Application of Biosheets as Right Ventricular Outflow Tract Repair Materials in a Rat Model.

Purposes: We report the experimental use of completely autologous biomaterials (Biosheets) made by "in-body tissue architecture" that could resolve problems in artificial materials and autologous pericardium. Here, Biosheets were implanted into full-thickness right ventricular outflow tract defects in a rat model. Their feasibility as a reparative material for cardiac defects was evaluated. Methods: As the evaluation of mechanical properties of the biosheets, the elastic moduli of the biosheets and RVOT-free walls of rats were examined using a tensile tester. Biosheets and expanded polytetrafluoroethylene sheet were used to repair transmural defects surgically created in the right ventricular outflow tracts of adult rat hearts (n = 9, each patch group). At 4 and 12 weeks after the operation, the hearts were resected and histologically examined. Results: The strength and elastic moduli of the biosheets were 421.3 ± 140.7 g and 2919 ± 728.9 kPa, respectively, which were significantly higher than those of the native RVOT-free walls (93.5 ± 26.2 g and 778.6 ± 137.7 kPa, respectively; P < 0.005 and P < 0.001, respectively). All patches were successfully implanted into the right ventricular outflow tract-free wall of rats. Dense fibrous adhesions to the sternum on the epicardial surface were also observed in 7 of 9 rats with ePTFE grafts, whereas 2 of 9 rats with biosheets. Histologically, the vascular-constructing cells were infiltrated into Biosheets. The luminal surfaces were completely endothelialized in all groups at each time point. There was also no accumulation of inflammatory cells. Conclusions: Biosheets can be formed easily and have sufficient strength and good biocompatibility as a patch for right ventricular outflow tract repair in rats. Therefore, Biosheet may be a suitable material for reconstructive surgery of the right ventricular outflow tract.

Case Report: Tricuspid Annuloplasty for Right-Sided Congestive Heart Failure Secondary to Pulmonary Hypertension in a Dog.

An 11-year-old, 12.3-kg, female Miniature Dachshund was presented to our institution with ascites of unknown etiology. The dog had been administered moxidectin for 3 years to treat a heartworm infection. Thoracic radiographs showed enlargement of the right heart. Echocardiography revealed right atrial and ventricular dilatation as well as flattening of the interventricular septum. Heartworm was identified in the main pulmonary artery, which was dilated. Tricuspid regurgitation (TR) was observed using color Doppler ultrasonography, and 2.5 L of ascites were removed. The dog was diagnosed with pulmonary hypertension, severe TR, and right-sided congestive heart failure. Except at the initial site, heartworm was not detected using echocardiography, and the antigen test was negative. However, pharmacological treatment did not improve the right-sided congestive heart failure. Instead, De Vega tricuspid annuloplasty (TAP) was performed on the beating heart under cardiopulmonary bypass with the owner's consent. Sutures terminated between the two commissures in the middle of the annulus and were secured using another pledget. Annular reduction was performed by tying down the plication suture while the cylindrical sizer was inserted into the tricuspid valve orifice. The size of the cylindrical sizer was 16 mm, which was set based on the height and width of the septal leaflet. A 6-month follow-up showed a reduction of TR and right-sided volume overload with no evidence of ascites retention/recurrence or any other complication. Our findings indicate that TAP may be a valid treatment option for dogs with right-sided congestive heart failure caused by secondary TR.

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

Modeling and Validation of Vertical Direction Force Estimation with a Three-Dimensional Force Measurement Instrument Based on a Zero-Compliance Mechanism.

