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1.  Structural Characterization of the Mechanosensitive Channel Candidate MCA2 from Arabidopsis thaliana 
PLoS ONE  2014;9(1):e87724.
Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca2+-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca2+ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures. Because of the abundance of protein in these cells, MCA2 was chosen for purification. Purified MCA2 in a detergent-solubilized state formed a tetramer, which was confirmed by chemical cross-linking. Single-particle analysis of cryo-electron microscope images was performed to depict the overall shape of the purified protein. The three-dimensional structure of MCA2 was reconstructed at a resolution of 26 Å from 5,500 particles and appears to comprise a small transmembrane region and large cytoplasmic region.
PMCID: PMC3903776  PMID: 24475319
2.  Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+ influx and modulates generation of reactive oxygen species in cultured rice cells 
BMC Plant Biology  2012;12:11.
Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.
We here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.
We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.
PMCID: PMC3313898  PMID: 22264357
3.  Polarized Morphogenesis Regulator Spa2 Is Required for the Function of Putative Stretch-Activated Ca2+-Permeable Channel Component Mid1 in Saccharomyces cerevisiae 
Eukaryotic Cell  2005;4(8):1353-1363.
Mid1 is a putative stretch-activated Ca2+ channel component and is required for the maintenance of viability in the mating process. In response to mating pheromone, the mid1 mutant normally forms a pointed mating projection but eventually dies. This phenotype is called the mid phenotype. To identify a protein regulating Mid1 or regulated by Mid1, we isolated a multicopy suppressor that rescues the mid1-1 mutant from mating pheromone-induced death and found that it encodes a truncated Spa2 protein lacking an amino-terminal region responsible for interaction with components of the mitogen-activated protein kinase cascades. One of these SPA2 alleles was SPA2ΔN, whose product lacked the region from Ser5 to Leu230. SPA2ΔN on a multicopy plasmid (YEpSPA2ΔN) complemented the mid phenotype but not another phenotype, low Ca2+ accumulation, of the mid1-1 mutant. Neither SPA2ΔN on a low-copy plasmid nor wild-type SPA2 on a multicopy plasmid had suppressive activity. The SPA2 gene is involved in the formation of a pointed mating projection, and cells of the spa2Δ mutant lacking Spa2 are viable and develop a peanut shell-like structure when exposed to mating pheromone. Like the spa2Δ mutant, the mid1-1 spa2Δ double mutant and the mid1-1/YEpSPA2ΔN strain developed the peanut shell-like structure. The mid1-1 spa2Δ double mutant did not have the mid phenotype, indicating that SPA2 is epistatic to MID1. Overexpression of Spa2ΔN abolished the localization of Spa2-green fluorescent protein to the tip of the mating projection. These results suggest that the Spa2ΔN protein interferes with the localization of the normal Spa2 protein and thereby prevents cells from entering the mating process. Therefore, we suggest that Mid1 function is influenced by Spa2 function through polarized morphogenesis.
PMCID: PMC1214535  PMID: 16087740
4.  A Polymer-Based Magnetic Resonance Tracer for Visualization of Solid Tumors by 13C Spectroscopic Imaging 
PLoS ONE  2014;9(7):e102132.
Morphological imaging precedes lesion-specific visualization in magnetic resonance imaging (MRI) because of the superior ability of this technique to depict tissue morphology with excellent spatial and temporal resolutions. To achieve lesion-specific visualization of tumors by MRI, we investigated the availability of a novel polymer-based tracer. Although the 13C nucleus is a candidate for a detection nucleus because of its low background signal in the body, the low magnetic resonance sensitivity of the nucleus needs to be resolved before developing a 13C-based tracer. In order to overcome this problem, we enriched polyethylene glycol (PEG), a biocompatible polymer, with 13C atoms. 13C-PEG40,000 (13C-PEG with an average molecular weight of 40 kDa) emitted a single 13C signal with a high signal-to-noise ratio due to its ability to maintain signal sharpness, as was confirmed by in vivo investigation, and displayed a chemical shift sufficiently distinct from that of endogenous fat. 13C-PEG40,000 intravenously injected into mice showed long retention in circulation, leading to its effective accumulation in tumors reflecting the well-known phenomenon that macromolecules accumulate in tumors because of leaky tumor capillaries. These properties of 13C-PEG40,000 allowed visualization of tumors in mice by 13C spectroscopic imaging. These findings suggest that a technique based on 13C-PEG is a promising strategy for tumor detection.
