Three dimensional microelectrodes enable high signal and spatial resolution for neural seizure recordings in brain slices and freely behaving animals.

Neural recordings made to date through various approaches-both in-vitro or in-vivo-lack high spatial resolution and a high signal-to-noise ratio (SNR) required for detailed understanding of brain function, synaptic plasticity, and dysfunction. These shortcomings in turn deter the ability to further design diagnostic, therapeutic strategies and the fabrication of neuro-modulatory devices with various feedback loop systems. We report here on the simulation and fabrication of fully configurable neural micro-electrodes that can be used for both in vitro and in vivo applications, with three-dimensional semi-insulated structures patterned onto custom, fine-pitch, high density arrays. These microelectrodes were interfaced with isolated brain slices as well as implanted in brains of freely behaving rats to demonstrate their ability to maintain a high SNR. Moreover, the electrodes enabled the detection of epileptiform events and high frequency oscillations in an epilepsy model thus offering a diagnostic potential for neurological disorders such as epilepsy. These microelectrodes provide unique opportunities to study brain activity under normal and various pathological conditions, both in-vivo and in in-vitro, thus furthering the ability to develop drug screening and neuromodulation systems that could accurately record and map the activity of large neural networks over an extended time period.

Periodontal Pathogens Promote Foam Cell Formation by Blocking Lipid Efflux.

Foam cells are one of the major cellular components of atherosclerotic plaques, within which the trace of periodontal pathogens has also been identified in recent studies. In line with these findings, the correlation between periodontitis and atherosclerotic cardiovascular incidences has been repetitively supported by evidence from a number of experimental studies. However, the direct role of periodontal pathogens in altered cellular signaling underlying such cardiovascular events has not been clearly defined. To determine the role of periodontal pathogens in the pathogenesis of atherosclerosis, especially in the evolution of macrophages into foam cells, we monitored the pattern of lipid accumulation within macrophages in the presence of periodontal pathogens, followed by characterization of these lipids and investigation of major molecules involved in lipid homeostasis. The cells were stained with the lipophilic fluorescent dye BODIPY 493/503 and Oil Red O to characterize the lipid profile. The amounts of Oil Red O-positive droplets, representing neutral lipids, as well as fluorescent lipid aggregates were prominently increased in periodontal pathogen-infected macrophages. Subsequent analysis allowed us to locate the accumulated lipids in the endoplasmic reticulum. In addition, the levels of cholesteryl ester in periodontal pathogen-infected macrophages were increased, implying disrupted lipid homeostasis. Further investigations to delineate the key messengers and regulatory factors involved in the altered lipid homeostasis have revealed alterations in cholesterol efflux-related enzymes, such as ABCG1 and CYP46A1, as contributors to foam cell formation, and increased Ca2+ signaling and reactive oxygen species (ROS) production as key events underlying disrupted lipid homeostasis. Consistently, a treatment of periodontal pathogen-infected macrophages with ROS inhibitors and nifedipine attenuated the accumulation of lipid droplets, further confirming periodontal pathogen-induced alterations in Ca2+ and ROS signaling and the subsequent dysregulation of lipid homeostasis as key regulatory events underlying the evolution of macrophages into foam cells.

Periodontal Pathogens Modulate Lipid Flux via Fatty Acid Binding Protein 4.

