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Aim. Intermittent positive pressure ventilation (IPPV) can adversely affect cardiopulmonary resuscitation outcomes by increasing the intrathoracic pressure. Continuous flow insufflation of oxygen (CFIO) has been investigated as a potential alternative, but evidence supporting its superiority over intermittent positive pressure ventilation in cases of cardiac arrest is scant. The aim of the current study was to compare the effects of continuous flow insufflation of oxygen using a one-way valve during cardiopulmonary-resuscitation with intermittent positive pressure ventilation in a rat model of respiratory arrest. Methods. Male Sprague-Dawley rats weighing 400∼450 g (from minimum to maximum) were randomly assigned to either a sham, IPPV, or CFIO group (n = 10 per group). Respiratory arrest was induced by blocking the endotracheal tube. Arterial blood gas analysis was performed during cardiopulmonary resuscitation to compare the oxygenation levels. Tissues were then harvested to compare the degrees of pulmonary barotrauma and ischemic brain injury. Results. Return of spontaneous circulation was observed in 6/10 rats in the IPPV group and 5/10 in the CFIO group. During cardiopulmonary resuscitation, the mean PaO2 was significantly higher in the CFIO group (83.10 mmHg) than in the IPPV group (56.10 mmHg). Lung biopsy revealed more inflammatory cells and marked thickening of the alveolar wall in the IPPV group; the group also exhibited a higher frequency of neuroglial cells and apoptotic bodies of pyramidal cells, resulting from ischemic injury. Conclusion. In a rat model of respiratory arrest, CFIO using a one-way valve resulted in a greater level of oxygenation and less lung and brain injuries than with IPPV.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with tumor progression in breast cancer. Although crosstalk between breast cancer cells and CAFs has been studied, the effect of CAFs on non-neoplastic breast epithelial cells is not fully understood to date. Here, we investigated the effect of CAFs on aggressive phenotypes in non-neoplastic MCF10A breast epithelial cells. CAFs induced epithelial-to-mesenchymal transition (EMT) and invasive phenotype in MCF10A cells. S100A8, a potential prognostic marker in several cancers, was markedly increased in MCF10A cells by CAFs. S100A8 was crucial for CAFs-induced invasive phenotype of MCF10A cells. Among cytokines increased by CAFs, interleukin (IL)-8 induced S100A8 through transcription factors p65 NF-κB and C/EBPβ. In a xenograft mouse model with MCF10A cells and CAFs, tumor was not developed, suggesting that coinjection with CAFs may not be sufficient for in vivo tumorigenicity of MCF10A cells. Xenograft mouse tumor models with MDA-MB-231 breast carcinoma cells provided an in vivo evidence for the effect of CAFs on breast cancer progression as well as a crucial role of IL-8 in tumor growth and S100A8 expression in vivo. Using a tissue microarray of human breast cancer, we showed that S100A8 expression was correlated with poor outcomes. S100A8 expression was more frequently detected in cancer-adjacent normal human breast tissues than in normal breast tissues. Together, this study elucidated a novel mechanism for the acquisition of invasive phenotype of non-neoplastic breast cells induced by CAFs, suggesting that targeting IL-8 and S100A8 may be an effective strategy against breast cancer.

A 31-year-old woman with a 15-year history of Takayasu's arteritis (TA) and a 13-year history of Hashimoto's thyroiditis presented with hematochezia. She received a diagnosis of Sjögren's syndrome at 1 month before her visit to Kyungpook National University Medical Center. Her colonoscopic findings were compatible with a diagnosis of ulcerative colitis (UC). She was treated with oral mesalazine, and her hematochezia symptoms subsequently disappeared. The coexistence of UC and TA has been reported; however, reports on the coexistence of UC and Sjögren's syndrome, or of UC and Hashimoto's thyroiditis are rare. Although the precise etiologies of these diseases are unknown, their presence together suggests that they may have a common pathophysiologic background. Furthermore, in patients with autoimmune or vascular diseases, including TA, systemic manifestations should be assessed with consideration of inflammatory bowel diseases including UC in the presence of gastrointestinal symptoms such as diarrhea and hematochezia.

