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INTRODUCTION: We performed in vitro experiments using whole human blood without anticoagulants to clarify the activity of anticoagulant proteins on membranes coated with acrylate-copolymer (ACP) with a hydrophilic blood-contacting layer compared to those coated by immobilizing heparin (IHP) in extracorporeal circulation. METHODS: Whole human blood from healthy volunteers was recirculated in two types of experimental circuits with an ACP-coated reservoir and tubes and an ACP-coated or IHP-coated membrane. To compare the fluctuation of anticoagulant proteins, the circuit pressure at the inlet and outlet of the membrane was measured every 5 min; antithrombin antigen (ATQ), antithrombin activity, protein-C quantitation (PCQ), protein-C activity, protein-S free antigen (PSQ), and protein-S activity were measured at 0, 30, 60, 120, and 180 min in each experiment (n = 5). RESULTS: The time taken to achieve high circuit pressure (> 300 mmHg) at the inlet of the membrane was significantly shorter in the ACP-coated membrane circuit (28 ± 2.7 min) than in the IHP-coated membrane circuit (54 ± 24 min); however, the ATQ, PCQ, and PSQ at 180 min of recirculation were significantly higher in the former than in the latter (all p < .05). CONCLUSIONS: ACP-coated membranes can prevent the consumption of anticoagulant proteins but cannot delay circuit thrombogenicity compared to IHP-coated membranes. Considering patient care during the post-extracorporeal circulation period, the use of ACP coating, which can preserve anticoagulant protein, is better in extracorporeal circulation circuits.
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Anticoagulantes , Heparina , Humanos , Anticoagulantes/farmacología , Heparina/farmacología , Polímeros/farmacología , Circulación Extracorporea , AntitrombinasRESUMEN
INTRODUCTION: The membrane oxygenator in extracorporeal circulation circuits is coated with acrylate-copolymer (ACP) or immobilized heparin (IHP) to enhance hemocompatibility. To evaluate the relative features of both coatings, we compared blood components circulated in the circuits with ACP-and IHP-coated membranes in vitro using whole human blood. METHODS: Whole human blood was heparinized and circulated in two experimental circuits with an ACP-coated reservoir, tubes, and an ACP- or IHP-coated membrane. Platelet (PLT) counts and the amount of total protein (TP), complement component 3 (C3), and complement component 4 (C4) were measured at 0, 8, 16, 24, and 32 h in each experiment (n = 5). RESULTS: The PLT count at 0-h circulation was lower in the IHP-coated than in the ACP-coated circuits (p = 0.034); however, no significant difference was observed at other time points. Reduction in TP at 8-h and 16-h circulation and in C3 at 32-h circulation was lesser in the ACP-coated than in the IHP-coated circuits (p = 0.004, 0.034, and 0.027, respectively); reduction in TP and C3 at other time points and C4 at each time point was not significantly different. There were significant interactions between coating type and circulation duration in the PLT, TP, and C3 transitions (p = 0.008, 0.020, and 0.043, respectively). CONCLUSIONS: Our findings suggest that ACP-coated membranes can prevent the initial drop in PLT count and C3 consumption over 32 h, whereas IHP-coated membranes could not prevent this drop in extracorporeal circulation. Therefore, ACP-coated membranes are suitable for short- and long-term extracorporeal life support.
