Search

Gammaretroviral vectors are widely used in cellular and gene therapy products because of the availability of stable vector producer cells. Accurately assessing vector copy number (VCN) is critical for selecting appropriate clones to avoid the risks of homologous recombination and complications in mutation detection. Traditional methods such as quantitative polymerase chain reaction (PCR) and Southern blotting have limitations in accuracy and throughput. This study presents a triplex droplet digital PCR (ddPCR) method for analyzing the VCN in gammaretroviral vector producer cells. We designed a universal primer– probe set targeting the packaging signal sequence common to murine leukemia virus– and murine stem cell virus– based gammaretroviral vectors. Two reference genes were selected after karyotyping the PG13 gammaretroviral vector packaging cell line to identify stable chromosomes. The triplex ddPCR assay was optimized and verified using PG13 cells transduced with constructs of different transgene and vector backbones. The assay showed high concordance with Southern blot. Using multiple reference genes ensures accurate and robust VCN assessment, especially in cell lines with chromosomal instability. This method improves the clone selection process for gammaretroviral vector producer cells, accelerates the development of novel cellular and gene therapy products, and ensures their rapid delivery to patients. • A triplex ddPCR was developed for accurate vector copy number (VCN) determination. • A universal primer–probe set for most gammaretroviral vectors was designed. • Multiple reference genes were used for VCN measurements in aneuploid cell lines. • ddPCR results matched Southern blot results, ensuring high reliability & throughput. [ABSTRACT FROM AUTHOR]

Kinetic and static properties of the coil-globule transition of poly(methyl methacrylate) (PMMA) in pure tert-butyl alcohol were determined by static light scattering and compared with those of PMMA in the mixed solvent tert-butyl alcohol+water(2.5 vol%) in order to examine the effect of water on the chain collapse. The measurements were carried out for the molecular weight M × 10[sup -6] = 4.1 and 12.2 in the concentration range of 0.6 × 10[sup -4]-2.6×10[sup -4] g/cm³, and the mean-square radius of gyration 〈s²〉 was determined as a function of the time after an abrupt decrease of temperature. PMMA chains collapsed to equilibrium globules within 90 min after quenching for M=1.22 × 10[sup 7] and within 30 min for M=4.1×10[sup 6]. Chain aggregation due to phase separation became noticeable after the collapse of the chain because of an increase of observed molecular weight. For PMMA in the mixed solvent tert-butyl alcohol+water(2.5 vol%), the chain collapse process has been observed for periods from hours to weeks depending on the molecular weight and temperature, and the chain aggregation was negligibly small in the chain collapse process. The expansion factor α²=〈s²〉/〈S²〉[sub 0] obtained for fully collapsed chains in pure tert-butyl alcohol was represented by the theoretical prediction α³-α-C(α[sup -3]-1)=B(1-θ/T)M[sup 1/2] with the coefficients of B=0.0179 and C=0.054. For PMMA in the mixed solvent, the coil-globule transition curve has been expressed by the same equation with B=0.0164 and C=0.049, close to the above values. The small amount of water in the mixed solvent caused a drastic slowdown in the chain-collapse rate but had little effect on the coil-globule transition curve. [ABSTRACT FROM AUTHOR]

Objective and design: To evaluate the potency of RORγt blockade for treatment of Inflammatory Bowel Disease (IBD), the efficacy of TAK-828F, a novel RORγt inverse agonist, in anti-TNF-α mAb non-responsive mouse colitis model and effect of TAK-828F on IL-17 production in peripheral mononuclear blood cells (PBMCs) of anti-TNF-α naive and treatment-failure patients of IBD was investigated. Methods and results: The colitis model showed Th17-dependent pathogenicity and response to anti-IL-12/23p40 monoclonal antibody (mAb), but no response to anti-TNF-α mAb. In the model, TAK-828F, at oral dosages of 1 and 3 mg/kg, inhibited progression of colitis and reduced the immune reaction that characterize Th17 cells. Anti-IL-17A mAb showed neither efficacy nor change in the T cell population and colonic gene expression in the model. In the normal mouse, a 4-week treatment of TAK-828F at 30 mg/kg did not severely reduce lymphocyte cell counts in peripheral and intestinal mucosa, which was observed in RORγ−/− mice. TAK-828F strongly inhibited IL-17 gene expression with IC50 values from 21.4 to 34.4 nmol/L in PBMCs from anti-TNF mAb naive and treatment-failure patients of IBD. Conclusions: These results indicate that RORγt blockade would provide an effective approach for treating refractory patients with IBD by blocking IL-23/Th17 pathway. [ABSTRACT FROM AUTHOR]

Objective and design: Protease activity of MALT lymphoma-translocation protein 1 (Malt1) plays an important role in the development of colitis, but the detailed mechanism has not been fully elucidated.Method: Effects of Malt1 protease on the activation of T cells and the development of experimental colitis was investigated using Malt1 protease-deficient (PD) mouse.Results: IL-2 production from CD4+ T cells of Malt1 PD mice was decreased compared with that of wild-type (WT) mice. Intraperitoneal injection of anti-CD3 antibody into Malt1 PD mouse induced less productions of IL-17 in the plasma, as well as the colonic gene expression of IL-17A, compared with WT mice, whereas IFN-γ production was not impaired. In naïve T-cell transfer colitis model, Malt1 PD T cells induced less disease severity than WT T cells. Then, reduction in the populations of Th17 and Th1/17 cells was observed in the mesenteric lymph nodes of the recipient mice transferred with Malt1 PD T cells, whereas those of Th1 cells were not impaired. IL-17A expression in the colon was also decreased in the mouse receiving Malt1 PD T cells.Conclusions: Inactivation of Malt1 protease activity abrogates Th17 and Th1/17 cell activation, resulting in the amelioration of experimental colitis. [ABSTRACT FROM AUTHOR]

IL-17-producing Th17 cells and IFN-γ and IL-17 double-producing Th1/17 cells have been identified as the pathogenic cells in inflammatory bowel disease (IBD). Retinoic acid-related orphan receptor γt (RORγt) is a master regulator for the differentiation and activation of Th17 and Th1/17 cells. We discovered a novel orally available TAK-828F, a strong and selective RORγt inverse agonist. To assess the potential of RORγt blockade in the therapy for IBD, the efficacy of TAK-828F in activated T cell transfer mouse colitis model was investigated. This model was highly sensitive to the prophylactic treatment of anti-TNF-α monoclonal antibody but partially susceptible to sulfasalazine, tacrolimus, and prednisolone. Oral administration of TAK-828F, at doses of 1 and 3 mg/kg, b.i.d, strongly protected the progression of colitis. TAK-828F decreased the population of Th17 and Th1/17 cells in a dose-dependent manner in the mesenteric lymph node. Moreover, expression of mRNA that are characteristic of the Th17 signature, such as IL-17A and IL-17F in the colon, were inhibited by TAK-828F, while the expression of IL-10, an anti-inflammatory cytokine, was increased. In the therapeutic treatment, TAK-828F lessened disease severity compared to the vehicle control mice. Interestingly, gene expression of zonula occludens-1 (ZO-1) and mucin 2 (Muc2), which play an important role in barrier function of the intestinal mucosa, was recovered by TAK-828F. These results indicate that blocking RORγt has promising pharmacological profile in the colitis model. RORγt blockade may provide a novel therapeutic paradigm for treatment of IBD with unique mechanism by which improves imbalance of the immune system. [ABSTRACT FROM AUTHOR]