Phosphatidylinositol 3-kinases (PI3Ks) have recently been implicated in apoptosis and ischemic cell death. We tested the efficacy of early intervention with a peptide PI3K activator in focal cerebral ischemia. After determining the most effective dose (24 μg/kg) and time window (2 h after MCAO) of treatment, a total of 48 rats were subjected to middle cerebral artery occlusion (MCAO). Diffusion weighted MRI (DWI) was performed 1 h after MCAO and rats with lesion sizes within a predetermined range were randomized to either PI3K activator or vehicle treatment arms. Fluid attenuated inversion recovery (FLAIR) MRI, neurological function, western blots, and immunohistochemistry were blindly assessed. Initial DWI lesion volumes were nearly identical between two groups prior to treatment. However, FLAIR showed significantly smaller infarct volumes in the PI3K activator group compared with vehicle (146 ± 81 mm3 and 211 ± 96 mm3, p = 0.045) at 48 h. The PI3K activator group also had better neurological function for up to 2 weeks. In addition, PI3K activator decreased the number of TUNEL-positive cells in the peri-infarct region compared with the control group. Western blot and immunohistochemistry showed increased expression of phosphorylated Akt (Ser473) and GSK-3β (Ser9) and decreased expression of cleaved caspase-9 and caspase-3. Our results suggest a neuroprotective role of early activation of PI3K in ischemic stroke. The use of DWI in the randomization of experimental groups may reduce bias.

Although the mortality rates for non-variceal upper gastrointestinal bleeding (NVUGIB) have recently decreased, it remains a significant medical problem. The main aim of this prospective multicenter database study was to construct a clinically useful predictive scoring system by using our predictors and compare its prognostic accuracy with that of the Rockall scoring system. Data were collected from consecutive patients with NVUGIB. Logistic regression analysis was performed to identify the independent predictors of 30-day mortality. Each independent predictor was assigned an integral point proportional to the odds ratio (OR) and we used the area under the curve to compare the discrimination ability between the new predictive model and the Rockall score. The independent predictors of mortality included age >65 years [OR 2.627; 95 % confidence interval (CI) 1.298–5.318], hemodynamic instability (OR 2.217; 95 % CI 1.069–4.597), serum blood urea nitrogen level >40 mg/dL (OR 1.895; 95 % CI 1.029–3.490), active bleeding at endoscopy (OR 2.434; 95 % CI 1.283–4.616), transfusions (OR 3.811; 95 % CI 1.640–8.857), comorbidities (OR 3.481; 95 % CI 1.405–8.624), and rebleeding (OR 10.581; 95 % CI 5.590–20.030). The new predictive model showed a high discrimination capability and was significantly superior to the Rockall score in predicting the risk of death (OR 0.837;95 % CI 0.818–0.855 vs. 0.761; 0.739–0.782; P = 0.0123). The new predictive score was significantly more accurate than the Rockall score in predicting death in NVUGIB patients. We need to prospectively validate the accuracy of this score for predicting mortality in NVUGIB patients.

AimsThis study aimed to investigate whether an aldehyde dehydrogenase (ALDH) activator (Alda-1®) reduces neuronal damage in a rat model of binge ethanol exposure.MethodsThirty-six adolescent male rats (130-150 g) were randomly assigned into three groups: sham, ethanol-only group (25% ethanol intragastrically thrice daily for four days, approximately 10 g/kg/day) and ethanol with Alda-1® group (10 mg/kg thrice daily for four days). The ALDH activity at baseline and 90 min after the last infusion in each group was measured. Brain damage was investigated using Luxol fast blue-Cresyl violet staining in the hippocampus, CA1 and CA2/3. The activation of astrocytes and microglia was examined using immunohistochemistry for antiglial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1).ResultsAfter a four-day binge, the ALDH activity level was doubled in the ethanol with Alda-1® group (mean: 7.87, SD: 0.67), whereas the levels in the sham group (mean: 4.07, SD: 0.53) and ethanol-only group (mean: 3.77, SD: 0.36) were slightly decreased. More significant neuronal shrinkage, fewer neurons, and loss of Nissl in the hippocampus were observed in the ethanol-only group compared to the ethanol with Alda-1® group. Astrocytosis and microgliosis of the hippocampus also showed increased activation in the ethanol only group compared with the ethanol with Alda-1® group.ConclusionAlda-1® administration reduces cytotoxic damage to the hippocampus in adolescent rats with binge ethanol exposure.