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INTRODUCTION: To understand the behavior of platelet volume indices and the von Willebrand factor (VWF), in vitro experiments using whole human blood were performed with extracorporeal circulation (ECC) circuits, including membrane oxygenators coated with acrylate copolymer (ACP) or immobilized heparin (IHP). METHODS: Heparinized blood was circulated through two distinct experimental circuits: an ACP-coated reservoir and tubes, as well as membranes coated with either ACP or IHP (comprising five pieces of each type). The platelet distribution width, mean platelet volume (MPV), platelet large cell ratio (P-LCR), VWF quantity (VWFQ), and VWF activity (VWFA) were measured at 0, 8, 16, 24, and 32 h in each experiment. A two-way analysis of variance (ANOVA) was performed to determine whether the coating type or circulation duration affected the transition of each measurement. RESULTS: Two-way ANOVA indicated that the transitions of MPV, P-LCR, and VWFA were significantly affected by the circulation duration (p = 0.030, 0.001, and <0.001, respectively) and that the transitions of VWFQ and VWFA were significantly affected by the coating type (p = 0.022 and 0.006, respectively). Factor interactions between the coating type and circulation duration were not observed for each transition (p > 0.05). CONCLUSIONS: Our findings suggest that P-LCR is a good index for platelet activation in blood-circulating ECC and that VWFA and VWFQ are significantly attenuated in blood-circulating ECC with ACP-coated membranes, indicating the advantage of IHP coating regarding platelet activation.
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Heparina , Oxigenadores de Membrana , Humanos , Factor de von Willebrand/metabolismo , Polímeros , Plaquetas/fisiologíaRESUMEN
To identify a gene responsible for the hooded phenotype in the rat, high-resolution linkage mapping for the hooded locus was performed using IS (non-hooded) and LEA (hooded) rats. The map revealed that only Kit gene existed in the critical region, suggesting that the Kit is a strong candidate gene. However, mutation was not found in the coding region of the LEA rat Kit gene. Further, the expressions of Kit mRNA were not different in fetal neural tubes and both neonatal and adult skins between IS and LEA rats. Furthermore, Kit-positive cells, possibly melanocytes, were found in the non-pigmented hair follicles of hooded phenotype rats. Several hypotheses are conceivable to account for mechanisms in the appearance of hooded phenotype.
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Mapeo Cromosómico , Ligamiento Genético/genética , Color del Cabello/genética , Pigmentación/genética , Ratas/genética , Animales , Regulación de la Expresión Génica , FenotipoRESUMEN
INTRODUCTION: A hemodialysis room has pipes connecting the console and central fluid equipment. While these pipes require disinfection, reports detailing disinfection strategies are unavailable. Therefore, we aimed to compare two easy disinfection strategies differing in sanitization frequency and sanitizer concentration. METHODS: Reverse osmosis water (ROW) purifying equipment and six dialysis consoles were connected by 20 m of pipes. Only ROW flowed through these pipes, because the dialysate solution was constituted at each console. The pipes were sanitized by two strategies: (1) strong and monthly (hypochlorite concentration: 100 ppm) or (2) weak and weekly (5 ppm). Both strategies were easy because the sodium hypochlorite was simply added to the ROW tank. To estimate sanitization efficacy, endotoxin counts at the ROW tank outlet, the end of the pipe, and the pipe after the endotoxin-cutting filter in each console were measured monthly for six continuous months. These counts were compared between the two sanitization strategies. RESULTS: The endotoxin counts at the ROW tank outlet and the end of the pipe were 0.004-0.017 and 0.012-0.081 EU/mL, respectively, in the strong and monthly strategy, and 0.001-0.003 and 0.001-0.005 EU/mL, respectively, in the weak and weekly strategy. The endotoxin counts at the pipe after the endotoxin-cutting filter were less than 0.001 EU/mL during the study period in both strategies. CONCLUSION: A weekly disinfection strategy was more effective than a monthly one, despite the lower hypochlorite concentration. The present study suggests that frequency is the most important factor in the disinfection of pipes in a dialysis room.
