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Abstract Background Mitochondria are known to synthesize adenosine triphosphate (ATP) through oxidative phosphorylation. Understanding and accurately measuring mitochondrial ATP synthesis rate can provide insights into the functional status of mitochondria and how it contributes to overall cellular energy homeostasis. Traditional methods only estimate mitochondrial function by measuring ATP levels at a single point in time or through oxygen consumption rates. This study introduced the relative mitochondrial ATP synthesis response against inhibiting and stimulating substrates (MitoRAISE), designed to detect real-time changes in ATP levels as the cells respond to substrates. Methods The sensitivity and specificity of the MitoRAISE assay were verified under various conditions, including the isolation of mitochondria, variations in cell numbers, cells exhibiting mitochondrial damage, and heterogeneous mixtures. Using peripheral blood mononuclear cells (PBMCs), we analyzed MitoRAISE data from 19 patients with breast cancer and 23 healthy women. Results The parameters observed in the MitoRAISE data increased depending on the quantity of isolated mitochondria and cell count, whereas it remained unmeasured in mitochondrial-damaged cell lines. Basal ATP, rotenone response, malonate response, and mitochondrial DNA copy numbers were lower in PBMCs from patients with breast cancer than in those from healthy women. Conclusions The MitoRAISE assay has demonstrated its sensitivity and specificity by measuring relative ATP synthesis rates under various conditions. We propose MitoRAISE assay as a potential tool for monitoring changes in the mitochondrial metabolic status associated with various diseases.

Detection of oncogenic fusion genes in cancers, particularly in the diagnosis of uncertain tumors, is crucial for determining effective therapeutic strategies. Although novel fusion genes have been discovered through sequencing, verifying their oncogenic potential remain difficult. Therefore, we evaluated the utility of targeted RNA sequencing in 165 tumor samples by identifying known and unknown fusions. Additionally, by applying additional criteria, we discovered eight novel fusion genes that are expected to process oncogenicity. Among the novel fusion genes, RAF1 fusion genes were detected in two cases. PTPRG-RAF1 fusion led to an increase in cell growth; while dabrafenib, a BRAF inhibitor, reduced the growth of cells expressing RAF1. This study demonstrated the utility of RNA panel sequencing as a theragnostic tool and established criteria for identifying oncogenic fusion genes during post-sequencing analysis.

Small cell lung cancer (SCLC) is a fast-growing and malignant cancer that responds well to chemotherapy; however, the survival rate is less than 15% after 2 years of diagnosis. Therefore, novel therapeutic agents for treating SCLC patients need to be evaluated. This study aims to identify the therapeutic targets based on the comprehensive genomic profiling of SCLC patients. Among the molecular-profiled SCLC samples obtained using targeted sequencing, the array-based comparative genomic hybridization (array CGH) identified focal insulin receptor substrate 2 (IRS2) amplification in the SCLC patients. IRS2 amplification was confirmed in 5% of 73 SCLC patients. To determine whether IRS2 amplification could act as a therapeutic target, we generated a patient-derived xenograft (PDX) model and subsequently screened 43 targeted agents using the PDX-derived cells (PDCs). Ceritinib significantly inhibited the cell growth and impaired the tumor sphere formation in IRS2-expressing PDCs. Its effects were confirmed in various in vitro assays and were further validated in the mouse xenograft models. In this study, we present that IRS2 amplification and/or expression serve as preclinical implications for a novel therapeutic target in SCLC progression. Furthermore, we suggest that insulin-like growth factor-1 (IGF-1) receptor inhibitor-based therapy could be used for treating SCLC with IRS2 amplification. Keywords: IRS2, small cell lung cancer, SCLC, patient-derived xenograft, PDX, targeted therapy, Ceritinib

Most NTRK fusions occur at very low frequencies in various common cancers. Recent recommendations on NTRK testing recommend immunohistochemistry (IHC) as the initial test for tumor types with a low frequency of NTRK fusions. This study investigated the accuracy of an IHC assay to detect NTRK fusions and characterize the clinicopathological and molecular features of NTRK-rearranged tumors. This retrospective study was conducted on 1113 solid tumor samples known to harbor no oncogenic driver alterations, including 510 non-small cell lung cancers (NSCLC), 503 colorectal cancers (CRC), and 79 inflammatory myofibroblastic tumors (IMT). Additionally, 21 ALK expression-positive cases were included. TRK expression was evaluated using a pan-Trk IHC assay, and positive cases were validated using NGS. TRK expression was observed in three NSCLCs (0.6%), six CRCs (1.2%), and six IMTs (6%). NTRK fusions were finally detected in two NSCLCs (0.4%), six CRCs (1.2%), and one IMT (1%). In NSCLC and CRC, the majority of NTRK fusions were readily discernible due to diffuse moderate-to-strong cytoplasmic staining on pan-Trk IHC. In IMT, focal weak nuclear staining indicated the presence of NTRK fusion. Therefore, the utility of pan-Trk IHC should be assessed considering that the difference in performance depends on tumor type.