C-reactive protein (CRP) is an acute phase protein synthesized upon the inflammatory responses, associated with breast cancer. The process of tumor cell invasion and metastasis involves the adherence of cells to the extracellular matrix via integrin as a receptor for matrix molecules. The present study investigated the role of CRP in the adhesive phenotype of breast cells and the underlying mechanisms. Here, we first showed that CRP induces adhesion of MCF10A human breast epithelial cells through the activation of integrin α2 signaling. Expression of integrin α2 was induced by CRP in which transcription factors c-fos and SP1 may be involved. Binding of CRP with integrin α2 leads to the activation of focal adhesion kinase (FAK), paxillin and ERKs. CRP also binds to an Fcγ receptor Fcγ receptor I (FcγRI), and induces activation of paxillin, FAK and ERKs. Integrin α2 and FAK have crucial roles in the adhesive and invasive phenotypes as well as MMP-9 upregulation induced by CRP in MCF10A cells. Treatment with an inflammatory lipid sphingosine-1-phosphate induced CRP, which may be secreted and exert an autocrine effect by binding to FcγRI and integrin α2. Involvement of CRP in adhesion, invasion, anchorage-independent growth and upregulation of integrin α2, paxillin and FAK was observed in MDA-MB-231 triple-negative human breast cancer (TNBC) cells. Using an in vivo invasion model and an orthotopic mouse tumor model with MDA-MB-231 cells, we showed that CRP has an important role in intravasation and tumor growth in vivo, demonstrating the in vivo relevance of our in vitro results. The present study elucidates a critical molecular basis between CRP, integrin α2 and FcγRI pathways in MCF10A breast cells and MDA-MB-231 TNBC cells, thereby providing useful information on CRP-induced aggressiveness of breast cells in the inflammatory microenvironment.

Effective treatment of diabetic neuropathy (DN) remains unsolved. We serendipitously observed dramatic relief of pain in several patients with painful DN receiving granulocyte-colony stimulating factor (G-CSF). The aim of this study was to determine if G-CSF could treat DN in an animal model and to ascertain its mechanism of action. In a rodent model of DN, G-CSF dramatically recovered nerve function, retarded histological nerve changes and increased the expression of neurotrophic factors within nerve. A sex-mismatched bone marrow transplantation (BMT) study revealed that G-CSF treatment increased the abundance of bone marrow (BM)-derived cells in nerves damaged by DN. However, we did not observe evidence of transdifferentiation or cell fusion of BM-derived cells. The beneficial effects of G-CSF were dependent on the integrity of BM. In conclusion, G-CSF produced a therapeutic effect in a rodent model of DN, which was attributed, at least in part, to the actions of BM-derived cells.

This study presents an optical frequency switching scheme for a high-speed broadband terahertz (THz) measurement system based on the photomixing technique. The proposed system can achieve high-speed broadband THz measurements using narrow optical frequency scanning of a tunable laser source combined with a wavelength-switchable laser source. In addition, this scheme can provide a larger output power of an individual THz signal compared with that of a multi-mode THz signal generated by multiple CW laser sources. A swept-source THz tomography system implemented with a two-channel wavelength-switchable laser source achieves a reduced time for acquisition of a point spread function and a higher depth resolution in the same amount of measurement time compared with a system with a single optical source. (C) 2017 Optical Society of America

Measurement of the mechanical properties of human skin in vivo is challenging. Moreover, those with regard to excitation frequency have been rarely reported thus far. In this study, a vibration-based experimental method was employed to measure the viscoelastic properties with regard to the excitation frequency. Pieces of human skin and skeletal muscle excised from cadavers immediately post mortem were stored in a sealed container. As the experiment began, they were removed from the container and used to measure the viscoelastic properties as time elapsed. Young's moduli of the samples of human skin tissue that were immediately removed from the container were found to be similar to those obtained with in-vivo indentation methods. They were also found to be approximately one-third of those of human skeletal muscles. The viscoelastic properties of human skin were found to remain almost constant within the frequency range up to 120 Hz and are similar to those of porcine tissue. Young's moduli of the human skin and skeletal muscle were also found to reach the maximum values approximately five days post mortem. However, the loss factor of the human skin did not vary significantly as time elapses.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial-mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial-mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.

FUS is distributed in the cytoplasm in microglia but is absent in astrocytes. FUS (green) is (a) apparently not expressed in GFAP-positive astrocytes, (red) and is (b) cytoplasmic in Iba-1-positive microglia (red, arrows) in the precentral gyrus of a normal control (CTL 4, top), sporadic ALS patient (middle), and FUS (p.G504Wfs*12) patient (bottom). Boxed region in the left panel is enlarged in the right panels. Cells were counter stained with the nuclear marker DAPI (blue). Scale bars = (a) 50 μm for the merged left panels and 10 μm for the right panels, and (b) 25 μm. Cells were counter stained with the nuclear marker DAPI (blue). (PDF 594 kb)

