Identification of Common Prognostic Gene Expression Signatures with Biological Meanings from Microarray Gene Expression Datasets

Numerous prognostic gene expression signatures for breast cancer were generated previously with few overlap and limited insight into the biology of the disease. Here we introduce a novel algorithm named SCoR (Survival analysis using Cox proportional hazard regression and Random resampling) to apply random resampling and clustering methods in identifying gene features correlated with time to event data. This is shown to reduce overfitting noises involved in microarray data analysis and discover functional gene sets linked to patient survival. SCoR independently identified a common poor prognostic signature composed of cell proliferation genes from six out of eight breast cancer datasets. Furthermore, a sequential SCoR analysis on highly proliferative breast cancers repeatedly identified T/B cell markers as favorable prognosis factors. In glioblastoma, SCoR identified a common good prognostic signature of chromosome 10 genes from two gene expression datasets (TCGA and REMBRANDT), recapitulating the fact that loss of one copy of chromosome 10 (which harbors the tumor suppressor PTEN) is linked to poor survival in glioblastoma patients. SCoR also identified prognostic genes on sex chromosomes in lung adenocarcinomas, suggesting patient gender might be used to predict outcome in this disease. These results demonstrate the power of SCoR to identify common and biologically meaningful prognostic gene expression signatures.     

Probing high affinity sequences of DNA aptamer against VEGF165

Vascular endothelial growth factor (VEGF(165)) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF(165) protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF(121), an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF(165) protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.     

Low resolution of mitochondrial COI barcodes for identifying species of the genus Larus (Charadriiformes: Laridae)

We evaluated whether cytochrome c oxidase subunit I (COI) barcodes that have been previously suggested for birds are useful for identifying species of the genus Larus, which are resident or migratory birds in Korea. We found 31 intra- or interspecific COI haplotypes from 12 of 13 Larus species in Korea. Haplotype analyses showed that the COI barcodes could not distinguish some interspecific haplotypes from 6 of 12 Larus species because there were no nucleotide substitutions among their COI haplotypes. The neighbor-joining tree formed shallow branches in the clades expected for L. saundersi, L. crassirostris, L. canus, L relictus, and L. ridbundus. In the nine Larus species, COI haplotypes were not grouped as distinct entities that were correctly assigned to their corresponding species, resulting in polytypic clades. These results indicate that the COI sequences need to be cautiously selected as a DNA barcode for identifying species of Korean Larus birds.     

The Loss of RGS Protein-Gαi2 Interactions Results in Markedly Impaired Mouse Neutrophil Trafficking to Inflammatory Sites

Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gα_i subunits to exchange GDP for GTP. By limiting the duration that Gα_i subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gα_i2 interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gα_i activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.     

Activation of ras-dependent signaling pathways by G(14) -coupled receptors requires the adaptor protein TPR1

Many G_q‐coupled receptors mediate mitogenic signals by stimulating extracellular signal‐regulated protein kinases (ERKs) that are typically regulated by the small GTPase Ras. Recent studies have revealed that members of the Gα_q family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gα_q family, the highly promiscuous Gα₁₄ can relay signals from numerous receptors. Here, we examined if Gα₁₄ interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα₁₄QL mutant in HEK293 cells led to the formation of GTP‐bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IκB kinase. Stimulation of endogenous G₁₄‐coupled somatostatin type 2 and α₂‐adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co‐immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Gα₁₄QL than Gα₁₄, suggesting that TPR1 preferentially binds to the GTP‐bound form of Gα₁₄. Activated Gα₁₄ also interacted with the Ras guanine nucleotide exchange factors SOS1 and SOS2. Expression of a dominant negative mutant of TPR1 or siRNA‐mediated knockdown of TPR1 effectively abolished the ability of Gα₁₄ to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα₁₄QL‐induced phosphorylations of ERK and IκB kinase, it did not affect Gα₁₄QL‐induced stimulation of phospholipase Cβ or c‐Jun N‐terminal kinase. Our results suggest that TPR1 is required for Gα₁₄ to stimulate Ras‐dependent signaling pathways, but not for the propagation of signals along Ras‐independent pathways. J. Cell. Biochem. 113: 3486–3497, 2012. © 2012 Wiley Periodicals, Inc.     

Depigmenting activities of kojic acid derivatives without tyrosinase inhibitory activities

We synthesized benzoate ester derivatives of kojic acid with and without adamantane moiety. Benzoate derivatives 2a-e that did not contain an adamantane moiety showed potent tyrosinase inhibitory activities. However, depigmenting activity was not noted in a cell-based assay. Contrasting results were obtained for benzoate derivatives (3a-e) containing an adamantane moiety. Compounds 3a-e showed potent depigmenting activities without tyrosinase inhibitory activities. To the best of our knowledge, this is the first study showing the depigmenting activities of kojic acid derivatives without tyrosinase inhibitory activities.     