MALT lymphoma-translocation protein 1 (Malt1) protease activity is triggered by stimulation of various immune receptors. Activation of Malt1 protease induces cleavage of negative regulators for immune responses, resulting in lymphocytes activation. Although Malt1 protease mediates the signaling process downstream of the T cell, B cell, and dectin receptors, its contribution in Fcγ receptor (FcγR) signaling has not been elucidated. In this study, we investigated the role of Malt1 protease activity in FcγR signaling using Malt1 protease-deficient (PD) mouse. In addition, role of Malt1 protease for the development of FcγR-mediated autoimmune disease was also investigated in vivo . Malt1 protease cleaves their substrates, such as RelB and cylindromatosis (CYLD). However, the Malt1 proteolytic activity was silenced in the Malt1 PD mice. Production of inflammatory cytokines via FcγR stimulation was decreased on dendritic cells prepared from Malt1 PD mice. In FcγR-dependent murine immune thrombocytopenia (ITP) model, gene expressions of the inflammatory cytokines in the spleen of Malt1 PD mice were lower than those of WT mice. Then, Malt1 PD mice protected the development of thrombocytopenia. These results clearly figured out that Malt1 protease activity plays an important role in the activation of innate immune cells via FcγR, and the development of FcγR-mediated autoimmune diseases. Therefore, Malt1 is an attractive target for the treatment of inflammatory diseases mediated by FcγR. [ABSTRACT FROM AUTHOR]

Supercritical CO 2 is a promising zero-surface-tension, highly diffusive solvent, which creates a uniform reaction environment that dissolves low-volatility metal compounds. Here we report a novel etching technique for transition metal (Cu, Ni, Co, and Fe) thin films in supercritical CO 2 (scCO 2 ). The metals were removed by reacting with the chelating agent, hexafluoroacetylacetone, by dissolving the product compounds in scCO 2 . The operating temperatures were 100–250 °C and pressure was fixed at 10 MPa. The changes in the optical transmittance and thickness of the films indicated etching in the scCO 2 . Scanning electron microscope observation and surface probe microscope analysis confirmed that there was no significant film agglomeration, in particular, and that the Fe and Co films were uniformly etched. [ABSTRACT FROM AUTHOR]

For private enterprises, it has become increasingly important and indispensable to utilize and purchase an advanced information system from a software provider. The system helps firms to keep an eye on items such as the volatile market conditions, competitor changes and changing consumer needs. Generally speaking, installation of the specific information system starts when both a private enterprise and a provider in coordination begin to discuss planning for the installation with a "Requirement for Proposal (RFP)" being issued. This process eventually proceeds to testing of the system before it is finally put into place. Later, software maintenance becomes an important issue. Recently, it has become evident that, for many private firms, the expenditure for the software maintenance seems increasingly more expensive than that of the original installation and development of the software. With this in mind, this paper attempts to focus on software maintenance and the problem thereof. This paper deals with two related issues. First, it discusses system maintenance and tries to come up with a model best suited for this purpose. The paper attempts to study system repatriation. When requiring system maintenance, a company usually wants to know the cost and the timing of the maintenance work. The information on these issues is estimated in this paper as "the additional dates of the system repatriation." A logistic curve is applied to evaluate the expenditure. The second issue this paper examines is development of a system to measure the approximate cost of repatriation. In developing this information, the paper highlights a decision-tree method. Taking advantage of these two different information sets, administrators in a given firm are given a rough idea as to when maintenance work starts and how much the costs of this process for the enterprise, thus allowing them to be well prepared for software maintenance. [ABSTRACT FROM AUTHOR]

Chain-expansion processes from knotted globules have been measured for poly (methyl methacrylate) (PMMA) in the mixed solvent tert-butyl alcohol (TBA) + water (2.5 vol %) by static light scattering. The solution was quenched from the Θ temperature of 41.5 °C to 37.0 °C, aged there for a time period tp and then returned rapidly to the Θ temperature. The chain-expansion process was determined as a time evolution of the expansion factor &agr;2 after the temperature increase. The measurement was carried out by changing the aging time tp from 240 to 7200 min, and the molecular weight from Mw = 4.0 x 106 to 1.5 x 107, by taking advantage of the extremely slow chain aggregation in the solution. The chain-expansion process obtained for Mw -- 1.22 x 107 became slow with increasing tp, which revealed the knot formation in single globules. The characteristic time of the chain expansion from globules aged for tp = 7200 min was found to depend on the molecular weight as M2.7w. This exponent, which is close to 3, demonstrated a disentanglement process due to self-reptation. The present data were compared with the previous data of the chain expansion from compact globules aged at 25.0 °C. The comparison made at Mw = 1.22 x 107 and at the same values of Tp revealed that the chain expansion from the globules aged at 25.0 °C was much faster than that from the globules at 37.0 °C, indicating a lower knot density in the more compact globules. It was conjectured that the knot formation due to self-reptation would be suppressed in a compact globule because an entire conformational change required by knot formation would become difficult to occur in the confined space of high segment concentration, particularly for a long polymer chain. The chain collapse of PMMA in the mixed solvent has been observed to occur extremely slowly at the later stage. This slow process was explained by the suppressed self-reptation. [ABSTRACT FROM AUTHOR]

This study adopts the modelling approach described by Kusaka and co-authors in previous papers. It aims at specifying two fundamental approaches for product development (PD) in order to gain long-range and short-range competitive advantages and to create decision support information. The long-range approach introduces product functions and technologies over successive generations of products, as and when they become appropriate; the short-range approach enables the latest information, based on present market needs and current technological advances, to be flexibly and quickly introduced into PD decision-making. Thus, the study builds a PD model that optimally selects product functions and technologies for successive generations. The system builds on previous research by introducing a time factor. A graphic information tool, which we call a generalised cost performance curve, enables decision-makers to see changing PD patterns for successive generations. It also clarifies product evolutionary patterns and their characteristics under planned roadmaps. [ABSTRACT FROM AUTHOR]

Abstract: Symptomatic spontaneous intratumoral hemorrhage is a rare event in a patient with a brain tumor (BT). Although the treatment of choice in such a case is surgical removal of both the tumor and the hemorrhage, the optimal timing for surgical intervention has not been clearly established, particularly in those who present with minimal neurological deficits and a small hemorrhage volume. Two cases of primary BTs manifesting as an intracerebral hemorrhage (ICH) are described, in which rebleeding from the tumor occurred shortly after the initial hemorrhage. The patients each presented with the sudden onset of a headache and minimal neurological deficits, and the neuroradiological workup was consistent with a diagnosis of hemorrhagic BT. Each patient remained neurologically stable, and elective surgery had been planned within 7 days of their admission, but rebleeding occurred 5 and 6 days, respectively, after admission. A BT manifesting as an ICH may rebleed shortly after the initial bleeding, and should be treated on an emergency basis instead of an elective basis regardless of the patient''s neurological status on admission or hematoma volume on the initial CT scans. [Copyright &y& Elsevier]