A strong correlation between chronic periodontitis and systemic diseases (e.g., cardiovascular disease, metabolic disorders) has been suggested for several decades. However, the evidence supporting this correlation is restricted primarily to epidemiologic studies, with only a few experimental outcomes confirming such a correlation and providing information about the underlying molecular mechanisms. To reveal a correlation between periodontitis and systemic diseases as well as a relevant molecular pathway, we investigated the effects of Porphyromonas gingivalis and Fusobacterium nucleatum, which play roles in chronic periodontitis progression, on Raw264.7 and THP-1 macrophages. Infection with P. gingivalis or F. nucleatum significantly induced the expression of fatty acid binding protein 4 (FABP4), one of the most important adipokines that play a role in the progression of systemic diseases such as atherosclerosis and type 2 diabetes. Periodontal pathogen-induced FABP4 expression in macrophages promoted lipid uptake by these cells, as demonstrated by the diminished lipid accumulation in cells treated with an FABP4 inhibitor, BMS309403, or with knockdown of FABP4 expression. This periodontal pathogen-induced FABP4 expression was dependent on the JNK pathway, and JNK inhibition reduced lipid uptake by reducing FABP4 expression. Serum levels of antibodies against P. gingivalis correlated with serum FABP4 levels in humans, whereas no association occurred between F. nucleatum antibody titers and FABP4 levels. To our knowledge, this report is the first to experimentally demonstrate that periodontal pathogens stimulate lipid uptake in macrophages by modulating FABP4 expression. These findings strongly support the hypothesis that periodontitis may affect the progression of various systemic diseases.

Development of Rapid and Specific Detection for the Human Aichivirus A Using the Loop-Mediated Isothermal Amplification from Water Samples.

Human Aichivirus A (AiV-A) is classified as a Kobuvirus, group IV positive sense single strand RNA viruses. The first outbreak of AiV-A was reported from Aichi Prefecture, Japan in 1989. AiV-A exists not only among clinical patients, such as diarrhea, but also in a variety of water environments, as its occurrence is reported across a wide geographical range, from developing to advanced countries. For diagnose of AiV-A from water samples, mostly polymerase chain reaction (PCR) system have been developed. However, loop-mediated isothermal amplification (LAMP) assay has not been applied. In this study, developed a LAMP method to achieve a rapid, specific and highly sensitive detection of AiV-A. The method developed in this study is aimed specifically at AiV-A. Through a specific and non-specific selection and sensitivity test process for the five prepared LAMP primer sets, one primer set and optimum reaction temperature were selected. A newly developed method was more rapid (approximately 2-8 h), specific and equivalent detection of AiV-A than with the conventional PCRs. In addition, confirm system of positive LAMP reaction was developed by using the restriction enzyme Aci I and Hae III. For evaluation and verification of developing LAMP assay, a was applied to twenty cDNA from groundwater samples. This study proved rapid and specific diagnosis of AiV-A from water samples, and it is also demanded to be applicable to other environmental, clinical and food samples.

Interobserver reproducibility of maximal axial diameter and tumour volume measurements from CT of patients with head and neck squamous cell carcinoma.

AIM: To evaluate the interobserver reproducibility of computed tomography (CT) measurements of maximum tumour diameter and tumour volume for head and neck squamous cell carcinoma. MATERIALS AND METHODS: Eighty consecutive patients who underwent neck CT for the initial evaluation of head and neck squamous cell carcinoma were included in this retrospective study. Two radiologists independently measured the maximal axial diameter and volume of tumours. The reproducibility between the two observers was assessed using 95% Bland-Altman limits of agreement, reproducibility coefficient, within-subject coefficient of variation, and intraclass correlation coefficient with subgroup analysis according to tumour location. Logistic regression analysis was performed to identify the risk factors for high variability in tumour volume. RESULTS: The 95% limits of agreement for maximal axial diameter and tumour volume were ±22.3% and ±42.8%, respectively. The within-subject coefficient of variation and reproducibility coefficient were 7.9% and 0.564 for maximal axial diameter and 22.9% and 5.069 for tumour volume. All intraclass correlation coefficients for maximal axial diameter and tumour volume demonstrated excellent agreement (all intraclass correlation coefficients >0.9). Peritumoural infiltration (odds ratio: 7.189; confidence interval: 1.815-28.469; p=0.005) was an independent risk factor for high interobserver variability. CONCLUSION: Changes in maximum axial diameter and tumour volume of <22.3% and 42.8%, respectively, were in the range of measurement error on CT. The presence of peritumoural infiltration on CT increases the error in tumour volume measurement.

Development and Optimization of a Reverse Transcription Hemi-Nested PCR Primer for the Detection of Potato Mop-Top Virus at Quarantine Inspection Sites in Korea.

