 expression. We found that claudin-7 expressing cells showed a reduced response to hepatocyte growth factor (HGF) treatment, were less motile, and formed fewer foot processes than the control cells did. In addition, cells transfected with claudin-7 dramatically decreased their invasive ability after HGF treatment. These effects were mediated through the MAPK signaling pathway since the phosphorylation level of ERK1/2 was significantly lower in claudin-7 transfected cells than in control cells. PD98059, a selective inhibitor of ERK/MAPK pathway, was able to block the motile effect. Claudin-7 formed stable complexes with claudin-1 and -3 and was able to recruit them to the cell-cell junction area in claudin-7 transfected cells. When control and claudin-7 transfected cells were inoculated into nude mice, claudin-7 expressing cells produced smaller tumors than the control cells. Taken together, our study demonstrates that claudin-7 inhibits cell migration and invasion through ERK/MAPK signaling pathway in response to growth factor stimulation in human lung cancer cells.
Claudin-7; Tight junctions; Cell migration and invasion; ERK1/2; Human lung cancer cells
We previously demonstrated that macrophage LRP1 deficiency increases atherosclerosis despite anti-atherogenic changes including decreased uptake of remnants and increased secretion of apoE. Thus, our objective was to determine whether the atheroprotective effects of LRP1 require interaction with apoE, one of its ligands with multiple beneficial effects.
Methods and Results
We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE−/−MΦLRP1−/−) and in LDLR−/− mice reconstituted with apoE−/−MΦLRP1−/− bone marrow. The combined absence of apoE and LRP1 promoted atherogenesis more than did macrophage apoE deletion alone in both apoE-producing LDLR−/− (+88%) and apoE−/− mice (+163%). The lesions of both mouse models with apoE−/−LRP1−/− macrophages had increased macrophage content. In vitro, apoE and LRP1 additively inhibit macrophage apoptosis. Furthermore, there was excessive accumulation of apoptotic cells in lesions of both LDLR−/− (+110%) and apoE−/−MΦLRP1−/− mice (+252%). The apoptotic cell accumulation was partially due to decreased efferocytosis as the ratio of free to cell-associated apoptotic nuclei was 3.5-fold higher in lesions of apoE−/−MΦLRP1−/− versus apoE−/− mice. Lesion necrosis was also increased (6-fold) in apoE−/−MΦLRP1−/− versus apoE−/− mice. Compared to apoE−/− mice, the spleens of apoE−/−MΦLRP1−/− mice contained 1.6 – and 2.4 –fold more total and Ly6-Chigh monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-Chigh and CCR2+ cells in lesions of apoE−/−MΦLRP1−/− versus apoE−/− mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes.
LRP1 exerts anti-atherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.
atherosclerosis; apoE; LRP1; Ly6-Chigh monocytes; apoptosis
Multiple cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem Cell Factor (SCF) is a key niche component that maintains HSCs. Using Scfgfp knock-in mice we found Scf was primarily expressed by perivascular cells throughout bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, Nestin-Cre, or Nestin-CreER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or Leptin receptor (Lepr)-expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.
10-Hydroxycamptothecin (HCPT) elicits strong anti-cancer effects and is less toxic making it widely used in recent clinical trials. However, its low solubility limits its application as an effective anti-cancer therapy. In this study, we investigate the hypothesis that the unique water dispersible oleic acid-Triton X-100-coated Fe3O4 nanoparticles loaded with HCPT disrupt epithelial cell-cell junctions and induce human lung cancer cell apoptosis through caspase-8 pathway. We characterized the HCPT-loaded nanoparticles and determined their effects on lung cancer cell viability and apoptosis by using immunofluorescence light microscopy and SDS-PAGE/immunoblots. We found that HCPT-loaded nanoparticles elicited an anti-proliferative effect in a dose-dependent manner. HCPT-loaded nanoparticles reduced the expression of cell-cell junction protein claudins, E-cadherin, and ZO-1, and transmission electron microcopy demonstrated a disrupted tight junction ultrastructure. Transepithelial electric resistance was also reduced indicating the reduction of tight junction functions. HCPT increased phosphorylation of p38 and SAPK/Jun kinase while it showed no effects on p42/44 MAP kinase. Compared with void Fe3O4 nanoparticles or HCPT drug alone, HCPT drug-loaded nanoparticles evoked synergistic effects by increasing cell apoptosis with enhanced activation of caspase-8 pathway. Therefore, our current study highlights the potential of HCPT drug-loaded nanoparticles as a chemotherapeutic agent for increasing anti-cancer drug efficacy.
