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The introduction of a 5′-tailed duplex (5′-TD) fragment into cells corrects a base-substitution mutation in a target DNA. We previously reported that the gene correction efficiency was improved when a frameshift type of second mismatch was present ∼330 bases distant from the target position, between the target DNA and the 5′-TD fragment. In this study, the effects of the second mismatches on the gene correction were further examined. Base–base mismatches 332 bases distant from the target position slightly enhanced gene correction, but less efficiently than the previously studied frameshift mismatches. The gene correction efficiency was also increased when the distance between the target position and the second frameshift mismatch was changed to ∼270 bases. These results suggested that the introduction of an appropriate second frameshift mismatch into the 5′-TD fragment improves the gene correction efficiency.

Background Minimally invasive cardiac surgery via right lateral minithoracotomy is a well-described approach. However, reports on isolated tricuspid valve surgery (TVS) in this technique are rare. Therefore, we like to give a contribution by reporting our experience. Methods We retrospectively reviewed 25 tricuspid valve operations via right lateral minithoracotomy with femoral cannulation between August 2009 and September 2013 (18 repairs, 7 replacements, and 72% repair rate). Three patients (12%) presented for a re-do operation, and nine patients (36%) suffered from active endocarditis at admission. All patients underwent TVS as single valve procedure. Ten patients received additional procedures such as removal of infected leads, resection of atrial tumors, or closure of atrial septal defects. An annuloplasty ring was inserted in 12 cases. We investigated the short-term morbidity and mortality with regard to the surgical procedure. Results Repair rate was 72%. Thirty-day and 1-year mortality were 4 and 20%, respectively. The only patient with early mortality received the surgical procedure on the tricuspid valve as fourth cardiac-related surgery and postoperative mortality was due to intracranial air embolism. Perioperative morbidity included reoperation for bleeding (8%) and stroke (4%). No disturbance of wound healing occurred. Durations of intensive care unit stay and hospital stay were 2.3 ± 2.4 and 17.4 ± 13.1 days, respectively. Endocarditis-caused surgery did not reveal any significant difference in the intra- or perioperative course compared with other indications. Conclusion Minimally invasive TVS via right lateral minithoracotomy is feasible with good results. Even in a cohort of patients suffering from elevated rate of active endocarditis, a high repair rate can be achieved.

8-Oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA is one of the most important oxidatively damaged bases and causes G:C → T:A substitution mutations. The Werner syndrome protein (WRN) is a cancer-related RecQ DNA helicase and plays many roles in DNA replication and repair. To examine the relationships between G(O)-induced mutations and WRN, shuttle plasmid DNA containing a G(O):C pair in the supF gene was transfected into human U2OS cells, in which WRN was knocked down. The plasmid DNA replicated in the knockdown cells was introduced into an Escherichia coli indicator strain. The knockdown of WRN increased the mutant frequency of the G(O)-plasmid DNA. Unexpectedly, however, the WRN knockdown only slightly enhanced the targeted G:C → T:A mutation. Instead, base-substitution mutations at various positions were more frequently detected, with statistical significance. The results obtained in this study suggested that the reduction of the cancer-related WRN induced action-at-a-distance mutagenesis by the G(O):C pair in human cells. In addition, the WRN knockdown decreased the G(O):A-induced A:T → C:G mutations, suggesting that WRN may enhance the mutations caused by G(O) in the nucleotide pool.

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies. A comprehensive analysis of the transcription of 4'-thioDNA templates to natural RNA is herein reported. The modifications of DNA with 2'-deoxy-4'-thionucleosides, so-called 4'-thioDNA, were found to act as templates for transcription with RNA polymerases both in vitro and in mammalian cells.

In recent years economic sense is required in hospital management, the national university hospital is also no exception. We performed duty improvement efforts as well as the current state analysis in the department of cardiac surgery for the purpose of revenue optimization in our hospital. At result, we succeeded in the improvement of the medical index as well as medical profit and found the key of solution to the serious problem that national university hospital has.

出版社版, Background: The aims of this study are (1) to investigate the occurrence rate of postoperative complications in patients ≥ 80 years old after cardiac surgery and (2) to elucidate the impact of the most common postoperative complications on mortality.Methods: Between January 1998 and December 2007, 649 patients aged over 80 years received isolated first-time coronary artery bypass graft (CABG), isolated aortic valve replacement (AVR) or a combination of both in our institute. Prospectively entered patient data were analyzed with respect to major complications and outcome parameters.Results: Acute renal failure (55.0% vs. 7.5%, p = 0.0001), low cardiac out-put syndrome (43.1% vs. 8.8%, p = 0.0001), sepsis (52.0% vs. 10.3%, p = 0.0001), prolonged respiratory failure with tracheotomy (29.0% vs. 11.0%, p = 0.002), re-thoracotomy due to bleeding (26.9% vs. 10.6%, p = 0.0001), and postoperative laparotomy (30.8% vs. 11.5%, p = 0.033) had a significant impact on mortality. A multivariate analysis revealed that advanced age (OR 1.130, 95%CI; 1.017–1.256, p = 0.023), low output syndrome (OR 5.094, 95%CI; 1.1635–15.871, p = 0.005), renal failure (OR 8.128, 95%CI; 3.347–19.742, p = 0.0001) and sepsis (OR 4.975, 95%CI; 1.420–17.426, p = 0.012) as independent risk factors.Conclusions: The present study demonstrates that among major complications, low output syndrome, renal failure requiring renal replacement therapy and sepsis, dramatically impaired the postoperative course patients aged over 80 years undergoing CABG, AVR or combined CABG and AVR.