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Desinfección , Diálisis Renal , Soluciones para Diálisis , Endotoxinas , Contaminación de Equipos/prevención & control , FiltraciónRESUMEN
OBJECTIVE: Currently, the foreign surfaces of extracorporeal circulation devices are coated with an acrylate-based copolymer that creates a hydrophilic blood-contacting layer to enhance biocompatibility. Several reports of acrylate-based copolymer with respect to biocompatibility have been published; however, the adsorption of peptide compounds on acrylate-based copolymer-coated membranes still requires clarity. In this study, we aimed to understand the adsorption of several peptide compounds of various molecular weights, including albumin, lysozyme, and vancomycin, on acrylate-based copolymer-coated membranes using in vitro studies. METHODS: Six experimental circuits consisting of acrylate-based copolymer-coated tubes and membranes, and six comprising acrylate-based copolymer-coated tubes and non-coated membranes were prepared for comparison. An experimental solution, composed of albumin, lysozyme, vancomycin, and saline, was continuously stirred in a reservoir, recirculated in each experimental circuit, and then filtered. Concentrations of albumin, lysozyme, and vancomycin were measured after 0, 15, 30, 45, 60, 90, and 120 min of recirculation. Similar experiments were performed in all the prepared circuits. RESULTS: The ratio of measured values at each time point to those at 0 min was not significantly different between acrylate-based copolymer-coated and non-coated membranes for albumin and lysozyme, but differed significantly for vancomycin; the ratios were higher in acrylate-based copolymer-coated than in non-coated membranes. CONCLUSION: This study suggests that albumin is not adsorbed on either acrylate-based copolymer-coated or non-coated membranes, that lysozyme is not adsorbed on either membrane or is adsorbed at a similar rate on both membranes, and that vancomycin is less adsorbed on acrylate-based copolymer-coated membranes. Thus, acrylate-based copolymer coating could inhibit the adsorption of various peptide compounds.
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Circulación Extracorporea/métodos , Polímeros/química , Adsorción , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Propiedades de SuperficieRESUMEN
Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250 degrees C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130 degrees C for 60 min or at 140 degrees C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130 degrees C for 60 min or at 140 degrees C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments.
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Endotoxinas/antagonistas & inhibidores , Calor , Vapor , Endotoxinas/análisis , Prueba de Limulus/métodos , Factores de TiempoRESUMEN
OBJECTIVE:: Extracorporeal circulation devices are coated with a biocompatible polymer coating agent (BPCA) that has a hydrophilic blood-contacting layer, but hemofilters are not. We aimed to investigate the antithrombotic properties of a BPCA-coated hemofilter. METHODS:: Four experiments using BPCA-coated circuits and non-coated hemofilters and four experiments using BPCA-coated circuits and BPCA-coated hemofilters were performed with whole human blood and compared by measuring the circuit pressure every 5 min, antithrombin activity every 40 min, and thrombin-antithrombin complex every 40 min, for a total of 240 min of recirculation. RESULTS:: The mean time required for the pressure at the inlet of the hemofilter to increase sharply was longer in BPCA-coated than in non-coated hemofilters (66 ± 11 min vs 25 ± 9 min, p < 0.01). The mean antithrombin activity value at 200 and 240 min of recirculation was significantly higher in the experiments with BPCA-coated versus non-coated hemofilters (43.3 ± 2.87 vs 33.3 ± 5.74, p = 0.04; 42.8 ± 3.59 vs 31.0 ± 5.35, p = 0.01, respectively); the antithrombin activity values at the other time points were not significantly different. Furthermore, all thrombin-antithrombin complex values in experiments with the BPCA-coated hemofilters achieved overrange at 80 min of recirculation, whereas those with the non-coated hemofilter achieved overrange at 40 min. CONCLUSION:: This study suggests that BPCA-coated hemofilters can inhibit antithrombin consumption, contributing to antithrombotic effects in extracorporeal circulation circuits.