Summary: Exome and transcriptome analyses of clinically homogeneous early-stage never-smoker female patients with lung adenocarcinoma were performed to understand tumor-T cell interactions and immune escape points. Using our novel gene panels of eight functional categories in the cancer-immunity cycle, three distinct subgroups were identified in this immune checkpoint blockade-refractory cohort by defective gene expression in two major domains, i.e., type I interferon production/signaling pathway and antigen-presenting machinery. Our approach could play a critical role in understanding immune evasion mechanisms, developing a method for effective selection of rare immune checkpoint blockade responders, and finding new treatment strategies.

The supramammillary area (SUM) of the hypothalamus has wide spread connection with numerous brain structures. It is known that the SUM can control the frequency of the hippocampal theta rhythm, which plays a role in the cognitive functions of the hippocampal formation. In order to examine the role of the specific cells of the SUM in learning and memory, selective cholinergic neurotoxic or excitotoxic lesioned rats of the SUM were tested for spatial memory on the Morris water maze (MWM) test. After the behavior tests, the expression of acetylcholinesterase (AChE) in the hippocampus was studied using the immunohistochemistry. In the MWM test, both lesion of the SUM with 192 IgG-saporin or ibotenic acid produced the impairment of spatial learning and memory. The expression of AChE immunreactive neurons in the hippocampal CA3 region was decreased after injections of 192 IgG-saporin into the SUM. These findings suggest that cholinoceptive cells of the SUM area may play a critical role in the process of learning and memory.

Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain-conjugated luciferase (PTD-Luc) for measuring cellular uptake efficacy. In this study, we evaluated the applicability of PTD-Luc to an intracellular ATP assay of live cells. The predominant fluorescence of Alexa 647-PTD-Luc was in the cytosol, whereas the fluorescence of Alexa 647-Luc was visualized surrounding the cell membrane, as confirmed by Western blot analysis. In vitro, PTD-Luc could detect less than 10–9 M ATP, and the correlation between the luciferase activity of PTD-Luc and the ATP content was strong (R = 0.999, p < 0.001). In vivo, luminescence signals of PTD-Luc detected intracellular ATP in as few as 50 HeLa cells, with a strong correlation between luminescence and cell number, suggesting high sensitivity and reliability. Furthermore, two blockers of the glycolytic pathway (2-deoxyglucose and iodoacetic acid) inhibited the signal in a dose-dependent manner, whereas potassium cyanide, an inhibitor of oxidative phosphorylation, had no effect on intracellular ATP in vivo, as seen with the PTD-Luc sensor. These data show that PTD-Luc can directly measure the intracellular ATP content in live cells, allowing real-time kinetic studies, suggesting that it is a promising tool for high-throughput drug screening and cytotoxicity assays.

Animal models using cell lines that are dual-labeled with luciferase and green fluorescent protein (GFP) are powerful tools for performing simultaneous quantitative and qualitative analysis of metastatic tumors. However, the applications of such dual-labeled tumor models have been limited due to the technical challenges associated with low bioluminescent signaling from tumor cells. Here, we used lentiviral vector (LV) encoding firefly luciferase and GFP and engineered a more sensitive and highly metastatic prostate cancer cell line (MLL-Luc/GFP cells), which allows simultaneous fluorescence and luminescence imaging. The light emission of MLL-Luc/GFP cells was 33.5 fold higher than that of PC-3-luc2-GFP prostate cancer cells which showed 750 p/s light emission per cell. Furthermore, the MLL-Luc/GFP cells showed 3.9 fold higher luciferase activities than did 4T1-luc2, which was previously recognized as exhibiting the highest luciferase activity. An in vivo evaluation with optical imaging showed pinpoint localization of GFP-positive cells in a metastatic lung as well as easy detection of early metastatic spreading. The newly engineered MLL-Luc/GFP cells provide an appropriate metastatic animal model system for future studies of metastasis and the testing of anti-metastatic therapies specifically aimed at prostatic cancer.

