Structures of the wild-type MexAB-OprM tripartite pump reveal its complex formation and drug efflux mechanism.

In Pseudomonas aeruginosa, MexAB-OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB-OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available. In this study, we present the structure of wild-type MexAB-OprM in the presence or absence of drugs at near-atomic resolution. The structure reveals that OprM does not interact with MexB directly, and that it opens its periplasmic gate by forming a complex. Furthermore, we confirm the residues essential for complex formation and observed a movement of the drug entrance gate. Based on these results, we propose mechanisms for complex formation and drug efflux.

Functional ectodermal organ regeneration as the next generation of organ replacement therapy.

In this decade, substantial progress in the fields of developmental biology and stem cell biology has ushered in a new era for three-dimensional organ regenerative therapy. The emergence of novel three-dimensional cell manipulation technologies enables the effective mimicking of embryonic organ germ formation using the fate-determined organ-inductive potential of epithelial and mesenchymal stem cells. This advance shows great potential for the regeneration of functional organs with substitution of complete original function in situ. Organoids generated from multipotent stem cells or tissue stem cells via establishment of an organ-forming field can only partially recover original organ function owing to the size limitation; they are considered 'mini-organs'. Nevertheless, they hold great promise to realize regenerative medicine. In particular, regeneration of a functional salivary gland and an integumentary organ system by orthotopic and heterotopic implantation of organoids clearly points to the future direction of organ regeneration research. In this review, we describe multiple strategies and recent progress in regenerating functional three-dimensional organs, focusing on ectodermal organs, and discuss their potential and future directions to achieve organ replacement therapy as a next-generation regenerative medicine.

SMART pass will prevent inappropriate operation of S-ICD.

BACKGROUND: Compared to screening ECG before implantation of a subcutaneous implantable cardioverter-defibrillator (S-ICD), selectable vectors without T-wave oversensing increase after S-ICD implantation. Newer algorithms have recently become available to reduce T-wave oversensing, such as SMART pass (SP). With this function, more selectable vectors are identified after S-ICD implantation. However, this improvement in eligibility utilizing SP has not yet been well validated. We aimed to clarify S-ICD eligibility before and after S-ICD implantation with and without SP. METHODS: Participants comprised 34 patients implanted with an S-ICD at Okayama University Hospital and its affiliated hospitals between February 2016 and August 2017. A total of 102 S-ICD vectors were assessed for eligibility before and after S-ICD implantation, at rest and during exercise testing. Vector availability was evaluated in the presence and absence of SP after S-ICD implantation. RESULTS: Subcutaneous implantable cardioverter-defibrillator eligibility was significantly better after implantation even without SP than S-ICD screening before S-ICD implantation, both at rest (before 65.7% vs after 95.1%, P < 0.01) and during exercise (before 59.3% vs after 90.6%, P < 0.01). SP improved S-ICD eligibility during exercise (SP on 97.9% vs off 90.6%, P = 0.03). Multivariate analysis showed the prevalence of S-ICD eligibility increased significantly after S-ICD implantation compared to screening before implantation. SP further increased selectable vectors in multivariate analysis. CONCLUSION: Available vectors increased significantly after S-ICD implantation compared to preoperative vectors as assessed by S-ICD screening ECG. T-wave oversensing during exercise has been an unresolved issue for S-ICD, but SP will help prevent inappropriate operation with S-ICD.

Insulin-like growth factor 1 modulates bioengineered tooth morphogenesis.

Regenerative therapy to replace missing teeth is a critical area of research. Functional bioengineered teeth have been produced by the organ germ method using mouse tooth germ cells. However, these bioengineered teeth are significantly smaller in size and exhibit an abnormal crown shape when compared with natural teeth. The proper sizes and shapes of teeth contribute to their normal function. Therefore, a method is needed to control the morphology of bioengineered teeth. Here, we investigated whether insulin-like growth factor 1 (IGF1) can regulate the sizes and shapes of bioengineered teeth, and assessed underlying mechanisms of such regulation. IGF1 treatment significantly increased the size of bioengineered tooth germs, while preserving normal tooth histology. IGF1-treated bioengineered teeth, which were developed from bioengineered tooth germs in subrenal capsules and jawbones, showed increased sizes and cusp numbers. IGF1 increased the number of fibroblast growth factor (Fgf4)-expressing enamel knots in bioengineered tooth germs and enhanced the proliferation and differentiation of dental epithelial and mesenchymal cells. This study is the first to reveal that IGF1 increases the sizes and cusp numbers of bioengineered teeth via the induction of enamel knot formation, as well as the proliferation and differentiation of dental epithelial and mesenchymal cells.

