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Recent evidence suggests that glia maturation factor β (GMFβ) is important in the pathogenesis of pulmonary arterial hpertension (PAH), but the underlying mechanism is unknown. To clarify whether GMFβ can be involved in pulmonary vascular remodeling and to explore the role of the IL‐6‐STAT3 pathway in this process, the expression of GMFβ in PAH rats is examined and the expression of downstream molecules including periostin (POSTN) and interleukin‐6 (IL‐6) is measured using real‐time quantitative polymerase chain reaction (RT‐qPCR) and western blot analysis. The location and expression of POSTN is also tested in PAH rats using immunofluorescence. It is proved that GMFβ is upregulated in the lungs of PAH rats. Knockout GMFβ alleviated the MCT‐PAH by reducing right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), and pulmonary vascular remodeling. Moreover, the inflammation of the pulmonary vasculature is ameliorated in PAH rats with GMFβ absent. In addition, the IL‐6‐STAT3 signaling pathway is activated in PAH; knockout GMFβ reduced POSTN and IL‐6 production by inhibiting the IL‐6‐STAT3 signaling pathway. Taken together, these findings suggest that knockout GMFβ ameliorates PAH in rats by inhibiting the IL‐6‐STAT3 signaling pathway. [ABSTRACT FROM AUTHOR]

Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3-5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.

Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart. Here we report that ASC tumors frequently harbor somatically acquired mutations in the UPF1 gene, which encodes the core component of the nonsense-mediated RNA decay (NMD) pathway. These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs. UPF1 mutations are, to our knowledge, the first known unique molecular signatures of pancreatic ASC.

Voltage-gated [K.sup.+] channel activities are involved in regulating growth factor-stimulated cell proliferation in a variety of cell types. Here we report that suppression of a voltage-gated [K.sup.+] channel with 4-aminopyridine (4-AP), barium, and tetraethylammonium inhibited both EGF- and insulin-stimulated myeloblastic leukemia ML-1 cell proliferation in a concentration-dependent manner. Both MAPK/ERK and Akt pathways are known to mediate cell proliferative signals of a variety of growth factors including insulin. In serum-starved ML-1 cells, insulin rapidly stimulated phosphorylation of ERK1/2 and Akt, and the phosphorylation levels peaked ~30 min after treatment. Pretreatment of ML-1 cells with 4-AP potently and dose-dependently prevented phosphorylation of ERK1/2 and Akt. However, insulin-induced activation of the Akt pathway also played a role in promoting ML-1 cell proliferation. Flow cytometry analysis revealed that although ML-1 cells were primarily arrested at [G.sub.1] phase by serum starvation for 36 h, they reentered the cell cycle after treatment with serum or insulin for 24 h. However, concomitant 4-AP treatment was able to attenuate cell cycle progression in synchronized ML-1 cells stimulated with growth factors. Our results strongly suggest that a 4-AP-sensitive [K.sup.+] channel activity plays an important role in controlling proliferation of ML-1 cells by affecting the activation of multiple signal transduction processes induced by insulin. growth factors; myeloblastic cells; signaling; ion channel blocker

Abstract Background Diabetic retinopathy is a major complication of dysregulated hyperglycemia. Retinal vascular endothelial cell dysfunction is an early event in the pathogenesis of diabetic retinopathy. Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by docosahexaenoic acid (DHA, 22:6 ω-3) and eicosapentaenoic acid (EPA, 20:5 ω-3). The influence of dietary omega-3 PUFA on brain zinc metabolism has been previously implied. Zn2+ is essential for the activity of Δ6 desaturase as a co-factor that, in turn, converts essential fatty acids to their respective long chain metabolites. Whether essential fatty acids (EFAs) α-linolenic acid and linoleic acid have similar beneficial effect remains poorly understood. Methods RF/6A cells were treated with different concentrations of high glucose, α-linolenic acid and linoleic acid and Zn2+. The alterations in mitochondrial succinate dehydrogenase enzyme activity, cell membrane fluidity, reactive oxygen species generation, SOD enzyme and vascular endothelial growth factor (VEGF) secretion were evaluated. Results Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by both linoleic acid (LA) and α-linolenic acid (ALA), while the saturated fatty acid, palmitic acid was ineffective. A dose–response study with ALA showed that the activity of the mitochondrial succinate dehydrogenase enzyme was suppressed at all concentrations of glucose tested to a significant degree. High glucose enhanced fluorescence polarization and microviscocity reverted to normal by treatment with Zn2+ and ALA. ALA was more potent that Zn2+. Increased level of high glucose caused slightly increased ROS generation that correlated with corresponding decrease in SOD activity. ALA suppressed ROS generation to a significant degree in a dose dependent fashion and raised SOD activity significantly. ALA suppressed high-glucose-induced VEGF secretion by RF/6A cells. Conclusions These results suggest that EFAs such as ALA and LA may have beneficial action in the prevention of high glucose-induced cellular damage.