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl4-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFβ1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.

Lindera obtusiloba extracts are commonly used as an alternative medicine due to its numerous health benefits in Korea. However, the antidepressant-like effects of L. obtusiloba extracts have not been fully elucidated. In this study, we aimed to determine whether L. obtusiloba extracts exhibited antidepressant-like activity in rats subjected to forced swim test (FST)-induced depression. Acute treatment of rats with L. obtusiloba extracts (200 mg/kg, p.o.) significantly reduced immobility time and increased swimming time without any significant change in climbing. Rats treated with L. obtusiloba extracts also exhibited a decrease in the limbic hypothalamic-pituitary-adrenal (HPA) axis response to the FST, as indicated by attenuation of the corticosterone response and decreased c-Fos immunoreactivity in the hippocampus CA3 region. In addition, L. obtusiloba extracts, at concentrations that were not affected by cell viability, significantly decreased luciferase activity in response to cortisol in a concentration-dependent manner by the glucocorticoid binding assay in HeLa cells. Our findings suggested that the antidepressant-like effects of L. obtusiloba extracts were likely mediated via the glucocorticoid receptor (GR). Further studies are needed to evaluate the potential of L. obtusiloba extracts as an alternative therapeutic approach for the treatment of depression.

TIMP-2 protein has been intensively studied as a promising anticancer candidate agent, but the in vivo mechanism underlying its anticancer effect has not been clearly elucidated by previous works. In this study, we investigated the mechanism underlying the anti-tumor effects of a TIMP-2 fusion protein conjugated with human serum albumin (HSA/TIMP-2). Systemic administration of HSA/TIMP-2 effectively inhibited tumor growth at a minimum effective dose of 60 mg/kg. The suppressive effect of HSA/TIMP-2 was accompanied by a marked reduction of in vivo vascularization. The anti-angiogenic activity of HSA/TIMP-2 was directly confirmed by CAM assays. In HSA/TIMP-2-treated tumor tissues, MMP-2 expression was profoundly decreased without a change in MT1-MMP expression of PECAM-1-positive cells. MMP-2 mRNA was also decreased by HSA/TIMP-2 treatment of human umbilical vein endothelial cells. Zymographic analysis showed that HSA/TIMP-2 substantially decreased extracellular pro-MMP-2 activity (94-99% reduction) and moderately decreased active MMP-2 activity (10-24% reduction), suggesting MT1-MMP-independent MMP-2 modulation. Furthermore, HSA/TIMP-2 had no effect on in vitro active MMP-2 activity and in vivo MMP-2 activity. These studies show that HSA/TIMP-2 potentiates anti-angiogenic activity by modulating MMP-2 expression, but not MMP-2 activity, to subsequently suppress tumor growth, suggesting an important role for MMP-2 expression rather than MMP-2 activity in anti-angiogenesis.

Purpose TIMP-2 has been studied as an attractive cancer therapeutic candidate, and a TIMP-2 fusion protein (HSA/TIMP-2) displayed effective anticancer activity, despite a lack of information about its pharmacokinetics (PK) and biodistribution. The purpose of this work was to assess the PK and biodistribution of HSA/TIMP-2 as well as to quantify accumulated HSA/TIMP-2 in tumors. Methods Cy5.5 near-infrared (NIR) fluorescence was conjugated to the HSA/TIMP-2 protein (Cy5.5–HSA/TIMP-2) for monitoring spatio-temporal changes in vivo. For PK and biodistribution analysis, 0.2 μg/g body weight of Cy5.5–HSA/TIMP-2 was injected into MAT-LyLu prostate tumor xenografts, which were then imaged using an IVIS-200 optical imaging system. To quantify the accumulated HSA/TIMP-2 in tumors, we introduced a standard curve with depth-corrected fluorescence measurement. Results In the vascular tube formation assay with human umbilical vein endothelial cells (HUVECs), Cy5.5–HSA/TIMP-2 showed an antiangiogenic effect. In prostate cancer xenografts, Cy5.5–HSA/TIMP-2 exhibited a prolongation of blood half-life to 19.6 h and relatively preferential distribution to the tumor. The amount of tumor-accumulated Cy5.5–HSA/TIMP-2 was calculated to be 4.5 ± 0.5 ng/g body weight at 2 days, representing 2.25 ± 0.25% of the initial dose. Conclusions We evaluated the pharmacokinetic profile and biodistribution of HSA/TIMP-2 with favorable results, providing new information for more effective approaches to cancer therapeutics using HSA/TIMP-2. Additionally, real-time in vivo fluorescence imaging analysis using a depth-corrected standard curve may serve as a platform to quantify biodistributed drug in anticancer therapeutic studies. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Mediastinal vascular malformations are rare and their diagnosis can be challenging. Imaging is vital for diagnosing mediastinal vascular malformations and can help avoid unnecessary invasive procedures. Herein, we report the detailed CT and MRI findings of a rare low-flow mediastinal vascular malformation in an asymptomatic 63-year-old male. [ABSTRACT FROM AUTHOR]