Biosafety, stability, and osteogenic activity of novel implants made of Zr70Ni16Cu6Al8 bulk metallic glass for biomedical application.

Superior mechanical and chemical properties of Zr70Ni16Cu6Al8 bulk metallic glass (BMG) demonstrate its promise as a novel biomaterial for fabrication of implants. The aim of the present study was to validate mechanical, chemical, and biological properties of Zr70Ni16Cu6Al8 BMG through comparison with titanium (Ti). Our data indicated higher tensile strength, lower Young's modulus, and reduced metal ion release of Zr70Ni16Cu6Al8 BMG compared with Ti. Biosafety of bone marrow mesenchymal cells on Zr70Ni16Cu6Al8 BMG was comparable to that of Ti. Next, screw-type implant prototypes made of Zr70Ni16Cu6Al8 BMG were fabricated and inserted into rat long bones. Zr70Ni16Cu6Al8 BMG implants indicated a higher removal-torque value and lower Periotest value compared with Ti implants. In addition, higher amounts of new bone formation and osseointegration were observed around Zr70Ni16Cu6Al8 BMG implants compared with Ti implants. Moreover, gene expression analysis displayed higher expression of osteoblast- and osteoclast-associated genes in the Zr70Ni16Cu6Al8 BMG group compared with the Ti group. Importantly, loading to implants upregulated bone formation, as well as osteoblast- and osteoclast-associated gene expression in the peri-implant area. No significant difference in concentrations of Ni, Al, Cu, and Zr in various organs was shown between in the Zr70Ni16Cu6Al8 BMG and Ti groups. Collectively, these findings suggest that Zr70Ni16Cu6Al8 BMG is suitable for fabricating novel implants with superior mechanical properties, biocompatibility, stability, and biosafety compared with Ti. STATEMENT OF SIGNIFICANCE: Titanium is widely used to fabricate orthopedic and dental implants. However, Titanium has disadvantages for biomedical applications in regard to strength, elasticity, and biosafety. Recently, we developed a novel hypoeutectic Zr70Ni16Cu6Al8 BMG, which has superior mechanical and chemical properties. However, the validity of Zr70Ni16Cu6Al8 BMG for biomedical application has not been cleared. The aim of the present study was to validate the mechanical, chemical, and biological properties of Zr70Ni16Cu6Al8 BMG for biomedical applications through comparison with Titanium. The present study clarifies that Zr70Ni16Cu6Al8 BMG has good mechanical properties, corrosion resistance, and osteogenic activity, which are necessary features for biomedical applications. The present study provides for the first time the superiority of Zr70Ni16Cu6Al8 BMG implants to Titanium implants for biomedical applications.

The impact of the CartoSound® image directly acquired from the left atrium for integration in atrial fibrillation ablation.

PURPOSE: Intracardiac echocardiographic (ICE) imaging might be useful for integrating three-dimensional computed tomographic (CT) images for left atrial (LA) catheter navigation during atrial fibrillation (AF) ablation. However, the optimal CT image integration method using ICE has not been established. METHODS: This study included 52 AF patients who underwent successful circumferential pulmonary vein isolation (CPVI). In all patients, CT image integration was performed after the CPVI with the following two methods: (1) using ICE images of the LA derived from the right atrium and right ventricular outflow tract (RA-merge) and (2) using ICE images of the LA directly derived from the LA added to the image for the RA-merge (LA-merge). The accuracy of these two methods was assessed by the distances between the integrated CT image and ICE image (ICE-to-CT distance), and between the CT image and actual ablated sites for the CPVI (CT-to-ABL distance). RESULTS: The mean ICE-to-CT distance was comparable between the two methods (RA-merge = 1.6 ± 0.5 mm, LA-merge = 1.7 ± 0.4 mm; p = 0.33). However, the mean CT-to-ABL distance was shorter for the LA-merge (2.1 ± 0.6 mm) than RA-merge (2.5 ± 0.8 mm; p < 0.01). The LA, especially the left-sided PVs and LA roof, was more sharply delineated by direct LA imaging, and whereas the greatest CT-to-ABL distance was observed at the roof portion of the left superior PV (3.7 ± 2.8 mm) after the RA-merge, it improved to 2.6 ± 1.9 mm after the LA-merge (p < 0.01). CONCLUSIONS: Additional ICE images of the LA directly acquired from the LA might lead to a greater accuracy of the CT image integration for the CVPI.

Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: an importance of the N-terminal domain for an allosteric regulatory property.

Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.

A potential pitfall of the modified 12 lead electrocardiogram (Mason-Likar modification) in catheter ablation of idiopathic ventricular arrhythmias originating from the outflow tract.

AIMS: The Mason-Likar modified electrocardiogram (ML-ECG) can be interchanged with standard 12 lead ECG electrode positions (standard ECG) without affecting the diagnostic interpretation during sinus rhythm, but the morphological differences during ventricular arrhythmias have not been sufficiently evaluated. This study aimed to elucidate the morphological changes in the ML-ECG precordial leads. METHODS AND RESULTS: In 53 consecutive patients with premature ventricular contractions predicted to originate from the outflow tract (OT-PVCs), the arrhythmias were analysed by those two ECG methods. The OT-PVC origin sites, which were predicted by currently published criteria with the respective ECG methods prior to catheter ablation, were compared with the successful ablation sites. Compared with the standard-ECG, S-waves in the ML-ECG became shallower in leads V1-4 (P < 0.05 in lead V1; P < 0.001 in leads V2-4), and pseudo-R-waves in lead V1 appeared in seven patients. The precordial leads transition zone shifted counter-clockwise in 18 patients in the ML-ECG. In leads I and aVL, the negative deflection amplitudes of the ML-ECG were greater than those of the standard ECG (P < 0.001), and polarity reversals in lead I appeared in 18 patients. The R-wave amplitudes in all ML-ECG inferior leads were greater than those in the standard-ECG leads (all for P < 0.001). Those changes had an effect on the diagnostic indexes for the localization, and the specificity of the criteria for the ML-ECG was poorer than that for the standard-ECG. CONCLUSION: Great differences were found between those two ECG methods. Predicting OT-PVC origins by diagnostic criteria with the ML-ECG might result in a misdiagnosis and inefficient ablation.

Functional tooth regeneration using a bioengineered tooth unit as a mature organ replacement regenerative therapy.

Donor organ transplantation is currently an essential therapeutic approach to the replacement of a dysfunctional organ as a result of disease, injury or aging in vivo. Recent progress in the area of regenerative therapy has the potential to lead to bioengineered mature organ replacement in the future. In this proof of concept study, we here report a further development in this regard in which a bioengineered tooth unit comprising mature tooth, periodontal ligament and alveolar bone, was successfully transplanted into a properly-sized bony hole in the alveolar bone through bone integration by recipient bone remodeling in a murine transplantation model system. The bioengineered tooth unit restored enough the alveolar bone in a vertical direction into an extensive bone defect of murine lower jaw. Engrafted bioengineered tooth displayed physiological tooth functions such as mastication, periodontal ligament function for bone remodeling and responsiveness to noxious stimulations. This study thus represents a substantial advance and demonstrates the real potential for bioengineered mature organ replacement as a next generation regenerative therapy.

Hard tissue-forming potential of stem/progenitor cells in human dental follicle and dental papilla.