The principle of a zero-compliance mechanism was used to develop a three-dimensional force measurement instrument. In each axis, the point of force is suspended by a zero-compliance mechanism. A vertical axis force estimation operation imitates the structure of a double series magnetic suspension system. An electromagnet directly controls the movement of the first suspended object (floator), which is denoted as a detection point, and indirectly controls the motion of the second floator, which is denoted as a point of force. Indirect control of the point of force is executed by the attractive force of a permanent magnet that is fixed to the bottom part of the detection point. To achieve zero-compliance, a Proportional-Integral-Derivative (PID) control is applied to the point of force, and to make the system stable, a Proportional-Derivative (PD) control is also applied to the detection point. In such suspension conditions, when force is exerted on the point of force, the displacement of the second floator is regulated to maintain its primary position while the detection point displaces in proportion to the applied force. Thus, a zero-compliance condition is maintained at the point of force, and the external force is measured from the linear displacement of the detection point. To restrict the motions of the detection point and the point of force in translation only, they are supported with leaf springs. This paper presents the modeling of the vertical direction force measurement operation of the developed three-axis force estimation instrument, and the theoretical analyses were validated by experiments of force measurement in both the millinewton and micronewton ranges.

Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.

Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.

The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing.

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.

Efficacy of pimobendan on survival and reoccurrence of pulmonary edema in canine congestive heart failure.

The aim of this study was to evaluate the efficacy of pimobendan with conventional therapies on survival and reocurrence of pulmonary edema in dogs with congestive heart failure (CHF) caused by myxomatous mitral valve disease (MMVD). Records of 197 client-owned dogs from 14 veterinary hospitals were included in this study. Dogs were administered conventional treatments with or without pimobendan. Sixty-four dogs received a standard dose of pimobendan (0.20-0.48 mg/kg every 12 hr (q12hr)), 49 dogs received a low dose of pimobendan (0.05-0.19 mg/kg q12hr), and 84 dogs received conventional therapy alone. Dogs in the standard-dose and low-dose pimobendan groups had significantly longer median survival times than dogs in the conventional group (334, 277 and 136 days, respectively; P<0.001). The reoccurrence rate of pulmonary edema in the standard-dose group was significantly lower than in the low-dose and conventional groups (43%, 59% and 62%, respectively; P<0.05). Combination of pimobendan with a conventional treatment regimen significantly prolonged survival time after an initial episode of pulmonary edema in dogs with CHF caused by MMVD. There was no difference in survival between dogs administered standard and low doses of pimobendan, but pimobendan did prevent the reoccurrence of pulmonary edema in a dose-dependent manner.

In-body tissue-engineered collagenous connective tissue membranes (BIOSHEETs) for potential corneal stromal substitution.

There is a severe shortage of donor cornea for transplantation in many countries. Collagenous connective tissue membranes, named BIOSHEETs, grown in vivo were successfully implanted in rabbit corneal stroma for in vivo evaluation of their suitability as a corneal stromal substitute to solve this global donor shortage. BIOSHEETs were prepared by embedding silicone moulds into dorsal subcutaneous pouches in rabbits for 1 month and stored in glycerol. After re-swelling in saline and trephining, disk-shaped BIOSHEETs (4 mm diameter) were allogeneically implanted into stromal pockets prepared in the right cornea of seven rabbits. Clinical tests for corneal thickness and transparency, and tissue analyses were performed. Because the BIOSHEETs (thickness, 131 ± 14 µm) obtained were opaque immediately after implantation, the transparency of the cornea decreased. The total thickness of the BIOSHEET-implanted cornea increased from 364 ± 21.0 µm to 726 ± 131 µm. After 4 weeks' implantation, the thickness of the cornea stabilized (493 ± 80 µm at 4 weeks and 447 ± 46 µm at 8 weeks). The transparency of the cornea increased progressively with time of implantation. The random orientation of collagen fibrils in the original BIOSHEETs tended to be homogeneous, similar to that of the native stroma. No inflammatory cells accumulated and fibroblast-like cells infiltrated the implant. The BIOSHEETs showed high biocompatibility with stromal tissues; however, further studies are needed to test its functional aspects. Although this research is only intended as a proof of concept, BIOSHEETs may be considered a feasible corneal stromal replacement, especially for treating visual impairment caused by stromal haze. Copyright © 2013 John Wiley & Sons, Ltd.

Electric-field-assisted formation of an interfacial double-donor molecule in silicon nano-transistors.

