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Purpose: Pulmonary large cell neuroendocrine carcinoma (LCNEC) is a high-grade lung neuroendocrine tumor with a poor prognosis, similar to small cell lung cancer (SCLC). However, it remains unclear whether to treat LCNEC as non-small-cell lung cancer (NSCLC) or as SCLC. We reviewed our experiences to suggest appropriate treatment strategy for resected pulmonary LCNEC.Materials and Methods: Forty-four patients were treated for pathologically diagnosed pulmonary LCNEC during 2005‒2018. We considered curative surgery first in early-stage or some locally advanced tumors, unless medically inoperable. Adjuvant treatments were decided considering patient’s clinical and pathological features. After excluding two stage I tumors with radiotherapy alone and three stage III tumors with upfront chemotherapy, we analyzed 39 patients with stage I‒III pulmonary LCNEC, who underwent curative resection first. Results: Adjuvant chemotherapy (NSCLC-based 91%, SCLC-based 9%) was performed in 62%, and adjuvant radiotherapy was done in three patients for pN2 or positive margin. None received prophylactic cranial irradiation (PCI). With a median follow-up of 30 months, the 2- and 5-year overall survival (OS) rates were 68% and 51%, and the 2- and 5-year recurrence-free survival (RFS) rates were 49% and 43%, respectively. Aged ≥67 years and SCLC-mixed pathology were significant poor prognostic factors for OS or RFS (p < 0.05). Among 17 recurrences, regional failures were most common (n = 6), and there were five brain metastases. Conclusions: Surgery and adjuvant treatment (without PCI) could achieve favorable outcomes in pulmonary LCNEC, which was more similar to NSCLC, although some factors worsened the prognosis. The importance of intensified adjuvant therapies with multidisciplinary approach remains high.

During the COVID-19 pandemic, vaccines were developed based on various platform technologies and were approved for emergency use. However, the comparative analysis of immunogenicity and durability of vaccine-induced antibody responses depending on vaccine platforms or vaccination regimens has not been thoroughly examined for mRNA- or viral vector-based vaccines. In this study, we assessed spike-binding IgG levels and neutralizing capacity in 66 vaccinated individuals prime-boost immunized either by homologous (BNT162b2-BNT162b2 or ChAdOx1-ChAdOx1) or heterologous (ChAdOx1-BNT162b2) vaccination for six months after the first vaccination. Despite the discrepancy in intervals for the prime-boost vaccination regimen of different COVID-19 vaccines, we found stronger induction and relatively rapid waning of antibody responses by homologous vaccination of the mRNA vaccine, while weaker boost effect and stable maintenance of humoral immune responses were observed in the viral vector vaccine group over 6 months. Heterologous vaccination with ChAdOx1 and BNT162b2 resulted in an effective boost effect with the highest remaining antibody responses at six months post-primary vaccination.

ObjectiveThis study aimed to update the possible clinical benefits of radiation therapy in recurrent ovarian cancer.MethodsThe medical records of 495 patients with recurrent ovarian cancer after initially undergoing maximal cytoreductive surgery and adjuvant platinum-based chemotherapy based on the pathologic stage between January 2010 and December 2020 were analyzed: 309 and 186 patients were treated without and with involved-field radiation therapy, respectively. Involved-field radiation therapy is defined as radiation therapy only to the areas of the body involved by tumor. The prescribed doses were ≥45 Gy (equivalent dose in 2 Gy/fraction). Overall survival was compared between patients treated with and without involved-field radiation therapy. The favorable group was defined as patients who satisfied at least four of the following factors: good performance, no ascites, normal CA-125, platinum-sensitive tumor, and nodal recurrence.ResultsThe median age of the patients was 56 years (range 49–63) and median time to recurrence was 11.1 months (range 6.1–15.5). 217 patients (43.8%) were treated at a single site. Radiation therapy, performance status, CA-125, platinum sensitivity, residual disease, and ascites were all significant prognostic factors. The 3-year overall survival of all patients, patients treated without radiation therapy, and patients treated with radiation therapy was 54.0%, 44.8%, and 69.3%, respectively. Radiation therapy was associated with higher overall survival rates in the unfavorable and favorable patient groups. Patient characteristics showed higher rates of normal CA-125, lymph node metastasis only, lower platinum sensitivity, and higher rates of ascites in the radiation therapy group. After propensity score matching, the radiation therapy group showed superior overall survival to the non-radiation therapy group. Normal CA-125, good performance status, and platinum sensitivity were associated with a good prognosis in patients treated with radiation therapy.ConclusionOur study showed that higher overall survival was observed in patients treated with radiation therapy in recurrent ovarian cancer.