PMCID: PMC4090184  PMID: 25007334
Nuclear EGFR (nEGFR) has been identified in various human tumor tissues, including cancers of the breast, ovary, oropharynx, and esophagus, and has predicted poor patient outcomes. We sought to determine if protein expression of nEGFR is prognostic in early stage non-small cell lung cancer (NSCLC).
Resected stage I and II NSCLC specimens were evaluated for nEGFR protein expression using immunohistochemistry (IHC). Cases with at least one replicate core containing ≥5% of tumor cells demonstrating strong dot-like nucleolar EGFR expression were scored as nEGFR positive.
Twenty-three (26.1% of the population) of 88 resected specimens stained positively for nEGFR. Nuclear EGFR protein expression was associated with higher disease stage (45.5% of stage II vs. 14.5% of stage I; p=0.023), histology (41.7% in squamous cell carcinoma vs. 17.1% in adenocarcinoma; p=0.028), shorter progression-free survival (PFS) (median PFS 8.7 months [95% CI 5.1–10.7 mo] for nEGFR positive vs. 14.5 months [95% CI 9.5–17.4 mo] for nEGFR negative; hazard ratio (HR) of 1.89 [95% CI 1.15–3.10]; p=0.011), and shorter overall survival (OS) (median OS 14.1 months [95% CI 10.3–22.7 mo] for nEGFR positive vs. 23.4 months [95% CI 20.1–29.4 mo] for nEGFR negative; HR of 1.83 [95% CI 1.12–2.99]; p=0.014).
Expression of nEGFR protein was associated with higher stage and squamous cell histology, and predicted shorter PFS and OS, in this patient cohort. Nuclear EGFR serves as a useful independent prognostic variable and as a potential therapeutic target in NSCLC.
PMCID: PMC3679338  PMID: 23628526
non-small cell lung cancer; nuclear; epidermal growth factor receptor; prognosis; biomarker; survival analysis
6.  Early Events in Lymphopoiesis, an Update 
Current opinion in hematology  2013;20(4):265-272.
Recent Findings & Summary
We can now appreciate that new lymphoid progenitors are drawn from a heterogeneous collection of hematopoietic stem cells (HSC) through asynchronous patterns of gene expression. Complex interactions then occur between the gene products. While transcription factors have long been a focus of investigation, micro RNAs are also being implicated in lymphopoiesis. Lymphocytes are normally replaced in correct proportion to other blood cells, but ratios change dramatically during infections. Long standing issues relating to T versus B lineage divergence remain but have been enriched with remarkable new findings about thymus seeding. Finally, knowledge obtained from studies of mice is slowly being extended to humans.
PMCID: PMC3676435  PMID: 23594693
Hematopoietic cell; B lymphopoiesis; Infection
7.  Hemoglobin Receptor Protein from Porphyromonas gingivalis Induces Interleukin-8 Production in Human Gingival Epithelial Cells through Stimulation of the Mitogen-Activated Protein Kinase and NF-κB Signal Transduction Pathways 
Infection and Immunity  2014;82(1):202-211.
Periodontitis is an inflammatory disease of polymicrobial origin affecting the tissues supporting the tooth. The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, triggers a series of host inflammatory responses that promote the destruction of periodontal tissues. Among the virulence factors of P. gingivalis, hemoglobin receptor protein (HbR) is a major protein found in culture supernatants. In this study, we investigated the roles of HbR in the production of inflammatory mediators. We found that HbR induced interleukin-8 (IL-8) production in the human gingival epithelial cell line Ca9-22. p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (Erk1/2) were activated in HbR-stimulated Ca9-22 cells. Inhibitors of p38 MAPK (SB203580) and Erk1/2 (PD98059) blocked HbR-induced IL-8 production. Additionally, HbR stimulated the translocation of NF-κB-p65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA (siRNA) targeting activating transcription factor 2 (ATF-2) or cyclic AMP-response element-binding protein (CREB) inhibited HbR-induced IL-8 production. Moreover, pretreatment with SB203580 and PD98059 reduced HbR-induced phosphorylation of CREB and ATF-2, respectively. Combined pretreatment with an inhibitor of NF-κB (BAY11-7082) and SB203580 was more efficient in inhibiting the ability of HbR to induce IL-8 production than pretreatment with either BAY11-7082 or SB203580 alone. Thus, in Ca9-22 cells, the direct activation of p38 MAPK and Erk1/2 by HbR caused the activation of the transcription factors ATF-2, CREB, and NF-κB, thus resulting in the induction of IL-8 production.
PMCID: PMC3911866  PMID: 24126532
8.  Over-the-scope-clipping system for anastomotic leak after colorectal surgery: Report of two cases 
An anastomotic leak is one of the major complications following colorectal surgery. Standard treatments for anastomotic leak are total parenteral nutrition or temporary ileostomy. The over-the-scope-clipping (OTSC) system was originally developed to treat intestinal perforation or to close the tissue after natural orifice transluminal endoscopic surgery. Two cases of successful management of an anastomotic leak after colorectal surgery using the OTSC system are reported. One patient avoided a temporary ileostomy. In the other, hospitalization was shortened by the use of the OTSC system. The OTSC system can be a potential option in the management of anastomotic leaks after colorectal surgery.
PMCID: PMC4069327  PMID: 24976736
Over-the-scope-clipping; Colorectal cancer; Anastomotic leak; Colorectal surgery; Double stapling technique
9.  The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages 
Immunity  2013;38(6):1105-1115.
How hematopoietic stem cells (HSCs) produce particular lineages is insufficiently understood. We searched for key factors that direct HSC to lymphopoiesis. Comparing gene expression profiles for HSCs and early lymphoid progenitors revealed that Satb1, a global chromatin regulator, was markedly induced with lymphoid lineage specification. HSCs from Satb1-deficient mice were defective in lymphopoietic activity in culture and failed to reconstitute T lymphopoiesis in wild-type recipients. Furthermore, Satb1 transduction of HSCs as well as embryonic stem cells robustly promoted their differentiation toward lymphocytes. Whereas genes that encode Ikaros, E2A, and Notch1 were unaffected, many genes involved in lineage decisions were regulated by Satb1. Satb1 expression was reduced in aged HSCs with compromised lymphopoietic potential, but forced Satb1 expression partly restored that potential. Thus, Satb1 governs the initiating process central to the replenishing of lymphoid lineages. Such activity in lymphoid cell generation may be of clinical importance and useful to overcome immunosenescence.
PMCID: PMC3777575  PMID: 23791645
Satb1; hematopoietic stem cells; early lymphoid differentiation; aging
10.  Draft Genome Sequences of Geomicrobium sp. Strains JCM 19037, JCM 19038, JCM 19039, and JCM 19055, Isolated from Aquatic Samples 
Genome Announcements  2014;2(3):e00622-14.
Haloalkaliphilic strains JCM 19037, JCM 19038, JCM 19039, and JCM 19055, closely related to Geomicrobium sediminis, were isolated from aquatic samples, and their draft genome sequences were determined. The genome information of these four strains will be useful for studies of their physiology and ecology.
PMCID: PMC4064037  PMID: 24948772
11.  Draft Genome Sequences of Vibrio sp. Strains Isolated from Tetrodotoxin-Bearing Scavenging Gastropod 
Genome Announcements  2014;2(3):e00623-14.
Vibrio sp. strains JCM 18905 and JCM 19053 were isolated from a tetrodotoxin (TTX)-bearing scavenging gastropod, and Vibrio sp. strain JCM 18904 was isolated from a sea cucumber. All these are closely related to Vibrio alginolyticus. Their comparative genome information is useful for studies of TTX production in bacteria.
PMCID: PMC4064038  PMID: 24948773
12.  Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer 
We previously reported the clinical efficacy of adoptive immunotherapy (AIT) with dendritic cells (DCs) pulsed with mucin 1 (MUC1) peptide and cytotoxic T lymphocytes (CTLs). We also reported that gemcitabine (GEM) enhances anti-tumor immunity by suppressing regulatory T cells. Therefore, in the present study, we performed combination therapy with AIT and GEM for patients with unresectable or recurrent pancreatic cancer.
Patients and methods
Forty-two patients with unresectable or recurrent pancreatic cancer were treated. DCs were generated by culture with granulocyte macrophage colony-stimulating factor and interleukin-4 and then exposed to tumor necrosis factor-α. Mature DCs were transfected with MUC1-mRNA by electroporation (MUC1-DCs). MUC1-CTLs were induced by co-culture with YPK-1, a human pancreatic cancer cell line, and then with interleukin-2. Patients were treated with GEM, while MUC1-DCs were intradermally injected, and MUC1-CTLs were intravenously administered.
Median survival time (MST) was 13.9 months, and the 1-year survival rate was 51.1%. Of 42 patients, one patient had complete response (2.4%), three patients had partial response (7.1%) and 22 patients had stable disease (52.4%). The disease control ratio was 61.9%. The MST and 1-year survival rate of 35 patients who received more than 1 × 107 MUC1-DCs per injection was 16.1 months and 60.3%, respectively. Liver metastasis occurred in only 5 patients among 35 patients without liver metastasis before treatment. There were no severe toxicities associated with AIT.
AIT with MUC1-DCs and MUC1-CTLs plus GEM may be a feasible and effective treatment for pancreatic cancer.
PMCID: PMC4074851  PMID: 24947606
Pancreatic cancer; MUC1; Dendritic cell; Cytotoxic lymphocyte; Gemcitabine; Immunotherapy
13.  Twitching in Sensorimotor Development from Sleeping Rats to Robots 
Current biology : CB  2013;23(12):R532-R537.
It is still not known how the “rudimentary” movements of fetuses and infants are transformed into the coordinated, flexible, and adaptive movements of adults. In addressing this important issue, we consider a behavior that has been perennially viewed as a functionless by-product of a dreaming brain: the jerky limb movements called myoclonic twitches. Recent work has identified the neural mechanisms that produce twitching as well as those that convey sensory feedback from twitching limbs to the spinal cord and brain. In turn, these mechanistic insights have helped inspire new ideas about the functional roles that twitching might play in the self-organization of spinal and supraspinal sensorimotor circuits. Striking support for these ideas is coming from the field of developmental robotics: When twitches are mimicked in robot models of the musculoskeletal system, basic neural circuitry self-organizes. Mutually inspired biological and synthetic approaches promise not only to produce better robots, but also to solve fundamental problems concerning the developmental origins of sensorimotor maps in the spinal cord and brain.
PMCID: PMC3709969  PMID: 23787051
sleep; myoclonic twitch; robot; spinal reflex; sensorimotor integration; embodiment; spontaneous activity; development; fetus; neonate
14.  Successful percutaneous treatment for massive hemorrhage due to infectious pseudoaneurysm in the abdominal wall after percutaneous endoscopic gastrostomy: a case report 
BMC Research Notes  2014;7:354.
Percutaneous endoscopic gastrostomy (PEG) is often performed for alimentation and to prevent weight loss in patients with feeding problems due to central neurologic diseases such as cerebral infarction or intracranial hemorrhage. Although infection at the skin site after PEG placement is a typical late complication of PEG, a ruptured infectious pseudoaneurysm caused massive bleeding adjacent to the tract is rare. Prompt treatment is required to avoid the hemorrhage shock, however surgical ligation is difficult to obtain the arrest of bleeding in damaged skin due to the infection.
Case presentation
A 70-year-old male was bedridden due a cerebral infarction suffered 1 year previously. APEG was placed because of feeding problems, and a push-type, 20-Fr gastrostomy tube was inserted through the anterior abdominal wall. On day 16 after PEG placement, the patient had massive bleeding from the PEG site due to the rupture of infectious pseudoaneurysm and developed a decreased level of consciousness and hypotension. Treatment by percutaneous direct injection of a mixture of n-butyl-cyanoacrylate (NBCA)-lipiodol was performed and achieved good hemostasis is obtained.
A rare case of an infectious pseudoaneurysm that developed in the abdominal wall and caused massive bleeding at a PEG placement site was described. Percutaneous injection of a mixture of n-butyl-cyanoacrylate (NBCA)-lipiodol under ultrasound guidance is an effective treatment in this case.
PMCID: PMC4057582  PMID: 24915936
n-butyl-cyanoacrylate-lipiodol; Infectious pseudoaneurysm; Abdominal wall; Percutaneous endoscopic gastrostomy
15.  Fabrication of single-crystalline microspheres with high sphericity from anisotropic materials 
Scientific Reports  2014;4:5186.
Microspheres with high sphericity exhibit unique functionalities. In particular, their high symmetry makes them excellent omnidirectional optical resonators. As such perfect micrometre-sized spheres are known to be formed by surface tension, melt cooling is a popular method for fabricating microspheres. However, it is extremely difficult to produce crystalline microspheres using this method because their surfaces are normally faceted. Only microspheres of polymers, glass, or ceramics have been available, while single-crystalline microspheres, which should be useful in optical applications, have been awaiting successful production. Here we report the fabrication of single-crystalline semiconductor microspheres that have surfaces with atomic-level smoothness. These microspheres were formed by performing laser ablation in superfluid helium to create and moderately cool a melt of the anisotropic semiconductor material. This novel method provides cooling conditions that are exceptionally suited for the fabrication of single-crystalline microspheres. This finding opens a pathway for studying the hidden mechanism of anisotropy-free crystal growth and its applications.
PMCID: PMC4046134  PMID: 24898213
16.  Histologic Evaluation of Human Alveolar Sockets Treated With an Artificial Bone Substitute Material 
This study involved a histologic, enzyme histologic, immunohistologic, and three-dimensional microstructure evaluating the extent of osteogenesis and repair in the human alveolar extraction socket achievable with an artificial bone substitute. After tooth extraction in 7 patients, extraction sockets were filled with Mastergraft (15% hydroxyapatite, 85% β-tricalcium phosphate complex). Radio-micrographs and histologic examinations were performed on samples obtained during dental implant placement procedure. On micro– computed tomography, new bone was observed in all collected samples, and osteogenesis was observed to have taken place around the artificial bone substitute. Histologically, active osteogenesis was found throughout the region observed. Addition of new bone around the Mastergraft was observed, and osteoblast-like cells were present. Cells that had partially invaded the artificial bone included tartrate-resistant acid phosphate–positive and CD34-positive cells. These findings indicate that the Mastergraft artificial bone induced osteogenesis in the jawbone and seemed effective for repairing bone defects.
PMCID: PMC4045110  PMID: 21415629
β-Tricalcium phosphate; hydroxyapatite; osteoblast; osteoclast; human alveolar socket repair
17.  Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab 
Cancer Biology & Therapy  2013;14(6):481-491.
The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (CtxR) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in CtxR clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all CtxR clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of CtxR cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.
PMCID: PMC3813564  PMID: 23760490
AKT; EGFR; MK-2206; cetuximab; acquired cetuximab-resistance; non-small cell lung cancer; MAPK
18.  Liver transplantation: Fifty years of experience 
Since 1963, when the first human liver transplantation (LT) was performed by Thomas Starzl, the world has witnessed 50 years of development in surgical techniques, immunosuppression, organ allocation, donor selection, and the indications and contraindications for LT. This has led to the mainstream, well-established procedure that has saved innumerable lives worldwide. Today, there are hundreds of liver transplant centres in over 80 countries. This review aims to describe the main aspects of LT regarding the progressive changes that have occurred over the years. We herein review historical aspects since the first experimental studies and the first attempts at human transplantation. We also provide an overview of immunosuppressive agents and their potential side effects, the evolution of the indications and contraindications of LT, the evolution of survival according to different time periods, and the evolution of methods of organ allocation.
PMCID: PMC4017051  PMID: 24833866
Liver transplantation; History; Survival; Indications; Organ allocation
19.  Draft Genome Sequence of Lactobacillus sucicola JCM 15457T, a Motile Lactic Acid Bacterium Isolated from Oak Sap 
Genome Announcements  2014;2(3):e00403-14.
Here, we report the draft genome sequence of a motile lactic acid bacterium, Lactobacillus sucicola JCM 15457T, isolated from oak sap. Motility-related genes and their organization in the annotated genome were broadly similar to those in the sequenced genomes of related lactobacilli.
PMCID: PMC4014693  PMID: 24812225
20.  Oral pathobiont induces systemic inflammation and metabolic changes associated with alteration of gut microbiota 
Scientific Reports  2014;4:4828.
Periodontitis has been implicated as a risk factor for metabolic disorders such as type 2 diabetes, atherosclerotic vascular diseases, and non-alcoholic fatty liver disease. Although bacteremias from dental plaque and/or elevated circulating inflammatory cytokines emanating from the inflamed gingiva are suspected mechanisms linking periodontitis and these diseases, direct evidence is lacking. We hypothesize that disturbances of the gut microbiota by swallowed bacteria induce a metabolic endotoxemia leading metabolic disorders. To investigate this hypothesis, changes in the gut microbiota, insulin and glucose intolerance, and levels of tissue inflammation were analysed in mice after oral administration of Porphyromonas gingivalis, a representative periodontopathogens. Pyrosequencing revealed that the population belonging to Bacteroidales was significantly elevated in P. gingivalis-administered mice which coincided with increases in insulin resistance and systemic inflammation. In P. gingivalis-administered mice blood endotoxin levels tended to be higher, whereas gene expression of tight junction proteins in the ileum was significantly decreased. These results provide a new paradigm for the interrelationship between periodontitis and systemic diseases.
PMCID: PMC4010932  PMID: 24797416
21.  Paliperidone extended release for the treatment of pediatric and adolescent patients with Tourette's disorder 
A subgroup of patients with Tourette's disorder (TD) has symptoms refractory to haloperidol, a standard therapeutic drug for TD.
We report on three cases of pediatric and adolescent patients who were treated with paliperidone extended release.
In two cases, TD symptoms were remarkably improved by switching from haloperidol to paliperidone extended release, and in another case, paliperidone extended release showed significant efficacy in treating TD symptoms as the first-line drug. In all cases, no significant adverse side effects were detected.
Paliperidone extended release may be a strong candidate for the treatment of pediatric and adolescent patients with TD.
PMCID: PMC4020572  PMID: 24829608
Tourette's disorder; Paliperidone extended release; Haloperidol; Tics
22.  Spatial and Temporal Analysis of Extracellular Matrix Proteins in the Developing Murine Heart: A Blueprint for Regeneration 
Tissue Engineering. Part A  2013;19(9-10):1132-1143.
The extracellular matrix (ECM) of the embryonic heart guides assembly and maturation of cardiac cell types and, thus, may serve as a useful template, or blueprint, for fabrication of scaffolds for cardiac tissue engineering. Surprisingly, characterization of the ECM with cardiac development is scattered and fails to comprehensively reflect the spatiotemporal dynamics making it difficult to apply to tissue engineering efforts. The objective of this work was to define a blueprint of the spatiotemporal organization, localization, and relative amount of the four essential ECM proteins, collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN) in the left ventricle of the murine heart at embryonic stages E12.5, E14.5, and E16.5 and 2 days postnatal (P2). Second harmonic generation (SHG) imaging identified fibrillar collagens at E14.5, with an increasing density over time. Subsequently, immunohistochemistry (IHC) was used to compare the spatial distribution, organization, and relative amounts of each ECM protein. COLIV was found throughout the developing heart, progressing in amount and organization from E12.5 to P2. The amount of COLI was greatest at E12.5 particularly within the epicardium. For all stages, FN was present in the epicardium, with highest levels at E12.5 and present in the myocardium and the endocardium at relatively constant levels at all time points. ELN remained relatively constant in appearance and amount throughout the developmental stages except for a transient increase at E16.5. Expression of ECM mRNA was determined using quantitative polymerase chain reaction and allowed for comparison of amounts of ECM molecules at each time point. Generally, COLI and COLIII mRNA expression levels were comparatively high, while COLIV, laminin, and FN were expressed at intermediate levels throughout the time period studied. Interestingly, levels of ELN mRNA were relatively low at early time points (E12.5), but increased significantly by P2. Thus, we identified changes in the spatial and temporal localization of the primary ECM of the developing ventricle. This characterization can serve as a blueprint for fabrication techniques, which we illustrate by using multiphoton excitation photochemistry to create a synthetic scaffold based on COLIV organization at P2. Similarly, fabricated scaffolds generated using ECM components, could be utilized for ventricular repair.
PMCID: PMC3609645  PMID: 23273220
23.  Effects of Magnetic Fields of up to 9.4 T on Resolution and Contrast of PET Images as Measured with an MR-BrainPET 
PLoS ONE  2014;9(4):e95250.
Simultaneous, hybrid MR-PET is expected to improve PET image resolution in the plane perpendicular to the static magnetic field of the scanner. Previous papers have reported this either by simulation or experiment with simple sources and detector arrangements. Here, we extend those studies using a realistic brain phantom in a recently installed MR-PET system comprising a 9.4 T MRI-scanner and an APD-based BrainPET insert in the magnet bore. Point and line sources and a 3D brain phantom were filled with 18F (low-energy positron emitter), 68Ga (medium energy positron emitter) or 120I, a non-standard positron emitter (high positron energies of up to 4.6 MeV). Using the BrainPET insert, emission scans of the phantoms were recorded at different positions inside and outside the magnet bore such that the magnetic field was 0 T, 3 T, 7 T or 9.4 T. Brain phantom images, with the ‘grey matter’ compartment filled with 18F, showed no obvious resolution improvement with increasing field. This is confirmed by practically unchanged transaxial FWHM and ‘grey/white matter’ ratio values between at 0T and 9.4T. Field-dependent improvements in the resolution and contrast of transaxial PET images were clearly evident when the brain phantom was filled with 68Ga or 120I. The grey/white matter ratio increased by 7.3% and 16.3%, respectively. The greater reduction of the FWTM compared to FWHM in 68Ga or 120I line-spread images was in agreement with the improved contrast of 68Ga or 120I images. Notwithstanding elongations seen in the z-direction of 68Ga or 120I point source images acquired in foam, brain phantom images show no comparable extension. Our experimental study confirms that integrated MR-PET delivers improved PET image resolution and contrast for medium- and high-energy positron emitters even though the positron range is reduced only in directions perpendicular to the magnetic field.
PMCID: PMC3995683  PMID: 24755872
24.  Steroid sulphatase and oestrogen sulphotransferase in human non-small-cell lung carcinoma 
British Journal of Cancer  2013;108(7):1415-1424.
Steroid sulphatase (STS) is one of the steroid-metabolising enzymes involved in desulphating inactive steroid sulphates and oestrogen sulphotransferase (EST) sulphates active oestrogen. The roles of both STS and EST have not been examined in oestrogen-dependent non-small-cell lung cancer (NSCLC).
We evaluated the immunoreactivity of STS and EST in NSCLC cases using immunohistochemistry. The function of STS and EST was further demonstrated using NSCLC cell lines.
The immunoreactivity of STS and EST was detected in 49.5% and 27.8% of NSCLC cases, respectively. The immunoreactivity of STS was significantly higher in female adenocarcinoma cases. The STS-positive NSCLCs were also significantly correlated in an inversed manner with tumour size and cell proliferation and tended to be associated with better clinical outcome. However, the immunoreactivity of EST was significantly correlated with intracellular oestradiol concentration. Results of in vitro analysis demonstrated that oestrone sulphate (E1-S) induced and pregnenolone sulphate (Preg-S) inhibited the proliferation in STS-expressing cell lines. The inhibition by Preg-S was reversed by a specific progesterone receptor blocker. Simultaneous addition of E1-S and Preg-S significantly suppressed the proliferation.
In NSCLC patients, STS is considered a good prognostic factor. Results of our present study also indicated the benefits of potential progesterone therapy for NSCLC patients.
PMCID: PMC3629433  PMID: 23531699
non-small-cell lung carcinoma; steroid sulphatase; oestrogen sulphotransferase; pregnenolone sulphate; progesterone receptor
25.  Application of MicroRNA in Diagnosis and Treatment of Ovarian Cancer 
BioMed Research International  2014;2014:232817.
Ovarian cancer has a poor prognosis because early detection is difficult and recurrent ovarian cancer is usually drug-resistant. The morbidity and mortality of ovarian cancer are high worldwide and new methods of diagnosis and therapy are needed. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression that are involved in carcinogenesis, metastasis, and invasion. Thus, miRNAs are likely to be useful as diagnostic and prognostic biomarkers and for cancer therapy. Many miRNAs have altered expression in ovarian cancer compared to normal ovarian tissues and these changes may be useful for diagnosis and treatment. For example, deficiencies of enzymes including Dicer and Drosha that are required for miRNA biogenesis may be adverse prognostic factors; miRNAs such as miR-214 and miR-31, which are involved in drug resistance, and the miR-200 family, which is implicated in metastasis, may serve as biomarkers; and transfection of downregulated miRNAs and inhibition of upregulated miRNAs may be effective for treatment of ovarian cancer. Chemotherapy targeting epigenetic mechanisms associated with miRNAs may also be effective to reverse gene silencing.
PMCID: PMC4009316  PMID: 24822185

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