OBJECTIVE: The existence of stem/progenitor cells in dental tissue has been suggested but their characterization in the human tooth germ remains elusive. The purpose of this study was to investigate these cells in human dental follicles and dental papillae at the crown-forming stage and compare their potential for hard tissue formation. DESIGN: We used dental follicle cells (DFCs) and dental papilla cells (DPCs) derived from dental follicles and dental papillae at the crown-forming stage and compared their proliferative capacity, cell surface antigens and ability to form hard tissue in vitro and in vivo. RESULTS: Both DFCs and DPCs had extensive proliferation ability, expressed similar cell surface antigens and were capable of forming hard tissue in vivo as well as in vitro. However, there were two differences between DFCs and DPCs. First, DPCs had a significantly higher calcium accumulation than that in DFCs. Second, DFCs expressed a cementoblast marker, whereas DPCs expressed an odontoblast marker. CONCLUSIONS: We propose that dental follicles and dental papillae at the crown-forming stage contain different types of stem/progenitor cells and may have hard tissue-forming ability in a possibly origin-specific lineage direction.

Fully functional bioengineered tooth replacement as an organ replacement therapy.

Current approaches to the development of regenerative therapies have been influenced by our understanding of embryonic development, stem cell biology, and tissue engineering technology. The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs which work in cooperation with surrounding tissues to replace organs that were lost or damaged as a result of disease, injury, or aging. Here, we report a successful fully functioning tooth replacement in an adult mouse achieved through the transplantation of bioengineered tooth germ into the alveolar bone in the lost tooth region. We propose this technology as a model for future organ replacement therapies. The bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication, and response to noxious stimulations such as mechanical stress and pain in cooperation with other oral and maxillofacial tissues. This study represents a substantial advance and emphasizes the potential for bioengineered organ replacement in future regenerative therapies.

Morphologically unique feature of recurrent ampulla (takotsubo) cardiomyopathy.

Two similar rare cases of recurrent ampulla (takotsubo) cardiomyopathy, which was induced by physical stress of recurrent rhabdomyolysis in case 1 and aggravation of respiratory disease in case 2, are presented. At the initial admission, both patients had typical ampulla cardiomyopathy, which was indicated by transient left ventricular (LV) apical ballooning, but at the second admission, they both had atypical ampulla cardiomyopathy, as diagnosed by transient basal midventricular ballooning. Electrocardiograms at each admission showed a specific T-wave inversion, which might indicate the region of LV asynergy, and prolongation of the QT interval. In both cases, the plasma level of endogenous catecholamines was high. It is possible that excessive sympathetic stimulation induced by physical stress was the cause of this cardiomyopathy, but the cause of the differences in wall motion abnormalities between the first and second admissions was not identified. Appropriate management and treatment of the underlying disease and determining the mechanisms of recurrent ampulla cardiomyopathy might prevent its recurrence.

Additional effects of bosentan in patients with idiopathic pulmonary arterial hypertension already treated with high-dose epoprostenol.

BACKGROUND: Combination therapy has been proposed in treatment algorithms for idiopathic pulmonary arterial hypertension (IPAH), so the additional effects of bosentan in IPAH patients already treated with high-dose epoprostenol (EPO) was evaluated in the present study. METHODS AND RESULTS: Bosentan (62.5 mg twice daily) was administered to 8 IPAH patients already being treated with high-dose EPO (average dose 99.6+/-43.4 ng . kg(-1) . min(-1)). Hemodynamics were assessed at baseline and at 2 days and then 1 year after the initiation of bosentan. Because a remarkable elevation of mixed venous oxygen saturation was observed at the initiation of bosentan, the dosage of EPO was reduced in 7 patients (from 99.6+/-43.4 to 82.8+/-31.3 ng . kg(-1) . min(-1), p<0.05). There was a significant decrease from the baseline value for systolic pulmonary artery pressure (80.1+/-19.3 to 66.8+/-16.5 mmHg, p<0.05). These effects were maintained for 1 year without progression of PAH in 6 patients whose condition had been stabilized at baseline. CONCLUSIONS: The additional use of bosentan for IPAH patients whose condition has been stabilized by high-dose EPO is safe and effective.

Growing bioengineered teeth from single cells: potential for dental regenerative medicine.