Alkaline phosphatase (ALP) is anchored to the outer leaflet of the lipid bilayer via phosphatidylinositol (PI) and ALP activity has been localized in the plasma membrane of numerous tissues. In the periodontal ligament ALP activity is found in the collagen fibers in addition to the plasma membrane of the osteoblasts and fibroblasts. In this study, we examined the distribution of ALP activity in the periodontal ligament of rat molars and also examined whether the bond between ALP and collagen fibers is dependent on PI by using phosphatidylinositol-specific phospholipase C (PI-PLC). ALP activity was distributed in the periodontal ligament. The activity mirrored the distribution of collagen fibers in the periodontal ligament. Cytochemical analysis also demonstrated that ALP activity was located not only in the plasma membrane of fibroblasts, but also in the collagen fiber bundles and fibrils in the periodontal ligament. After treatment with PI-PLC, the loss of ALP activity in the periodontal ligament was observed histochemically, and the loss of ALP activity in the fibroblasts as well as in the collagen fiber bundles and fibrils was observed cytochemically. These results strongly indicate that the bond between ALP and the collagen fibers is also dependent on PI. [ABSTRACT FROM AUTHOR]

Not infrequently, cerebral angiography performed immediately after the onset of subarachnoid hemorrhage (SAH) fails to reveal any causative lesion. Although the cause of the SAH in most of these cases remains unknown, repeat angiography sometimes discloses a lesion not detected by the initial angiography. The frequency of finding a ruptured aneurysm by repeat angiography and the angiographic characteristics of such aneurysms have been investigated retrospectively. Nineteen among 316 non-traumatic SAH patients (6%) showed initially negative angiogram, and 7 of 19 patients (36%) were identified as harboring an “initially occult” aneurysm. It was possible to identify the expected cause of these initial false-negative angiograms in four of those seven patients. The aneurysm was located in the anterior communicating artery (ACoA) complex in four of the seven patients. When interpreting the angiograms of patients with SAH, neurologists and neurosurgeons should bear in mind the finding that the ACoA complex is the most frequent site of “initially occult” aneurysms. One patient had a dissecting aneurysm of the internal carotid artery, and although such lesions have been considered rare, they should be included in the differential diagnosis of SAH of unknown origin. [Copyright &y& Elsevier]

Postoperative communicating hydrocephalus in adult patients with supratentorial malignant glioma is a relatively uncommon condition that occurs months after the initial operation of tumor excision. It occurred in only five of 50 consecutive cases treated in our department during the past 10 years. The hydrocephalus appeared to be attributable to leptomeningeal dissemination of tumor cells and subsequent impairment in cerebrospinal fluid (CSF) absorption. The tumors were located adjacent to the lateral ventricles in all five patients, and the proximity of the tumor to the cerebral ventricles may have facilitated dissemination of the tumor cells into the CSF space, resulting in hydrocephalus. The hydrocephalus was treated by a shunt surgery in all five cases, and the symptoms temporarily improved. None of the five patients experienced shunt malfunction or abdominal symptoms attributable to metastasis to the peritoneal cavity, and all five patients died of regrowth of the intracranial tumor or of pneumonia. [Copyright &y& Elsevier]

The use of induced hypothermia in the treatment of traumatic spinal cord injury (SCI) has been studied extensively between the 1960s and 1970s. Although the treatment showed some promise, it became less popular by the 1980s, mainly because of its adverse effects. However, a revival of hypothermia in the treatment of traumatic brain injury (TBI) in the last decade has encouraged neuroscientists to conduct experiments to reevaluate the potential benefits of hypothermia in traumatic SCI. All laboratory investigations studying the mechanisms of action and/or the efficacy of induced hypothermia in treating experimental traumatic SCI published in the last decade were reviewed. Although efficacy of hypothermia in improving functional outcome of mild to moderate traumatic SCI has been demonstrated, hypothermia may not be protective against severe traumatic SCI. At present, induced hypothermia has yet to be recognized or approved as a potential treatment having therapeutic value for traumatic SCI in humans. The continued search for a possible synergistic effect of induced hypothermia and pharmacological therapy may yield some promise. It has also been deduced from these laboratory studies that hyperthermia is deleterious and rigorous measures to prevent hyperthermia should be taken to minimize the propagation of secondary neuronal damage after traumatic SCI. [Copyright &y& Elsevier]

Severe metabolic acidosis develops following prolonged periods of cardiopulmonary arrest (CPA), and excessive hydrogen ions derived from lactate and other noxious acids cause marked hyperkalemia in most CPA patients. This study investigated whether the serum electrolyte imbalance in resuscitated CPA patients is affected by the etiology of the CPA. Between 1999 and 2000, return of spontaneous circulation (ROSC) was achieved and serum electrolyte concentration measurements and blood gas analysis (BGA) were performed in 65 of 270 CPA patients treated. Of the 65 patients, subarachnoid hemorrhage (SAH) was the cause of the CPA in ten, cardiac attack was the cause in 16 and asphyxia was the cause in nine patients. The clinical and laboratory data of these 35 patients were retrospectively compared among the three groups. The SAH group had significantly lower serum potassium concentrations than the other two groups and significantly higher glucose concentrations than the asphyxia group. Massive amounts of catecholamines are released into the systemic circulation of SAH patients and our results may indicate that the amount of catecholamines released in resuscitated SAH patients is greater than in heart attack or asphyxia patients, resulting in a lower serum potassium concentration despite the presence of severe metabolic acidosis. It should be clarified in a prospective study whether the presence of normokalemia and hyperglycemia in resuscitated CPA patients reliably predicts the presence of SAH. [Copyright &y& Elsevier]

We report prolonged unilateral vasodilatation and hemispheric brain edema in a 49-year-old man with fulminant hepatic failure (FHF). The patient presented with a tonic–clonic seizure caused by a hypertensive subcortical hemorrhage in the left parietal lobe. Serial computed tomography (CT) scans showed progressive darkening of the ipsilateral hemisphere, suggesting hemispheric cerebral infarction, but the patient did not show clinical signs of deterioration. Brain magnetic resonance angiography showed dilation of the large arteries of the left hemisphere. Evaluation of cerebral blood flow 7 days postictus with single photon emission CT revealed marked ipsilateral hyperperfusion. The darkening of the hemisphere was brain edema elicited by hyperperfusion. Brain edema was reversible, disappearing 14 days postictus. Hemispheric brain edema was caused by unilateral cerebral vasodilatation and resultant hyperperfusion. Although brain edema is a major complication in FHF patients and cerebral hyperperfusion is responsible for edema formation, CT findings of these patients almost invariably show a bilateral lesion. Unilateral vasodilatation and subsequent hemispheric hyperperfusion may be due to overproduction of vasodilators, already abundant in the brains of patients with severe hepatic failure, by seizure activity. [Copyright &y& Elsevier]

Reports on endovascular treatment of ruptured vertebral artery (VA) dissection in the acute stage. Outcomes of the Glasgow Outcome Scale; Clinical and angiographic characteristics of 16 patients with ruptured VA dissection treated endovascularly; Conclusions.

