Diverse Shape Design and Physical Property Evaluation of In-Body Tissue Architecture-Induced Tissues.

Autologous-engineered artificial tissues constitute an ideal alternative for radical surgery in terms of natural anticoagulation, self-repair, tissue regeneration, and the possibility of growth. Previously, we focused on the development and practical application of artificial tissues using "in-body tissue architecture (iBTA)", a technique that uses living bodies as bioreactors. This study aimed to further develop iBTA by fabricating tissues with diverse shapes and evaluating their physical properties. Although the breaking strength increased with tissue thickness, the nominal breaking stress increased with thinner tissues. By carving narrow grooves on the outer periphery of an inner core with narrow grooves, we fabricated approximately 2.2 m long cord-shaped tissues and net-shaped tissues with various designs. By assembling the two inner cores inside the branched stainless-steel pipes, a large graft with branching was successfully fabricated, and its aortic arch replacement was conducted in a donor goat without causing damage. In conclusion, by applying iBTA technology, we have made it possible, for the first time, to create tissues of various shapes and designs that are difficult using existing tissue-engineering techniques. Thicker iBTA-induced tissues exhibited higher rupture strength; however, rupture stress was inversely proportional to thickness. These findings broaden the range of iBTA-induced tissue applications.

Carotid Artery Bypass Surgery of In-Body Tissue Architecture-Induced Small-Diameter Biotube in a Goat Model: A Pilot Study.

Biotubes are autologous tubular tissues developed within a patient's body through in-body tissue architecture, and they demonstrate high potential for early clinical application as a vascular replacement. In this pilot study, we used large animals to perform implantation experiments in preparation for preclinical testing of Biotube. The biological response after Biotube implantation was histologically evaluated. The designed Biotubes (length: 50 cm, internal diameter: 4 mm, and wall thickness: 0.85 mm) were obtained by embedding molds on the backs of six goats for a predetermined period (1-5 months). The same goats underwent bypass surgery on the carotid arteries using Biotubes (average length: 12 cm). After implantation, echocardiography was used to periodically monitor patency and blood flow velocity. The maximum observation period was 6 months, and tissue analysis was conducted after graft removal, including the anastomosis. All molds generated Biotubes that exceeded the tensile strength of normal goat carotid arteries, and eight randomly selected Biotubes were implanted. Thrombotic occlusion occurred immediately postoperatively (1 tube) if anticoagulation was insufficient, and two tubes, with insufficient Biotube strength (<5 N), were ruptured within a week. Five tubes maintained patency for >2 months without aneurysm formation. The spots far from the anastomosis became stenosed within 3 months (3 tubes) when Biotubes had a wide intensity distribution, but the shape of the remaining two tubes remained unchanged for 6 months. The entire length of the bypass region was walled with an αSMA-positive cell layer, and an endothelial cell layer covered most of the lumen at 2 months. Complete endothelial laying of the luminal surface was obtained at 3 months after implantation, and a vascular wall structure similar to that of native blood vessels was formed, which was maintained even at 6 months. The stenosis was indicated to be caused by fibrin adhesion on the luminal surface, migration of repair macrophages, and granulation formation due to the overproliferation of αSMA-positive fibroblasts. We revealed the importance of Biotubes that are homogeneous, demonstrate a tensile strength > 5 N, and are implanted under appropriate antithrombotic conditions to achieve long-term patency of Biotube. Further, we clarified the Biotube regeneration process and the mechanism of stenosis. Finally, we obtained the necessary conditions for a confirmatory implant study planned shortly.

Three cases of xanthinuria identified by gas chromatography/mass spectrometry-based urine metabolomics.

Introduction: Early diagnosis of patients with urolithiasis or hypouricemia owing to inborn errors of hypoxanthine metabolism is important in preventing renal failure or drug-induced toxicity. Case presentation: We identified three patients with xanthinuria using gas chromatography/mass spectrometry-based urine metabolomics: a 72-year-old male with bladder stone, a severe hypouricemic 59-year-old female with type 2 diabetes mellitus, and an 8-year and 9-month-old female who was first discovered to harbor a mutation in the xanthine dehydrogenase gene using whole-exome sequencing, but had a normal molybdenum cofactor sulfurase gene. Hydantoin-5-propionate was detected in the first and third patients but not in the second, suggesting that the first and second patients had type I and II xanthinuria, respectively. Conclusion: Gas chromatography/mass spectrometry-based metabolomics can be used for undiagnosed patients with xanthinuria, identification of the type of xanthinuria without allopurinol loading, and the quick functional evaluation of mutations in the xanthinuria-related genes.

