Recently microRNAs (miRNAs) have been attractive targets with their key roles in biological regulation through post-transcription to control mRNA stability and protein translation. Though melatonin was known as an anti-angiogenic agent, the underlying mechanism of melatonin in PC-3 prostate cancer cells under hypoxia still remains unclear. Thus, in the current study, we elucidated the important roles of miRNAs in melatonin-induced anti-angiogenic activity in hypoxic PC-3 cells. miRNA array revealed that 33 miRNAs (>2 folds) including miRNA3195 and miRNA 374b were significantly upregulated and 16 miRNAs were downregulated in melatonin-treated PC-3 cells under hypoxia compared to untreated control. Melatonin significantly attenuated the expression of hypoxia-inducible factor (HIF)-1 alpha, HIF-2 alpha and vascular endothelial growth factor (VEGF) at mRNA level in hypoxic PC-3 cells. Consistently, melatonin enhanced the expression of miRNA3195 and miRNA 374b in hypoxic PC-3 cells by qRT-PCR analysis. Of note, overexpression of miRNA3195 and miRNA374b mimics attenuated the mRNA levels of angiogenesis related genes such as HIF-1alpha, HIF-2 alpha and VEGF in PC-3 cells under hypoxia. Furthermore, overexpression of miRNA3195 and miRNA374b suppressed typical angiogenic protein VEGF at the protein level and VEGF production induced by melatonin, while antisense oligonucleotides against miRNA 3195 or miRNA 374b did not affect VEGF production induced by melatonin. Also, overexpression of miR3195 or miR374b reduced HIF-1 alpha immunofluorescent expression in hypoxic PC-3 compared to untreated control. Overall, our findings suggest that upregulation of miRNA3195 and miRNA374b mediates anti-angiogenic property induced by melatonin in hypoxic PC-3 cells.
melatonin; miRNA3195; miRNA374b; VEGF; HIF-1 alpha; PC-3 cells.
Though ergosterol peroxide (EP) derived from Neungyi mushrooms (Sarcodon aspratus) was known to have cytotoxic, apoptotic, anti-inflammatory and antimycobacterial effects, the underlying molecular mechanism of EP still remains unclear. Thus, in the present study, the apoptotic mechanism of EP was elucidated in DU 145 prostate cancer cells.
Cell viability of prostate cancer cells was measured by MTT assay. To see whether EP induces the apoptosis, FACS, western blot and TUNEL assay were performed. To determine the role of Death receptor (DR) 5 molecules in EP-induced apoptosis in DU 145 prostate cancer cells, the silencing of DR 5 was performed by using siRNAs.
EP showed significant cytotoxicity against DU 145, PC 3, M2182 prostate cancer cells. Also, EP effectively increased the sub G1 population and terminal deoxynucleotidyl transferase DUTP nick end labeling (TUNEL) positive cells in DU 145 prostate cancer cells. Furthermore, western blotting revealed that EP cleaved poly (ADP-ribose) polymerase (PARP) and caspase 8/3, attenuated the expression of fluorescence loss in photobleaching (FLIP), Bcl-XL and Bcl-2 as well as activated Bax, Fas-associated death domain (FADD) and DR 5 in a concentration dependent manner in DU 145 prostate cancer cells. Conversely, caspase 8 inhibitor Z-IETD-FMK blocked the apoptotic ability of EP to cleave PARP and an increase of sub G1 population in DU 145 prostate cancer cells. Likewise, the silencing of DR 5 suppressed the cleavages of PARP induced by EP in DU 145 prostate cancer cells.
Overall, our findings suggest that ergosterol peroxide induces apoptosis via activation of death receptor 5 and caspase 8/3 in DU 145 prostate cancer cells as a cancer chemopreventive agent or dietary factor.