Introduction: The detailed mechanism of the process during bone healing of drill-hole injury has been elucidated, but a crucial factor in regulating drill-hole healing has not been identified. The transcription factor p53 suppresses osteoblast differentiation through inhibition of osterix expression. In present study, we demonstrate the effects of p53 deficiency on the capacity of MSCs and osteoblasts during drill-hole healing. Materials and methods: Mesenchymal stromal cells (MSCs) and osteoblasts were collected from bone marrow and calvaria of p53 knockout (KO) mice, respectively. The activities of cell mobility, cell proliferation, osteoblast differentiation, and wound healing of MSCs and/or osteoblasts were determined by in vitro experiments. In addition, bone healing of drill-hole injury in KO mice was examined by micro-CT and immunohistological analysis using anti-osterix, Runx2, and sclerostin antibodies. Results: KO MSCs stimulated cell mobility, cell proliferation, and osteoblast differentiation. Likewise, KO osteoblasts enhanced cell proliferation and wound healing. KO MSCs and osteoblasts showed high potency in the inflammation and callus formation phases compared to those from wild-type (WT) mice. In addition, increased expression of osterix and Runx2 was observed in KO MSCs and osteoblasts that migrated in the drill-hole. Conversely, sclerostin expression was inhibited in KO mice. Eventually, KO mice exhibited high repairability of drill-hole injury, suggesting a novel role of p53 in MSCs and osteoblasts in improving bone healing. Conclusion: p53 Deficiency promotes bone healing of drill-hole injury by enhancing the bone-regenerative ability of MSCs and osteoblasts. [ABSTRACT FROM AUTHOR]

We investigated the potency of TAK-828F, a RORγt inverse agonist, in murine experimental autoimmune encephalomyelitis (EAE) model. TAK-828F inhibited the differentiation of Th17 and Th1/17 cells in inguinal lymph node. Increase of these cells in central nervous system (CNS) was also inhibited by TAK-828F. Prophylactic and therapeutic treatments of TAK-828F were efficacious in the model. Plasma concentration of TAK-828F was higher than that in CNS. These results indicate that TAK-828F mainly acts at peripheral and results in the reduction of Th17- and Th1/17-dependent inflammation in CNS. Blocking RORγt may be a promising strategy for treatment of multiple sclerosis. Unlabelled Image • RORγt inverse agonist, TAK-828F, suppressed Th17 and Th1/17 cells in lymph node. • TAK-828F was prophylactically and therapeutically efficacious in murine EAE model. • Increase in Th17 and Th1/17 cell in CNS tissue was inhibited by TAK-828F treatment. • Plasma concentration of TAK-828F was higher than that in spinal cord of EAE mice. • TAK-828F acts at peripheral and results in the reduction of immune reaction in CNS. [ABSTRACT FROM AUTHOR]

Knotting in a polymer chain was investigated by observing the chain expansion process from a collapsed chain for the solution of poly(methyl methacrylate)(PMMA) with M w = 1.22 × 107 in the mixed solvent tert-butyl alcohol + water(2.5 vol%). Because of very slow phase separation in the solution the chain collapse after a temperature drop and chain expansion after a jump were determined by static light scattering. PMMA chain was shown to collapse rapidly to an unknotted globule, which subsequently contracted slowly to a knotted globule. The chain expansion process from the knotted globule was dominated by the chain compactness and the degree of knotting of the initial globule. A complete chain expansion from a heavily knotted globule often required a very long time indicating an existence of long-lived knots. The chain expansion from the unknotted globule occurred too fast to be followed by scattering experiment. Image 1 • Collapse and expansion of PMMA chain can be observed far below the θ-point. • Chain collapse occurs rapidly by crumpling first and slowly by knotting. • Degree of Knotting in a globule changes with the quench depth and aging time. • Observed chain expansion process reflects unknotting process in a globule. [ABSTRACT FROM AUTHOR]

Examines the kinetics of chain aggregation of poly(methyl methacrylate) (PMMA) in isoamyl acetate. Light-scattering measurements on dilute solutions of PMMA in isoamyl acetate; Observed phase separation process; Phase separation temperature of the solution at the same concentrations as in the light-scattering measurement.

Objective The aim of the present study was to examine the effect of extracellular heat shock protein (HSP) 72 on human periodontal ligament fibroblast cells (hPDLFs) in vitro. Design hPDLFs were stimulated by recombinant human HSP72 (rhHSP72). TAK-242 was used to inhibit toll-like receptor 4 (TLR4) activity. Interleukin ( IL ) -6, IL-8 and tumor necrosis factor ( TNF ) -α mRNA levels were analyzed by real-time PCR and protein levels were analyzed by enzyme-linked immunosorbent assay. p65/RelA phosphorylation was analyzed by western blot. Results IL-6, IL-8 and TNF-α mRNA and protein levels were significantly increased by rhHSP72 stimulation. These effects were inhibited by TAK-242 treatment. Additionally, p65/RelA phosphorylation was increased after 5-min rhHSP72 stimulation, which was inhibited by TAK-242 treatment. Conclusion Extracellular HSP72 induces proinflammatory cytokines through TLR4/NF-κB in hPDLFs. [ABSTRACT FROM AUTHOR]

Background. Large craniotomy for acute subdural hematoma is sometimes too invasive. We report good outcomes for two cases of neuroendoscopic evacuation of hematoma and contusion by 1 burr hole surgery. Case Presentation. Both patients arrived by ambulance at our hospital with disturbed consciousness after falling. Case 1 was an 81-year-old man who took antiplatelet drugs for brain infarction. Case 2 was a 73-year-old alcoholic woman. CT scanning showed acute subdural hematoma and frontal contusion in both cases. In the acute stage, glycerol was administered to reduce edema; CTs after 48 and 72 hours showed an increase of subdural hematoma and massive contusion of the frontal lobe. Disturbed consciousness steadily deteriorated. The subdural hematoma and contusion were removed as soon as possible by neuroendoscopy under local anesthesia, because neither patient was a good candidate for large craniotomy considering age and past history. 40%~70% of the hematoma was removed, and the consciousness level improved. Conclusion. Neuroendoscopic removal of acute subdural hematoma and contusion has advantages and disadvantages. For patients with underlying medical issues or other risk factors, it is likely to be effective. [ABSTRACT FROM AUTHOR]