Nanoparticles; anti-cancer drug; apoptosis; HCC827 cells; caspase-8; tight junctions
The adipocyte/macrophage fatty acid-binding proteins, aP2 (FABP4) and Mal1 (FABP5), are intracellular lipid chaperones that modulate systemic glucose metabolism, insulin sensitivity and atherosclerosis. Combined deficiency of aP2 and Mal1 has been shown to reduce the development of atherosclerosis, but the independent role of macrophage Mal1 expression in atherogenesis remains unclear.
Methods and Results
Here we transplanted wild type (WT), Mal1-/- or aP2-/- bone marrow into LDLR-/- mice and fed them a Western diet for 8 weeks. Mal1-/-→LDLR-/- mice had significantly reduced (36%) atherosclerosis in the proximal aorta compared to control WT→LDLR-/- mice. Interestingly, peritoneal macrophages isolated from Mal1-deficient mice displayed increased PPARγ activity and up-regulation of a PPARγ-related cholesterol trafficking gene CD36. Mal1-/- macrophages showed suppression of inflammatory genes such as COX-2 and IL6. Mal1-/-→LDLR-/- mice had significantly decreased macrophage numbers in the aortic atherosclerotic lesions compared to WT→LDLR-/- mice, suggesting that monocyte recruitment may be impaired. Indeed, blood monocytes isolated from Mal1-/-→LDLR-/- mice on a high-fat diet had decreased CC chemokine receptor 2 (CCR2) gene and protein expression levels compared to WT monocytes.
Taken together our results demonstrate that Mal1 plays a pro-atherogenic role by suppressing PPARγ activity, which increases expression of CCR2 by monocytes promoting their recruitment to atherosclerotic lesions.
Atherosclerosis; Macrophages; PPARγ; CD36; CCR2 receptor
Predominant components in electro- or magneto-encephalography (EEG/MEG) are scalp projections of synchronized neuronal electrical activity distributed over cortical structures. Reconstruction of cortical sources underlying EEG/MEG can thus be achieved with the use of the cortical current density (CCD) model. We have developed a sparse electromagnetic source imaging method based on the CCD model, named as the variation-based cortical current density (VB-SCCD) algorithm, and have shown that it has much enhanced performance in reconstructing extended cortical sources in simulations (Ding, 2009). The present study aims to evaluate the performance of VB-SCCD, for the first time, using experimental data obtained from six participants. The results indicate that the VB-SCCD algorithm is able to successfully reveal spatially distributed cortical sources behind motor potentials induced by visually cued repetitive finger movements, and their dynamic patterns, with millisecond resolution. These findings of motor sources and cortical systems are supported by the physiological knowledge of motor control and evidence from various neuroimaging studies with similar experiments. Furthermore, our present results indicate the improvement of cortical source resolvability of VB-SCCD, as compared with two other classical algorithms. The proposed solver embedded in VB-SCCD is able to handle large-scale computational problems, which makes the use of high-density CCD models possible and, thus, reduces model misspecifications. The present results suggest that VB-SCCD provides high resolution source reconstruction capability and is a promising tool for studying complicated dynamic systems of brain activity for basic neuroscience and clinical neuropsychiatric research.
Sparse electromagnetic source imaging; cortical current density model; EEG; VB-SCCD; motor potential; thresholding; large-scale computation
Injuries to articular cartilage result in the development of lesions that form on the surface of the cartilage. Such lesions are associated with articular cartilage degeneration and osteoarthritis. The typical injury response often causes collateral damage, primarily an effect of inflammation, which results in the spread of lesions beyond the region where the initial injury occurs.