Purpose NUT carcinoma (NC) is a solid tumor caused by the rearrangement of NUTM1 that usually develops in midline structures, such as the thorax. No standard treatment has been established despite high lethality. Thus, we investigated whether targeting the junction region of NUTM1 fusion breakpoints could serve as a potential treatment option for NC. Materials and Methods We designed and evaluated a series of small interfering RNAs (siRNAs) targeting the junction region of BRD4-NUTM1 fusion (B4N), the most common form of NUTM1 fusion. Droplet digital polymerase chain reaction using the blood of patients was also tested to evaluate the treatment responses by the junction sequence of the B4N fusion transcripts. Results As expected, the majority of NC fusion types were B4N (12 of 18, 67%). B4N fusion-specific siRNA treatment on NC cells showed specific inhibitory effects on the B4N fusion transcript and fusion protein without affecting the endogenous expression of the parent genes, resulting in decreased relative cell growth and attenuation of tumor size. In addition, the fusion transcript levels in platelet-rich-plasma samples of the NC patients with systemic metastasis showed a negative correlation with therapeutic effect, suggesting its potential as a measure of treatment responsiveness. Conclusion This study suggests that tumor-specific sequences could be used to treat patients with fusion genes as part of precision medicine for a rare but deadly disease. [ABSTRACT FROM AUTHOR]

Purpose Tropomyosin receptor kinase (TRK) inhibitors are approved for the treatment of neurotrophic receptor tyrosine kinase (NTRK) fusion-positive tumors. The detection of NTRK fusion using a validated method is required before therapeutic application. An interlaboratory comparison study of next-generation sequencing (NGS)–based NTRK gene fusion detection with validated clinical samples was conducted at six major hospitals in South Korea. Materials and Methods A total of 18 samples, including a positive standard reference and eight positive and nine negative clinical samples, were validated using the VENTANA pan-TRK (EPR17341) and TruSight Oncology 500 assays. These samples were then tested using four different NGS panels currently being used at the six participating institutions. Results NTRK fusions were not detected in any of the nine negative clinical samples, demonstrating 100% specificity in all six participating institutions. All assays showed 100% analytical sensitivity to identify the NTRK fusion status in formalin-fixed paraffinembedded (FFPE) samples, although with variable clinical sensitivity. False-negative results were due to low tumor purity, poor RNA quality, and DNA-based sequencing panel. The RNA-based targeted NGS assay showed an overall high success rate of identifying NTRK fusion status in FFPE samples. Conclusion This study is the first to test the proficiency of NGS-based NTRK detection in South Korea with the largest participating institutions. RNA-based NGS assays to detect NTRK fusions can accurately characterize fusion transcripts if sufficient RNA of adequate quality is available. The comparative performance data will support the implementation of targeted NGS-based sequencing assays for NTRK fusion detection in routine diagnostics. [ABSTRACT FROM AUTHOR]

Purpose To find biomarkers for disease, there have been constant attempts to investigate the genes that differ from those in the disease groups. However, the values that lie outside the overall pattern of a distribution, the outliers, are frequently excluded in traditional analytical methods as they are considered to be 'some sort of problem.' Such outliers may have a biologic role in the disease group. Thus, this study explored new biomarker using outlier analysis, and verified the suitability of therapeutic potential of two genes (TM4SF4 and LRRK2). Materials and Methods Modified Tukey's fences outlier analysis was carried out to identify new biomarkers using the public gene expression datasets. And we verified the presence of the selected biomarkers in other clinical samples via customized gene expression panels and tissue microarrays. Moreover, a siRNA-based knockdown test was performed to evaluate the impact of the biomarkers on oncogenic phenotypes. Results TM4SF4 in lung cancer and LRRK2 in breast cancer were chosen as candidates among the genes derived from the analysis. TM4SF4 and LRRK2 were overexpressed in the small number of samples with lung cancer (4.20%) and breast cancer (2.42%), respectively. Knockdown of TM4SF4 and LRRK2 suppressed the growth of lung and breast cancer cell lines. The LRRK2 overexpressing cell lines were more sensitive to LRRK2-IN-1 than the LRRK2 under-expressing cell lines. Conclusion Our modified outlier-based analysis method has proved to rescue biomarkers previously missed or unnoticed by traditional analysis showing TM4SF4 and LRRK2 are novel target candidates for lung and breast cancer, respectively. [ABSTRACT FROM AUTHOR]