Background Mutations in the fused in sarcoma (FUS) gene have been linked to amyotrophic lateral sclerosis (ALS). ALS patients with FUS mutations exhibit neuronal cytoplasmic mislocalization of the mutant FUS protein. ALS patients’ fibroblasts or induced pluripotent stem cell (iPSC)-derived neurons have been developed as models for understanding ALS-associated FUS (ALS-FUS) pathology; however, pathological neuronal signatures are not sufficiently present in the fibroblasts of patients, whereas the generation of iPSC-derived neurons from ALS patients requires relatively intricate procedures. Results Here, we report the generation of disease-specific induced neurons (iNeurons) from the fibroblasts of patients who carry three different FUS mutations that were recently identified by direct sequencing and multi-gene panel analysis. The mutations are located at the C-terminal nuclear localization signal (NLS) region of the protein (p.G504Wfs*12, p.R495*, p.Q519E): two de novo mutations in sporadic ALS and one in familial ALS case. Aberrant cytoplasmic mislocalization with nuclear clearance was detected in all patient-derived iNeurons, and oxidative stress further induced the accumulation of cytoplasmic FUS in cytoplasmic granules, thereby recapitulating neuronal pathological features identified in mutant FUS (p.G504Wfs*12)-autopsied ALS patient. Importantly, such FUS pathological hallmarks of the patient with the p.Q519E mutation were only detected in patient-derived iNeurons, which contrasts to predominant FUS (p.Q519E) in the nucleus of both the transfected cells and patient-derived fibroblasts. Conclusions Thus, iNeurons may provide a more reliable model for investigating FUS mutations with disrupted NLS for understanding FUS-associated proteinopathies in ALS. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0075-6) contains supplementary material, which is available to authorized users.

We propose a Terahertz tomography system using self-heterodyne detection with phase noise compensation. The phase noise compensation actively reduces phase noise from laser sources and offers improved resolution and reproducibility of the tomography system.

Endogenous FUS is partially redistributed to the cytoplasm in response to sorbitol. Primary fibroblasts of a representative control and the patient with the Q519E mutation shows intense staining for FUS (green) in the nuclei (DAPI) and the stress granule markers eIF4G (red) in the cytoplasm. Patients with the G504Wfs*12 and R495* mutations also show that a majority of FUS protein in the nuclei with a slight increase of cytoplasmic FUS (left panel). Cells treated with 0.4 M sorbitol for 1 hr are shown on the right panel. In response to sorbitol stress, slight decrease of nucleus FUS and increase of cytoplasmic FUS-positive inclusion bodies co-localized with eIF4G stress granules were observed. The accumulation of cytoplasmic FUS granules in mutant fibroblasts were much greater than that in healthy controls. Cells were counter stained with the nuclear marker DAPI (blue). Scale bars = 10 μm. (PDF 821 kb)

Structures of FUS-Transportin-1 complexes. (a) Overall structure of FUS-Transportin-1 complexes are presented in blue sphere and white surface, respectively. The position of FUS (p.Q519) is marked by red sphere models. (b) The focused view around the mutation (p.Q519). The structure of FUS and Transportin-1 complexes are consisted of a ball-and-stick representation. Stick models are colored by atom (N: blue, O: red, C: gray, respectively). The important position of the mutation (p.Q519) is depicted in a red ball-and-stick representation. A possible hydrogen bonding between Q519 of FUS and E509 of Transportin-1 is shown in the red circle with the acceptor-donor distance (3.21 Å). (PDF 69 kb)

FUS is distributed in the nucleus in ALS-FUS patient postcentral gyrus and dorsal horn. (a) DAB staining depicts predominant nucleus localization of FUS (as indicated by their morphology) in the postcentral gyrus of a normal control (CTL 4, top), sporadic ALS patient (middle), and FUS (p.G504Wfs*12) patient (bottom). The enlarged images are shown in the right panels. Scale bars = 10 μm. (b) The dorsal horn of the spinal cord sections from normal control (top), sporadic ALS patient (top), and FUS (p.G504Wfs*12) patient (bottom) were compared. The same predominant nucleus staining of FUS were observed by DAB staining in the dorsal horn neurons (as indicated by their morphology) of a normal control (top), sporadic ALS patient (middle), and FUS (p.G504Wfs*12) patient (bottom). Scale bars = 10 μm. (PDF 378 kb)

This study included two parts: 1) cadaver dissection to elucidate the perfusion of toenail flaps by the fibro-osseous hiatus branch (FHB), and 2) clinical application of the toenail flap for reconstruction of a fingernail defect. Four second toes of two fresh Korean cadavers were dissected. The plantar digital artery (PDA) and terminal segment branch (TSB) were ligated, and red latex was injected distally into the ligated PDA. Perfusion of the dye into the toenail bed through the FHB was observed. From Oct 2004 to Sep 2009, eight toenail flaps based on the FHB pedicle with or without the distal phalanx and pulp were applied to seven patients for finger nail reconstruction. The toenail flap was marked at 5 mm distal to the nail fold and 5 mm lateral to the paronychium. The toenail complex based on the FHB was elevated and transferred to the finger. The nail and matrix were elevated with or without including the distal phalanx. The results of perfusion study showed that one side of the unilateral FHB was identified and traced proximal to the PDA, which was ligated. The distal toenail bed was perfused by the dye through the FHB. In clinical application, all the toenail flaps flourished and survived. We suggest that the toenail flap based on the FHB may be useful for fingernail reconstruction with minimal donor morbidity.