Control of coupling of dopant atoms in silicon nanostructures is a fundamental challenge for dopant-based applications. However, it is difficult to find systems of only a few dopants that can be directly addressed and, therefore, experimental demonstration has not yet been obtained. In this work, we identify pairs of donor atoms in the nano-channel of a silicon field-effect transistor and demonstrate merging of the donor-induced potential wells at the interface by applying vertical electric field. This system can be described as an interfacial double-donor molecule. Single-electron tunneling current is used to probe the modification of the potential well. When merging occurs at the interface, the gate capacitance of the potential well suddenly increases, leading to an abrupt shift of the tunneling current peak to lower gate voltages. This is due to the decrease of the system's charging energy, as confirmed by Coulomb blockade simulations. These results represent the first experimental observation of electric-field-assisted formation of an interfacial double-donor molecule, opening a pathway for designing functional devices using multiple coupled dopant atoms.

Tunneling in Systems of Coupled Dopant-Atoms in Silicon Nano-devices.

Following the rapid development of the electronics industry and technology, it is expected that future electronic devices will operate based on functional units at the level of electrically active molecules or even atoms. One pathway to observe and characterize such fundamental operation is to focus on identifying isolated or coupled dopants in nanoscale silicon transistors, the building blocks of present electronics. Here, we review some of the recent progress in the research along this direction, with a focus on devices fabricated with simple and CMOS-compatible-processing technology. We present results from a scanning probe method (Kelvin probe force microscopy) which show direct observation of dopant-induced potential modulations. We also discuss tunneling transport behavior based on the analysis of low-temperature I-V characteristics for devices representative for different regimes of doping concentration, i.e., different inter-dopant coupling strengths. This overview outlines the present status of the field, opening also directions toward practical implementation of dopant-atom devices.

The plant circadian clock looks like a traditional Japanese clock rather than a modern Western clock.

Life cycle adaptation to seasonal changes in photoperiod and ambient temperature is a major determinant of the ecological success behind the widespread domestication of flowering plants. The circadian clock plays a role in the underlying mechanism for adaptation through generating endogenous rhythms that allow plants to adapt and adjust to both the 24 h diurnal rotation and 365 d seasonal revolution. Nevertheless, the mechanism by which the circadian clock tracks seasonal changes in photoperiod and temperature is a longstanding subject in the field. Recently, we have begun to understand the question of how the light and ambient temperature signals feed into the circadian clock transcriptional circuitry in day-night cycles in order to track seasonal changes in photoperiod and ambient temperature. (1-4) Our results collectively indicate that the evening complex (EC) nighttime repressor consisting of LUX-ELF3-ELF4 plays a crucial role in this respect. Here, we discuss about these recent studies to add further implications.

Plasma cytokine levels in dogs undergoing cardiopulmonary bypass.

UNLABELLED: Currently, there are no reports of inflammatory responses to CPB in dogs. We investigated the time course of pro- and anti-inflammatory cytokine levels during and after CPB. ANIMALS: The study group included 11 dogs that underwent mitral valve repair with CPB, and the control group included 7 healthy dogs that underwent ovariohysterectomy. METHODS: Blood samples from the study group dogs were collected before, during and after surgery and analyzed for plasma levels of interleukin-6 (IL-6), tissue necrosis factor-α (TNF-α), interleukin-10 (IL-10), white blood cells (WBC), and C-reactive protein (CRP). Each inflammatory parameter was also compared with that of the control group dogs. RESULTS: After CPB, plasma levels of IL-6, WBC counts, and CRP levels were significantly higher than preoperative levels, and IL-6 levels in the study group were significantly higher than those in the control group. CONCLUSIONS: CPB induces a systemic inflammatory response in dogs.

Insight into the mechanism of end-of-day far-red light (EODFR)-induced shade avoidance responses in Arabidopsis thaliana.

Shade avoidance responses are changes in plant architecture to reduce the part of a body that is in the shade in natural habitats. The most common warning signal that induces shade avoidance responses is reduction of red/far-red light ratio perceived by phytochromes. A pair of basic helix-loop-helix transcription factors, named PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PIF5, is crucially involved in the shade avoidance-induced hypocotyl elongation in Arabidopsis thaliana. It has been recently reported that PIF7 also plays a role in this event. Here, we examined the involvement of these PIFs in end-of-day far-red light (EODFR) responses under light and dark cycle conditions. It was shown that PIF7 played a predominant role in the EODFR-dependent hypocotyl elongation. We propose the mechanism by which PIF7 together with PIF4 and PIF5 coordinately transcribes a set of downstream genes to promote elongation of hypocotyls in response to the EODFR treatment.