BACKGROUND: The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs that can replace organs lost or damaged due to disease, injury or aging. Dental regenerative medicine has made the most progress and is the most useful model for the consideration of strategies in future organ replacement therapies. OBJECTIVE: This review describes strategies that have been pursued to date and experiments currently being conducted to bioengineer teeth in anticipation of the production of fully functional organs. METHODS: To realize the practical application of 'bioengineered tooth' transplantation therapy, four major hurdles must be overcome. The present status of the hurdles to this therapy are described and discussed in this review. RESULTS/CONCLUSION: The bioengineering techniques developed for tooth regeneration will in the future make substantial contributions to the ability to grow primordial organs in vitro and also to grow fully functioning organs, such as the liver, kidney and heart.

[Application of mesenchymal stem cells to liver regenerative medicine].

Stem cell-based therapy has received attention as a possible alternative to organ transplantation, owing to the ability of stem cells to repopulate and differentiate at the engrafted site. We transplanted bone marrow-derived mesenchymal stem cells (BMSCs) into liver-injured rats to test the therapeutic effect. Rat bone marrow cells were cultured in the presence of hepatocyte growth factor (HGF). RT-PCR and immunocytochemical analysis indicated that the BMSCs expressed the albumin mRNA and the production of protein after cultivation with HGF for 2 weeks. The BMSCs appeared to differentiate into hepatocyte-like cells in response to the culture with HGF. After labeling with a fluorescent marker, the BMSCs were transplanted into CCl(4)-injured rats by injection through the caudal vein. The liver was excised and blood samples were collected 4 weeks later. Engraftment of the transplanted BMSCs was seen with significant fluorescence in the injured liver. Transplantation of the BMSCs into liver-injured rats restored their serum albumin level and suppressed transaminase activity and liver fibrosis. Therefore, BMSCs were shown to have a therapeutic effect on liver injury. Recently, we have been trying to use mesenchymal stem cells isolated from dental papilla of discarded human wisdom teeth. Autologous transplantation of mesenchymal stem cells from bone marrow and dental papilla could be ethically and functionally promising for stem cell-based therapy.

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease.

Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.

Osteogenic differentiation of human dental papilla mesenchymal cells.

We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.

[Takotsubo cardiomyopathy manifesting as no reflow pattern in coronary flow by transthoracic Doppler echocardiography and prolonged recovery of regional left ventricular wall motion abnormality: a case report].

A 80-year-old woman was admitted to our hospital because of chest pain. Electrocardiography revealed ST segment elevation in the I, aVL, and V1-V5 leads. Echocardiography revealed left ventricular apical aneurysmal change with ejection fraction of 31%. Coronary angiography showed no abnormalities. Creatine kinase was not elevated in her clinical course. The diagnosis was takotsubo cardiomyopathy. Transthoracic Doppler echocardiography was performed on the 2nd hospital day. Coronary flow velocity pattern in the left anterior descending artery revealed shortened diastolic deceleration time (108 msec) and systolic retrograde flow. Asynergy of the left ventricle gradually improved, but still persisted slightly at 6 months after discharge. Most patients with takotsubo cardiomyopathy have normal coronary flow velocity pattern in the acute phase. In this case, no reflow pattern of coronary flow was observed during prolonged recovery from left ventricular regional wall motion abnormality.

Expression of hepatocyte growth factor mRNA in rat liver cirrhosis induced by N-nitrosodimethylamine as evidenced by in situ RT-PCR.

Hepatocyte growth factor (HGF) is a potent inducer of hepatocyte proliferation and is expressed during liver failure. In this study we used the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect HGF mRNA expression in normal rat livers and cirrhotic rat livers induced by treatment with N-nitrosodimethylamine (DMN). In normal control livers, in situ RT-PCR detected HGF mRNA expression in Ito cells and Kupffer cells, both of which showed rounded morphologies. However, in the cirrhotic livers induced by DMN, HGF mRNA-positive cells were spindle-shaped and surrounded the hepatocytes located around the sinusoids. These cells appeared to be sinusoidal endothelial cells as well as Ito and Kupffer cells. Because it has been suggested that HGF expression is related to transforming growth factor-beta (TGF-beta) levels that may play an essential role in disease progression in cirrhotic livers, TGF-beta mRNA expression in normal and cirrhotic livers was also compared using in situ RT-PCR. Our results confirmed that expression of TGF-beta mRNA co-localized with HGF mRNA expression in the cirrhotic liver.