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

Dendroaspin (Den) and rhodostomin (Rho) are snake venom proteins containing a PRGDMP motif. Although Den and Rho have different 3D structures, they are highly potent integrin inhibitors. To study their structure, function, and dynamics relationships, we expressed Den and Rho in Pichia pastoris. The recombinant Den and Rho inhibited platelet aggregation with the K_I values of 149.8 and 83.2 nM. Cell adhesion analysis showed that Den was 3.7 times less active than Rho when inhibiting the integrin αIIbβ3 and 2.5 times less active when inhibiting the integrin αvβ3. In contrast, Den and Rho were similarly active when inhibiting the integrin α5β1 with the IC₅₀ values of 239.8 and 256.8 nM. NMR analysis showed that recombinant Den and Rho have different 3D conformations for their arginyl‐glycyl‐aspartic acid (RGD) motif. However, the comparison with Rho showed that the docking of Den into integrin αvβ3 resulted in a similar number of contacts. Analysis of the dynamic properties of the RGD loop in Den and Rho showed that they also had different dynamic properties. These results demonstrate that protein scaffolds affect the function, structure, and dynamics of their RGD motif.     

Ethyl pyruvate attenuates ventilation‐induced diaphragm dysfunction through high‐mobility group box‐1 in a murine endotoxaemia model

Mechanical ventilation (MV) can save the lives of patients with sepsis. However, MV in both animal and human studies has resulted in ventilator‐induced diaphragm dysfunction (VIDD). Sepsis may promote skeletal muscle atrophy in critically ill patients. Elevated high‐mobility group box‐1 (HMGB1) levels are associated with patients requiring long‐term MV. Ethyl pyruvate (EP) has been demonstrated to lengthen survival in patients with severe sepsis. We hypothesized that the administration of HMGB1 inhibitor EP or anti‐HMGB1 antibody could attenuate sepsis‐exacerbated VIDD by repressing HMGB1 signalling. Male C57BL/6 mice with or without endotoxaemia were exposed to MV (10 mL/kg) for 8 hours after administrating either 100 mg/kg of EP or 100 mg/kg of anti‐HMGB1 antibody. Mice exposed to MV with endotoxaemia experienced augmented VIDD, as indicated by elevated proteolytic, apoptotic and autophagic parameters. Additionally, disarrayed myofibrils and disrupted mitochondrial ultrastructures, as well as increased HMGB1 mRNA and protein expression, and plasminogen activator inhibitor‐1 protein, oxidative stress, autophagosomes and myonuclear apoptosis were also observed. However, MV suppressed mitochondrial cytochrome C and diaphragm contractility in mice with endotoxaemia (P < 0.05). These deleterious effects were alleviated by pharmacologic inhibition with EP or anti‐HMGB1 antibody (P < 0.05). Our data suggest that EP attenuates endotoxin‐enhanced VIDD by inhibiting HMGB1 signalling pathway.     

Hydrangenol suppresses VEGF-stimulated angiogenesis by targeting p27KIP1-dependent G1-cell cycle arrest,VEGFR-2-mediated signaling,and MMP-2 expression*

We previously reported that hydrangenol has potent antitumor activity against human bladder cancer EJ cells. Here, we investigated the antiangiogenic activity of hydrangenol using in vitro and ex vivo models. Treatment with hydrangenol significantly inhibited the proliferation of vascular endothelial growth factor (VEGF)-induced HUVECs in a concentration-dependent manner (EC_50?=?10?μM). Flow cytometry analysis revealed that hydrangenol suppressed the VEGF-induced inhibition of G1-cell cycle phase and also decreased cyclin D1, cyclin E, CDK2, and CDK4 levels. Hydrangenol-mediated arrest in the G1-cell cycle phase was associated with p27KIP1 level, but not p21WAF1 or p53 level. Hydrangenol also significantly inhibited VEGFR-2-mediated signaling pathways including ERK1/2, AKT, and endothelial nitric oxide synthase. Interestingly, immunoprecipitation assay demonstrated that the inhibition of VEGFR-2 activation was independent of VEGF binding, thereby suggesting an allosteric regulation of hydrangenol against VEGFR-2. Additionally, hydrangenol inhibited migration, invasion, and capillary-like tubular formation in VEGF-stimulated HUVECs. Zymography and immunoblot analyses revealed that these inhibitory activities were partially owing to the VEGF-induced inhibition of matrix metalloproteinase-2 activity. Finally, VEGF-mediated microvessel sprouting was inhibited in the presence of hydrangenol in ex vivo aortic ring assay. Taken together, hydrangenol possesses a potent antiangiogenesis potential; thus we believe that hydrangenol may be developed as a therapeutic reagent to treat angiogenesis-mediated diseases.     

Visible light regulates neurite outgrowth of nerve cells

The neurite outgrowth of PC12 cells on collagen-coated glass plates under light emitting diode (LED) irradiation at several wavelengths (i.e., 455, 470, 525, 600, 630, 880 and 945 nm) was investigated. No neurite outgrowth was observed during cultivation under irradiation from the lamp of an inverted light microscope through filters (yielding mixed light at ca. 525 nm and more than 800 nm), whereas neurite outgrowth was observed during cultivation in the dark. When these cells were irradiated with monochromatic LED light, neurite outgrowth was slightly, but not completely, suppressed at 455, 525, 600, 630, 880 and 945 nm, as was observed in the case of mixed light. Long connected neuronal outgrowths (e.g., 3 mm length) were observed with LED light at 470 nm and 1.8 mW/cm² intensity. No such outgrowths were observed at other LED light wavelengths (i.e., 455, 525, 600, 630, 880 and 945 nm). Irradiation at 470 nm may have caused specific responses to transductional signals in these cells that led to the connection of neuronal outgrowths between cells. Not only suppressed neurite outgrowth but also long connected neurite outgrowths were observed when PC12 cells were cultured under several different wavelengths of light.