Heart failure (HF) patients should be systematically educated before discharge on how to manage with standard written materials for patient self-management. However, because of the absence of readily available written materials to reinforce their learned knowledge, patients with HF feel inadequately informed in terms of the discharge information provided to them. This study aimed to develop core content to prepare patients with HF for transition from hospital to home care. The content was validated by expert panelists using Delphi methods. Nineteen draft items based on literature review were developed. We established a consensus on four core sections, including 47 categories and 128 subcategories through the Delphi survey: (1) understanding HF (five categories and 23 subcategories), (2) HF medication (19 categories and 45 subcategories), (3) HF management (20 categories and 47 subcategories), and (4) HF diary (three categories and 13 subcategories). Each section provided easy-to-understand educational contents using cartoon images and large or bold letters for older patients with HF. The developed core HF educational contents showed high consensus between the experts, along with clinical validity. The contents can be used as an educational booklet for both planning discharge education of patients with HF and for post-discharge management when transitioning from hospital to home. Based on this study, a booklet series for HF patients was first registered at the National Library of Korea. Future research should focus on delivering the core content to patients with HF in convenient and accessible format through various media.

Body transportation is defined as moving the body of the victim from the scene of a homicide to another location. We analyzed data on 513 South Korean homicide cases with convictions to compare 104 homicides involving the transportation of victims' bodies with 409 homicides that did not. Offenders who transported their victims' bodies were significantly younger and more likely to be related to the victims and were less likely to use sharp instruments and more likely to use blunt instruments or strangle the victims than offenders who did not. Victims of homicide involving body transportation were more likely to suffer injuries to the neck and head and less likely to suffer injuries to the torso. Finally, offenders who transported victims' bodies were more likely to have planned the offense and destroyed evidence. The present findings have practical implications for police investigations.

An ever-increasing number of computing devices interconnected through wireless networks encapsulated in the cyber-physical-social systems and a significant amount of sensitive network data transmitted among them have raised security and privacy concerns. Intrusion detection system (IDS) is known as an effective defence mechanism and most recently machine learning (ML) methods are used for its development. However, Internet of Things (IoT) devices often have limited computational resources such as limited energy source, computational power and memory, thus, traditional ML-based IDS that require extensive computational resources are not suitable for running on such devices. This study thus is to design and develop a lightweight ML-based IDS tailored for the resource-constrained devices. Specifically, the study proposes a lightweight ML-based IDS model namely IMPACT (IMPersonation Attack deteCTion using deep auto-encoder and feature abstraction). This is based on deep feature learning with gradient-based linear Support Vector Machine (SVM) to deploy and run on resource-constrained devices by reducing the number of features through feature extraction and selection using a stacked autoencoder (SAE), mutual information (MI) and C4.8 wrapper. The IMPACT is trained on Aegean Wi-Fi Intrusion Dataset (AWID) to detect impersonation attack. Numerical results show that the proposed IMPACT achieved 98.22% accuracy with 97.64% detection rate and 1.20% false alarm rate and outperformed existing state-of-the-art benchmark models. Another key contribution of this study is the investigation of the features in AWID dataset for its usability for further development of IDS.

A general strategy for visible-light-enabled site-selective trifluoromethylative pyridylation of unactivated alkenes has been developed using pyridines and triflic anhydride (Tf2 O). Intriguingly, the N-triflylpyridinium salts, generated in situ from pyridines and Tf2 O, serve as effective modular bifunctional reagents to install both CF3 and pyridyl groups to various olefins while controlling C4-selectivity in radical addition to the pyridine core. This synthetic route exhibited broad substrate scope under metal-free and mild photocatalytic conditions, granting efficient access to valuable C4-alkylated pyridines and quinolines without requiring prefunctionalization of the reaction site.