C-terminus of PIEZO1 governs Ca2+ influx and intracellular ERK1/2 signaling pathway in mechanotransduction.

Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca2+ influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca2+ influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs.

The fecal carriage rate of extended-spectrum ß-lactamase-producing or carbapenem-resistant Enterobacterales among Japanese infants in the community at the 4-month health examination in a rural city.

Background: Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) is a great public health concern globally not only in hospitals but also in the community. To our knowledge, there have been few studies on the prevalence of ESBL-E and much less about carbapenem-resistant Enterobacterales (CRE) among children in the community, and there is no such study in Japan despite such situations. This study aimed to clarify their carriage status among Japanese infants in the community by taking the opportunity of the 4-month health checkup. Methods: This prospective analysis was conducted from April 2020 to March 2021 in Shimabara City, Nagasaki Prefecture, Japan. The research-related items were mailed to all subjects with official documents for the checkup. The fecal samples were obtained from the diaper by guardians beforehand and were collected with the questionnaire and then screened for ESBL-E and CRE by a clinical laboratory company with selective agars followed by identification and confirmation. Only the positive samples were analyzed about resistant genotypes. Results: One hundred fifty infants aged 4-5 months, over half of the subjects, participated in this study. The overall ESBL-E carriage rate was 19.3% (n = 29), and no CRE carrier was detected among them. All identified ESBL-E were E. coli except for one K. pneumoniae. A significantly higher carriage rate was recorded among the infants born at "Hospital A" (25.0%) than the others (11.3%). Enterobacterales producing CTX-M-9 ± TEM were broadly distributed among the positive samples (65.5%), whereas the CTX-M-1 group was exclusively detected among those from "Hospital A". Recursive partitioning analysis suggested that delivery facilities might be an important factor for ESBL-E colonization, although the effect could be decreased as they grow. In contrast, no significant effect was observed for other factors such as parent(s) as healthcare worker(s), having a sibling(s), and the mode of delivery. Conclusion: This study revealed the ESBL-E and CRE carriage status of Japanese infants in the community for the first time, although the setting is somewhat limited. Our findings indicated that environmental factors, especially delivery facilities, influenced ESBL-E colonization among infants aged 4-5 months, implying the need for strengthening countermeasures against antimicrobial resistance at delivery facilities and communities outside the hospitals.

Pre-implantation evaluation of a small-diameter, long vascular graft (Biotube®) for below-knee bypass surgery in goats.

There are no small-diameter, long artificial vascular grafts for below-knee bypass surgery in chronic limb-threatening ischemia. We have developed tissue-engineered vascular grafts called "Biotubes®" using a completely autologous approach called in-body tissue architecture (iBTA). This study aimed at pre-implantation evaluation of Biotube and its in vivo preparation device, Biotube Maker, for use in below-knee bypass surgery. Forty nine makers were subcutaneously embedded into 17 goats for predetermined periods (1, 2, or 3 months). All makers produced Biotubes as designed without inflammation over all periods, with the exception of a few cases with minor defects (success rate: 94%). Small hole formation occurred in only a few cases. All Biotubes obtained had an inner diameter of 4 mm and a length of 51 to 52 cm with a wall thickness of 594 ± 97 µm. All Biotubes did not kink when completely bent under an internal pressure of 100 mmHg and did not leak without any deformation under a water pressure of 200 mmHg. Their burst strength was 2409 ± 473 mmHg, and suture retention strength was 1.75 ± 0.27 N, regardless of the embedding period, whereas tensile strength increased from 7.5 ± 1.3 N at 1 month to 9.7 ± 2.0 N at 3 months with the embedding period. The amount of water leakage from the needle holes prepared in the Biotube wall was approximately 1/7th of that in expanded polytetrafluoroethylene vascular grafts. The Biotubes could be easily connected to each other without cutting or anastomosis leaks. They could be stored for at least 1 year at room temperature. This study confirmed that even Biotubes formed 1 month after embedding of Biotube Makers had properties comparable to arteries.