Infective endocarditis caused by Pasteurella multocida is a rare disease with a high mortality rate. Most patients have a previous history of animal contact. The most common comorbid condition is a chronic liver disease, which is attributable to the high mortality rate. Over half of the patients who survived the disease had required valve replacement surgery. We report the case of a 70-year-old man diagnosed with prosthetic valve endocarditis caused by Pasteurella multocida and complicated with multiple peripheral arterial embolism. He had neither any underlying immunocompromising diseases nor previous history of animal contact. He was successfully cured after antibiotic therapy for 6 weeks without valve replacement surgery.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron loss. Although the underlying cause of the disease remains unclear, a variety of pathogenic mechanisms have been proposed. Despite promising preclinical studies showing the modification of the disease progression, most trials have failed to demonstrate any significant improvement in outcome. Stem cell therapy therefore has been proposed as an alternative therapy for ALS. In this study, we evaluated the dose-dependent effects of human bone marrow mesenchymal stem cells (hMSCs) obtained from an ALS patient (ALS-hMSCs) on SOD1 mice via intrathecal injection and showed its practicality for hMSCs. We transplanted different doses (1 × 10 4 , 2 × 10 5 , and 1 × 10 6 ) of ALS-hMSCs into the cisterna magna and performed clinical observations including symptom onset, survival time, and locomotor performance using the rotarod test. Nissl staining was performed to evaluate motor neurons in lumbar spinal cord sections at 109 days, and transplanted cells were evaluated by immuno-fluorescence staining at the end stage. A cell dose of 1 × 10 6 cells significantly prolonged life span and delayed the decline of motor performance. At this dose, the average number of motor neurons was significantly higher than those of the untreated and 1 × 10 4 cell treated groups. Most injected hMSCs distributed in the ventricular system and subarachnoid space, while some migrated into the brain and spinal cord. These data suggest that intrathecal injection with an optimized cell number could be a potential route for stem cell therapy in ALS patients.

Glycogen synthase kinase (GSK)-3 has recently been implicated in the pathogenesis of neurodegenerative diseases. Although the neuroprotective effects of GSK-3 inhibitors in Alzheimer's disease have been established, their effects on amyotrophic lateral sclerosis (ALS) have not been well defined. This study was undertaken to evaluate the effects of GSK-3 inhibition in the G93A-SOD1 mouse model of ALS. Groups of G93A-SOD1 mice were treated with varying concentrations of GSK-3 inhibitor VIII, a specific GSK-3 inhibitor that crosses the BBB, intraperitoneally 5 days a week after 60 days of age. The GSK-3 inhibitor VIII treatment significantly delayed the onset of symptoms and prolonged the life span of the animals, and inhibited the activity of GSK-3 in a concentration-dependent manner. Furthermore, this treatment preserved survival signals and attenuated death and inflammatory signals. These data suggest that GSK-3 plays an important role in the pathogenic mechanisms of ALS and that inhibition of GSK-3 could be a potential therapeutic candidate for ALS.

The present study aimed to elucidate the molecular link between S1P and CRP during the invasive process of breast epithelial cells. This is the first report showing that transcription of CRP was ma...

We propose and demonstrate a continuous-wave vector THz imaging system utilizing a photonic generation of two-tone THz signals and self-mixing detection. The proposed system measures amplitude and phase information simultaneously without the local oscillator reference or phase rotation scheme that is required for heterodyne or homodyne detection. In addition, 2π phase ambiguity that occurs when the sample is thicker than the wavelength of THz radiation can be avoided. In this work, THz signal having two frequency components was generated with a uni-traveling-carrier photodiode and electro-optic modulator on the emitter side and detected with a Schottky barrier diode detector used as a self-mixer on the receiver side. The proposed THz vector imaging system exhibited a 50-dB signal to noise ratio and 0.012-rad phase fluctuation with 100-μs integration time at 325-GHz. With the system, we demonstrate two-dimensional THz phase contrast imaging. Considering the recent use of two-dimensional arrays of Schottky barrier diodes as a THz image sensor, the proposed system is greatly advantageous for realizing a real-time THz vector imaging system due to its simple receiver configuration.