Anatomically terminal parts of the urinary, reproductive, and digestive systems of birds all connect to the cloaca. As the feces drain through the cloaca in chickens, the cloacal bacteria were previously believed to represent those of the digestive system. To investigate similarities between the cloacal microbiota and the microbiota of the digestive and reproductive systems, microbiota inhabiting the colon, cloaca, and magnum, which is a portion of the chicken oviduct of 34-week-old, specific-pathogen-free hens were analyzed using a 16S rRNA metagenomic approach using the Ion torrent sequencer and the Qiime2 bioinformatics platform. Beta diversity via unweighted and weighted unifrac analyses revealed that the cloacal microbiota was significantly different from those in the colon and the magnum. Unweighted unifrac revealed that the cloacal microbiota was distal from the microbiota in the colon than from the microbiota in the magnum, whereas weighted unifrac revealed that the cloacal microbiota was located further away from the microbiota in the magnum than from the microbiota inhabiting the colon. Pseudomonas spp. were the most abundant in the cloaca, whereas Lactobacillus spp. and Flavobacterium spp. were the most abundant species in the colon and the magnum. The present results indicate that the cloaca contains a mixed population of bacteria, derived from the reproductive, urinary, and digestive systems, particularly in egg-laying hens. Therefore, sampling cloaca to study bacterial populations that inhabit the digestive system of chickens requires caution especially when applied to egg-laying hens. To further understand the physiological role of the microbiota in chicken cloaca, exploratory studies of the chicken's cloacal microbiota should be performed using chickens of different ages and types.

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells.

Orthohantaviruses are negative-sense, single-stranded RNA viruses harbored by rodents, shrews, moles, and bats. Of the shrew-borne orthohantaviruses in the Republic of Korea (ROK), Jeju orthohantavirus (Jeju virus, JJUV) was found on Jeju Island. This small-scale epidemiologic survey investigated the geographic distribution and molecular phylogeny of JJUV in the ROK. In 32 trapping sites, tissues of 84 Crocidura shantungensis were analyzed for JJUV RNA. JJUV RNA was detected in seven (8.3%) shrews captured on the Korean peninsula. The molecular epidemiologic survey demonstrated the prevalence of JJUV by geographic distribution. The RNA loads of JJUV were evaluated in various tissues. Entire coding sequences of tripartite genomes were recovered from two JJUV strains on the mainland. Phylogenetic relationships of the JJUV revealed a distinct geographic lineage of mainland strains from the strains on Jeju Island. This study sheds light on the molecular epidemiology, phylogeographic diversity, and virus-host co-divergence of JJUV, ROK.

Fibrosis is characterized by the increased accumulation of extracellular matrix (ECM), which drives abnormal cell proliferation and progressive organ dysfunction in many inflammatory and metabolic diseases. Studies have shown that halofuginone, a racemic halogenated derivative, inhibits glutamyl-prolyl-transfer RNA-synthetase (EPRS)-mediated fibrosis. However, the mechanism by which this occurs is unclear. We explored the mechanistic aspects of how EPRS could develop liver fibrotic phenotypes in cells and animal models. Treatment with TGF-β1 up-regulated fibronectin and collagen I levels in LX2 hepatic stellate cells. This effect was inhibited in prolyl-transfer RNA synthetase (PRS)-suppressed LX2 cells. Using the promoter luciferase assay, TGF-β1-mediated collagen I, α1 chain transcription and γ2 basal laminin transcription in LX2 cells were down-regulated by EPRS suppression, suggesting that EPRS may play roles in ECM production at transcriptional levels. Furthermore, signal transducer and activator of transcription (STAT) signaling activation was involved in the effects of TGF-β1 on ECM expression in a PRS-dependent manner. This was mediated via a protein-protein complex formation consisting of TGF-β1 receptor, EPRS, Janus kinases, and STAT6. Additionally, ECM expression in fibrotic livers overlapped with EPRS expression along fibrotic septa regions and was positively correlated with STAT6 activation in carbon tetrachloride-treated mice. This was less obvious in livers of Eprs-/+ mice. These findings suggest that, during fibrosis development, EPRS plays roles in nontranslational processes of ECM expression via intracellular signaling regulation upon TGF-β1 stimulation.-Song, D.-G., Kim, D., Jung, J. W., Nam, S. H., Kim, J. E., Kim, H.-J., Kim, J. H., Lee, S.-J., Pan, C.-H., Kim, S., Lee, J. W. Glutamyl-prolyl-tRNA synthetase induces fibrotic extracellular matrix via both transcriptional and translational mechanisms.