On rare occasions, rupture of a cerebral aneurysm causes acute subdural hematoma (SDH) in addition to subarachnoid hemorrhage (SAH). The frequency of SDH resulting from aneurysmal rupture is summarized, and its clinical and radiological characteristics, as well as potential pitfalls in the diagnosis and the management of this life-threatening condition are described. Among 641 patients with nontraumatic SAH treated between 1992 and 2000, 12 patients (1.9%) presented with SDH due to aneurysmal rupture. The SAH grade on admission was grade II in one patient, grade IV in three patients, and grade V in eight patients. Four underwent both hematoma evacuation and clipping of the aneurysm, four underwent hematoma evacuation alone, and the other four patients were treated conservatively. The outcome was good recovery in two patients, severe disability in one patient, and death in nine patients. Patients with a good outcome had a better SAH grade on admission, smaller midline shift, and smaller SDH volume on the initial CT scan, and they had been treated by both SDH evacuation and clipping of the aneurysm. Emergency one-stage operations may be beneficial for aneurysmal SDH patients who are in good SAH grade, or those who are in poor SAH grade but show some neurological recovery after resuscitation. It should also be mentioned that there are cases of aneurysmal SDH without recognizable SAH on the CT scans, and that a case of aneurysmal SDH may present as a case of ‘head trauma’ after an accident, because of the disturbance of consciousness resulting from aneurysmal rupture. [Copyright &y& Elsevier]

Occlusal disharmony leads to morphological changes in the hippocampus and osteopenia of the lumbar vertebra and long bones in mice, and causes stress. Various types of stress are associated with increased incidence of cardiovascular disease, but the relationship between occlusal disharmony and cardiovascular disease remain poorly understood. Therefore, in this work, we examined the effects of occlusal disharmony on cardiac homeostasis in bite-opening (BO) mice, in which a 0.7 mm space was introduced by cementing a suitable applicance onto the mandibular incisior. We first examined the effects of BO on the level of serum corticosterone, a key biomarker for stress, and on heart rate variability at 14 days after BO treatment, compared with baseline. BO treatment increased serum corticosterone levels by approximately 3.6-fold and the low frequency/high frequency ratio, an index of sympathetic nervous activity, was significantly increased by approximately 4-fold by the BO treatment. We then examined the effects of BO treatment on cardiac homeostasis in mice treated or not treated with the non-selective β-blocker propranolol for 2 weeks. Cardiac function was significantly decreased in the BO group compared to the control group, but propranolol ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage were significantly increased in the BO group, but propranolol blocked these changes. The BO-induced cardiac dysfunction was associated with increased phospholamban phosphorylation at threonine-17 and serine-16, as well as inhibition of Akt/mTOR signaling and autophagic flux. These data suggest that occlusal disharmony might affect cardiac homeostasis via alteration of the autonomic nervous system. [ABSTRACT FROM AUTHOR]

Mastication is closely related to brain function. Animal experiments have revealed that tooth loss has a negative influence on brain function. Clinical studies also suggest that normal occlusion is an essential factor for favorable brain function. Mandibular prognathism (MP) usually results in occlusal dysfunction. However, the relationship between MP and brain function remains unclear. In the present study, we examined the relationship between MP and brain function by measuring brain blood flow (BBF). Seventeen subjects with normal occlusion (NORM) and 25 patients with MP participated in this study. The number of occlusal contacts were counted. Electromyography of the masseter muscles during clenching was also recorded. BBF was measured with non-invasive functional near-infrared spectroscopy during calculation task and chewing task. The number of the occlusal contacts and masseter muscle activity were lower in MP compared with NORM. The calculation task increased BBF in both groups. The chewing task also increased BBF in the inferior frontal gyrus in both groups, although the increase in MP was smaller than in NORM. We discovered that patients with MP exhibited a smaller increase in BBF at the inferior frontal gyrus during chewing as compared with NORM. As such, MP would negatively affect brain function. [ABSTRACT FROM AUTHOR]

In skeletal muscle, the major isoform of β-adrenergic receptor (β-AR) is β2-AR and the minor isoform is β1-AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the β-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic β1- or β2-AR activation with a specific β1-AR agonist, dobutamine (DOB), or a specific β2-AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic β1-AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic β2-AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic β1-AR stimulation did not induce muscle hypertrophy, but did induce fibrosis and apoptosis concomitantly with increased levels of p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), calmodulin kinase II (Thr-286) and mammalian target of rapamycin (mTOR) (Ser-2481) phosphorylation. On the other hand, chronic β2-AR stimulation in masseter muscle induced muscle hypertrophy without histological abnormalities, as in the case of cardiac muscle, concomitantly with phosphorylation of Akt (Ser-473) and mTOR (Ser-2448) and increased expression of microtubule-associated protein light chain 3-II, an autophagosome marker. These results suggest that the β1-AR pathway is deleterious and the β2-AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness. [ABSTRACT FROM AUTHOR]

Abstract Introduction The purpose of this study was to examine growth-promoting effects of myo-inositol nutritional supplementation on the mandible in experimental animals. Methods Mice were fed on diets that contained various concentration of myo-inositol for 3 to 12 weeks. The length of the mandible, maxilla, and femur were measured on μCT images. The mandible and tibia were examined histologically and immunohistochemically. The effects of myo-inositol on cell proliferation and chondrocytic differentiation were examined using ATDC5 cells. Results Myo-inositol supplementation had no effects on body weight, length, and maxilla and femur lengths. However, the length of mandible and the thickness of the mandibular condylar cartilage (MCC) were increased by myo-inositol supplement. Microarray analysis revealed that Pik3cd was highly expressed in MCC as compared to that in the cartilage of the tibial growth plate, which was confirmed by real-time RT-PCR and immunohistochemistry. ATDC5 cells also highly expressed Pik3CD. Myoinositol induced increases in cell proliferation and chondrocytic differentiation in ATDC5 cells. The addition of a PIK3CD inhibitor blocked the induction of cell proliferation by myo-inositol in ATDC5 cells. Conclusions Nutritional supplementation with myo-inositol in growing mice augmented mandibular endochondral growth without any systemic effects. The specific promotion of mandibular growth by myoinositol is primarily dependent on the specific intensive expression of PIK3CD in the MCC. Highlights • Myo-inositol augmented mandibular endochondral growth in mice specifically. • Myo-inositol did not augment another site of endochondral ossification. • PIK3CD is specifically highly expressed in mandibular condylar cartilage. • PIK3CD inhibition reduced cell proliferation induced by myo-inositol. • The promotion of mandibular growth is due to the intense PIK3CD expression. [ABSTRACT FROM AUTHOR]

Leptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells. Bone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers. Bone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly. LRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue. • LRP1 deficiency inhibits osteogenic potential and reduces alveolar bone mass. • LRP1 deficiency in LepR+ cells promotes osteoclast activity. • cKO PDLs possess low osteogenic potential. • LRP1 expressed in PDLSCs inhibits bone resorption and promotes bone formation. • LRP1 performs these roles by stimulating osteoblast differentiation. [ABSTRACT FROM AUTHOR]

C-C chemokine receptor 9 (CCR9) is the homing receptor for C-C motif chemokine ligand 25 (CCL25), and contributes to the maintenance of mucosal immunity and pathogenesis of inflammatory bowel disease (IBD) through the recruitment of T cells into the gut mucosa. Recent reports suggest that the interaction of CCR9 and CCL25 in the large intestine correlate with disease severity of colonic IBD. MLN3126 is an orally available small molecular compound with potent and selective CCR9 antagonist activity. MLN3126 inhibited CCL25-induced calcium mobilization in human CCR9 transfected cells and CCL25-induced chemotaxis of mouse primary thymocytes in a dose-dependent manner. The potential effect of MLN3126 in an activated T cell transfer mouse colitis model was compared with that of an anti-tumor necrosis factor (TNF)-α antibody. CCL25 protein was detected in the colon of mucosal epithelial cells and CCR9 + CD4 + T cells were observed in the lamina propria of the colon of mice with colitis. Dietary administration of MLN3126 to the mice maintained sufficient concentration of the compound in the plasma and dose-dependently inhibited progression of colitis compared to the vehicle control group. Anti-TNF-α antibody, a surrogate for a standard of care for IBD treatment, was also efficacious in the colitis model. These results suggest that MLN3126 would be a promising orally available CCR9 antagonist to treat colonic IBD. [ABSTRACT FROM AUTHOR]