Results and discussion
We present a minimal mathematical model based on known mechanisms to investigate the spread and abatement of such lesions. The first case corresponds to the parameter values listed in Table 1, while the second case has parameter values as in Table 2. In particular we represent the "balancing act" between pro-inflammatory and anti-inflammatory cytokines that is hypothesized to be a principal mechanism in the expansion properties of cartilage damage during the typical injury response. We present preliminary results of in vitro studies that confirm the anti-inflammatory activities of the cytokine erythropoietin (EPO). We assume that the diffusion of cytokines determine the spatial behavior of injury response and lesion expansion so that a reaction diffusion system involving chemical species and chondrocyte cell state population densities is a natural way to represent cartilage injury response. We present computational results using the mathematical model showing that our representation is successful in capturing much of the interesting spatial behavior of injury associated lesion development and abatement in articular cartilage. Further, we discuss the use of this model to study the possibility of using EPO as a therapy for reducing the amount of inflammation induced collateral damage to cartilage during the typical injury response.
Model Parameter Values for Results in Figure 5
Model Parameter Values for Results in Figure 6
The mathematical model presented herein suggests that not only are anti-inflammatory cy-tokines, such as EPO necessary to prevent chondrocytes signaled by pro-inflammatory cytokines from entering apoptosis, they may also influence how chondrocytes respond to signaling by pro-inflammatory cytokines.
This paper has been reviewed by Yang Kuang, James Faeder and Anna Marciniak-Czochra.
SLE is an autoimmune inflammatory disease in which various pro- and anti-inflammatory cytokines, including TGF-β, IL-10, BAFF, IL-6, IFN-α, IFN-γ, IL-17, and IL-23, play crucial pathogenic roles. Virtually, all these cytokines can be generated by both innate and adaptive immune cells and exert different effects depending on specific local microenvironment. They can also interact with each other, forming a complex network to maintain delicate immune homeostasis. In this paper, we elaborate on the abnormal secretion and functions of these cytokines in SLE, analyze their potential pathogenic roles, and probe into the possibility of them being utilized as targets for therapy.
The common carp is one of the most important cultivated species in the world of freshwater aquaculture. The cultivation of this species is particularly productive due to its high skeletal muscle mass; however, the molecular mechanisms of skeletal muscle development in the common carp remain unknown. It has been shown that a class of non-coding ∼22 nucleotide RNAs called microRNAs (miRNAs) play important roles in vertebrate development. They regulate gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNAs and thereby cause translational repression or mRNA destabilization. Intriguingly, the role of miRNAs in the skeletal muscle development of the common carp remains unknown. In this study, a small-RNA cDNA library was constructed from the skeletal muscle of the common carp, and Solexa sequencing technology was used to perform high throughput sequencing of the library. Subsequent bioinformatics analysis identified 188 conserved miRNAs and 7 novel miRNAs in the carp skeletal muscle. The miRNA expression profiling showed that, miR-1, miR-133a-3p, and miR-206 were specifically expressed in muscle-containing organs, and that miR-1, miR-21, miR-26a, miR-27a, miR-133a-3p, miR-206, miR-214 and miR-222 were differentially expressed in the process of skeletal muscle development of the common carp. This study provides a first identification and profiling of miRNAs related to the muscle biology of the common carp. Their identification could provide clues leading towards a better understanding of the molecular mechanisms of carp skeletal muscle development.
Tight junctions (TJs) are the most apical component of the junctional complexes in mammalian epithelial cells and form selective paracellular barriers restricting the passage of solutes and ions across the epithelial sheets. Claudins, a TJ integral membrane protein family, play a critical role in regulating paracellular barrier permeability. In the in vitro cell culture system, transepithelial electrical resistance (TER) measurement and the flux of radioisotope or fluorescent labeled molecules with different sizes have been widely used to determine the TJ barrier function. In the in vivo system, the tracer molecule Sulfo-NHS-Biotin was initially used in Xenopus embryos system and subsequently was successfully applied to a number of animal tissues in situ and in different organisms under the experimental conditions to examine the functional integrity of TJs by several laboratories. In this chapter, we will describe the detailed procedures of applying biotin as a paracellular tracer molecule to different in vivo systems to assay TJ barrier function.
Tight Junctions; Permeability barrier; Biotin tracer; Claudin-7; Epithelial cells
To determine the activation of MAP kinases in and around cartilage subjected to mechanical damage and to determine the effects of their inhibitors on impaction induced chondrocyte death and cartilage degeneration.
The phosphorylation of MAP kinases was examined with confocal microscopy and immunoblotting. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan loss were determined with fluorescent microscopy and DMMB assay. The expression of catabolic genes at mRNA levels was examined with quantitative real time PCR.
Early p38 activation was detected at 20 min and 1 hr post-impaction. At 24 hr, enhanced phosphorylation of p38 and ERK1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and proteoglycan losses. Quantative Real-time PCR analysis revealed that blunt impaction significantly up-regulated MMP-13, TNF-α, and ADAMTS-5 expression.
These findings implicate p38 and ERK MAPKs in the post injury spread of cartilage degeneration and suggest that the risk of PTOA following joint trauma could be decreased by blocking their activities, which might be involved in up-regulating expressions of MMP-13, ADAMTS-5, and TNF-α.
Impact; Cartilage damage; MAP kinases; Inhibitors; Post-traumatic Osteoarthritis
The balance between apoptosis susceptibility and efferocytosis of macrophages is central to plaque remodeling and inflammation. LRP1 and its ligand, apoE, have been implicated in efferocytosis and apoptosis in some cell types. We investigated the involvement of the macrophage LRP1/apoE axis in controlling plaque apoptosis and efferocytosis.
Method and Results
LRP1-/- macrophages displayed nearly 2-fold more TUNEL positivity compared to WT cells in the presence of DMEM alone or with either LPS or oxidized LDL. The survival kinase, pAkt, was barely detectable in LRP1-/- cells, causing decreased pBad and increased cleaved caspase-3. Regardless of the apoptotic stimulation and degree of cell death, LRP1-/- macrophages displayed enhanced inflammation with increased IL-1β, IL-6, and TNFα expression. Efferocytosis of apoptotic macrophages was reduced by 60% in LRP1-/- versus WT macrophages despite increased apoE expression by both LRP1-/- phagocytes and WT apoptotic cells. Compared to WT macrophage lesions, LRP1-/- lesions had 5.7-fold more necrotic core with more dead cells not associated with macrophages.
Macrophage LRP1 deficiency increases cell death and inflammation by impairing pAkt activation and efferocytosis. Increased apoE expression in LRP1-/- macrophages suggests that the LRP1/apoE axis regulates the balance between apoptosis and efferocytosis thereby preventing necrotic core formation.
LRP1; apoptosis; efferocytosis; apolipoprotein E; necrosis; inflammation
Electro- or magnetoencephalography (EEG/MEG) are of the utmost advantage in studying transient neuronal activity and its timing with respect to behavior in the working human brain. Direct localization of the neural substrates underlying EEG/MEG is commonly achieved by modeling neuronal activity as dipoles. However, the success of neural source localization with the dipole model has only been demonstrated in relatively simple localization tasks owing to the simplified model and its insufficiency in differentiating cortical sources with different extents. It would be of great interest to image complex neural activation with multiple sources of different cortical extensions directly from EEG/MEG. We have investigated this crucial issue by adding additional parameters to the dipole model, leading to the multipole model to better represent the extended sources confined to the convoluted cortical surface. The localization of multiple cortical sources is achieved by the use of the subspace source localization method with the multipole model. Its performance is evaluated with simulated data as compared with the dipole model, and further illustrated with the real data obtained during visual stimulations in human subjects. The interpretation of the localization results is fully supported by our knowledge about their anatomic locations and functional magnetic resonance imaging (fMRI) data in the same experimental setting. Methods for estimating multiple neuronal sources at cortical areas will facilitate our ability to characterize the cortical electrical activity from simple early sensory components to more complex networks, such as in visual, motor and cognitive tasks.
source model; cortical source; extent; electroencephalography; multipole
Heterotrimeric GTP-binding proteins, which consist of Gα, Gβ, and Gγ subunits, play important roles in transducing extracellular signals perceived by cell surface receptors into intracellular physiological responses. In addition to a single prototypical Gα protein (GPA1), Arabidopsis has three unique Gα-like proteins, known as XLG1, XLG2, and XLG3, that have been found to be localized in nuclei, although their functions and mode of action remain largely unknown. Through a transcriptomic analysis, we found that XLG2 and XLG3 were rapidly induced by infection with the bacterial pathogen Pseudomonas syringae, whereas the XLG1 transcript level was not affected by pathogen infection. A reverse genetic screen revealed that the xlg2 loss-of-function mutation causes enhanced susceptibility to P. syringae. Transcriptome profiling revealed that the xlg2 mutation affects pathogen-triggered induction of a small set of defense-related genes. However, xlg1 and xlg3 mutants showed no difference from wild-type plants in resistance to P. syringae. In addition, the xlg2 xlg3 double mutant and the xlg1 xlg2 xlg3 triple mutant were not significantly different from the xlg2 single mutant in the disease resistance phenotype, suggesting that the roles of XLG1 and XLG3 in defense, if any, are less significant than for XLG2. Constitutive overexpression of XLG2 leads to the accumulation of abnormal transcripts from multiple defense-related genes. Through co-immunoprecipitation assays, XLG2 was found to interact with AGB1, the sole Gβ subunit in Arabidopsis, which has previously been found to be a positive regulator in resistance to necrotrophic fungal pathogens. However, no significant difference was found between three xlg single mutants, the xlg2 xlg3 double mutant, the xlg triple mutant, and wild-type plants in resistance to the necrotrophic fungal pathogens Botrytis cinerea or Alternaria brassicicola. These results suggest that XLG2 and AGB1 are components of a G-protein complex different from the prototypical heterotrimeric G-protein and may have distinct functions in modulating defense responses.
Defense responses; disease resistance; plant–microbe interactions; Arabidopsis; G-protein
Prostagladin (PG) E2, a major product of activated macrophages, has been implicated in atherosclerosis and plaque rupture. The PGE2 receptors, EP2 and EP4, are expressed in atherosclerotic lesions and are known to inhibit apoptosis in cancer cells. To examine the roles of macrophage EP4 and EP2 in apoptosis and early atherosclerosis, fetal liver cell transplantation was used to generate LDLR−/− mice chimeric for EP2−/− or EP4−/− hematopoietic cells. After 8-weeks on a Western diet, EP4−/− → LDLR−/− mice, but not EP2−/− → LDLR−/− mice, had significantly reduced aortic atherosclerosis with increased apoptotic cells in the lesions. EP4−/− peritoneal macrophages had increased sensitivity to pro-apoptotic stimuli, including palmitic acid and free cholesterol loading, which was accompanied by suppression of activity of p-Akt, p-Bad and NF-kB-regulated genes. Thus, EP4 deficiency inhibits the PI3K/Akt and NF-kB pathways compromising macrophage survival and suppressing early atherosclerosis, identifying macrophage EP4 signaling pathways as molecular targets for modulating the development of atherosclerosis.
Atherosclerosis; Cyclooxygenase; EP4 signaling; NF-κB; Macrophages; Apoptosis
It is desirable to estimate both location and extent information of epileptogenic zones from noninvasive EEG. In the present study, we use a subspace source localization method, i.e. FINE, combined with a local thresholding technique to achieve such tasks. We have evaluated the performance of this method in interictal spikes from three pediatric patients with medically intractable partial epilepsy. The present results suggest that the thresholded subspace correlation, which is obtained from FINE scanning, is a favorable marker, which implies the extents of current sources associated with epileptic activities. Our findings were validated through comparison to invasive ECoG recordings during interictal spikes. The surgical resections in these three patients are well correlated with the epileptogenic zones identified from both EEG sources and ECoG potential distributions. The value of the proposed noninvasive technique for estimating epileptiform activity was supported by satisfactory surgery outcomes.