Onychodactylus koreanus, a hynobiid salamander species endemic to the Korean Peninsula, can be regarded as a strict ecological specialist, probably vulnerable to anthropogenic environmental modifications and climate change. We used mitochondrial cytochrome b gene to analyze the genetic diversity and phylogenetic structure of O. koreanus from 19 populations collected in an attempt to cover its major distribution within South Korea. A total of 76 haplotypes of O. koreanus obtained in our analyses could be subdivided into three phylogenetic clades, KR, NE and SE. Clade KR haplotypes occur in most of the regions throughout the Korean Peninsula with four distinct subclades (KR I–IV). Clade NE and SE haplotypes were only observed in two populations YY and YS, respectively. Haplotype sharing was scarce even among populations in geographical proximity, and most of the populations were represented by a single clade or subclade, indicating the low level of gene flow among populations. O. koreanus likely originated from the historical southward dispersal of its ancestral lineages following divergence from Chinese O. zhaoermii that was recovered as the sister of O. koreanus in our phylogenetic analysis. Our results have critical implications for the taxonomic status of O. koreanus and its long-term management plan.

Purpose Early prediction of treatment outcomes represents an essential step towards increased treatment efficacy and survival in patients with hepatocellular carcinoma (HCC). In this study, we performed two-dimensional electrophoresis (2-DE) followed by protein profiling to identify biomarkers predictive of therapeutic outcomes in patients with HCC who received liver-directed therapy (LDTx) involving local radiotherapy (RT), and studied the underlying mechanisms of the identified proteins. Materials and methods 2-DE analysis was conducted by pooling sera from patients with a good or poor prognosis; serum proteomic profiles of the two groups were compared and analyzed using matrixassisted laser desorption/ionization time-of-flight mass spectrometry. Identified proteins were confirmed via enzyme-linked immunosorbent assay. An invasion assay was performed after overexpression and knockdown of target protein in Huh7 cells. Results Levels of inter-alpha inhibitor H4 (ITIH4), fibrinogen gamma chain, keratin 9/1 complex, carbonic anhydrase I, and carbonmonoxyhemoglobin S were changed by more than 4-fold in response to LDTx. In particular, pre-LDTx ITIH4 expression was more than 5-fold higher in patients with a good prognosis, compared to patients with a poor prognosis. The migration ability of Huh7 cells was significantly suppressed and enhanced by ITIH4 overexpression and knockdown, respectively. The tumors of patients with HCC and a good prognosis expressed high levels of ITIH4, compared to those of patients with a poor prognosis. Conclusion Taken together, ITIH4 may be a potential therapeutic target that could inhibit cancer metastasis, as well as a prognostic marker for patients with HCC who are receiving LDTx.

Tissue inhibitors of metalloproteinases (TIMPs) have been known to be involved in tumorigenesis in both matrix metalloproteinase (MMP)-dependent and MMP-independent manner. This manuscript highlights key findings from our recent research describing the mechanism by which TIMP-2 stimulates lung adenocarcinoma cell proliferation. Our study showed for the first time that TIMP-2 induces lung adenocarcinoma cell proliferation through c-Src kinase activation, independent of MMP inhibition. c-Src kinase activity, induced by TIMP-2, concomitantly in­­­creased FAK, phosphoinositide 3-kinase (PI3-kinase)/AKT, and ERK1/2 activation. Furthermore, we showed from multiple cohorts that high TIMP-2 expression in lung adenocarcinomas is associated with a worse prognosis, especially for stage I lung adenocarcinoma. Through integrated analysis of The Cancer Genome Atlas data, Reverse Phase Protein Assay data showed that Src phosphorylation at Y418 significantly increased when TIMP-2 was highly expressed. TIMP-2 expression was significantly associated with the alteration of driving genes and activation of the PI3-kinase/AKT pathway. Taken together, our results suggest that TIMP-2 may play a key role in tumorigenesis of lung adenocarcinoma.

Charcot-Marie-Tooth disease type 2D is a hereditary axonal and glycyl-tRNA synthetase (GARS)-associated neuropathy that is caused by a mutation in GARS. Here, we report a novel GARS-associated mouse neuropathy model using an adenoviral vector system that contains a neuronal-specific promoter. In this model, we found that wild-type GARS is distributed to peripheral axons, dorsal root ganglion (DRG) cell bodies, central axon terminals, and motor neuron cell bodies. In contrast, GARS containing a G240R mutation was localized in DRG and motor neuron cell bodies, but not axonal regions, in vivo. Thus, our data suggest that the disease-causing G240R mutation may result in a distribution defect of GARS in peripheral nerves in vivo. Furthermore, a distributional defect may be associated with axonal degradation in GARS-associated neuropathies.