Ergosterol peroxide; Apoptosis; Caspase 8/3; Z-IETD-FMK; DR 5; DU 145 prostate cancer cells
The transcription factor Stat3 is an activator of systemic inflammatory genes. Two isoforms of Stat3 are generated by alternative splicing, Stat3α and Stat3β. The β isoform lacks the transactivation domain but retains other functions, including dimerization and DNA binding. Stat3β-deficient mice exhibit elevated expression of systemic inflammatory genes and are hyperresponsive to lipopolysaccharide, suggesting that Stat3β functions predominantly as a suppressor of systemic inflammation. To test whether Stat3β deficiency would provoke pathologic effects associated with chronic inflammation, we asked whether selective removal of Stat3β would exacerbate the development of atherosclerosis in apolipoprotein E-deficient mice. In apoE−/−Stat3β−/− mice atherosclerotic plaque formation was significantly enhanced relative to apoE−/−Stat3β+/+ controls. The ability of Stat3β deficiency to promote atherosclerosis was more pronounced in female mice, but could be unmasked in males by feeding a high fat diet. Infiltrating macrophages were not increased in aortas of apoE−/−Stat3β−/− mice. In contrast, the proportion of pro-inflammatory TH17 cells was significantly elevated in aortic infiltrates from apoE−/−Stat3β−/− mice, relative to paired apoE−/−Stat3β+/+ littermates. These observations indicate that Stat3β can suppress pathologic sequelae associated with chronic inflammation. Our findings further suggest that in Stat3β-deficient mice the unopposed action of Stat3α may enhance atherogenesis in part by promoting differentiation of TH17 cells.
Stat3; Atherosclerosis; Inflammation; Acute phase response
We present a novel method of targeted gene disruption that involves direct injection of recombinant Cas9 protein complexed with guide RNA into the gonad of the nematode Caenorhabditis elegans. Biallelic mutants were recovered among the F1 progeny, demonstrating the high efficiency of this method.
Caenorhabditis elegans; Cas9–sgRNA ribonucleoprotein; NHEJ; genome editing
Although many critical roles of the RUNX family proteins have already been identified, little attention has been given to how these proteins interact with other factors. Elucidating RUNX protein interactions will help extend our understanding of their roles in normal development and tumorigenesis. In this study, we performed large-scale RNAi screening to identify genes that genetically interact with rnt-1, the sole homolog of RUNX protein in the nematode Caenorhabditis elegans. To this end, we took advantage of the fact that C. elegans can survive a severe loss of RNT-1 function with only mild phenotypes, and we looked for genes that caused a synthetic phenotype in the rnt-1 mutant background. We identified seven genes, three of which (cdk-8, cic-1, and sur-2) are involved in transcription, two of which (pgp-2 and cct-5) are involved in stress response, and two of which (D2045.7 and W09D10.4) are involved in signaling cascades, according to their functional gene ontology terms. We further confirmed that the CDK8-containing mediator complex genetically interacts with RNT-1 by showing that knockdown of each component of the CDK8 mediator complex caused a synthetic phenotype, that is, the exploded intestine through the vulva (Eiv) phenotype, in the rnt-1 mutant background. We also identified a putative target gene, acs-4, which is regulated by the RNT-1 and CDK8 mediator complex. Our results strengthen the notion that the CDK8 mediator complex may also act together with RUNX proteins in mammals.
RNAi; RUNX; CDK8; mediator; genetic interaction
The neutrophil elastase (NE) gene encodes a powerful serine protease that is involved in the process of normal tissue turnover, natural host defense or tissue damage in acute and chronic inflammatory disorders. Furthermore, NE was suggested as one of the determinant factors of individual susceptibility to lung cancer resulting from imbalance between α1-antitrypsin (AT) and NE. To determine whether NE plays a role in risk for lung cancer, we screened polymorphisms in the promoter region of the NE gene and assessed the role of the NE polymorphisms in the risk for lung cancer. We confirmed three previously identified polymorphisms which are located at −903, −741, and extra 52 bp STS relative to the transcription initiation site. In addition, two new polymorphisms at −832 (G/T) and −789 (C/T) were identified. Their rare allelic frequencies of new polymorphism are 0.02 and 0.01, respectively, among Caucasians. The prevalence of the NE −903 (T/T) and (T/G) genotypes were 0.88 and 0.12 in controls as compared to 0.96 and 0.04 in lung cancer patients using genomic DNA isolated from 113 Caucasian lung cancer cases and 131 controls. A significant increase in lung cancer risk was observed for expected high NE activity genotypes (OR = 3.2, 95% CI = 1.02–10.3) as compared to low NE activity genotypes. These results were consistent with previous in vitro functional analysis, which reported an approximately two-fold increase enzyme expression with the −903T/−741G allele as compared to the −903G/−741A variant. These results confirm that the NE promoter region polymorphisms may influence in risk for lung cancer.