Effectiveness of a photoscreener in identifying undiagnosed unilateral amblyopia at vision screening of 3-year-old children in Japan.

PURPOSE: In Japan a basic preparatory ophthalmic examination is routinely performed for 3-year-old children. This study aimed to determine the value of incorporating a photoscreener into the examination and evaluate parents' satisfaction with the photoscreener examination. STUDY DESIGN: Prospective study. METHODS: Children aged 42-47 months in Nagasaki City, Japan, underwent a visual acuity test by a parent at home and by automated vision screening using a photoscreener at their local municipal health center between October 2018 and March 2019. Subjects were children referred to Nagasaki University Hospital for examination after failing either test. Children previously diagnosed with strabismus and/or amblyopia were excluded. A questionnaire survey evaluated the level of satisfaction with the photoscreener-based screening by parents who attended these examinations at the local municipal health center. RESULTS: Of children who completed the two tests, 52 (failed visual acuity test, 3; failed photoscreener examination, 49) were referred for examination. Of the 49 photoscreener failures, 12 were diagnosed with amblyopia: unilateral amblyopia with anisometropic hyperopia in 10 (83.3%), and bilateral amblyopia with astigmatism and hyperopia in 2 (16.7%). The photoscreener detected all 12 cases of amblyopia, whereas the home-based visual acuity test detected only two cases. More than 80% of 1035 parents were satisfied with the photoscreener examination. CONCLUSION: Unilateral amblyopia with anisometropic hyperopia was easily overlooked with the home-based test but was detectable by photoscreener examination. The photoscreener proved to be an effective screening tool for amblyopia in children and was considered a satisfactory examination by a high proportion of parents.

A novel heterozygous GLI2 mutation in a patient with congenital urethral stricture and renal hypoplasia/dysplasia leading to end-stage renal failure.

Glioblastoma 2 (GLI2) is a mediator of Sonic hedgehog signaling pathway that plays an important role in development of the central nervous system and limbs. Heterozygous GLI2 mutations have been associated with postaxial polydactyly, various pituitary dysfunction, and holoprosencephaly-like phenotype. Herein, we report a Japanese boy who presented with isolated growth hormone deficiency with ectopic posterior pituitary, postaxial polydactyly, atrioventricular septal defect, intellectual disability and dysmorphic facial features including mid-facial hypoplasia. The patient was also complicated with congenital urethral stricture with megacystis, hydronephrosis, and renal hypoplasia/dysplasia, which led to end-stage renal failure by the age of 8 years. Trio-whole-exome sequencing showed a novel de novo heterozygous frameshift mutation in GLI2 (c.3369delG, p.Met1123Ilefs*7) in the patient. This is the first report of possible association between GLI2 mutation and the phenotype of congenital anomalies of the kidney and urinary tract, and subsequent end-stage renal failure. Further studies on the urogenital phenotype in patients with GLI2 mutations may clarify a role of GLI2 in embryonic development of the urinary tract.

[Clinical Study to Determine the Dose Intensity of BEP Chemotherapy for Ovarian Malignant Germ Cell Tumors].

A standard regimen for ovarian malignant germ cell tumors is bleomycin, etoposide, cisplatin(BEP)chemotherapy. Adherence to a treatment schedule of every 21 days has been reported to be important. However, the incidence of febrile neutropenia( FN)and the optimaluse of granulocyte-colony stimulating factor(G-CSF)are unclear because of the low incidence of ovarian malignant germ cell tumors. We experienced 2 cases of ovarian malignant germ cell tumors that received BEP therapy after fertility-conserving surgery. In 1 case, we delayed drug administration in the first cycle because of FN. However, in order to maintain dose intensity(DI), we performed chemotherapy every 21 days by shortening the rest period. Myelosuppression may be severe in the first cycle of BEP therapy; however, it may be possible to adhere to the treatment schedule by using primary prophylactic administration of G-CSF.

Factors associated with nurses' intention to leave their jobs after the Fukushima Daiichi Nuclear Power plant accident.