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Acrilatos , Coagulación Sanguínea/fisiología , Materiales Biocompatibles Revestidos , Hemofiltración/instrumentación , Interacciones Hidrofóbicas e Hidrofílicas , Polímeros , Trombosis/prevención & control , Antitrombina III , Antitrombinas , Pruebas de Coagulación Sanguínea , Humanos , Técnicas In Vitro , Péptido Hidrolasas , SulfonasRESUMEN
The growth-retarded (grt) mouse has an autosomal recessive, fetal-onset, severe thyroid hypoplasia related to TSH hyporesponsiveness. Through genetic mapping and complementation experiments, we show that grt is a missense mutation of a highly conserved region of the tyrosylprotein sulfotransferase 2 (Tpst2) gene, encoding one of the two Tpst genes implicated in posttranslational tyrosine O-sulfation. We present evidence that the grt mutation leads to a loss of TPST2 activity, and TPST2 isoform has a high degree of substrate preference for TSH receptor (TSHR). The expression of TPST2 can restore TSH-TSHR-mediated cAMP production in fibroblasts derived from grt mice. Therefore, we propose that the tyrosine sulfation of TSHR by TPST2 is crucial for TSH signaling and resultant thyroid gland function.
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Hipotiroidismo Congénito/enzimología , Hipotiroidismo Congénito/genética , Enanismo/enzimología , Enanismo/genética , Mutación Missense , Sulfotransferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Hipotiroidismo Congénito/complicaciones , Cartilla de ADN/genética , Enanismo/complicaciones , Femenino , Prueba de Complementación Genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Datos de Secuencia Molecular , Fenotipo , Procesamiento Proteico-Postraduccional , Receptores de Tirotropina/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad por Sustrato , Sulfotransferasas/deficiencia , Sulfotransferasas/metabolismo , Tirotropina/metabolismoRESUMEN
PURPOSE: Currently, the foreign surfaces of various extracorporeal circulation devices are coated with a biocompatible polymer coating agent (BPA), which creates a hydrophilic blood-contacting layer to reduce thrombogenicity, while the membranes in hemodialyzers are not. We aimed to clarify other side effects of BPA-coated membranes by examining the diffusion performance in in vitro experiments. METHODS: We used a polyethersulfone membrane (sieving coefficient of albumin is ≤0.01) coated with BPA product, SEC-1TM (Toyobo), in a hemodialyzer. To estimate the diffusion rates of a wide range of molecules, 2 L of saline containing vancomycin, lysozyme, and albumin were recirculated in the circuit configured with a hemodialyzer, and dialyzed continuously using water. The concentrations of sodium, vancomycin, lysozyme, and albumin were measured every 5 minutes for 30 minutes and compared in experiments with BPA-coated (n = 4) and BPA-noncoated (n = 4) membranes. RESULTS: The removal rates of sodium and vancomycin after 5 minutes of dialysis (n = 24) were significantly higher in BPA-coated than noncoated membranes, while those of lysozyme and albumin were not significantly different. The removal rates of sodium and vancomycin after 30 minutes of dialysis (n = 4) were significantly higher, and those of lysozyme were significantly lower in BPA-coated than noncoated membranes, while those of albumin were not significantly different. CONCLUSIONS: The preliminary study suggests that BPA-coated membranes enhanced the diffusion rate of molecules with low and middle molecular weight without affecting the sieving coefficient of albumin. Thus, BPA coating can enhance the dialysis performance of membranes.
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Circulación Extracorporea , Riñones Artificiales , Membranas Artificiales , Polímeros/farmacología , Diálisis Renal/instrumentación , Sulfonas/farmacología , Materiales Biocompatibles Revestidos/farmacología , Difusión/efectos de los fármacos , Circulación Extracorporea/instrumentación , Circulación Extracorporea/métodos , Humanos , Plásticos/farmacologíaRESUMEN
Mx is an interferon (IFN)-induced protein that shows antiviral activities against RNA viruses. We examined an expression of mRNA, an intracellular localization of protein, and an antiviral property of canine Mx1 and Mx2. Both Mx1 and Mx2 mRNAs were induced in a canine kidney cell line Madin-Darby canine kidney (MDCK), stimulated with an IFN-inducer, poly(I) x poly(C) for 12 h, suggesting the presence of regulatory mechanisms consistent with Mx genes in other species. By immunostaining BALB/3T3 fibroblasts transiently transfected FLAG epitope-tagged canine Mx1 and Mx2 cDNAs with an anti-FLAG tag, it was revealed that both Mx1 and Mx2 proteins are localized in cytoplasm. BALB/3T3 fibroblasts expressing stably Mx2 but not Mx1 had an antiviral activity against recombinant vesicular stomatitis virus (VSV). This is the first report demonstrating the functional analysis of canine Mx proteins.