It is generally required to use modeling or animation tools for inserting dynamic objects into augmented reality, and this process demands high expertise and complexity. This paper proposes a video object based authoring method that enables augmentation with dynamic video objects without such process. Integrated grab-cut and grow-cut method strips initial area of video target off the existing video clips, and snap-cut method is then applied to track objects` boundaries over frames so as to augment real world with continuous motion frames. Experiment shows video cut-out and authoring results achieved by only a few menu selections and simple correcting sketch.

The adenosine triphosphate (ATP) plays important roles under physiological and pathological conditions such as traumatic brain injury, neuroinflammation and neuropathic pain. In the present study, we set out to study the role of lysosomal vesicles on ATP release from the dorsal root ganglion neurons. We found that the lysosomal vesicles, which contain the quinacrine-positive fluorescence and express the vesicular nucleotide transporter (VNUT), were localized within the soma and growth cone of the cultured dorsal root ganglion neurons. In addition, the number of the quinacrine staining was decreased by application of lysosomal exocytosis activators, and this decrease was suppressed by the metformin and vacuolin-1, which suppressed lysosomal exocytosis. Thus, these findings suggest that ATP release via the lysosomal exocytosis may be one of the pathways for ATP release in response to stimulation.

In in vitro and in vivo systems, understanding localization and the functional role of ATP is essential, but effective methods to monitor ATP in cells and tissues are limited. Although quinacrine dihydrochloride is a well-known fluorescent dye used to detect ATP, it is limited in its use because it shows non-specific nuclear staining both in vitro and in vivo. A commercial luciferin-luciferase bioluminescence assay has also been used to detect ATP, but it can not be easily used in vivo. Thus, to effectively monitor ATP in vivo, we employed a novel two-photon ATP fluorescent probe, acedan-based Zn(DPA). Using the acedan-based Zn(DPA) probe, we show that this probe produces high quality images of ATP in lung, spleen, liver and spinal cord tissues.

The present study demonstrates that adenosine triphosphate (ATP) is released from Schwann cells through lysosomal exocytosis during Wallerian degeneration and in response to stimulation. In primary Schwann cell cultures, ATP was stored in lysosomal vesicles. ATP could then induce Ca(2+)-dependent lysosomal exocytosis. Among three stimulants of lysosomal exocytosis (glutamate, NH(4)Cl and zymosan), only NH(4)Cl was sufficient to induce ATP release from ex vivo sciatic nerve explants at 3 days in vitro. Lysosomal exocytosis inhibitors (metformin, chlorpromazine and vacuolin-1) reversed the effect of NH(4)Cl-enhanced ATP release, replicating the state of explants treated with NH(4)Cl in the absence of lysosomal exocytosis inhibitors. Furthermore, we observed ATP release through lysosomal exocytosis during Wallerian degeneration in sciatic explant cultures using the recently identified vesicular nucleotide transporter (VNUT). From these experiments, we conclude that the exocytosis of lysosomes in Schwann cells during Wallerian degeneration is Ca(2+)-dependent, and that it induces ATP release from Schwann cells.