Potato mop-top virus (PMTV) is classified as a plant quarantine virus in Korea. It is tested at import inspection sites. In this study, two sets of reverse transcription-hemi-nested polymerase chain reaction primers were developed to diagnose PMTV. In addition, modified- positive control plasmid was developed for the identification of false positive results in plant quarantine. The assay is expected to be useful for improving the detection and diagnosis of PMTV.

Sensitive method for measuring third order nonlinearities in compact dielectric and hybrid plasmonic waveguides.

We demonstrate a sensitive method for the nonlinear optical characterization of micrometer long waveguides, and apply it to typical silicon-on-insulator nanowires and to hybrid plasmonic waveguides. We demonstrate that our method can detect extremely small nonlinear phase shifts, as low as 7.5·10<(-4) rad. The high sensitivity achieved imparts an advantage when investigating the nonlinear behavior of metallic structures as their short propagation distances complicates the task for conventional methods. Our results constitute the first experimental observation of χ((3)) nonlinearities in the hybrid plasmonic platform and is important to test claims of hybrid plasmonic structures as candidates for efficient nonlinear optical devices.

Development of a Specific Diagnostic System for Detecting Turnip Yellow Mosaic Virus from Chinese Cabbage in Korea.

Turnip yellow mosaic virus (TYMV) is a plant pathogenic virus transmitted mainly through its host Brassica spp. TYMV is originated from Europe. Its infection cases have been reported in Australia, Brazil, Turkey, and Japan. Symptoms similar to those of TYMV infections were also reported in Korea in 2012. In this study, we developed RT-PCR primer pairs that were highly sensitive for detecting TYMV. The developed RT-PCR primer pairs offered about 10-100 times stronger detection sensitivity compared to primer pairs previously used in Korea. As a result, a 491 bp TYMV-specific band was identified. The specific band was confirmed to be TYMV based on sequencing results and phylogenetic analysis. The RT-PCR primer pairs developed in this study can be used to rapidly and precisely diagnose TYMV in agricultural products such as Chinese cabbage and other crops infected by TYMV.

Downregulation of microRNA-362-3p and microRNA-329 promotes tumor progression in human breast cancer.

p130Cas regulates cancer progression by driving tyrosine receptor kinase signaling. Tight regulation of p130Cas expression is necessary for survival, apoptosis, and maintenance of cell motility in various cell types. Several studies revealed that transcriptional and post-translational control of p130Cas are important for maintenance of its expression and activity. To explore novel regulatory mechanisms of p130Cas expression, we studied the effect of microRNAs (miRs) on p130Cas expression in human breast cancer MCF7 cells. Here, we provide experimental evidence that miR-362-3p and miR-329 perform a tumor-suppressive function and their expression is downregulated in human breast cancer. miR-362-3p and miR-329 inhibited cellular proliferation, migration, and invasion, thereby suppressing tumor growth, by downregulating p130Cas. Ectopic expression of p130Cas attenuated the inhibitory effects of the two miRs on tumor progression. Relative expression levels of miR-362-3p/329 and p130Cas between normal and breast cancer correlated inversely; miR-362-3p/329 expression was decreased, whereas that of p130Cas increased in breast cancers. Furthermore, we showed that downregulation of miR-362-3p and miR-329 was caused by differential DNA methylation of miR genes. Enhanced DNA methylation (according to methylation-specific PCR) was responsible for downregulation of miR-362-3p and miR-329 in breast cancer. Taken together, these findings point to a novel role for miR-362-3p and miR-329 as tumor suppressors; the miR-362-3p/miR-329-p130Cas axis seemingly has a crucial role in breast cancer progression. Thus, modulation of miR-362-3p/miR-329 may be a novel therapeutic strategy against breast cancer.

Leptin protects rat articular chondrocytes from cytotoxicity induced by TNF-α in the presence of cyclohexamide.