The transience of transgene expression is a major obstacle in the development of nonviral vectors. The CpG-free and pLIVE plasmids reportedly achieve long-term transgene expression in mouse liver. In this work, the anti-silencing elements within these plasmids were studied. The effects of plasmid that was being silenced on transgene expression from the CpG-free plasmid and those of transgene expression at early time points on silencing were also examined. The results suggested that the backbone sequence of the CpG-free plasmid and the 3' untranslated region of the albumin gene of the pLIVE plasmid contribute to durable expression. In addition, no influence of the silencing of another plasmid on the duration of CpG-free plasmid expression or of transgene expression at early time points on silencing was detected.

Veno-arterial extracorporeal membrane oxygenation (ECMO) may be implanted using peripheral ECMO (pECMO) or central ECMO (cECMO) cannulation techniques. The aim of this study was to compare the outcome between these two cannulation techniques. A retrospective study was performed at Dusseldorf University Hospital from October 2009 through June 2011. Inclusion criteria were patients with veno-arterial ECMO support ≥24 h. Various pre- and postimplantation variables were investigated including postimplantation hemodynamic and ECMO parameters, oxygenation/ventilation parameters at 3, 6, 12, 24, 48, 72 h, as well as renal and liver function tests at first and third postoperative days following implantation. Outcome data of patients receiving pECMO were compared with those who received cECMO. The inclusion criteria were met by 37 patients (25 pECMO and 12 cECMO). There were no significant differences in baseline characteristics between these two groups except for younger age in pECMO patients (P = 0.005). All postimplantation variables were comparable between the two groups except for higher PO2 and lower PCO2 values at the 3rd hour postimplantation in patients with pECMO (P = 0.007 and 0.01, respectively). Eleven (44%) of the pECMO patients required re-exploration for bleeding versus 100% of patients with cECMO (P = 0.01). Ischemic leg complication was observed in four pECMO and three cECMO patients. Thirty-day mortality in patients with pECMO and cECMO was 60% versus 67%, respectively (P = 1.00). In this study, no particular oxygenation/ventilation, hemodymanic, or end-organ function advantage was observed with either cannulation technique. However, more bleeding and resternotomy complications were observed in cECMO patients.

Percutaneous nephrolithotomy (PCNL) is considered the standard procedure for the removal of large renal calculi. The development of the "minimally invasive PCNL" (mini-PCNL) has reduced the complications of the surgery; it also appears to be associated with less morbidity than the conventional PCNL (con-PCNL). This study aimed at evaluating the efficacy of endoscopic intrarenal surgery, using the prone-split leg position, using flexible ureteroscopy and mini-PCNL (mini- endoscopic combined intrarenal surgery [ECIRS]) by retrospectively comparing this technique with mini-PCNL and con-PCNL.In total, 161 consecutive patients who were observed for the follow-up of large renal calculi between February 2004 and January 2013 were selected for mini-ECIRS (60), mini-PCNL (19), or con-PCNL (82). Mini-ECIRS was performed with patients in the prone split-leg position via 18F minipercutaneous tract and 14F ureteral access sheath. The mini-PCNL was performed via 18F percutaneous tract and con-PCNL performed via the 30F tract. Mean size of the renal calculi removed via mini-ECIRS, mini-PCNL, and con-PCNL were 39.2, 38.4, and 34.6 mm, respectively.Average surgical time for mini-ECIRS was shorter than that for mini-PCNL and con-PCNL (120.5 vs. 181.9 vs 134.1 min, respectively; P0.001). The stone-free rate for mini-ECIRS was significantly higher than that of the other procedures (initial rates 81.7% vs. 38.9% vs. 45.1%, respectively; P0.001; rates after further treatment 86.7% vs. 61.1% vs. 61.0%, respectively; P=0.002). Only one patient in the mini-ECIRS group needed blood transfusions. The decrease in hemoglobin during mini-ECIRS and mini-PCNL was significantly lower than that during con-PCNL (P=0.011).Mini-ECIRS is better than monotherapy with mini-PCNL or con-PCNL. The study results show that mini-ECIRS is a safe, efficient, and versatile procedure that can be effective for the management of renal calculi.