A crucial role of the inflammatory lipid sphingosine-1-phosphate (S1P) in breast cancer aggressiveness has been reported. Recent clinical studies have suggested that C-reactive protein (CRP) has a role in breast cancer development. However, limited information is available on the molecular basis for the expression of CRP and its functional significance in breast cell invasion. The present study aimed to elucidate the molecular link between S1P and CRP during the invasive process of breast epithelial cells. This is the first report showing that transcription of CRP was markedly activated by S1P in breast cells. Our data suggest that not only S1P treatment but also the endogenously produced S1P may upregulate CRP in breast carcinoma cells. Transcription factors CCAAT/enhancer-binding protein beta and c-fos were required for S1P-induced CRP expression. Coupling of S1P 3 to heterotrimeric G q triggered the expression of CRP, utilizing signaling pathways involving reactive oxygen species (ROS), Ca 2+ and extracellular signal-related kinases (ERKs). S1P-induced CRP expression was crucial for the transcriptional activation of matrix metalloproteinase-9 through ERKs, ROS and c-fos, leading to breast cell invasion. Using a xenograft mice tumor model, we demonstrated that S1P induced CRP expression both in vitro and in vivo. Taken together, our findings have revealed a molecular basis for S1P-induced transcriptional activation of CRP and its functional significance in the acquisition of the invasive phenotype of human breast epithelial cells under inflammatory conditions. Our findings may provide useful information on the identification of useful therapeutic targets for inflammatory breast cancer. © 2014 Macmillan Publishers Limited. Chemicals/CAS: 2 (2 amino 3 methoxyphenyl)chromone, 167869-21-8; C reactive protein, 9007-41-4; fingolimod, 162359-56-0; gelatinase B, 146480-36-6; mitogen activated protein kinase kinase, 142805-58-1; sphingosine 1 phosphate, 26993-30-6. Tradenames: pd 98059.

Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. Sphingosine 1-phosphate (S1P), acting on the G-protein-coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase-9 (MMP-9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP-9 by S1P. In addition, activation of extracellular-signal-regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine-protein kinase (Akt) are involved in the process of S1P-mediated induction of MMP-9 expression and invasion. Activation of the S1P receptor S1P3 and Gαq are required for S1P-induced invasive/migratory responses, suggesting that the enhancement of S1P-mediated invasiveness is triggered by the specific coupling of S1P3 to the heterotrimeric Gαq subunit. Activation of phospholipase C-β4 and intracellular Ca2+ release are required for S1P-induced MMP-9 upregulation. Taken together, this study demonstrated that S1P regulates MMP-9 induction and invasiveness through coupling of S1P3 and Gαq in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.

Bone marrow-derived mesenchymal stromal cells (MSCs) have been used successfully as a source of stem cells for treating neurodegenerative diseases. However, for reasons that are not clear, autologous MSC transplants have not yielded successful results in human trials. To test one possible reason, we compared the migratory ability of MSCs from amyotrophic lateral sclerosis (ALS) patients with those of healthy controls. We found that MSCs derived from ALS patients (ALS-MSCs) had a reduced ability to migrate, which may explain why autologous transplantation is not successful. We also found that expression of one of the intracellular factors implicated in migration, β-PIX, was significantly reduced in ALS-MSCs compared with healthy stem cells. Restoration of β-PIX expression by genetic manipulation restored the migratory ability of ALS-MSCs, and inhibition of β-PIX expression with shRNA reduced the migration of healthy MSCs. We suggest that transplantation of allogeneic or genetically modified autologous stem cells might be a more promising strategy for ALS patients than transplantation of autologous stem cells.

The serotonergic system is one of the major systems targeted in the pharmacological treatment of mood disorders including depression. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been reported to induce the expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin. The 14-3-3 protein family not only activates neuronal enzymes, including TPH, but also plays a role in a wide variety of cell signaling. The aim of the present study was to determine whether fluoxetine regulates both the interaction of TPH and 14-3-3 proteins as well as the increase of those proteins in the dorsal raphe nucleus and the hippocampus. Sprague-Dawley rats were administered fluoxetine or vehicle for 5 and 14 days and sacrificed at 5 and 14 days after initial treatment. The intensity of immunoreactivity for TPH and 14-3-3 proteins in the dorsal raphe nucleus of the midbrain and in the hippocampus was measured, and the colocalization of both proteins was observed with double-labeling immunofluorescence. At 5 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein increased in both the dorsal raphe nucleus and the hippocampus, while that of TPH did not change in either region. In addition, at 14 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein significantly increased in both the dorsal raphe nucleus and hippocampus, while that of TPH showed few changes in either region. Colocalization of TPH and 14-3-3 proteins was observed in the cell bodies of dorsal raphe nucleus, whereas it was not observed in the hippocampus. These results suggest that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with delayed pharmacological effects of SSRIs.