We conducted a survey among nurses who were working at the Fukushima Medical University Hospital at the time of the Fukushima Daiichi Nuclear Power Plant accident to clarify the factors associated with their intention to leave their jobs during the radiation emergency. We asked 345 nurses (17 men and 328 women) about their intention to leave their jobs after the accident. We also asked about relevant factors including the participants' demographic factors, living situation, working status, and knowledge of radiation health effects. We found that living with preschoolers (OR = 1.87, 95%CI: 1.02-3.44, p = 0.042), anxiety about life in Fukushima City after the accident (OR = 5.55, 95%CI: 1.18-26.13, p = 0.030), consideration of evacuation from Fukushima after the accident (OR = 2.42, 95%CI: 1.45-4.06, p = 0.001), consideration of the possible radiation health effects in children (OR = 1.90, 95%CI: 1.02-3.44, p = 0.042), and anxiety about relationships with colleagues in the hospital after the accident (OR = 3.23, p = 0.001) were independently associated with the nurses' intention to leave their jobs after the accident. On the other hand, the percentage of nurses with knowledge on radiation health effects was relatively low among those who had the intention to leave the job and among those who did not have the intention to leave the job after the accident, with no significant differences between the two groups. Our results suggest the need for an education program for nurses regarding radiation health effects.

Two-dimensional crystallization of intact F-ATP synthase isolated from bovine heart mitochondria.

Mitochondrial F-ATP synthase produces the majority of ATP for cellular functions requiring free energy. The structural basis for proton motive force-driven rotational catalysis of ATP formation in the holoenzyme remains to be determined. Here, the purification and two-dimensional crystallization of bovine heart mitochondrial F-ATP synthase are reported. Two-dimensional crystals of up to 1 µm in size were grown by dialysis-mediated detergent removal from a mixture of decylmaltoside-solubilized 1,2-dimyristoyl-sn-glycero-3-phosphocholine and F-ATP synthase against a detergent-free buffer. A projection map calculated from an electron micrograph of a negatively stained two-dimensional crystal revealed unit-cell parameters of a = 185.0, b = 170.3 Å, γ = 92.5°.

Bovine heart NADH-ubiquinone oxidoreductase contains one molecule of ubiquinone with ten isoprene units as one of the cofactors.

NADH-ubiquinone oxidoreductase (Complex I) is located at the entrance of the mitochondrial electron transfer chain and transfers electrons from NADH to ubiquinone with 10 isoprene units (Q(10)) coupled with proton pumping. The composition of Complex I, the largest and most complex proton pump in the mitochondrial electron transfer system, especially the contents of Q(10) and phospholipids, has not been well established. An improved purification method including solubilization of mitochondrial membrane with deoxycholate followed by sucrose gradient centrifugation and anion-exchange column chromatography provided reproducibly a heme-free preparation containing 1 Q(10), 70 phosphorus atoms of phospholipids, 1 zinc ion, 1 FMN, 30 inorganic sulfur ions, and 30 iron atoms as the intrinsic constituents. The rotenone-sensitive enzymatic activity of the Complex I preparation was comparable to that of Complex I in the mitochondrial membrane. It has been proposed that Complex I has two Q(10) binding sites, one involved in the proton pump and the other functioning as a converter between one and two electron transfer pathways [Ohnishi, T., Johnson, J. J. E., Yano, T., LoBrutto, R., and Widger, R. W. (2005) FEBS Lett. 579, 500-506]. The existence of one molecule of Q(10) in the fully oxidized Complex I suggests that the affinity of Q(10) to one of the two Q(10) sites is greatly dependent on the oxidation state and/or the membrane potential and that the Q(10) in the present preparation functions as the converter of the electron transfer pathways which should be present in any oxidation state.

Pathological influence of obesity on renal structural changes in chronic kidney disease.