OBJECTIVE: Although leptin appears to be an important local and systemic factor influencing cartilage homeostasis, the role of leptin in chondrocyte death is largely unknown. Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine that plays a central role in the pathogenesis of articular diseases. This study examines whether leptin modulates TNF-α-induced articular chondrocyte death. METHODS: Primary rat articular chondrocytes were isolated from knee joint cartilage slices. To induce cell death, the chondrocytes were treated with TNF-α. To examine whether leptin modulates the extent of TNF-α-mediated chondrocyte death, the cells were pretreated with leptin for 3 h before TNF-α treatment followed by viability analysis. To examine the mechanism by which leptin modulates the extent of TNF-α-mediated chondrocyte death, we utilized mitochondrial membrane potential (MMP) measurements, flow cytometry, nuclear morphology observation, co-immunoprecipitation, western blot analysis and confocal microscopy. RESULTS: We demonstrated that leptin suppresses TNF-α induced chondrocyte death. We further found that apoptosis partially contributes to TNF-α induced chondrocyte death while necroptosis primarily contributes to TNF-α induced chondrocyte death. In addition, we observed that leptin exerts anti-TNF-α toxicity via c-jun N-terminal kinase (JNK) in rat articular chondrocytes. CONCLUSION: Based on our findings, we suggest that the leptin present in the articular joint fluid protects articular chondrocytes against cumulative mechanical load and detrimental stresses throughout a lifetime, delaying the onset of degenerative changes in chondrocytes. We can further hypothesize that leptin protects articular chondrocytes against destructive stimuli even in the joints of osteoarthritis (OA) patients.

Development of PCR diagnostic system for detection of the seed-transmitted tobacco ringspot virus in quarantine.

Tobacco ringspot virus (TRSV) is a plant quarantine virus in Korea. As such, a TRSV examination is conducted when importing various crops. In this study, RT-PCR and nested PCR systems for TRSV detection in quarantine sites, and the modified-positive control plasmid for proving laboratory contamination and false positive reactions were developed. The developed diagnostic system was used to detect TRSV in the quarantine site. It revealed that from 2012 to August 2014, a total of 12 cases were detected in imported various crops. The system is expected to continue contributing to TRSV detection in plant quarantine.

Retinal pigment epithelial cells undergoing mitotic catastrophe are vulnerable to autophagy inhibition.

The increased mitochondrial DNA damage leads to altered functional capacities of retinal pigment epithelial (RPE) cells. A previous study showed the increased autophagy in RPE cells caused by low concentrations of rotenone, a selective inhibitor of mitochondrial complex I. However, the mechanism by which autophagy regulates RPE cell death is still unclear. In the present study, we examined the mechanism underlying the regulation of RPE cell death through the inhibition of mitochondrial complex I. We report herein that rotenone induced mitotic catastrophe (MC) in RPE cells. We further observed an increased level of autophagy in the RPE cells undergoing MC (RPE-MC cells). Importantly, autophagy inhibition induced nonapoptotic cell death in RPE-MC cells. These findings indicate that autophagy has a pivotal role in the survival of RPE-MC cells. We next observed PINK1 accumulation in the mitochondrial membrane and parkin translocation into the mitochondria from the cytosol in the rotenone-treated RPE-MC cells, which indicates that increased mitophagy accompanies MC in ARPE-19 cells. Noticeably, the mitophagy also contributed to the cytoprotection of RPE-MC cells. Although there might be a significant gap in the roles of autophagy and mitophagy in the RPE cells in vivo, our in vitro study suggests that autophagy and mitophagy presumably prevent the RPE-MC cells from plunging into cell death, resulting in the prevention of RPE cell loss.

The role of protein kinase CK2 in cyclosporine-induced nephropathy in rats.