// Kyoung-Jin Kim 1 , Ji-Hye Kim 1 , Seo Jin Lee 1 , Eun-Jung Lee 1 , Eui-Cheol Shin 2 and Jinsil Seong 1 1 Department of Radiation Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea 2 Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Techonology, Daejeon, Republic of Korea Correspondence to: Jinsil Seong, email: jsseong@yuhs.ac Keywords: hepatocellular carcinoma, anti-PD-L1, radiation, combination therapy, antitumor effect Received: October 21, 2016 Accepted: March 28, 2017 Published: April 17, 2017 ABSTRACT Background & aims: Although immunotherapy has emerged as an attractive therapy for refractory cancers, its limited efficacy in hepatocellular carcinoma (HCC) suggests the need for a combination strategy that can either enhance or complement therapeutic effect. We investigated whether combination of immune checkpoint blockade (ICB) and radiation could enhance antitumor effect in a murine HCC model. Methods: Using murine HCC, HCa-1, the effect of radiation on programmed death-ligand1 (PD-L1) expression was determined by real-time PCR, flow cytometry, and western blotting. Signaling pathways involved in altered PD-L1 expression were examined. Tumor growth and survival rate were evaluated for a combination of anti-PD-L1 and radiation. Immunological parameters in the tumor were assessed using flow cytometry and histological study. Results: Radiation upregulated PD-L1 expression in tumor cells through IFN-γ/STAT3 signaling, which could facilitate therapeutic action of anti-PD-L1. Combination of anti-PD-L1 and radiation significantly suppressed tumor growth compared to treatment with anti-PD-L1 alone or radiation alone group ( P< 0.01). Survival was significantly improved in the combination group compared to anti-PD-L1 alone or radiation alone group (7-week survival rate; 90% vs. 0% or 30%, respectively, P< 0.001). The underlying mechanism involved increasing apoptosis, decreasing tumor cell proliferation, as well as restoration of CD8 + T cell functions. Conclusions: The combination of anti-PD-L1 and radiation significantly improved the antitumor effect shown in tumor growth delay as well as in survival, supporting a novel combination strategy of immunoradiotherapy in HCC.

In Augmented Reality, it needs restoration and tracking of a real-time scene structure for the augmented 3D model from input video or images. Most of the previous approaches construct accurate 3D models in advance and try to fit them in real-time. However, it is difficult to measure 3D model accurately and requires long pre-processing time to construct exact 3D model specifically. In this research, we suggest a real-time scene structure analysis method for the wide indoor mobile augmented reality, using only generic models without exact pre-constructed models. Our approach reduces cost and time by removing exact modeling process and demonstrates the method for restoration and tracking of the indoor repetitive scene structure such as corridors and stairways in different scales and details.

In the present study we examine the effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the phosphorylation status of specific phosphotyrosine residues on the vascular endothelial cell growth factor receptor-2 (VEGFR-2) cytoplasmic tail and examine the effects on associated downstream signaling pathways. In order to focus on metalloproteinase-independent mechanisms, we utilized the TIMP-2 analog known as Ala+TIMP-2 that is deficient in matrix metalloproteinase (MMP) inhibitory activity. Our experiments are designed to compare the effects of VEGF-A stimulation with or without Ala+TIMP-2 pretreatment, as well as basal responses in human microvascular endothelial cells. Our results show that Ala+TIMP-2 selectively alters the phosphorylation pattern of VEGFR-2 following VEGF-A stimulation and disrupts the downstream activation of PLC-γ, Ca+2 flux, Akt, and eNOS, as well as decreasing cGMP levels. Moreover, we observed an Ala+TIMP-2-induced reduction in cGMP levels typically elevated by exogenous NO donors implicating Ala+TIMP-2 in the direct activation of an isobutylmethylxanthine (IBMX)-sensitive cGMP phosphodiesterase activity. TIMP-2 suppression of endothelial mitogenesis and angiogenesis involves at least two mechanisms, one mediated by protein tyrosine phosphatase inhibition of VEGFR-2 activation and downstream signaling and a second mechanism involving direct activation of an IBMX-sensitive phosphodiesterase activity.

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue offers significant diagnostic utility but is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed a reliable protein extraction method for FFPE tissue, based on heat-induced antigen retrieval within a pressure cooker. The protein extraction yield from archival FFPE tissue section is approximately 90% of that recovered from frozen tissue. This method demonstrates preservation of immuno-reactivity and recovery of full-length proteins by western blotting. Additionally we developed a well-based reverse-phase protein array platform utilizing an electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, remain stable over 1 month at room temperature. Theses data suggest that this new protein-profiling platform coupled with the protein extraction method can be used for molecular profiling analysis in FFPE tissue, and contribute to the validation and development of biomarkers in clinical studies.