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Proteínas de Unión al GTP/fisiología , Animales , Antivirales/metabolismo , Células 3T3 BALB , Secuencia de Bases , Línea Celular , Efecto Citopatogénico Viral , Citoplasma/metabolismo , ADN Complementario/genética , Perros , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Inductores de Interferón/farmacología , Ratones , Proteínas de Resistencia a Mixovirus , Poli I-C/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/metabolismo , Transfección , Virus de la Estomatitis Vesicular Indiana/patogenicidadRESUMEN
The CKH-Jsr (jumbled spine and ribs) mouse was found as a spontaneous mutant with malformation of vertebrae, that is, a short trunk and kinky tail. We examined Lunatic Fringe (Lfng) and Uncx4.1 expression in the presomitic mesoderm (PSM) and somites of Jsr-mutant (CKH-Jsr/+) embryos to elucidate pathogenesis of the Jsr mutation. Expression pattern of Lfng in the PSM of Jsr-mutant embryos was similar to that of the normal (C57BL/6) embryos. However, expression pattern of Uncx4.1 in the somites of Jsr-mutant embryos was impaired to be irregular and mosaic, suggesting that the anterior-posterior (A-P) polarity is disordered in the Jsr mutant. These results indicate that the Jsr mutation disrupts the A-P polarity of somites during the somitogenesis without altering Lfng expression pattern in the PSM.
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Regulación del Desarrollo de la Expresión Génica/fisiología , Glicosiltransferasas/biosíntesis , Proteínas de Homeodominio/biosíntesis , Ratones Mutantes/embriología , Animales , Tipificación del Cuerpo , Hibridación in Situ , Ratones , Ratones Endogámicos/embriología , Mutación , Somitos/metabolismoRESUMEN
Ribonuclease (RNase), which often represents molecular biological contamination, is a thermostable enzyme. When RNase is heated at 121 degrees C by autoclave sterilization for 20 min, it does not lose its activity. However, the nature of the molecular events by which the irreversible denaturation occurs remains unknown. The purpose of this study was to elucidate the molecular mechanisms of irreversible thermal denaturation of RNase A and to develop an advanced sterilization method using soft-hydrothermal processing, which has the advantages of improved safety and cost-efficiency. The enzymatic activity of RNase was measured using polyacrylamide gel electrophoresis with torula yeast RNA. We evaluated the temperature and time course of irreversible thermoinactivation of RNase by normal autoclaving, hot-air sterilization, and soft-hydrothermal processing that had been controlled to the desired steam saturation ratio. The results indicated that RNase A was deactivated by autoclave sterilization (121 degrees C, 20 min) immediately after treatment, but was reactivated over time. Hot-air sterilization (180 degrees C, atmospheric pressure, 60 min) produced results similar to that of autoclave sterilization. In contrast, RNase A was irreversibly thermoinactivated by soft-hydrothermal processing (110 degrees C, 20 min) at 100% steam saturation ratio. We also determined that the mechanism of irreversible thermoinactivation of RNase A involved hydrolysis and deamidation under this condition at a steam saturation ratio of more than 100%.