Retinoic acid-related orphan receptor γt (RORγt) is a key master regulator of the differentiation and activation of IL-17 producing CD4 + Th17, CD8 + Tc17 and IL-17/IFN-γ co-producing cells (Th1/17 cells). These cells play critical roles in the pathogenesis of autoimmune diseases such as inflammatory bowel disease and multiple sclerosis. Thus, RORγt is an attractive target for the treatment of these diseases. We discovered TAK-828F, an orally available potent and selective RORγt inverse agonist. The inhibitory effect on the activation and differentiation of Th17 cells by TAK-828F was evaluated in mouse and human primary cells. TAK-828F inhibited IL-17 production from mouse splenocytes and human peripheral blood mononuclear cells dose-dependently at concentrations of 0.01–10 μM without affecting the production of IFN-γ. Additionally, TAK-828F strongly inhibited Th17, Tc17 and Th1/17 cells’ differentiation from naive T cells and memory CD4 + T cells at 100 nM without affecting Th1 cells’ differentiation. In addition, TAK-828F improved Th17/Treg cells’ population ratio by inhibiting Th17 cells’ differentiation and up-regulating Treg cells. Furthermore, TAK-828F, at 100 nM, reduced the production of Th17-related cytokines (IL-17, IL-17F and IL-22) without affecting IFN-γ production in whole blood. These results demonstrate that TAK-828F has the potent and selective inhibitory activity against RORγt both in mouse and human cells. Additionally, oral administration of TAK-828F showed promising efficacy in naive T cell transfer mouse colitis model. TAK-828F may provide a novel therapeutic option to treat immune diseases by inhibiting Th17 and Th1/17 cells’ differentiation and improving imbalance between Th17 and Treg cells. [ABSTRACT FROM AUTHOR]

Teeth develop through interactions between epithelial and mesenchymal tissues mediated by a signaling network comprised of growth factors and transcription factors. However, little is known about how epigenetic modifiers affect signaling pathways and thereby regulate tooth formation. We previously reported that the histone 3 lysine 9 (H3K9) methyltransferase (MTase) G9a is specifically enriched in the tooth mesenchyme during mouse development. In this study, we investigated the functions of G9a in tooth development using G9a conditional knockout (KO) mice. We used Sox9-Cre mice to delete G9a in the tooth mesenchyme because Sox9 is highly expressed in the mesenchyme derived from the cranial neural crest. Immunohistochemical analyses revealed that G9a expression was significantly decreased in the mesenchyme of Sox9-Cre;G9afl/fl (G9a cKO) mice compared with that in Sox9-Cre;G9a fl/+(control) mice. Protein levels of the G9a substrate H3K9me2 were also decreased in the tooth mesenchyme. G9a cKO mice showed smaller tooth germ after embryonic day (E) 16.5 and E17.5, but not at E15.5. The developing cusp tips, which were visible in control mice, were absent in G9a cKO mice at E17.5. At 3 weeks after birth, small first molars with smaller cusps and unseparated roots were formed. Organ culture of tooth germs derived from E15.5 cKO mouse embryos showed impaired tooth development, suggesting that tooth development per se is affected independently of skull development. BrdU labeling experiments revealed that the proliferation rates were decreased in the mesenchyme in G9a cKO mice at E17.5. In addition, the proliferation rates in the tooth inner enamel epithelium were also decreased. In situ hybridization revealed altered localization of genes associated with tooth development. In cKO mice, intensively localized expression of mRNAs encoding bone morphogenic protein ( Bmp2 and Bmp4 ) was observed in the tooth mesenchyme at E17.5, similar to the expression patterns observed in control mice at E15.5. Localization of Shh and related signaling components, including Gli1 , Ptch1 , and Ptch2 , in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21 , both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development. [ABSTRACT FROM AUTHOR]

In orthodontics, it is important to reduce the static friction between brackets and wires in order to enable easy tooth movement. The goal of the present study was to deposit diamond-like carbon (DLC), fluorine-doped DLC (F-DLC), and silicon-doped DLC (Si-DLC) coatings onto the slot surface in stainless steel orthodontic brackets using the plasma-enhanced chemical vapor deposition (PECVD) method and to characterize the frictional property between the coated bracket and wire under dry and wet conditions. In order to characterize DLC-, F-DLC- or Si-DLC-coated surface, XPS, the surface roughness and surface wettability of three deferent surfaces were measured. A nanoindentation test and a scratch test were performed in order to measure the hardness and adhesiveness, respectively, of DLC-, F-DLC- or Si-DLC coatings. The static friction between DLC-, F-DLC-, Si-DLC-coated brackets and 0.019 × 0.025-in stainless steel (SS) orthodontic wires was measured for several angulations under dry and wet conditions using a universal testing machine equipped with a custom-made friction-testing device. The F 1s or Si 2p and Si 2s peaks were observed for F-DLC (27.8 at.%:F) or Si-DLC (26.8 at.%:Si), respectively. There were no significant differences in the surface roughness of the slot surface of the bracket among the four types of specimens. The F-DLC was significantly hydrophobic and Si-DLC was significantly hydrophilic as compared to DLC. Doping the DLC with fluorine or silicon caused the surface hardness to decrease significantly. The results of the present study indicate that DLC, F-DLC and Si-DLC coatings provided a significant reduction in static friction. Among the coatings examined herein, F-DLC-coated bracket exhibited the significantly lowest static friction between the bracket and wire under the wet condition, which was lower than that under the dry condition. The F-DLC coating is highly promising as a means of promoting effective tooth movement and shortening treatment time for orthodontic treatments. [ABSTRACT FROM AUTHOR]