Primary effusion lymphoma is a large cell non-Hodgkin lymphoma exclusively presenting with effusions in body cavities in absence of solid tumor masses. It is typically associated with human herpesvirus 8 infection in immunocompromised patients with human immunodeficiency virus (HIV) infection. We report herein a case of primary effusion lymphoma in a HIV-negative patient. [ABSTRACT FROM AUTHOR]

Purpose The discoidin domain-containing receptor tyrosine kinase 2 (DDR2) is known to contain mutations in a small subset of patients with squamous cell carcinomas (SCC) of the lung. Studying the DDR2 mutations in patients with SCC of the lung would advance our understanding and guide the development of therapeutic strategies against lung cancer. Materials and Methods We selected 100 samples through a preliminary genetic screen, including specimens from biopsies and surgical resection, and confirmed SCC by histologic examination. DDR2mutations on exons 6, 15, 16, and 18 were analyzed by Sanger sequencing of formalin-fixed, paraffin-embedded tissue samples. The functional effects of novel DDR2mutants were confirmed by in vitro assays. Results We identified novel somatic mutations of DDR2 in two of the 100 SCC samples studied. One mutation was c.1745T>A (p.V582E) and the other was c.1784T>C (p.L595P), and both were on exon 15. Both patients were smokers and EGFR/KRAS/ALK-triple negative. The expression of the mutant DDR2 induced activation of DDR2 by the collagen ligand and caused enhanced cell growth and tumor progression. Moreover, dasatinib, a DDR2 inhibitor, showed potential efficacy against DDR2 L595P mutant-bearing cells. Conclusion Our results suggest that a mutation in DDR2 occurs naturally with a frequency of about 2% in Korean lung SCC patients. In addition, we showed that each of the novel DDR2mutations were located in a kinase domain and induced an increase in cell proliferation rate. [ABSTRACT FROM AUTHOR]

The objectives of this study were twofold. The first was to compare differences in perceptions of the talent development environment, achievement goals, and perceived competence, in terms of an individual characteristic (i.e. gender), a cultural characteristic (i.e. country), and their interactions (i.e. gender by country). The second was to examine the moderating effects of perceived competence on the relationships between the talent development environment and achievement goals. Data were collected from 363 athletes in Singapore and 349 athletes in Korea. A series of MANOVAs and path analyses were employed for testing of the main hypotheses. First, in terms of the talent development environment, male athletes scored higher in long-term development focus, communication, support network, and long-term development fundamentals. Second, in terms of achievement goals and perceived competence, male athletes scored higher in competence and performance-approach goal, but female athletes scored higher in mastery-avoidance goal. While Singaporean athletes scored higher in perceived competence, mastery-approach, and mastery-avoidance, Korean athletes scored higher in performance-avoidance. Lastly, the path analysis provided empirical evidence supporting the moderation effects of perceived competence on the relationships between the talent development environment and achievement goal adoption. [ABSTRACT FROM AUTHOR]

Marine organisms are frequently used to be harmful and have lower side effects than synthetic drugs. The cognitive improving efficacy of gamma aminobutyric acid-enriched fermented Saccharina japonica (FSJ) on the memory deficient rats, which were induced by trimethyltin chloride (TMT), was investigated by assessing the Morris water maze test and by performing choline acetyltransferase (ChAT), cAMP response element binding protein (CREB), and brain derived neurotrophic factor (BDNF) immunohistochemistry. The neurite outgrowth of Neuro2a cells was assessed in order to examine the underlying mechanisms of the memory enhancing effects of FSJ. Treatment with FSJ tended to shorten the latency to find the platform in the acquisition test of the Morris water maze at the second and fourth day compared to the control group. In the probe trial, the FSJ treated group increased time spent in the target quadrant, compared to that of the control group. Consistent with the behavioral data, these treatments recovered the loss of ChAT, CREB, and BDNF immunepositive neurons in the hippocampus produced by TMT. Treatment with FSJ markedly stimulated neurite outgrowth of the Neuro2a cells as compared to that of the controls. These findings demonstrate that FSJ may be useful for improving the cognitive function via regulation of neurotrophic marker enzyme activity. [ABSTRACT FROM AUTHOR]