OBJECTIVES: Protein kinase casein kinase II (PKCK2) has multiple, overlapping roles in induction of apoptosis. Apoptosis can be a common pathway of renal injury caused by a nephrotoxic drug or an injury. We evaluated the role of PKCK2 in cyclosporine (CsA)-induced nephropathy in rats by inhibiting PKCK2 with emodin. METHODS: Male Sprague-Dawley rats fed a low-sodium diet were divided into four treatment groups: control (0.9% saline injection), CsA (15 mg/kg/d subcutaneously), CsA + emodin (CsA plus emodin 20 mg/kg/d subcutaneously), and emodin only. The expression levels of apoptosis-associated factors and of PKCK2 were examined by Western blot analysis. RESULTS: Overexpression of PKCK2 noted with CsA treatment was prevented by emodin, a low-molecular-weight PKCK2 inhibitor, which dampend drug-induced up-regulation phosphorylated p53 and activation of caspases 3, 7, and 8. In addition, emodin prevented increased Bax/Bcl-2 ratio induced by CsA. Emodin prevented up-regulation of PKCK2 by CsA treatment, suggesting that its apoptotic-preventing activity was mediated via PKCK2. CONCLUSIONS: Our findings indicated that PKCK2 may play a role in apoptotic injury associated with CsA-induced nephropathy in rats.

Upregulation of CXCR4 is functionally crucial for maintenance of stemness in drug-resistant non-small cell lung cancer cells.

The hypothesis of cancer stem cells has been proposed to explain the therapeutic failure in a variety of cancers including lung cancers. Previously, we demonstrated acquisition of epithelial-mesenchymal transition, a feature highly reminiscent of cancer stem-like cells, in gefitinib-resistant A549 cells (A549/GR). Here, we show that A549/GR cells contain a high proportion of CXCR4+ cells that are responsible for having high potential of self-renewal activity in vitro and tumorigenicity in vivo. A549/GR cells exhibited strong sphere-forming activity and high CXCR4 expression and SDF-1α secretion compared with parent cells. Pharmacological inhibition (AMD3100) and/or siRNA transfection targeting CXCR4 significantly suppressed sphere-forming activity in A549 and A549/GR cells, and in various non-small cell lung cancer (NSCLC) cell lines. A549/GR cells showed enhanced Akt, mTOR and STAT3 (Y705) phosphorylation. Pharmacological inhibition of phosphatidyl inositol 3-kinase or transfection with wild-type PTEN suppressed phosphorylation of Akt, mTOR and STAT3 (Y705), sphere formation, and CXCR4 expression in A549/GR cells, whereas mutant PTEN enhanced these events. Inhibition of STAT3 by WP1066 or siSTAT3 significantly suppressed the sphere formation, but not CXCR4 expression, indicating that STAT3 is a downstream effector of CXCR4-mediated signaling. FACS-sorted CXCR4+ A549/GR cells formed many large spheres, had self-renewal capacity, demonstrated radiation resistance in vitro and exhibited stronger tumorigenic potential in vivo than CXCR4- cells. Lentiviral-transduction of CXCR4 enhanced sphere formation and tumorigenicity in H460 and A549 cells, whereas introduction of siCXCR4 suppressed these activities in A549/GR cells. Our data indicate that CXCR4+ NSCLC cells are strong candidates for tumorigenic stem-like cancer cells that maintain stemness through a CXCR4-medated STAT3 pathway and provide a potential therapeutic target for eliminating these malignant cells in NSCLC.

Fat-forming variant of solitary fibrous tumour of the pleura: CT findings.

The fat-forming variant of solitary fibrous tumour (SFT) was previously called lipomatous haemangiopericytoma and is a rare variant of solitary fibrous tumour. It predominantly occurs in the deep soft tissues of the retroperitoneum and thigh. Only a handful of cases involving the perineum, spine, thoracic wall and pelvic cavity have been reported in the radiological literature and the fat-forming variant of SFT involving the pleura has not been previously reported. Herein, we report the CT findings of a case of the fat-forming variant of SFT involving the pleura that was treated by excision. Chest CT showed a large lobulated heterogeneous fatty mass with a multifocal enhancing soft-tissue component in the left lower hemithorax. Although rare, the fat-forming variant of SFT of the pleura should be added to the differential diagnosis of fat-containing pleural soft-tissue tumours.

A case of a giant mucocoele of the appendiceal stump presented with a palpable mass in the right thigh: pre-operative diagnosis based on characteristic multidetector CT findings.