Cell differentiation is controlled by specific transcription factors. The functions and expression levels of these transcription factors are regulated by epigenetic modifications, such as histone modifications and cytosine methylation of the genome. In tendon tissue, tendon-specific transcription factors have been shown to play functional roles in the regulation of tenocyte differentiation. However, the effects of epigenetic modifications on gene expression and differentiation in tenocytes are unclear. In this study, we investigated the epigenetic regulation of tenocyte differentiation, focusing on the enzymes mediating histone 3 lysine 9 (H3K9) methylation. In primary mouse tenocytes, six H3K9 methyltransferase (H3K9MTase) genes, i.e., G9a, G9a- like protein ( GLP), PR domain zinc finger protein 2 ( PRDM2), SUV39H1, SUV39H2, and SETDB1/ESET were all expressed, with increased mRNA levels observed during tenocyte differentiation. In mouse embryos, G9a and Prdm2 mRNAs were expressed in tenocyte precursor cells, which were overlapped with or were adjacent to cells expressing a tenocyte-specific marker, tenomodulin. Using tenocytes isolated from G9a-flox/flox mice, we deleted G9a by infecting the cells with Cre-expressing adenoviruses. Proliferation of G9a-null tenocytes was significantly decreased compared with that of control cells infected with GFP-expressing adenoviruses. Moreover, the expression levels of tendon transcription factors gene, i.e., Scleraxis ( Scx), Mohawk ( Mkx), Egr1, Six1, and Six2 were all suppressed in G9a-null tenocytes. The tendon-related genes Col1a1, tenomodulin, and periostin were also downregulated. Consistent with this, Western blot analysis showed that tenomodulin protein expression was significantly suppressed by G9a deletion. These results suggested that expression of the H3K9MTase G9a was essential for the differentiation and growth of tenocytes and that H3K9MTases may play important roles in tendinogenesis. [ABSTRACT FROM AUTHOR]

Background: Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. Methodology: We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. Results and Conclusion: Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy. [ABSTRACT FROM AUTHOR]

Tissue-specific gene expression is subjected to epigenetic and genetic regulation. Posttranslational modifications of histone tails alter the accessibility of nuclear proteins to DNA, thus affecting the activity of the regulatory complex of nuclear proteins. Methylation at histone 3 lysine 9 (H3K9) is a crucial modification that affects gene expression and cell differentiation. H3K9 is known to have 0-3 methylation states, and these four methylated states are determined by the expression of sets of histone methyltransferases. During development, teeth are formed through mutual interactions between the mesenchyme and epithelium via a process that is subjected to the epigenetic regulation. In this study, we examined the expression of all H3K9 methyltransferases (H3K9MTases) during mouse tooth development. We found that four H3K9MTases-G9a, Glp, Prdm2, and Suv39h1-were highly expressed in the tooth germ, with expression peaks at around embryonic days 16.5 and 17.5 in mice. Immunohistochemical and in situ hybridization analyses revealed that all four H3K9MTases were enriched in the mesenchyme more than in the epithelium. Substrates of H3K9MTases, H3K9me1, H3K9me2, and H3K9me3 were also enriched in the mesenchyme. Taken together, these data suggested that coordinated expression of four H3K9MTases in the dental mesenchyme might play important roles in tooth development. [ABSTRACT FROM AUTHOR]

It has been reported that reactive oxygen species (ROS) play a role as intracellular signaling molecules in RANKL stimulation. Previously we demonstrated that induction of cytoprotective enzyme expression by Nrf2-gene transfer successfully ameliorated RANKL-dependent osteoclastogenesis. In the present study, we hypothesized that Nrf2 activation by inhibiting ubiquitination and degradation of Nrf2 by ETGE-peptide would induce Nrf2-dependent cytoprotective enzyme expression, attenuate ROS signaling, and thereby inhibit RANKL-dependent osteoclastogenesis. ETGE-peptide containing a cell-permeable sequence (seven consecutive arginine; 7R-ETGE) was applied to a mouse macrophage cell-line RAW 264.7 cell or a primary macrophage culture. ETGE-peptide prevents Keap1 from binding to Nrf2. Nrf2 nuclear translocation and Nrf2-dependent cytoprotective enzyme induction was observed. The effects of 7R-ETGE on RANKL-dependent induction of intracellular ROS levels and osteoclastogenesis were examined. Finally, the protective effect of 7R-ETGE on RANKL-mediated bone destruction was investigated in mice. 7R-ETGE dose-dependently induced nuclear Nrf2, followed by the induction of cytoprotective enzyme expression at both the gene and protein level. 7R-ETGE inhibited upregulation of intracellular ROS levels by RANKL stimulation, and osteoclastogenesis was attenuated. Of particular interest was that local injection of 7R-ETGE ameliorated RANKL-mediated bone destruction. Local induction of nuclear Nrf2 by protein transduction is a potential novel therapeutic target for bone destruction diseases such as periodontitis and rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell-cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation. [ABSTRACT FROM AUTHOR]

The periodontal ligament (PDL) is one of the connective tissues located between the tooth and bone. It is characterized by rapid turnover. Periodontal ligament fibroblasts (PDLFs) play major roles in the rapid turnover of the PDL. Microarray analysis of human PDLFs (HPDLFs) and human dermal fibroblasts (HDFs) demonstrated markedly high expression of chemokine (CXC motif) ligand 12 (CXCL12) in the HPDLFs. CXCL12 plays an important role in the migration of mesenchymal stem cells (MSCs). The function of CXCL12 in the periodontal ligament was investigated in HPDLFs. Expression of CXCL12 in HPDLFs and HDFs was examined by RT-PCR, qRT-PCR and ELISA. Chemotactic ability of CXCL12 was evaluated in both PDLFs and HDFs by migration assay of MSCs. CXCL12 was also immunohistochemically examined in the PDL in vivo. Expression of CXCL12 in the HPDLFs was much higher than that in HDFs in vitro. Migration assay demonstrated that the number of migrated MSCs by HPDLFs was significantly higher than that by HDFs. In addition, the migrated MSCs also expressed CXCL12 and several genes that are familiar to fibroblasts. CXCL12 was immunohistochemically localized in the fibroblasts in the PDL of rat molars. The results suggest that PDLFs synthesize and secrete CXCL12 protein and that CXCL12 induces migration of MSCs in the PDL in order to maintain rapid turnover of the PDL. [ABSTRACT FROM AUTHOR]

In recent years, information systems have becomes important in many corporations. A maintenance process in an information system has more costs than a development process for an information system as a managed item. As one of the causes, it appears that the system is not delivered by the appointed date and a part of the development process is executed in the maintenance process. Therefore, the cost of the maintenance process increases. However, it is not clear what influence is caused on the maintenance process by a development project with a delayed delivery date. As the main reasons for the increase in the cost of the maintenance process, there appears to be 1) an increasing number of user inquiries for software operation, data validity and software bugs, and 2) a long processing time for the user inquiries. However, it is not obvious what influence these factors have on the increased cost of a maintenance process. In this study, in order to enable proper countermeasures, the tendencies in the occurrence of solved user inquiries are analyzed. In this analysis, actual data from a development site is used [ABSTRACT FROM AUTHOR]

Among the various kinds of fibroblasts existing in the human body, the periodontal ligament (PDL) fibroblasts have been suggested as multipotent cells. Periodontal ligament fibroblasts are characterized by rapid turnover, a high remodeling capacity and remarkable capacity for renewal and repair. They also differentiate into osteoblasts and cementoblasts. We established iPS cells from human PDL fibroblasts by introducing the ES cell markers Oct3/4, Sox2, Nanog, Klf4 and Lin28 by retrovirus transduction, even without the oncogene c- Myc. The iPS cells established in this study expressed the ES cell markers and formed teratomas in SCID mice. The c- Myc expression level in the PDL fibroblasts was higher than that in the iPS cells by quantitative RT-PCR. Therefore, we have concluded that PDL fibroblasts could be an optimal cell source for iPS cells. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The aim of the present study is to investigate the involvement of immunocompetent cells, pro-inflammatory cytokines and HSP, to evaluate a change of periodontal ligament during the initial stage of orthodontic tooth movement. Design: In the present study, we investigated the distributional density of immunocompetent cells, the localisation of cytokines, and the expression levels of their mRNA in the periodontal ligament during the initial stage of orthodontic tooth movement, using immunohistochemistry and real-time PCR. Results: Orthodontic tooth movement led to significant recruitment of OX6+ cells and ED1+ cells in the rat PDL. Double-immunofluorescence staining showed that some ED1+ cells expressed pro-inflammatory factors of IL-1β and TNF-α in the PDL during orthodontic tooth movement. Real-time PCR analysis revealed that the expression levels of IL-1β (Il1b) and TNF-α (Tnf) mRNA gradually increased following its decrease after 1h of orthodontic tooth movement. These findings suggest that ED1+ cells are involved in the expression of TNF-α and IL-1β and the subsequent regulation of bone resorption on pressure side. HSP27 (Hspb1) mRNA levels were significantly increased as compared with the control at 1h of the initial stage of treatment. Conclusion: ED1+ cells involved in the expression of TNF-α and IL-1β may play an important role in the initial reaction of the PDL and in the induction of the osteoclastic bone resorption during orthodontic tooth movement. [Copyright &y& Elsevier]

Heat shock proteins (HSPs) are molecular chaperones that maintain intracellular protein homeostasis and ensure survival of cells. Continuous orthodontic force on the tooth is considered to be a type of physical stress loaded to the periodontal ligament (PDL). However, little is known about the role of HSPs during tooth movement. This study was performed to examine the expression of HSPs in the PDL during tooth movement using laser microdissection, microarray analysis, real-time RT-PCR and immunohistochemistry. Gene expression of HSPA1A in the pressure zone of the PDL was higher during 6 h of tooth movement than in the control group. Expression of HSPA1A decreased with time. HSPA1A was also detected in the pressure zone of the PDL at the protein level 24 h after the initial tissue change. These results strongly suggest that expression of HSPA1A in the PDL during early stages of tooth movement is a critical factor for tissue reaction. [ABSTRACT FROM AUTHOR]

Background: Osteoclasts play a critical role in bone resorption due to orthodontic tooth movement (OTM). In OTM, a force is exerted on the tooth, creating compression of the periodontal ligament (PDL) on one side of the tooth, and tension on the other side. In response to these mechanical stresses, the balance of receptor activator of nuclear-factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) shifts to stimulate osteoclastogenesis. However, the mechanism of OPG expression in PDL cells under different mechanical stresses remains unclear. We hypothesized that compression and tension induce different microRNA (miRNA) expression profiles, which account for the difference in OPG expression in PDL cells. To study miRNA expression profiles resulting from OTM, compression force (2 g/cm2) or tension force (15% elongation) was applied to immortalized human PDL (HPL) cells for 24 h, and miRNA extracted. The miRNA expression in each sample was analyzed using a human miRNA microarray, and the changes of miRNA expression were confirmed by real-time RT-PCR. In addition, miR-3198 mimic and inhibitor were transfected into HPL cells, and OPG expression and production assessed. Results: We found that certain miRNAs were expressed differentially under compression and tension. For instance, we observed that miR-572, − 663, − 575, − 3679-5p, UL70-3p, and − 3198 were upregulated only by compression. Real-time RT-PCR confirmed that compression induced miR-3198 expression, but tension reduced it, in HPL cells. Consistent with previous reports, OPG expression was reduced by compression and induced by tension, though RANKL was induced by both compression and tension. OPG expression was upregulated by miR-3198 inhibitor, and was reduced by miR-3198 mimic, in HPL cells. We observed that miR-3198 inhibitor rescued the compression-mediated downregulation of OPG. On the other hand, miR-3198 mimic reduced OPG expression under tension. However, RANKL expression was not affected by miR-3198 inhibitor or mimic. Conclusions: We conclude that miR-3198 is upregulated by compression and is downregulated by tension, suggesting that miR-3198 downregulates OPG expression in response to mechanical stress. [ABSTRACT FROM AUTHOR]

Osteoclastic bone resorption enables orthodontic tooth movement (OTM) in orthodontic treatment. Previously, we demonstrated that local epigallocatechin gallate (EGCG) injection successfully slowed the rate of OTM; however, repeat injections were required. In the present study, we produced a liquid form of EGCG-modified gelatin (EGCG-GL) and examined the properties of EGCG-GL with respect to prolonging EGCG release, NF-E2-related factor 2 (Nrf2) activation, osteoclastogenesis inhibition, bone destruction, and OTM. We found EGCG-GL both prolonged the release of EGCG and induced the expression of antioxidant enzyme genes, such as heme oxygenase 1 (Hmox1) and glutamate-cysteine ligase (Gclc), in the mouse macrophage cell line, RAW264.7. EGCG-GL attenuated intracellular reactive oxygen species (ROS) levels were induced by the receptor activator of nuclear factor-kB ligand (RANKL) and inhibited RANKL-mediated osteoclastogenesis in vitro. An animal model of bone destruction, induced by repeat Lipopolysaccharide (LPS)-injections into the calvaria of male BALB/c mice, revealed that a single injection of EGCG-GL on day-1 could successfully inhibit LPS-mediated bone destruction. Additionally, experimental OTM of maxillary first molars in male mice was attenuated by a single EGCG-GL injection on day-1. In conclusion, EGCG-GL prolongs the release of EGCG and inhibits osteoclastogenesis via the attenuation of intracellular ROS signaling through the increased expression of antioxidant enzymes. These results indicate EGCG-GL would be a beneficial therapeutic approach both in destructive bone disease and in controlling alveolar bone metabolism. [ABSTRACT FROM AUTHOR]

Photocatalysts have multiple applications in air purifiers, paints, and self-cleaning coatings for medical devices such as catheters, as well as in the elimination of xenobiotics. In this study, a coating of a UV-responsive photocatalyst, titanium dioxide (TiO2), was applied to an orthodontic resin. The antibacterial activity on oral bacteria as well as hydrophilic properties and mechanical properties of the TiO2-coated resin were investigated. ultraviolet A (UVA) (352 nm) light was used as the light source. Antibacterial activity was examined with or without irradiation. Measurements of early colonizers and cariogenic bacterial count, i.e., colony forming units (CFU), were performed after irradiation for different time durations. Hydrophilic properties were evaluated by water contact angle measurements. While, for the assessment of mechanical properties, flexural strength was measured by the three-point bending test. In the coat(+)light(+) samples the CFU were markedly decreased compared to the control samples. Water contact angle of the coat(+)light(+) samples was decreased after irradiation. The flexural strength of the specimen irradiated for long time showed a higher value than the required standard value, indicating that the effect of irradiation was weak. We suggest that coating with the ultraviolet responsive photocatalyst TiO2 is useful for the development of orthodontic resin with antimicrobial properties. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which is concerned with the case of an 83 year old man who was referred to an otorhinolaryngology clinic after complaining of an unusual strange sensation in his throat.