source localization; extent; EEG; ECoG; FINE; epilepsy; interictal; MRI; surgical outcome
We have developed a new L1-norm based generalized minimum norm estimate (GMNE) and have fully characterized the concept of sparseness regularization inherited in the proposed algorithm, which is termed as sparse source imaging (SSI). The new SSI algorithm corrects inaccurate source field modeling in previously reported L1-norm GMNEs and proposes that sparseness a priori should only be applied to the regularization term, not to the data term in the formulation of the regularized inverse problem. A new solver to the newly developed SSI has been adopted and known as the second order cone programming (SOCP). The new SSI is assessed by a series of simulations and then evaluated using somatosensory evoked potential (SEP) data with both scalp and subdural recordings in a human subject. The performance of SSI is compared with other L1-norm GMNEs and L2-norm GMNEs using three evaluation metrics, i.e. localization error, orientation error, and strength percentage. The present simulation results indicate that the new SSI has significantly improved performance in all evaluation metrics, especially in the metric of orientation error. L2-norm GMNEs show large orientation errors because of the smooth regularization. The previously reported L1-norm GMNEs show large orientation errors due to the inaccurate source field modeling. The SEP source imaging results indicate that SSI has the best accuracy in the prediction of subdural potential field as validated by direct subdural recordings. The new SSI algorithm is also applicable to MEG source imaging.
sparse source imaging; source field modeling; sparseness regularization; EEG; GMNE; L1-norm; SCOP; LP
Genetic studies have demonstrated an important role for proprotein convertase subtilisin/kexin type 9 (PCSK9) as a determinant of plasma cholesterol levels. However, the underlying molecular mechanism is not completely understood. To this end, we have generated a mammalian cell expression system for human PCSK9 and its mutants and produced transgenic mice expressing human PCSK9. HEK293T cells transfected with the human PCSK9 DNA construct expressed and secreted PCSK9 and displayed decreased LDLR levels; functional PCSK9 protein was purified from the conditioned medium. In vitro studies showed that PCSK9 self-associated in a concentration-, temperature-, and pH-dependent manner. A mixture of PCSK9 monomers, dimers, and trimers displayed an enhanced LDLR degrading activity compared to monomeric PCSK9. A gain-of-function mutant, D374Y, displayed greatly increased self-association compared to wild-type PCSK9. Moreover, we demonstrated that the catalytic domain of PCSK9 is responsible for the self-association. Self-association of PCSK9 was enhanced by incubation with mouse apoE−/− VLDL and inhibited by incubation with both human and mouse HDL. When PCSK9 protein was incubated with total serum, it partially associated with LDL and HDL but not with VLDL. In transgenic mice, PCSK9 also associated with LDL and HDL but not with VLDL. We conclude that self-association is an intrinsic property of PCSK9, correlated to its LDLR-degrading activity and affected by plasma lipoproteins. These results provide a basis for developing strategies to manipulate PCSK9 activity in the circulation for the treatment of hypercholesterolemia.
AIM: To evaluate the effects of interferon-α-2b (IFN-α-2b) on expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma (HCC) inoculated in nude mice and to study the underlying mechanism of IFN-α-2b against HCC growth.
METHODS: Thirty-two nude mice bearing human HCC were randomly divided into four groups (n = 8). On the 10th day after implantation of HCC cells, the mice in test groups (groups A, B and C) received IFN-α-2b at a serial dose (10 000 IU for group A, 20 000 IU for group B, 40 000 IU for group C sc daily) for 35 d. The mice in control group received normal saline (NS). The growth conditions of transplanted tumors were observed. Both genes and proteins of COX-2 and VEGF were detected by RT-PCR and Western blot. Apoptosis of tumor cells in nude mice was detected by TUNEL assay after treatment with IFN-α-2b.
RESULTS: Tumors were significantly smaller and had a lower weight in the IFN-α-2b treatment groups than those in the control group (P < 0.01), and the tumor growth inhibition rate in groups A, B and C was 27.78%, 65.22% and 49.64%, respectively. The expression levels of both genes and proteins of COX-2 and VEGF were much lower in the IFN-α-2b treatment groups than in the control group (P < 0.01). The apoptosis index (AI) of tumor cells in the IFN-α-2b treatment groups was markedly higher than that in the control group (P < 0.01). Group B had a higher inhibition rate of tumor growth, a lower expression level of COX-2 and VEGF and a higher AI than groups A and C (P < 0.05), but there was no significant difference between groups A and C.