Insight into a Physiological Role for the EC Night-Time Repressor in the Arabidopsis Circadian Clock.

Life cycle adaptation to seasonal variation in photoperiod and temperature is a major determinant of ecological success of widespread domestication of Arabidopsis thaliana. The circadian clock plays a role in the underlying mechanism for adaptation. Nevertheless, the mechanism by which the circadian clock tracks seasonal changes in photoperiod and temperature is a longstanding subject of research in the field. We previously showed that a set of the target genes (i.e. GI, LNK1. PRR9 and PRR7) of the Evening Complex (EC) consisting of LUX-ELF3-ELF4 is synergistically induced in response to both warm-night and night-light signals. Here, we further show that the responses occur within a wide range of growth-compatible temperatures (16-28°C) in response to a small change in temperature (Δ4°C). A dim light pulse (<1 µmol m(-2) s(-1)) causes the enhanced effect on the transcription of EC targets. The night-light pulse antagonizes against a positive effect of the cool-night signal on the EC activity. The mechanism of double-checking external temperature and light signals through the EC nighttime repressor might enable plants to ignore (or tolerate) daily fluctuation of ambient temperature within a short time interval in their natural habitats. Taken together, the EC night-time repressor might play a physiological role in tracking seasonal variation in photoperiod and temperature by conservatively double-checking both the light and temperature conditions. Another EC target output gene PIF4 regulating plant morphologies is also regulated by both the temperature and light stimuli during the night. Hence, the EC night-time repressor is also implicated in a physiological output of the PIF4-mediated regulation of morphologies in response to seasonal variation in photoperiod and ambient temperature.

Successful implantation of autologous valved conduits with self-expanding stent (stent-biovalve) within the pulmonary artery in beagle dogs.

OBJECTIVES: To evaluate the functionality of an autologous heart valve with stent (Stent-biovalve or SBV) after implantation in the pulmonic valve position in beagle dogs. ANIMALS: Five beagle dogs. METHODS: A mold with an aperture of a tri-leaflet structure was constructed from a pair of concave and convex rods to which a nitinol (NiTi) stent was mounted. This mold was embedded in a dorsal subcutaneous pouch in beagle dogs for 4 weeks. At the time of the removal, the surfaces of the molds were completely covered with connective tissues, tri-leaflet valves were formed and the NiTi stent was tightly connected to the structure. RESULTS: The mean burst strength of the SBV leaflet was 2710 mmHg (range 2280-3116 mmHg), which was approximately equal to that of the native pulmonic valve leaflet. After implantation in the pulmonary position, the SBV showed good functionality as a pulmonic valve. At 84 days after implantation, the SBV was replaced with autologous fibroblasts and collagenous tissues, and showed organization similar to that of native heart valves. CONCLUSION: Stent-Biovalves achieved good valvular function with laminar flow in the pulmonic valve position of beagle dogs.

Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.

Implantation study of a tissue-engineered self-expanding aortic stent graft (bio stent graft) in a beagle model.

The use of stent grafts for endovascular aortic repair has become an important treatment option for aortic aneurysms requiring surgery. This treatment has achieved excellent outcomes; however, problems like type 1 endoleaks and stent graft migration remain. Bio stent grafts (BSGs), which are self-expanding stents covered with connective tissue, were previously developed using "in-body tissue architecture" technology. We assessed their early adaptation to the aorta after transcatheter implantation in a beagle model. BSGs were prepared by subcutaneous embedding of acryl rods mounted with self-expanding nitinol stents in three beagles for 4 weeks (n = 3/dog). The BSGs were implanted as allografts into infrarenal abdominal aortas via the femoral artery of three other beagles. After 1 month of implantation, aortography revealed no stenosis or aneurysmal changes. The luminal surface of the BSGs was completely covered with neointimal tissue, including endothelialization, without any thrombus formation. The cover tissue could fuse the luminal surface of the native aorta with tight conjunctions even at both ends of the stents, resulting in complete impregnation of the strut into the reconstructed vascular wall, which is expected to prevent endoleaks and migration in clinical applications.