Using the paradigm of in vitro differentiation of hESCs/iPSCs into retinal pigment epithelial (RPE) cells, we have recently profiled mRNA and miRNA transcriptomes to define a set of RPE mRNA and miRNA signature genes implicated in directed RPE differentiation. In this study, in order to understand the role of DNA methylation in RPE differentiation, we profiled genome-scale DNA methylation patterns using the method of reduced representation bisulfite sequencing (RRBS). We found dynamic waves of de novo methylation and demethylation in four stages of RPE differentiation. Integrated analysis of DNA methylation and RPE transcriptomes revealed a reverse-correlation between levels of DNA methylation and expression of a subset of miRNA and mRNA genes that are important for RPE differentiation and function. Gene Ontology (GO) analysis suggested that genes undergoing dynamic methylation changes were related to RPE differentiation and maturation. We further compared methylation patterns among human ESC- and iPSC-derived RPE as well as primary fetal RPE (fRPE) cells, and discovered that specific DNA methylation pattern is useful to classify each of the three types of RPE cells. Our results demonstrate that DNA methylation may serve as biomarkers to characterize the cell differentiation process during the conversion of human pluripotent stem cells into functional RPE cells. [ABSTRACT FROM AUTHOR]

Abstract: Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular processes. TAK1, a member of the MAPKKK family, is essential for TNFα-induced NF-κB activation. Phosphorylation and Lys63-linked polyubiquitination (polyUb) of TAK1 are critical for its activation. However, whether TAK1 is regulated by polyubiquitination-mediated protein degradation after its activation remains unknown. Here we report that TNFα induces TAK1 Lys48 linked polyubiquitination and degradation at the later time course. Furthermore, we provide direct evidence that TAK1 is modified by Lys48-linked polyubiquitination at lysine-72 by mass spectrometry. A K72R point mutation on TAK1 abolishes TAK1 Lys48-linked polyubiquitination and enhances TAK1/TAB1 co-overexpression-induced NF-κB activation. As expected, TAK1 K72R mutation inhibits TNFα-induced Lys48-linked TAK1 polyubiquitination and degradation. TAK1 K72R mutant prolongs TNFα-induced NF-κB activation and enhances TNFα-induced IL-6 gene expression. Our findings demonstrate that TNFα induces Lys48-linked polyubiquitination of TAK1 at lysine-72 and this polyubiquitination-mediated TAK1 degradation plays a critical role in the downregulation of TNFα-induced NF-κB activation. [Copyright &y& Elsevier]

How attention interacts with low-level visual representations to give rise to perception remains a central yet controversial question in neuroscience. While several previous studies suggest that the units of attentional selection are individual objects, other evidence points instead toward lower-level features, such as an attended color or direction of motion. We used both human fMRI and psychophysics to investigate the relationship between object-based and feature-based attention. Specifically, we focused on whether feature-based attention is modulated by object appearance, comparing three conditions: (a) features appearing as one object; (b) features appearing as two separate but identical objects; (c) features appearing as two different objects. Stimuli were two random-dot fields presented bilaterally to central fixation, and object appearance was induced by the presence of one or two boxes around the fields. In the fMRI experiment, participants performed a luminance discrimination task on one side, and ignored the other side, where we probed for enhanced activity when either it was perceived as belonging to a same object, or shared features with the task side. In the psychophysical experiments, participants performed luminance discrimination on both sides with overlapping red and green dots, now attending to either the same features (red/red or green/green) or different features (red/green or green/red) on both sides. Results show that feature-based attentional enhancement exists in all three conditions, i.e., regardless whether features appear as one object, two identical objects, or two different objects. Our findings indicate that feature-based attention differs from object-based attention in that it is not dependent upon object appearance. Thus feature-based attention may be mediated by earlier cortical processes independent of perceiving visual features into well-formed objects.

A molecular hyperspectral imaging (MHI) system was developed to evaluate the protective effects of erythropoietin (EPO) on early diabetic retinopathy in rats. The system was used to capture hyperspectral images of rat retinal sections selected from three groups: normal control, diabetic, and EPO. Three biochemical parameters were defined, namely, the spectral transmittance index, thickness of outer nuclear layer, and cell area percentage. The corresponding algorithms to calculate these were likewise presented. Experimental results show that, after treatment, the newly defined biochemical parameters of the EPO group become more similar to those of the normal control group compared with those of the diabetic group. This indicates that, to some degree, EPO provides protective effects on the retinal cells of chemically induced diabetic rats after it is injected intravitreally at the onset of diabetes. The results likewise show that the MHI system could provide useful quantitative information regarding retinal sections, which ophthalmologists can use to determine the pathogenesis of diabetic retinopathy and evaluate the protective effect of EPO on diabetic retinal cells.

Background: Heat-shock transcription factor 4 (HSF4) mutations are associated with autosomal dominant lamellar cataract and Marner cataract. Disruptions of the Hsf4 gene cause lens defects in mice, indicating a requirement for HSF4 in fiber cell differentiation during lens development. However, neither the relationship between HSF4 and crystallins nor the detailed mechanism of maintenance of lens transparency by HSF4 is fully understood., Results: In an attempt to determine how the underlying biomedical and physiological mechanisms resulting from loss of HSF4 contribute to cataract formation, we generated an Hsf4 knockout mouse model. We showed that the Hsf4 knockout mouse (Hsf4-/-) partially mimics the human cataract caused by HSF4 mutations. Q-PCR analysis revealed down-regulation of several cataract-relevant genes, including gamma S-crystallin (Crygs) and lens-specific beaded filament proteins 1 and 2 (Bfsp1 and Bfsp2), in the lens of the Hsf4-/- mouse. Transcription activity analysis using the dual-luciferase system suggested that these cataract-relevant genes are the direct downstream targets of HSF4. The effect of HSF4 on gamma S-crystallin is exemplified by the cataractogenesis seen in the Hsf4-/-,rncat intercross. The 2D electrophoretic analysis of whole-lens lysates revealed a different expression pattern in 8-week-old Hsf4-/- mice compared with their wild-type counterparts, including the loss of some alpha A-crystallin modifications and reduced expression of gamma-crystallin proteins., Conclusion: Our results indicate that HSF4 is sufficiently important to lens development and disruption of the Hsf4 gene leads to cataracts via at least three pathways: 1) down-regulation of gamma-crystallin, particularly gamma S-crystallin; 2) decreased lens beaded filament expression; and 3) loss of post-translational modification of alpha A-crystallin.