Radiotherapy (RT) is a potent anti-tumor modality. However, unwanted effects including increased recurrence and metastasis that involve factors such as cytokines, which induce complex molecular mechanisms, have also been reported. In a previous study, we showed that interleukin (IL)-12 and radiotherapy combination treatment suppressed tumor growth and metastasis in a hepatoma mouse model. In this study, we investigated the mechanism underlying the IL-12 anti-tumor effect during radiotherapy. In tumor-bearing mice, irradiation decreased IL-12 expression in the tumors and spleens. However, a number of dendritic cells infiltrated into the tumors in which IL-12 expression did not decrease. To further study the underlying detailed mechanism for this decrease in IL-12, LPS-stimulated bone marrow-derived dendritic cells (BMDCs) were irradiated, and then IL-12- and IL-6-associated molecules were examined in irradiated tumors and BMDCs. Irradiation resulted in IL-12 suppression and IL-6 increase. IL-6 and signal transducer and activator of transcription 3 (STAT3) inhibitors restored the irradiation-induced IL-12 decrease via suppression of C-Rel activation. Taken together, our study suggests that irradiation-induced IL-6 can decrease IL-12 production through the inhibition of C-Rel phosphorylation by the IL-6/STAT3 signaling pathway.

We previously showed that plasma gelsolin, a major component of the extracellular actin scavenging system, is an matrix metalloproteinase (MMP)-14 substrate. Here we confirmed that plasma gelsolin is cleaved by MMP-14 at the plasma level, and found that it was most efficiently digested by MMP-3 followed by MMP-2, MMP-1, MMP-14, and MMP-9, in that order. Plasma gelsolin (90 kDa) was cut into several fragments of 43-48 kDa by MMP-3. The MMP-3 cleavage sites in plasma gelsolin were determined by labeling the C termini generated by in-gel digestion with 50% H2 18O combined with peptide mass mapping, and sequencing of the N-terminal amino acids. Plasma gelsolin was cleaved at Asn416-Val417, Ser51-Met52, and Ala435-Gln436. Proteolytic cleavage by MMP-3 resulted in considerable loss of its actin filament-depolymerizing activity. This suggests that MMPs weaken the extracellular actin-scavenging system by cleaving plasma gelsolin and may, therefore, be involved in pathological conditions induced by extracellular actin, such as endothelial injury, respiratory distress syndrome, hepatic necrosis, and septic shock.

In addition to inhibiting matrix metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1) is involved in the regulation of cell growth and survival. To determine its mechanism of action, we investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signal-regulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. We found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These findings demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt.

Hollow SnO2 spheres functionalized by Au catalysts were synthesized via the use of Au-decorated block copolymer (Au-BCP) sphere templates. Uniformly distributed Au nanoparticles on BCP spheres were prepared by the infiltration of Au precursors into polystyrene-b-poly(4-vinylpyridine) (PS-b-P4VP) spheres. A thin SnO2 layer was coated on the Au-BCP spheres using RF sputtering at room temperature without morphological deformation of the spheres. The Au nanoparticles were uniformly transferred from the Au-BCP spheres to the inner shells of the hollow SnO2 spheres followed by decomposition of BCP spheres. The Au-loaded hollow SnO2 spheres exhibited a superior H2S sensitivity (Rair/Rgas = 17.4 at 5 ppm) with remarkably selective characteristics with a minor response (Rair/Rgas < 2.5 at 5 ppm) toward other interfering gases. Our results pave the way for a new catalyst loading method using Au-BCP spheres for the uniformly distributed Au NPs on the SnO2 layers.

Diagnostic sensing device using exhaled breath of human have critical advantages due to the noninvasive diagnosis and high potential for portable device with simple analysis process. Here, we report ultrafast as well as highly sensitive bumpy WO3 hemitube nanostructure assisted by O2 plasma surface modification with functionalization of graphene-based material for the detection of acetone (CH3COCH3) and hydrogen sulfide (H2S) which are biomarkers for the diagnosis of diabetes and halitosis, respectively. 0.1 wt % graphene oxide (GO)- and 0.1 wt % thin layered graphite (GR)- WO3 hemitube composites showed response times of 11.5 ± 2.5 s and 13.5 ± 3.4 s to 1 ppm acetone as well as 12.5 ± 1.9 s and 10.0 ± 1.6 s to 1 ppm of H2S, respectively. In addition, low limits of detection (LOD) of 100 ppb (Rair/Rgas = 1.7 for acetone and Rair/Rgas = 3.3 for H2S at 300 °C) were achieved. The superior sensing properties were ascribed to the electronic sensitization of graphene based materials by modulating space charged layers at the interfaces between n-type WO3 hemitubes and p-type graphene based materials, as identified by Kelvin Probe Force Microscopy (KPFM). Rapid response and superior sensitivity of the proposed sensing materials following cyclic thermal aging demonstrates good potential for real-time exhaled breath diagnosis of diseases.