Anaplastic lymphoma kinase (ALK) fusion is a common mechanism underlying pathogenesis of non-small cell lung carcinoma (NSCLC) where these rearrangements represent important diagnostic and therapeutic targets. In this study, we found a new ALK fusion gene, SEC31A-ALK, in lung carcinoma from a 53-year-old Korean man. The conjoined region in the fusion transcript was generated by the fusion of SEC31A exon 21 and ALK exon 20 by genomic rearrangement, which contributed to generation of an intact, in-frame open reading frame. SEC31A-ALK encodes a predicted fusion protein of 1,438 amino acids comprising the WD40 domain of SEC31A at the N-terminus and ALK kinase domain at the C-terminus. Fluorescence in situ hybridization studies suggested that SEC31A-ALK was generated by an unbalanced genomic rearrangement associated with loss of the 3!-end of SEC31A. This is the first report of SEC31A-ALK fusion transcript in clinical NSCLC, which could be a novel diagnostic and therapeutic target for patients with NSCLC. [ABSTRACT FROM AUTHOR]

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents. [ABSTRACT FROM AUTHOR]

TM4SF5 (transmembrane 4 L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5mRNAwas expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day postfertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5- related signalling. [ABSTRACT FROM AUTHOR]

TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway. [ABSTRACT FROM AUTHOR]

Caffeic acid phenethyl ester (CAPE) is an active component of propolis that has a variety of potential pharmacological effects. Although we previously demonstrated that propolis has antidepressant-like activity, the effect of CAPE on this activity remains unknown. The present study assessed whether treatment with CAPE (5, 10, and 20 μmol/kg for 21 days) has an antidepressant-like effect in mice subjected to chronic unpredictable stress via tail suspension (TST) and forced swim (FST) tests. CAPE administration induced behaviors consistent with an antidepressant effect, evidenced by decreased immobility in the TST and FST independent of any effect on serum corticosterone secretion. Western blots, conducted subsequent to behavioral assessment, revealed that CAPE significantly decreased glucocorticoid receptor phosphorylation at S234 (pGR(S234)), resulting in an increased pGR(S220/S234) ratio. We also observed negative correlations between pGR(S220)/(S234) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation, which was decreased by CAPE treatment. These findings suggest that CAPE treatment exerts an antidepressantlike effect via down regulation of p38 MAP phosphorylation, thereby contributing to enhanced GR function. [ABSTRACT FROM AUTHOR]

Using GRIMM-aerosol sampler, NOAA-HYSPLIT model, and 3D-WRF-3.3 model, the transportation of dusts from Gobi Desert toward Gangneung city, Korea was investigated from 09:00 LST October 27 to 04:00 LST October 28, 2003. Maximum PM10 (PM2.5, PM1) concentration was detected with 3.8 (3.4, 14.1) times higher magnitude than one in non-Yellow Dust period. The combination of dusts transported from the desert under westerly wind with particulate matters and gases from vehicles on the road of the city caused high PM concentrations near the ground surface at 09:00 LST and their maxima at 17:00 LST near sunset with further pollutants from heating boilers in the resident area. Positive geopotential tendency at the 500 hPa level of the city ((?F/?t;; m day-1) corresponding to negative vorticity of -4 x 10-5 sec-1 (-2.5 x 10-5 sec-1) at 0900 LST (21:00 LST; at night) was +83 m day-1 (+30 m day ¹) and it caused atmospheric depth between 500 hPa level and the ground surface to be vertically expanded. However, its net reduction to -53m/12hrs until 21:00 LST indicated synoptic-scale atmospheric layer to be vertical shrunken, resulting in the increase of PM concentrations at 17:00 LST. Simultaneously, much shallower microscale stable nocturnal surface inversion layer (NSIL) than daytime thermal internal boundary layer induced particulate matters to be merged inside the NSIL, resulting in maximum PM concentrations at 17:00 LST. [ABSTRACT FROM AUTHOR]