The pre-operative diagnosis of a mucocoele of the appendiceal stump (MAS) may be difficult owing to rarity and non-specific clinical presentation. However, a pre-operative diagnosis of a MAS is important to prevent widespread dissemination by inadvertent spillage of mucous contents. We describe a case of a MAS presenting with a palpable mass in the right thigh in which a pre-operative diagnosis was made by characteristic multidetector CT (MDCT) findings.

Modeling action potential generation during single and dual electrode stimulation of CA3 axons in hippocampal slice.

Despite widespread use of electrical stimulation of hippocampal brain slices, the precise sites of action potential initiation and propagation are unknown. To better understand these sites, we modeled axon recruitment by coupling stimulation-induced electric potential fields to biophysical models of CA3 axon arbors in mouse hippocampal brain slices. Geometrically realistic CA3 axon arbors were generated from prior anatomic observations. For single electrode stimulation, 95% of initiation sites were no further than 135microm away and propagation sites were no further than 875microm. Using dual electrode stimulation, additional axon arbor pieces were recruited, an effect that increased with distance between stimulating electrodes.

Histopathologic changes of acellularized xenogenic carotid vascular grafts implanted in a pig-to-goat model.

INTRODUCTION: The present study was designed to determine the in vivo patency and recellularization pattern of acellularized small-diameter xenogenic arterial grafts. We implanted acellularized porcine carotid arteries in bilateral carotid arteries of goats and microscopically analyzed the recellularization pattern of these grafts with the recipient's cells over time. MATERIAL AND METHODS: Carotid arteries of pigs weighing 30-40 kg were harvested and decellularized with hypertonic saline followed by sodium dodecyl sulfate. Acellularized porcine carotid vascular xenografts (0.4-0.5 cm in diameter) were prepared into 4 cm-long segments and implanted bilaterally in the carotid arteries of 10 black-haired goats. The in vivo patency of the implanted acellularized xenogenic grafts was evaluated at regular intervals by color Doppler ultrasonography. The goats were sacrificed at predetermined intervals (1 week, 1 month, 3 months, 6 months, 12 months after implantation), two animals at each interval. Upon retrieval, visual inspections and histopathologic examinations of the grafts were performed. To identify smooth muscle cells and functioning endothelial cells, immunohistochemical staining for alpha-smooth muscle actin and von Willebrand factor were also performed. RESULTS AND CONCLUSIONS: All experimental animals survived the observation period. Nineteen out of 20 implanted grafts showed patency with no thrombi. Microscopic analysis revealed that the grafts were completely covered with the hosts' endothelial cells, beginning from anastomotic sites. The grafts were gradually recellularized with recipients'cells including fibroblasts, myofibroblasts and smooth muscle cells. In conclusion, this study suggested that acellularized xenogenic vascular grafts can be a good alternative for the small-diameter vascular graft.

Ketones inhibit mitochondrial production of reactive oxygen species production following glutamate excitotoxicity by increasing NADH oxidation.

Dietary protocols that increase serum levels of ketones, such as calorie restriction and the ketogenic diet, offer robust protection against a multitude of acute and chronic neurological diseases. The underlying mechanisms, however, remain unclear. Previous studies have suggested that the ketogenic diet may reduce free radical levels in the brain. Thus, one possibility is that ketones may mediate neuroprotection through antioxidant activity. In the present study, we examined the effects of the ketones beta-hydroxybutyrate and acetoacetate on acutely dissociated rat neocortical neurons subjected to glutamate excitotoxicity using cellular electrophysiological and single-cell fluorescence imaging techniques. Further, we explored the effects of ketones on acutely isolated mitochondria exposed to high levels of calcium. A combination of beta-hydroxybutyrate and acetoacetate (1 mM each) decreased neuronal death and prevented changes in neuronal membrane properties induced by 10 microM glutamate. Ketones also significantly decreased mitochondrial production of reactive oxygen species and the associated excitotoxic changes by increasing NADH oxidation in the mitochondrial respiratory chain, but did not affect levels of the endogenous antioxidant glutathione. In conclusion, we demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD+/NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress.