Preparation and Observation of Fresh-frozen Sections of the Green Fluorescent Protein Transgenic Mouse Head

Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections

Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer

Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC

Superconductivity of FeSe0.5Te0.5 Thin Films Grown by Pulsed Laser Deposition

FeSe0.5Te0.5 thin films with PbO-type structure are successfully grown on MgO(100) and LaSrAlO4(001) substrates from FeSe0.5Te0.5 or FeSe0.5Te0.75 polycrystalline targets by pulsed-laser deposition. The film deposited on the MgO substrate (film thickness ~ 55 nm) shows superconductivity at 10.6 K (onset) and 9.2 K (zero resistivity). On the other hand, the film deposited on the LaSrAlO4 substrate (film thickness ~ 250 nm) exhibits superconductivity at 5.4 K (onset) and 2.7 K (zero resistivity). This suggests the strong influence of substrate materials and/or the c-axis length to superconducting properties of FeSe0.5Te0.5 thin films.Comment: 24 pages, 5 figure

Hemorrhagic shock and encephalopathy syndrome – the markers for an early HSES diagnosis

<p>Abstract</p> <p>Background</p> <p>The hemorrhagic shock and encephalopathy syndrome (HSES) is a devastating disease that affects young children. The outcomes of HSES patients are often fatal or manifesting severe neurological sequelae. We reviewed the markers for an early diagnosis of HSES.</p> <p>Methods</p> <p>We examined the clinical, biological and radiological findings of 8 patients (4 months to 9 years old) who met the HSES criteria.</p> <p>Results</p> <p>Although cerebral edema, disseminated intravascular coagulopathy (DIC), and multiple organ failure were seen in all 8 cases during their clinical courses, brain computed tomography (CT) scans showed normal or only slight edema in 5 patients upon admission. All 8 patients had normal platelet counts, and none were in shock. However, they all had severe metabolic acidosis, which persisted even after 3 hours (median base excess (BE), -7.6 mmol/L). And at 6 hours after admission (BE, -5.7 mmol/L) they required mechanical ventilation. Within 12 hours after admission, fluid resuscitation and vasopressor infusion for hypotension was required. Seven of the patients had elevated liver enzymes and creatine kinase (CK) upon admission. Twenty-four hours after admission, all 8 patients needed vasopressor infusion to maintain blood pressure.</p> <p>Conclusion</p> <p>CT scan, platelet count, hemoglobin level and renal function upon admission are not useful for an early diagnosis of HSES. However, the elevated liver enzymes and CK upon admission, hypotension in the early stage after admission with refractory acid-base disturbance to fluid resuscitation and vasopressor infusion are useful markers for an early HSES diagnosis and helpful to indicate starting intensive neurological treatment.</p

Effects of ezetimibe on glucose metabolism in patients with type 2 diabetes: A 12-week, open-label, uncontrolled, pilot study

AbstractBackground: A potential effect of ezetimibe, a novel cholesterol-absorption inhibitor, on insulin resistance has been reported in an animal model.Objective: The aim of this study was to evaluate the effects of ezetimibe on glucose metabolism in patients with type 2 diabetes mellitus (T2DM).Methods: Between March and June 2008, outpatients with T2DM who were being treated at Yokohama Sakae Kyosai Hospital, Yokohama, Japan, were enrolled in this pilot study if they had not achieved the target lipid levels recommended by the Japan Atherosclerosis Society Guidelines despite diet and exercise or a statin therapy for ≥3 months. At baseline and at 4 and 12 weeks after open-label treatment with ezetimibe 10 mg/d, the levels of lipid parameters, fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c), and high-sensitivity C-reactive protein were measured. Adverse effects (AEs) were assessed at each study visit by patient interviews and laboratory testing.Results: A total of 21 consecutive patients (10 men, 11 women; mean [SD] age, 72 [9] years; weight, 63.4 [10.5] kg; body mass index, 25.5 [3.2] kg/m2) were enrolled in this study. The mean (SD) level of LDL-C decreased significantly from 146 (31) to 114 (27) mg/dL (−21%; P < 0.001) after 12 weeks of treatment with ezetimibe. The mean level of remnant-like particle cholesterol also decreased significantly from 6.5 (3.8) to 4.8 (2.2) mg/dL (−15%; P = 0.03). Treatment with ezetimibe was associated with a reduction in FPG level from 127 (31) to 119 (30) mg/dL (P = 0.02), and HbAlc from 6.3% (0.6%) to 6.1% (0.7%) (P = 0.003). No AEs were observed or reported during the study period.Conclusion: In this small, open-label, uncontrolled, pilot study, ezetimibe was associated with a significant decrease in lipid parameters and improvement in glucose metabolism in these patients with T2DM

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two beta-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS. (c) 2013 Elsevier B.V. All rights reserved

Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate

Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS-risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5 mm (~3.5 mm3) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4 Å resolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported

Evaluation of Transduction Properties of an Adenovirus Vector in Neonatal Mice

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of life via retro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants

Protonation State and Hydration of Bisphosphonate Bound to Farnesyl Pyrophosphate Synthase

Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to FPP and is known to be a molecular target of osteoporosis drugs, such as risedronate (RIS), which is a nitrogen-containing bisphosphonate. The protonation states and hydration structure of RIS bound to FPPS were determined by neutron protein crystallography, which allows direct visualization of hydrogens and deuteriums. The structure analysis revealed that the phosphate groups of RIS were fully deprotonated with the abnormally decreased pKa, and that the roles of E93 and D264 consisted of canceling the extra negative charges upon the binding of ligands. Collectively, our neutron structures provided insights into the physicochemical properties during the bisphosphonate binding event