Neutrophil elastase; Lung cancer; Genetic polymorphism; Cancer susceptibility
The empirical use of a chemotherapy regimen shows different results in individual breast cancer patient treatment. Recent studies showed the effectiveness of the adenosine triphosphate-based chemotherapy response assay (ATP-CRA). However, little is known about the correlation between chemosensitivity and breast cancer molecular subtypes. Therefore, we investigated whether the result of ATP-CRA is associated with a molecular subtype of breast cancer.
Two hundred eighty-seven patients diagnosed with breast cancer and receiving ATP-CRA at Mokdong Hospital, Ewha Womans University between September 2007 and December 2010 were enrolled in this study. Hormone receptor status, HER2/neu expression, and results of chemosensitivity tests of the patients was analyzed.
In all of four subtypes, the combination of two agents showed significant higher mean cell death rate than a single agent. Within the breast cancer cell lines in this study, the range of chemosensitivity response was very wide and varied for each patient. For this reason, the molecular subtype of breast cancer is inconclusive in choosing an effective chemotherapeutic agent and in vitro chemosensitivity test, prior to therapy, could be a useful method for planning chemotherapy for each patient.
Chemosensitivity response to anticancer agents was found to vary depending on the individual breast cancer patients. The molecular subtype of breast cancer is inconclusive to choose the effective chemotherapeutic agent and the in vitro chemosensitivity test, prior to therapy, could be more useful for planning chemotherapy for each patient.
Adjuvant chemotherapy; Breast neoplasms; Adenosine triphosphate
Control of the motion of domain walls in magnetic nanowires is at the heart of various recently proposed three-dimensional (3D) memory devices. However, fabricating 3D nanostructures is extremely complicated using standard lithography techniques. Here we show that highly pure 3D magnetic nanowires with aspect-ratios of ~100 can be grown using focused electron-beam-induced-deposition. By combining micromanipulation, Kerr magnetometry and magnetic force microscopy, we determine that the magnetisation reversal of the wires occurs via the nucleation and propagation of domain walls. In addition, we demonstrate that the magnetic switching of individual 3D nanostructures can be directly probed by magneto-optical Kerr effect.
To investigate the structural anisotropy in bovine septal cartilage, quantitative procedures in microscopic magnetic resonance imaging (μMRI), polarized light microscopy (PLM), and mechanical indentation were used to measure the tissue in three orthogonal planes: vertical, medial, and caudocephalic. The quantitative T2 imaging experiments in μMRI found strong anisotropy in the images of both vertical and caudocephalic planes but little anisotropy in the images from the medial plane. The PLM birefrigent experiments found that the retardation values in the medial section were only about 10% of these in the vertical and caudocephalic sections and that the angle values in all three sections followed the rotation of the tissue section in the microscope stage. The stress relaxation experiments in mechanical indentation showed reduced stiffness in the medial plane compared to stiffness in either the vertical or caudocephalic planes. Collectively, the results in this project coherently indicate a marked structural anisotropy in cartilage from the nasal septum, where the long axis of the collagen fibrils is oriented in parallel with the medial axis.