Propolis is a natural product made by honeybees that has been widely used in folk medicine with a broad spectrum of biological activities. To investigate the antidepressant-like activity of propolis extract, CD-I mice were administered an ethanol extract of propolis (50, 100, or 200 mg/kg, p.o.) prior to the behavioral test. The propolis extract-treated group showed a dose-dependent decrease in immobility time in the FST and tail suspension test without altering locomotor activity. Propolis extract decreased the limbic hypothalamic-pituitary-adrenal axis response to the FST as indicated by an attenuated corticosterone response and decreased in c-fos immunoreactive neurons in the hippocampal dentate gyrus. Western blot analysis revealed a reduction in hippocampal glucocorticoid receptor (GR) expression following the FST, which was reversed by propolis extract. Propolis extract also increased pGR(S220)/(S234) ratio by a differential phosphorylation in S220 and S234. FST-induced downregulation of cAMP-responsive element binding protein phosphorylation at S133 (pCREB) was restored by propolis extract, showing a strong and positive relationship between pCREB and pGR(S220)/(S234) ratio. These findings suggest that the propolis extract potentiates antidepressant-like activity by enhancing GR function which is one of the therapeutic mechanisms of antidepressant; thus, propolis extract may provide a novel therapy for depression. [ABSTRACT FROM AUTHOR]

Abstract: Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain-conjugated luciferase (PTD-Luc) for measuring cellular uptake efficacy. In this study, we evaluated the applicability of PTD-Luc to an intracellular ATP assay of live cells. The predominant fluorescence of Alexa 647-PTD-Luc was in the cytosol, whereas the fluorescence of Alexa 647-Luc was visualized surrounding the cell membrane, as confirmed by Western blot analysis. In vitro, PTD-Luc could detect less than 10-9 M ATP, and the correlation between the luciferase activity of PTD-Luc and the ATP content was strong (R = 0.999, p < 0.001). In vivo, luminescence signals of PTD-Luc detected intracellular ATP in as few as 50 HeLa cells, with a strong correlation between luminescence and cell number, suggesting high sensitivity and reliability. Furthermore, two blockers of the glycolytic pathway (2-deoxyglucose and iodoacetic acid) inhibited the signal in a dose-dependent manner, whereas potassium cyanide, an inhibitor of oxidative phosphorylation, had no effect on intracellular ATP in vivo, as seen with the PTD-Luc sensor. These data show that PTD-Luc can directly measure the intracellular ATP content in live cells, allowing real-time kinetic studies, suggesting that it is a promising tool for high-throughput drug screening and cytotoxicity assays. [ABSTRACT FROM AUTHOR]

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion- mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration. [ABSTRACT FROM AUTHOR]

The EMT (epithelial-mesenchymal transition) is involved in fibrosis and cancer, and is regulated by different signalling pathways mediated through soluble factors, actin reorganization and transcription factor actions. Because the tetraspan (also called tetraspanin) TM4SF5 (transmembrane 4 L6 family member 5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that TGFβ1 (transforming growth factor β1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the EGFR [EGF (epidermal growth factor) receptor] pathway. Inhibition of EGFR activity following TGFβ1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFβ1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad-overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFβ1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features, suggesting that TM4SF5 induction may be involved in the development of liver pathologies. [ABSTRACT FROM AUTHOR]