Development of tissue-engineered self-expandable aortic stent grafts (Bio stent grafts) using in-body tissue architecture technology in beagles.

In this study, we aimed to describe the development of tissue-engineered self-expandable aortic stent grafts (Bio stent graft) using in-body tissue architecture technology in beagles and to determine its mechanical and histological properties. The preparation mold was assembled by insertion of an acryl rod (outer diameter, 8.6 mm; length, 40 mm) into a self-expanding nitinol stent (internal diameter, 9.0 mm; length, 35 mm). The molds (n = 6) were embedded into the subcutaneous pouches of three beagles for 4 weeks. After harvesting and removing each rod, the excessive fragile tissue connected around the molds was trimmed, and thus tubular autologous connective tissues with the stent were obtained for use as Bio stent grafts (outer diameter, approximately 9.3 mm in all molds). The stent strut was completely surrounded by the dense collagenous membrane (thickness, ∼150 µm). The Bio stent graft luminal surface was extremely flat and smooth. The graft wall of the Bio stent graft possessed an elastic modulus that was almost two times higher than that of the native beagle abdominal aorta. This Bio stent graft is expected to exhibit excellent biocompatibility after being implanted in the aorta, which may reduce the risk of type 1 endoleaks or migration.

The EC night-time repressor plays a crucial role in modulating circadian clock transcriptional circuitry by conservatively double-checking both warm-night and night-time-light signals in a synergistic manner in Arabidopsis thaliana.

During the last decade, significant research progress has been made in Arabidopsis thaliana in defining the molecular mechanisms behind the plant circadian clock. The circadian clock must have the ability to integrate both external light and ambient temperature signals into its transcriptional circuitry to regulate its function properly. We previously showed that transcription of a set of clock genes including LUX (LUX ARRHYTHMO), GI (GIGANTEA), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED GENE 1), PRR9 (PSEUDO-RESPONSE REGULATOR 9) and PRR7 is commonly regulated through the evening complex (EC) night-time repressor in response to both moderate changes in temperature (Δ6°C) and differences in steady-state growth-compatible temperature (16-28°C). Here, we further show that a night-time-light signal also feeds into the circadian clock transcriptional circuitry through the EC night-time repressor, so that the same set of EC target genes is up-regulated in response to a night-time-light pulse. This light-induced event is dependent on phytochromes, but not cryptochromes. Interestingly, both the warm-night and night-time-light signals negatively modulate the activity of the EC night-time repressor in a synergistic manner. In other words, an exponential burst of transcription of the EC target genes is observed only when these signals are simultaneously fed into the repressor. Taken together, we propose that the EC night-time repressor plays a crucial role in modulating the clock transcriptional circuitry to keep track properly of seasonal changes in photo- and thermal cycles by conservatively double-checking the external light and ambient temperature signals.

Transport spectroscopy of coupled donors in silicon nano-transistors.

The impact of dopant atoms in transistor functionality has significantly changed over the past few decades. In downscaled transistors, discrete dopants with uncontrolled positions and number induce fluctuations in device operation. On the other hand, by gaining access to tunneling through individual dopants, a new type of devices is developed: dopant-atom-based transistors. So far, most studies report transport through dopants randomly located in the channel. However, for practical applications, it is critical to control the location of the donors with simple techniques. Here, we fabricate silicon transistors with selectively nanoscale-doped channels using nano-lithography and thermal-diffusion doping processes. Coupled phosphorus donors form a quantum dot with the ground state split into a number of levels practically equal to the number of coupled donors, when the number of donors is small. Tunneling-transport spectroscopy reveals fine features which can be correlated with the different numbers of donors inside the quantum dot, as also suggested by first-principles simulation results.