Human post-mortem brain studies have revealed reduced density and size of neurons and glial cells in the dorsolateral prefrontal cortex (dlPFC) in major depressive disorder (MDD). However, the basis of these cytoarchitectural abnormalities and the relationship between them are not understood. We hypothesized that the reduced density of GABAergic neurons and glial cells was associated with altered glutamate neurotransmission in the dlPFC. In order to test this hypothesis, we examined a specific marker type (i.e., calretinin, CR: as a marker of GABAergic neurons) and also attempted to identify the neuropathological markers that correlate with the density of CR-immunoreactive (IR) GABAergic neurons in the dlPFC, using the Stanley Neuropathology Consortium Integrative Database (SNCID, http://sncid.stanleyresearch.org/), which is a web-based tool used to integrate Stanley Medical Research Institute (SMRI) data sets. We found that the density of CR-IR GABAergic neurons was significantly lower in layer I of the dlPFC of MDD patients (n=15) than in that of unaffected controls (n=15) (p=0.021). CR-IR GABAergic neuronal changes were positively correlated with changes in several markers for glial cells and pyramidal neurons in the dlPFC of all SNC subjects (n=60). We also found that the glutamate changes negatively correlated with glial fibrillary acidic protein (GFAP) expression levels and CR-IR GABAergic neuronal density in the prefrontal cortex of all SNC subjects (P0.05). These findings yield some insight into the mechanism by which increased glutamatergic neurotransmission leads to excitotoxic damage both in neurons and glial cells in the dlPFC of MDD patients.

Corticotropin-releasing factor (CRF) is a peptide involved in the activation of the hypothalamic-pituitary-adrenal (HPA) axis. CRF is distributed not only along the HPA axis but also throughout pain-relevant anatomical sites. CRF elicits potent antinociception at the three main levels of pain transmissions: namely, the brain, spinal cord, and peripheral sensory neurons. The widespread distribution of CRF receptors 1 and 2 in the brain offers several targets wherein CRF could alter pain, some of which may be independent of the HPA axis. In this study, we assessed the expression of CRF and its receptors, CRF receptor type (CRFR)1 and CRFR2, in the spinal dorsal horn and dorsal root ganglion (DRG) in a rat model of neuropathic pain induced by spinal nerve injury (SNI). CRF was expressed in a few DRG neurons and primary afferent fibers in the dorsal horns of naїve rats, and the CRF-positive neurons in DRG and fibers in the spinal dorsal horn were found to have increased after SNI. CRFR1 was not expressed in DRG or the dorsal horn and CRFR2 was expressed weakly in the small neurons in DRG in the naїve rats. After SNI, CRFR1 was expressed in the activated microglia in the ipsilateral dorsal horn, and immunoreaction for CRFR2 was increased in the contralateral DRG following SNI. Consequently, it has been suggested that the increased expression of CRF and CRFR2 in DRG neurons and primary afferent fibers in dorsal horn, and CRFR1 in the activated microglia, may be involved in the mediation of stress responses as well as in microglial activation in the neuropathic pain state following SNI.

This study included two parts: 1) cadaver dissection to elucidate the perfusion of toenail flaps by the fibro-osseous hiatus branch (FHB), and 2) clinical application of the toenail flap for reconstruction of a fingernail defect. Four second toes of two fresh Korean cadavers were dissected. The plantar digital artery (PDA) and terminal segment branch (TSB) were ligated, and red latex was injected distally into the ligated PDA. Perfusion of the dye into the toenail bed through the FHB was observed. From Oct 2004 to Sep 2009, eight toenail flaps based on the FHB pedicle with or without the distal phalanx and pulp were applied to seven patients for finger nail reconstruction. The toenail flap was marked at 5 mm distal to the nail fold and 5 mm lateral to the paronychium. The toenail complex based on the FHB was elevated and transferred to the finger. The nail and matrix were elevated with or without including the distal phalanx. The results of perfusion study showed that one side of the unilateral FHB was identified and traced proximal to the PDA, which was ligated. The distal toenail bed was perfused by the dye through the FHB. In clinical application, all the toenail flaps flourished and survived. We suggest that the toenail flap based on the FHB may be useful for fingernail reconstruction with minimal donor morbidity.

T cells play a pivotal role in the initiation and progression of multiple sclerosis. We have found that 1,4-aryl-2-mercaptoimidazole (KRM-III) inhibited T-cell antigen receptor- and phorbol myristate acetate/ionomycin-induced activation of nuclear factor of activated T cells (NFAT) and T-cell proliferation with an IC(50) of 5 microM. The KRM-III-mediated inhibitory effect was specific for NFAT activation but not for nuclear factor kappaB. Oral administration of 90 mg/kg KRM-III resulted in complete abrogation of anti-CD3 antibody-induced T-cell activation and a 45.8% reduction in footpad swelling in bovine serum albumin-induced delayed-type hypersensitivity. In the murine experimental autoimmune encephalomyelitis (EAE) model, oral administration of KRM-III significantly attenuated the severity of disease when given before or after disease onset. Draining lymph node cells from KRM-III-treated mice showed markedly reduced proliferation in response to myelin oligodendrocyte glycoprotein peptide. Histological analysis indicated that KRM-III reduced the infiltration of inflammatory cells to the white matter of spinal lumbar cords. These results demonstrate that KRM-III efficiently inhibits T-cell activation and inflammatory responses and lessens EAE clinical signs, which suggest KRM-III as a potential lead compound for the treatment of T-cell-driven autoimmune diseases.