The hypothalamus-pituitary-adrenocortex (HPA) axis is the central mediator of the stress response. The supramammillary (SuM) region is relatively unique among the hypothalamic structures in that it sends a large, direct projection to the hippocampal formation. It has been shown that mild stress could activate the SuM cells that project to the hippocampus. However, the role of these cell populations in modulating the stress response is not known. The present study examined the effect of stress on different populations of SuM cells that project to the hippocampus by injecting the fluorescent retrograde tracer, fluorogold (FG), into the hippocampus and utilizing the immunohistochemistry of choline acetyltransferase (ChAT), corticotrophin releasing factor (CRF), serotonin (5-HT), glutamate decarboxylase (GAD), tyrosine hydroxylase (TH) and NADPH-d reactivity. Immobilization (IMO) stress (2 hr) produced an increase in the expression of ChAT- immunoreactivity, and tended to increase in CRF, 5-HT, GAD, TH-immunoreactivity and nitric oxide (NO)-reactivity in the SuM cells. Fifty-three percent of 5-HT, 31% of ChAT and 56% of CRF cells were double stained with retrograde cells from the hippocampus. By contrast, a few retrogradely labeled cells projecting to the hippocampus were immunoreactive for dopamine, γ-aminobutyric acid (GABA) and NO. These results suggest that the SuM region contains distinct cell populations that differentially respond to stress. In addition, the findings suggest that serotonergic, cholinergic and corticotropin releasing cells projecting to the hippocampus within the SuM nucleus may play an important role in modulating stress-related behaviors. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess the relationships between femoral neck angle and bone mineral density (BMD) and lower extremity injury incidence, and to show what differences exist between various sports populations based on gender. 129 athletes from the Korea National Sports University (81 males, 48 females) participated in this study. DXA hip joint scan was conducted and an injury history questionnaire was used. There were no significant differences in age, weight, height, and leg length in either gender. There was no significant gender difference in femoral neck angle (55.05±4.11°in males, 55.17±2.83° in females). Femoral neck BMD showed a significant gender difference (1.27±0.20 g/cm² in males, 1.14±0.15 g/cm²in females). There was no interaction in femoral angle based upon sports type and gender; however, there was a significant interaction in femoral neck BMD based upon sports type and gender (F(11,4)=4.29, p=0.03). Although lower limb injury incidence increased by 12% in male and 74% in female when femoral neck angle was over 54°, there was no significant relationship in femoral neck angle and injury incidence. Lower extremity injury incidence was decreased by 28% in males and 47% in females when male athletes had a BMD over 1.19 g/cm² and females over 1.08 g/cm² without a significant difference. Although there was no significant difference in femoral neck angle based upon sports type and gender, there was a significant difference in femoral neck BMD. In addition, there was no significant relationship in femoral neck angle and BMD related to injury in this study. [ABSTRACT FROM AUTHOR]

The diversity of Palaearctic chipmunks was analysed using a set of morphological (705 skulls from entire range) and genetic features (144 specimens, cytochrome b gene). Based on the results, we propose three taxa within Eutamias sibiricus. These are E. s. sibiricus, inhabiting Russia, the extreme northeast of the Korea Peninsula, Mongolia, Hokkaido Island, and northeast China; E. s. barberi, inhabiting the Korea Peninsula except for the extreme northeast region, and E. s. senescens, inhabiting central China. [ABSTRACT FROM AUTHOR]

Purpose - The purpose of this paper is to examine whether cultural differences could explain differences between Korean and American apparel web sites. Design/methodology/approach - The contents of American and Korean apparel web sites are compared. The chi-square test of independence is used to determine significant differences between the contents of the two countries apparel web sites. Findings - The paper finds that American web sites provide specific information related to products and online purchase of products, while Korean web sites provide information related to consumers' relationships to the larger community. This difference could be due to cultural differences: the Korean culture tends to reflect collectivism while the American culture tends to reflect individualism. Research limitations/implications - The fundamental limitation of this research is that it is descriptive. Practical implications - This research suggests that transcultural web sites may not be appropriate and that e-tailers need to be sensitive to cultural differences. Originality/value - The primary contribution of this paper is the identification of clear differences between Korean and American apparel web sites and that Hofstede's collectivism/individualism cultural value is a possible explanation of the difference between the two countries. [ABSTRACT FROM AUTHOR]

Previous research has discussed the interaction of hand preference, eye dominance, and sport performance. In this study, the relation of eye dominance with performance and subjective ratings in golf putting was investigated. 47 right-handed Japanese students from a college of physical education putted 10 balls to a drawn circle 3 m away, each under right-handed and left-handed stance conditions. Putting performance was measured by the number of successful puns. After putting in each condition, they rated subjective visibility and feelings of hitting. Analyses indicated that right-eyed subjects had significantly better performance using the right-handed stance than the left-handed stance, whereas left-eyed subjects showed the opposite. Most subjective ratings were more positive with right-handed stance for both right-eyed and left-eyed subjects. These findings suggest that eye dominance could have some influence on putting performance of Japanese novice golfers. [ABSTRACT FROM AUTHOR]

In response to the recent economic crisis in Korea and its negative effects on families, the current study examined the interrelationships among economic pressure, emotional distress, marital conflict, and marital satisfaction for 236 Korean couples. The family stress model (Conger & Elder, 1994; Conger, Rueter, & Conger, 2000; Conger, Rueter, & Elder, 1999) was tested using structural equation modeling. The results generally supported the theoretical model, showing that economic pressure negatively affects marital satisfaction via emotional distress and marital conflict. The results also implied cultural differences in the process of family stress. Korean husbands' emotional distress did not affect marital conflict or marital satisfaction, suggesting that Korean husbands may differ from their wives in their reaction to emotional distress from economic pressure. [ABSTRACT FROM AUTHOR]