bovine nasal cartilage; collagen anisotropy; MRI; PLM; biomechanical indentation
To investigate the dependency of T1 relaxation on mechanical strain in articular cartilage, quantitative MRI T1 imaging experiments were carried out on cartilage before/after the tissue was immersed in gadolinium contrast agent and when the tissue was being compressed (up to ~ 48% strains). The spatial resolution across the cartilage depth was 17.6μm. The T1 profile in native tissue (without the presence of gadolinium ions) was strongly strain-dependent, which is also depth-dependent. At the modest strains (e.g., 14% strain), T1 reduced by up to 68% in the most surface portion of the tissue. Further compression (e.g., 45% strain) reduced T1 mostly in the middle and deep portions of the tissue. For the gadolinium-immersed tissue, both modest and heavy compressions (up to 48% strain) increased T1 slightly but significantly, although the overall shapes of the T1 profiles remained approximately the same regardless of the amount of strains. The complex relationships between the T1 profiles and the mechanical strains were a direct consequence of the depth-dependent proteoglycan concentration in the tissue, which determined the tissue’s mechanical properties. This finding has potential implications in the use of gadolinium contrast agent in clinical MRI of cartilage (the dGEMRIC procedure), when the loading or loading history of patients is considered.
MRI; articular cartilage; T1 relaxation; strain; dGEMRIC; gadolinium contrast agent
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder that presents with hemolytic anemia, marrow failure and thrombophilia. During acute attacks, corticosteroid can alleviate the hemolytic paroxysm, but the prolonged administration induces serious toxicity including immunosuppression. So American thoracic society (ATS) for tuberculosis (TB) recommends prophylactic anti-TB medication in patients with a long-term steroid therapy. However, in the patient who was treated for active TB in the past, there are no guidelines of the test for determining patients who have latent TB infection (LTBI) and no recommendations of TB prophylaxis if there is no evidence of reactivation at present. A 40-year-old male patient presented with fever and aggravated weakness for a week. He was diagnosed with PNH a month ago and took corticosteroid for 3 weeks. In the past, he was diagnosed with pulmonary TB and completely cured after treatment. According to guideline, he was not indicated with TB prophylaxis. However, he caught miliary TB, progressed to acute respiratory distress syndrome. We experience this embarrassing case, and emphasize the need to investigate multicentral TB prevalence and to make the guidelines of anti-TB medication in subgroups of hematologic diseases including PNH.
Paroxysmal nocturnal hemoglobinuria; Miliary tuberculosis (TB); Tuberculosis (TB) prophylaxis
The hippocampus is important for spatial navigation. Literature shows that allocentric visual contexts in the animal's background are critical for making conditional response selections during navigations. In a traditional maze task, however, it is difficult to identify exactly which subsets of visual contexts are critically used. In the current study, we tested in rats whether making conditional response selections required the hippocampus when using computer-generated visual contextual stimuli in the animal's background as in primate and human studies. We designed a new task, visual contextual response selection (VCRS) task, in which the rat ran along a linear track and encountered a touchscreen monitor at the end of the track. The rat was required to touch one of the adjacent rectangular box images depending on the visual contextual stimuli displayed in the two peripheral monitors positioned on both sides of the center touchscreen monitor. The rats with a GABA-A receptor agonist, muscimol (MUS), infused bilaterally in the dorsal hippocampi showed severe performance deficits in the VCRS task and the impairment was completely reversible with vehicle injections. The impairment in contextual response selection with hippocampal inactivations occurred regardless of whether the visual context was presented in the side monitors or in the center touchscreen monitor. However, when the same visual contextual stimuli were pitted against each other between the two side monitors and as the rats simply ran toward the visual context associated with reward on a T-shaped track, hippocampal inactivations with MUS showed minimal disruptions, if any, in performance. Our results suggest that the hippocampus is critically involved in conditional response selection using visual stimuli in the background, but it is not required for the perceptual discrimination of those stimuli.
hippocampus; context; navigation; episodic memory; response selection; choice behavior; decision making
Background and Purpose:
To investigate the duration of seizures and its relationship to seizure type, epilepsy syndrome, and seizure clustering.