Sensitive detection of acetone and hydrogen sulfide levels in exhaled human breath, serving as breath markers for some diseases such as diabetes and halitosis, may offer useful information for early diagnosis of these diseases. Exhaled breath analyzers using semiconductor metal oxide (SMO) gas sensors have attracted much attention because they offer low cost fabrication, miniaturization, and integration into portable devices for noninvasive medical diagnosis. However, SMO gas sensors often display cross sensitivity to interfering species. Therefore, selective real-time detection of specific disease markers is a major challenge that must be overcome to ensure reliable breath analysis. In this work, we report on highly sensitive and selective acetone and hydrogen sulfide detection achieved by sensitizing electrospun SnO2 nanofibers with reduced graphene oxide (RGO) nanosheets. SnO2 nanofibers mixed with a small amount (0.01 wt %) of RGO nanosheets exhibited sensitive response to hydrogen sulfide (Rair/Rgas = 34 at 5 ppm) at 200 °C, whereas sensitive acetone detection (Rair/Rgas = 10 at 5 ppm) was achieved by increasing the RGO loading to 5 wt % and raising the operation temperature to 350 °C. The detection limit of these sensors is predicted to be as low as 1 ppm for hydrogen sulfide and 100 ppb for acetone, respectively. These concentrations are much lower than in the exhaled breath of healthy people. This demonstrates that optimization of the RGO loading and the operation temperature of RGO-SnO2 nanocomposite gas sensors enables highly sensitive and selective detection of breath markers for the diagnosis of diabetes and halitosis.

After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

Summary of univariate analysis results Patients with RLS Patients without RLS p Female patients 23 49 0.326Diabetes 33 91 0.471Hepatitis 02 10 0.362ESS 0005.70±3.84 0005.20±3.63 0.272Daytime sleepiness* 08 19 0.841Age (years) 00059.2±13.1 00063.4±12.4 0.057Duration of dialysis (years) 0005.13±5.43 0005.10±4.60 0.974Body weight (kg) ±00059.7 11.7 00060.5±10.3 0.657BMI (kg/m 2 ) ±00022.7 3.6 00022.4±3.9 0.679BUN (mg/dL ) 0058.70 ±22.78 0058.65 24.69 0.990Urea reduction ratio (%) 00072.9±5.1 00071.9±7.4 0.715Creatinine (mg/dL ) ±0008.94±2.63 0009.56 4.37 0.373Phosphate (mmoL/L) ±0004.57±1.77 0004.61 1.59 0.876Hb (g/L) 0010.98±1.46 0010.63±1.46 0.172Iron (μg/dL) ±0070.44 24.92 0062.41±31.23 0.127Ferritin (mg/L ) ±0291.89 264.53 0293.49±322.77 0.977TIBC (μg/dL) ±0226.29 41.03 0217.51±47.54 0.281PTH (pg/mL) 0177.34±162.6 00149.2±168.0 0.399Vitamin B 12 (pg/mL) 1792.91±1042.6 02045.9±1225.1 0.398Folate (ng/mL) 00112.8±94.4 00129.7±100.3 0.405Total bilirubin (mg/dL ) 0000.50±0.24 0000.46±0.18 0.217ALT (IU/L) 00023.7±30.7 00016.4±13.4 0.125*Patients with an ESS score greater than 9 were considered tohave daytime sleepiness. RLS: restless legs syndrome, ESS: Epworth sleep scale, BMI: body mass index, BUN: blood urea nitrogen, Hb: hemoglobin, TIBC:total iron-binding capacity, PTH: parathyroid hormone, ALT: al-anine aminotransferase.

We previously reported that endostatin inhibits endothelial and tumor cellular invasion by blocking activation and catalytic activity of matrix metalloproteinase (MMP)-2. Here we have examined the domain of proMMP-2 responsible for the binding of endostatin using surface plasmon resonance. ProMMP-2 and proMMP-2deltaHP lacking the hinge and hemopexin-like (HP) domains bound little to the immobilized endostatin. The active MMP-2 and MMP-2deltaHP, but not the HP domain of MMP-2, bound to endostatin at similar levels. In addition, preincubation of MMP-2 and MMP-2deltaHP with the MMP inhibitor actinonin, which binds to the active site of MMP-2, abolished their bindings to endostatin. These results indicate that endostatin binds to neither the latent proMMP-2 nor the HP domain but to the catalytic domain of MMP-2.