CONCLUSION: The inhibitory effects of IFN-α-2b on implanted tumor growth and apoptosis may be associated with the down-regulation of COX-2 and VEGF expression. There is a dose-effect relationship. The medium dose of IFN-α-2b for inhibiting tumor growth is 20 000 IU/d.
Hepatocellular carcinoma; Interferon-α-2b; Cyclooxygenase-2; Vascular endothelial growth factor; Apoptosis
MicroRNAs (miRNAs) regulate gene expression for diverse functions, but only a limited number of mRNA targets have been experimentally identified. We show that GW182 family proteins AIN-1 and AIN-2 act redundantly to regulate the expression of miRNA targets, but not miRNA biogenesis. Immunoprecipitation (IP) and mass spectrometry indicate that AIN-1 and AIN-2 interact only with miRNA-specific Argonaute proteins ALG-1 and ALG-2 and with components of the core translational initiation complex. Known miRNA targets are enriched in AIN-2 complexes, correlating with the expression of corresponding miRNAs. Combining IP with pyrosequencing and microarray analysis of RNAs associated with AIN-1/AIN-2, we identified 106 previously annotated miRNAs plus 9 new candidate miRNAs, but nearly no siRNAs, and more than 3500 potential miRNA targets including nearly all known ones. Our results demonstrate an effective biochemical approach to systematically identify miRNA targets and provide valuable insights regarding the properties of miRNA effector complexes.
microRNA; heterochronic gene; GW182; lin-28; hbl-1; alg-1; alg-2; Argonaute
In the present study, we have validated the cortical potential imaging (CPI) technique for estimating cortical potentials from scalp EEG using simultaneously recorded electrocorticogram (ECoG) in the presence of strong local inhomogeneity, i.e. Silastic ECoG grid(s). The finite element method (FEM) was used to model the realistic post-operative head volume conductor, which includes the scalp, skull, cerebrospinal fluid (CSF) and brain, as well as the Silastic ECoG grid(s) implanted during the surgical evaluation in epilepsy patients, from the co-registered magnetic resonance (MR) and computer tomography (CT) images. A series of computer simulations were conducted to evaluate the present FEM-based CPI technique, and to assess the effect of the Silastic ECoG grid on the scalp EEG forward solutions. The present simulation results show that the Silastic ECoG grid has substantial influence on the scalp potential forward solution due to the distortion of current pathways in the presence of the extremely low conductivity materials. On the other hand, its influence on the estimated cortical potential distribution is much less than that on the scalp potential distribution. With appropriate numerical modeling and inverse estimation techniques, we have demonstrated the feasibility of estimating the cortical potentials from the scalp EEG with the implanted Silastic ECoG gird(s), in both computer simulations and in human experimentation. In an epilepsy patient undergoing surgical evaluation, the cortical potentials were reconstructed from the simultaneously recorded scalp EEG, in which main features of spatial patterns during interictal spike were preserved and over 0.75 correlation coefficient value was obtained between the recorded and estimated cortical potentials. The FEM-based CPI technique provides a means of connecting the simultaneous recorded ECoG and the scalp EEG, and promises to become an effective tool to evaluate and validate CPI techniques using clinic data.
finite element method; cortical potential imaging; electrophysiological neuroimaging; brain mapping; EEG; ECoG; interictal spike; epilepsy
The present study aims to accurately localize epileptogenic regions which are responsible for epileptic activities in epilepsy patients by means of a new subspace source localization approach, i.e. First-principle-vectors (FINE), using scalp EEG recordings.