We examined 1,251 seizures from 152 patients who underwent video-electrocorticographic monitoring with subdural electrodes. Their seizure duration, seizure types, epilepsy syndromes, and seizure clusters were analyzed.
The median seizure duration was 91.5s (4–1016s). There were 34 (2.7%) seizures lasting > 5 minutes in 20 (13.2%) patients. There was a significant difference in seizure duration according to seizure types (p < 0.0001), but not to epilepsy syndromes. There were 99 seizure clusters in 67 (44.1%) patients. The first seizure in a cluster of seizures tended to last longer than non-cluster seizures (median 98s versus 89s, p = 0.033). Seizure duration was significantly longer in mesial temporal lobe epilepsy than in neocortical lobe epilepsy (median 103s versus 87s, p = 0.041). Rate of seizure cluster was lower in mTLE (38.0%) than in NLE (47.1%), but this difference was not significant.
Seizure durations were different among seizure types. Seizure clustering also differ between patients with mTLE and those with NLE, which suggests different seizure generation and propagation among different epileptogenic foci. This study has implications for the identification of abnormally prolonged seizures.
Epilepsy; Seizure duration; Seizure cluster; Subdural grid
Gene set analysis is a powerful method of deducing biological meaning for an a priori defined set of genes. Numerous tools have been developed to test statistical enrichment or depletion in specific pathways or gene ontology (GO) terms. Major difficulties towards biological interpretation are integrating diverse types of annotation categories and exploring the relationships between annotation terms of similar information.
GARNET (Gene Annotation Relationship NEtwork Tools) is an integrative platform for gene set analysis with many novel features. It includes tools for retrieval of genes from annotation database, statistical analysis & visualization of annotation relationships, and managing gene sets. In an effort to allow access to a full spectrum of amassed biological knowledge, we have integrated a variety of annotation data that include the GO, domain, disease, drug, chromosomal location, and custom-defined annotations. Diverse types of molecular networks (pathways, transcription and microRNA regulations, protein-protein interaction) are also included. The pair-wise relationship between annotation gene sets was calculated using kappa statistics. GARNET consists of three modules - gene set manager, gene set analysis and gene set retrieval, which are tightly integrated to provide virtually automatic analysis for gene sets. A dedicated viewer for annotation network has been developed to facilitate exploration of the related annotations.
GARNET (gene annotation relationship network tools) is an integrative platform for diverse types of gene set analysis, where complex relationships among gene annotations can be easily explored with an intuitive network visualization tool (http://garnet.isysbio.org/ or http://ercsb.ewha.ac.kr/garnet/).
DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.
PDZ-GEF is a novel guanine nucleotide exchange factor for Rap1 GTPase. Here we isolated Drosophila melanogaster PDZ-GEF (dPDZ-GEF), which contains the all-conserved domains of mammalian and nematode PDZ-GEF including cyclic nucleotide monophosphate-binding, Ras exchange motif, PDZ, RA, and GEF domains. dPDZ-GEF loss-of-function mutants were defective in the development of various organs including eye, wing, and ovary. Many of these phenotypes are strikingly similar to the phenotype of the rolled mutant, implying that dPDZ-GEF functions upstream of the mitogen-activated protein (MAP) kinase pathway. Indeed, we found that dPDZ-GEF is specifically involved in photoreceptor cell differentiation, facilitating its neuronal fate via activation of the MAP kinase pathway. Rap1 was found to link dPDZ-GEF to the MAP kinase pathway; however, Ras was not involved in the regulation of the MAP kinase pathway by dPDZ-GEF and actually had an inhibitory function. The analyses of ovary development in dPDZ-GEF-deficient mutants also demonstrated another role of dPDZ-GEF independent of the MAP kinase signaling pathway. Collectively, our findings identify dPDZ-GEF as a novel upstream regulator of various morphogenetic pathways and demonstrate the presence of a novel, Ras-independent mechanism for activating the MAP kinase signaling pathway.