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Ribonucleasa Pancreática/química , Análisis de Varianza , Animales , Bovinos , Desaminación , Contaminación de Medicamentos , Activación Enzimática , Reactivadores Enzimáticos , Estabilidad de Enzimas , Hidrólisis , Presión , Desnaturalización Proteica , Ribonucleasa Pancreática/metabolismo , Vapor , TemperaturaRESUMEN
Animal facilities generate a large amount of used bedding containing excrement as medical waste. We developed a recycling system for used bedding that involves soft hydrothermal processing. In this study, we examined the effects of bedding type on growth, hematologic and serum biochemical values, and organ weights of female and male mice reared on either recycled or fresh bedding from 3 to 33 wk of age. Neither growth nor physiology differed between mice housed on recycled bedding compared with fresh bedding. When 14-wk-old mice were bred, litter size and total number of weaned pups showed no significant differences between animals raised on recycled or fresh bedding. Because bedding type influences the environment within cages and animal rooms, we evaluated particulate and ammonia data from cages and animal rooms. Values were significantly lower from cages and rooms that used recycled bedding than from those using fresh bedding, thus indicating that recycled bedding has the potential to improve the environment within both cages and animal rooms. Overall, this study revealed that recycled bedding is an excellent material for use in housing laboratory rodents. Specifically, recycled bedding may reduce medical waste and maintain healthy environments within cages and animal rooms.
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Bienestar del Animal , Conservación de los Recursos Naturales , Vivienda para Animales/tendencias , Ratones , Animales , Femenino , Masculino , Ratones/crecimiento & desarrollo , Ratones/fisiología , Ratones Endogámicos ICR , Reproducción/fisiología , MaderaRESUMEN
We investigate the structural, electronic, and transport properties of mechanically deformed Mo6S6 nanowires using a density-functional based tight binding method extended with a Green's functions formalism. We present two interesting results: first, the properties of the wire are not affected by bending, and second, a metal-insulator transition occurs when the wire is twisted. This indicates that molybdenum sulfide nanowires can be used as a nanocable to flexibly transfer information between electromechanical switches, which can be also constructed from the same wires. Hence, our results suggest the Mo6S6 nanowires as unique building blocks for future nanodevices.
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We propose to synthesize a new class of single-walled nanotubular compounds (SWNCs) and investigate the interplay between their structural and electronic properties using ab initio density functional calculations. SWNCs are composed of cyclacenes of variable diameter interconnected by various linker compounds. Cyclacenes map directly onto and can be viewed as the shortest segments of (n,0) zigzag carbon nanotubes. We focus on cyclacenes with n=6-12 fused benzene rings interconnected by biphenyl, tetrazine, or acetylene linkers. Depending upon the nature and the orientation of the linkers, we find it possible to change the systems from narrow-gap to wide-gap semiconductors, and to modulate the band dispersion, suggesting the possibility of band gap engineering.
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We investigate the equilibrium geometry and electronic structure of Mo12S9I9 nanowires using ab initio density functional calculations. The skeleton of these unusually stable nanowires consists of rigid, functionalized Mo octahedra, connected by flexible, bistable sulfur bridges. This structural flexibility translates into a capability to stretch up to approximately 20% at almost no energy cost. The nanowires change from conductors to narrow-gap magnetic semiconductors in one of their structural isomers.
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The jumbled spine and ribs (Jsr) mouse was derived from a spontaneous mutation. As the phenotype, a shortened trunk and kinky tail are characteristic Jsr traits. In this study, on high resolution mapping it was found that Lunatic fringe (Lfng) mapped at the same position as Jsr. Lfng was identified as the candidate gene for Jsr, but sequence analysis of this gene revealed no substitution in the coding region of cDNA. Therefore, we adopted the strategy of positional cloning for Jsr using a mouse bacterial artificial chromosome (BAC) library. A BAC contig was constructed from three BAC clones showing positive signals of Lfng and 11MMHAP75FRD8.seq near the Jsr locus on chromosome 5. Based on the genetic mapping of both T7 and sp6 ends of a clone of BAC382-O-7 (BAC382), the Jsr gene was considered to exist in BAC382 and to be positioned near the sp6 side.