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 by HMDM. The involvement of nuclear factor (NF)-kappaB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-kappaB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-kappaB activation and TNF-alpha production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-kappaB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-alpha. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-alpha, and NO. In particular, we showed that T. vaginalis induced TNF-alpha production in macrophages through NO-dependent activation of NF-kappaB, which might be closely involved in inflammation caused by T. vaginalis.

The vanilloid receptor VR1 renders a group of primary afferents that express it sensitive to noxious heat and capsaicin, and is thus an important marker for nociceptors. We use double immunofluorescence and confocal microscopy to show that the density of VR1-positive fibers and boutons in the dorsal horn increases progressively from spinal segments L4 to L6 and that the colocalization of VR1 with the neuropeptide substance P (SP) in lamina I and along the lateral collateral path, where the majority of visceral afferents terminate, is negligible at L4, but substantial at L6. We conclude that VR1 is expressed by visceral afferents to the lower lumbar spinal cord in the rat, which also express SP.

Hepatocellular carcinoma (HCC) has a poor prognosis due to its aggressive nature. Ligand activation of G-protein coupled receptor with enhanced G-protein activity may contribute to tumorigenesis and tumor progression in microenvironments. Here, we report the overexpression of Gα12 in HCC and its effect on deregulation of microRNAs in association with epithelial-mesenchymal transition. Gα12 expression levels were higher in human HCC than surrounding normal tissue. Array analyses using Huh7 cells stably transfected with an active mutant of Gα12 (Gα12QL) enabled us to extract down-regulated microRNAs. Of them, decreases of miR-200b/a, -192 and -215 contributed to ZEB induction through the up-regulation of MDM2. Transfection with miR-200b, -192 or miR-215 mimic antagonized HCC cell migration and invasion facilitated by Gα12QL. In tumor xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1, a mesenchymal liver tumor cell line. In human HCC samples, levels of the identified microRNAs were all decreased, and correlated with the induction of mesenchymal markers. Moreover, decreases of the miRNAs discriminated microvascular invasion versus non-microvascular invasion of HCC. Conclusion: Increase of Gα12 in HCC deregulates a set of miRNAs promoting epithelial-mesenchymal transition of HCC and may be responsible for tumor microvascular invasion, implying that Gα12 signaling and the downstream mediators may be utilized as therapeutic targets and/or prognostic markers. Citation Format: Sang Geon Kim, Yoon Mee Yang, Tae Hyun Kim, Chan Gyu Lee, Jihyun An, Sung Hoon Kim, Chang Ho Lee, Sejin Hwang, Seung Jin Lee, Kang Mo Kim. G12 overexpression in hepatocellular carcinoma promotes epithelial-mesenchymal transition through microRNAs deregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 515. doi:10.1158/1538-7445.AM2014-515

Endogenous FUS cytoplasmic incorporation into stress granule marker eIF4G in response to arsenite in patient iNeurons. Immunocytochemistry performed on vehicle treated iNeurons (left panel) as compared to cells treated with 0.5 mM arsenite for 30 min (right panel) at day 10 are shown. A representative control shows FUS protein predominantly localized to the nuclei. All three ALS-FUS patients show a majority of FUS protein (green) in the cytoplasm of iNeurons. Cytoplasmic FUS-positive inclusion bodies (green) were detectable in eIF4G-positive stress granules (red) in patients. Cells were fixed and probed by immunofluorescence for DAPI (blue). Scale bars = 25 μm. (PDF 751 kb)

Endogenous FUS cytoplasmic incorporation into stress granule marker eIF4G in response to arsenite in patient iNeurons. Immunocytochemistry performed on vehicle treated iNeurons (left panel) as compared to cells treated with 0.5 mM arsenite for 30 min (right panel) at day 10 are shown. A representative control shows FUS protein predominantly localized to the nuclei. All three ALS-FUS patients show a majority of FUS protein (green) in the cytoplasm of iNeurons. Cytoplasmic FUS-positive inclusion bodies (green) were detectable in eIF4G-positive stress granules (red) in patients. Cells were fixed and probed by immunofluorescence for DAPI (blue). Scale bars = 25 μm. (PDF 751 kb)