BACKGROUND: Role of obesity in renal pathological and structural changes remains poorly investigated, and this study was designed to examine the pathological effects of obesity on renal structural components in patients with chronic kidney diseases (CKD). METHODS: The study subjects were obese (body mass index, BMI > or = 25 kg/m2) patients with nonglomerulonephritis (non-GN, n = 26), IgA nephropathy (IgAN, n = 19), benign nephrosclerosis (BNS, n = 15), and thin basement membrane disease (TMD, n = 6), and 65 nonobese controls (n = 20, 20, 10, and 15, respectively). Patients were evaluated for glomerular lesions (mesangial proliferation and focal segmental/global glomerulosclerosis), glomerular size, and thickness of glomerular basement membrane (GBM). RESULTS: Urinary protein was higher in obese non-GN, IgAN, and BNS groups than in the respective controls. Focal segmental glomerulosclerosis (FSGS) lesions were noted in all obesity groups. The glomeruli were larger in size in obese than in nonobese patients of the non-GN and IgAN groups. The glomeruli of nonobese TMD and BNS patients were significantly larger in size than those of nonobese non-GN patients. GBM were thicker in obese than in nonobese patients irrespective of types of glomerular diseases, but only significantly so in non-GN and BNS groups. CONCLUSION: In non-GN, IgAN, and BNS, obesity worsens proteinuria and is associated with structural changes such as glomerulomegaly and GBM thickening, similar to changes observed in obesity-related nephropathy. Obesity seems to worsen the renopathological state in CKD.

Effect of the side chain structure of coenzyme Q on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase.

Steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase using coenzyme Q with two isoprenoid unit (Q2) or with a decyl group (DQ) show an ordered sequential mechanism in which the order of substrate binding and product release is NADH-Q2 (DQ) -Q2H2 (DQH2)-NAD+ in contrast to the order determined using Q1 (Q1-NADH-NAD(+)-Q1H2) (Nakashima et al., J. Bioenerg. Biomembr. 34, 11-19, 2002). The effect of the side chain structure of coenzyme Q suggests that NADH binding to the enzyme results in a conformational change, in the coenzyme Q binding site, which enables the site to accept coenzyme Q with a side chain significantly larger than one isoprenoid unit. The side chains of Q2 and DQ bound to the enzyme induce a conformational change in the binding site to stabilize the substrate binding, while the side chain of Q1 (one isoprenoid unit) is too short to induce the conformational change.

Effect of pH on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase.

Complete initial steady state kinetics of NADH-decylubiquinone (DQ) oxidoreductase reaction between pH 6.5 and 9.0 show an ordered sequential mechanism in which the order of substrate bindings and product releases is NADH-DQ-DQH2-NAD+. NADH binding to the free enzyme is accelerated by protonation of an amino acid (possibly a histidine) residue. The NADH release is negligibly slow under the turnover conditions. The rate of DQ binding to the NADH-bound enzyme and the maximal rate at the saturating concentrations of the two substrates, which is determined by the rates of DQH2 formation in the active site and releases of DQH2 and NAD+ from the enzyme, are insensitive to pH, in contrast to clear pH dependencies of the maximal rates of cytochrome c oxidase and cytochrome bc1 complex. Physiological significances of these results are discussed.

The second coenzyme Q1 binding site of bovine heart NADH: coenzyme Q oxidoreductase.

The rotenone sensitivity of bovine heart NADH: coenzyme Q oxidoreductase (Complex I) depends significantly on coenzyme Q1 concentration. The rotenone-insensitive Complex I reaction in Q1 concentration range above 300 microM indicates an ordered sequential mechanism with Q1 and reduced Q1 (Q1H2) as the initial substrate to bind to the enzyme and the last product to be released from the enzyme product complex, respectively. This is the case in the rotenone-sensitive reaction although both Km and Vmax values of the rotenone-insensitive reaction for Q1 are significantly higher than those of the rotenone-sensitive reaction (Nakashima et al., 2002, J. Bioenerg. Biomemb. 34, 11-19). This rigorous control mechanism between the nucleotide and ubiquinone binding sites strongly suggests that the rotenone-insensitive reaction is also physiologically relevant.

Steady-state kinetics of NADH:coenzyme Q oxidoreductase isolated from bovine heart mitochondria.

Steady-state kinetics of the bovine heart NADH:coenzyme Q oxidoreductase reaction were analyzed in the presence of various concentrations of NADH and coenzyme Q with one isoprenoid unit (Q1). Product inhibitions by NAD+ and reduced coenzyme Q1 were also determined. These results show an ordered sequential mechanism in which the order of substrate binding and product release is Q1-NADH-NAD+-Q1H2. It has been widely accepted that the NADH binding site is likely to be on the top of a large extramembrane portion protruding to the matrix space while the Q1 binding site is near the transmembrane moiety. The rigorous controls for substrate binding and product release are indicative of a strong, long range interaction between NADH and Q1 binding sites.