Computer simulations were first performed to assess source localization accuracy of FINE under the clinical electrode set-up. The source localization results from FINE were compared with the results from a classic subspace source localization approach, i.e. MUSIC, and their differences were tested statistically using the paired t-test. Other influence factors to source localization accuracy were assessed statistically by ANOVA. The interictal epileptiform spike data from three adult epilepsy patients with medically intractable partial epilepsy and well-defined symptomatic MRI lesions were then studied using both FINE and MUSIC. The comparison between the electrical sources estimated by the subspace source localization approaches and MRI lesions were made through the co-registration between the EEG recordings and MRI scans. The accuracy of estimations made by FINE and MUSIC was also evaluated and compared by R2 statistic, which was used to indicate the goodness-of-fit of the estimated sources to the scalp EEG recordings. The 3-concentric-spheres head volume conductor model was build for each patient with different radii of three spheres which takes the individual head size and thickness of individual skull into the consideration.
The results from computer simulations indicate that the improvement of source spatial resolvability and localization accuracy of FINE as compared with MUSIC is significant when simulated sources are closely spaced, deep, or signal-to-noise-ratio is low in a clinical electrode set-up. The interictal electrical generators estimated by FINE and MUSIC are in concordance with the patients’ structural abnormality, i.e. MRI lesions, in all three patients. The higher R2 values achieved by FINE than MUSIC indicate that FINE provides a more satisfactory fitting of the scalp potential measurements than MUSIC in all patients.
The present results suggest that FINE provides a useful brain source imaging technique, from clinical EEG recordings, for identifying and localizing epileptogenic regions in epilepsy patients with focal partial seizures.
The present study may lead to establishment of a high resolution source localization technique from the scalp recorded EEGs for aiding presurgical planning in epilepsy patients.
source localization; high-resolution EEG; FINE; MUSIC; epilepsy; interictal; presurgical planning
The subspace source localization approach, i.e. first principle vectors (FINE), is able to enhance the spatial resolvability and localization accuracy for closely-spaced neural sources from EEG and MEG measurements. Computer simulations were conducted to evaluate the performance of the FINE algorithm in an inhomogeneous realistic geometry head model under a variety of conditions. The source localization abilities of FINE were examined at different cortical regions and at different depths. The present computer simulation results indicate that FINE has enhanced source localization capability, as compared with MUSIC and RAP-MUSIC, when sources are closely spaced, highly noise-contaminated, or inter-correlated. The source localization accuracy of FINE is better, for closely-spaced sources, than MUSIC at various noise levels, i.e. SNR from 6 dB to 16 dB, and RAP-MUSIC at relatively low noise levels, i.e. 6 dB to 12 dB. The FINE approach has been further applied to localize brain sources of motor potentials, obtained during the finger tapping tasks in a human subject. The experimental results suggest that the detailed neural activity distribution could be revealed by FINE. The present study suggests that FINE provides enhanced performance in localizing multiple closely-spaced, and inter-correlated sources under low signal-to-noise ratio, and may become an important alternative to brain source localization from EEG or MEG.
Source localization; electrophysiological neuroimaging; subspace; MUSIC; FINE; brain mapping; inverse problem; EEG; MEG
We propose a new integrative approach to characterize the structure of seizures in the space, time, and frequency domains. Such characterization leads to a new technical development for ictal source analysis for the presurgical evaluation of epilepsy patients. The present new ictal source analysis method consists of three parts. First, a three-dimensional source scanning procedure is performed by a spatio-temporal FINE source localization method to locate the multiple sources responsible for the time evolving ictal rhythms at their onsets. Next, the dynamic behavior of the sources is modeled by a multivariate autoregressive process (MVAR). Lastly, the causal interaction patterns among the sources as a function of frequency are estimated from the MVAR modeling of the source temporal dynamics. The causal interaction patterns indicate the dynamic communications between sources, which are useful to distinguish the primary sources responsible for the ictal onset from the secondary sources caused by the ictal propagation. The present ictal analysis strategy has been applied to a number of seizures from five epilepsy patients, and their results are consistent with observations from either MRI lesions or SPECT scans, which indicate its effectiveness. Each step of the ictal source analysis is statistically evaluated in order to guarantee the confidence in the results.
ictal source analysis; source localization; causal interaction; MVAR; EEG; MRI; SPECT; lesion; statistical assessment; presurgical evaluation; interictal