Current techniques for mitral valve repair (MVR) in Barlow’s disease require high level of surgical expertise due to a complex anatomy. A novel and simple standardized technique that particularly considers the pathological changes of the mitral valve in Barlow’s disease has been developed. Between 2009 and 2013, 22 patients underwent minimally invasive MVR for Barlow’s disease and severe mitral regurgitation (MR). A simple, standardized technique was applied, including resection of P2 segment of posterior mitral leaflet (PML) with preservation of the shortest chordae, transfer of the preserved chordae to A2, and implantation of a semi-rigid open ring. In 2015, all patients were contacted for follow-up by transthoracic echocardiography (TTE) and interviewed for their clinical status. During follow-up (mean 2.8 ± 1.1 years; 100% complete), one patient died due to abdominal bleeding 4 months after the initial MVR and one patient with severe calcification of PML underwent valve replacement due to recurrence of MR. Among the remaining cohort (mean follow-up 3.0 ± 1.0 years), NYHA class I, II and III was present in 13, 6, and 1, respectively. TTE demonstrated MR grade 0, 1+, or 2+ in 40, 55, and 5%, respectively, with mean and maximum transvalvular gradients ranging at 1.9 ± 1.7 and 4.7 ± 3.3 mmHg, respectively. A simple and standardized technique facilitates the repair of MR in the presence of Barlow’s, simultaneously addressing the height of PML and the position of the anterior leaflet. This technique has proven durable in the mid-term follow-up in our small series and warrants further validation in larger cohorts.

An emergency thoracic endovascular aortic repair (TEVAR) with zone 2 landing without revascularization of the left subclavian artery was performed due to the impending rupture of a distal arch aneurysm in an old patient presenting hemoptysis. Two months later, the patient had recurrent massive hemoptyses and continued after additional zone 0 TEVAR. The lung parenchyma was considered to be the bleeding source and transcatheter pulmonary artery embolization was performed, and the episodes of massive hemoptysis appeared to have ceased. However, the patient died of sudden recurrent massive hemoptysis 40 days later. Inflammation and/or infection of the lung parenchyma adjunct to the aortic aneurysm could be cause of fatal hemoptysis, and aggressive therapy such as lung resection should be considered in such patients.

ObjectiveTo clarify prognostic factors for idiopathic inflammatory myopathy (IIM)-associated interstitial lung disease (ILD).DesignSystematic review and meta-analysis using the Grades of Recommendation, Assessment, Development and Evaluation system.Data sourcesMedline, EMBASE and Science Citation Index Expanded were searched through 9 August 2018.Eligibility criteria for selecting studiesThe review includes primary studies addressing all-cause mortality of IIM-associated ILD. Potential prognostic factors were any clinical information related to the outcome.Data extraction and synthesisTwo reviewers extracted relevant data independently and assessed risk of bias using the Quality in Prognostic Studies tool. Meta-analysis was conducted using a random effects model and if inappropriate the results were reported qualitatively. Prognostic factors were determined based on statistically significant results derived from multivariate analysis.ResultsOf a total of 5892 articles returned, 32 were deemed eligible for analysis and cumulatively, these studies reported 28 potential prognostic factors for all-cause mortality. Each study was subject to certain methodological constraints. The four prognostic factors, which demonstrated statistically significant results on both univariate and multivariate analyses, were as follows: age (MD 5.90, 3.17–8.63/HR 1.06, 1.02–1.10 and 2.31, 1.06–5.06), acute/subacute interstitial pneumonia (A/SIP) (OR 4.85, 2.81–8.37/HR 4.23, 1.69–12.09 and 5.17, 1.94–13.49), percentage of predicted forced vital capacity (%FVC) (OR 0.96, 0.95–0.98/HR 0.96, 0.93–0.99) and anti-Jo-1 antibody (OR 0.35, 0.18–0.71/HR 0.004, 0.00003–0.54) (univariate/multivariate, 95% CI). Other prognostic factors included ground glass opacity/attenuation (GGO/GGA) and extent of radiological abnormality. The quality of the presented evidence was rated as either low or very low.ConclusionsOlder age, A/SIP, lower value of %FVC, GGO/GGA and extent of radiological abnormality were demonstrated to predict poor prognosis for IIM-associated ILD while a positive test for anti-Jo-1 antibody indicated better prognosis. However, given the weak evidence they should be interpreted with caution.Trial registration numberCRD42016036999.

Decellularization is a promising option to diminish immune and inflammatory response against donor grafts. In order to accelerate the autologous in vivo recellularization of aortic conduits for an enhanced biocompatibility, we tested fibronectin surface coating in a standardized rat implantation model. Detergent-decellularized rat aortic conduits (n = 36) were surface-coated with covalently Alexa488-labeled fibronectin (50 μg/ml, 24 h) and implanted into the systemic circulation of Wistar rats for up to 8 weeks (group FN; n = 18). Uncoated implants served as controls (group C; n = 18). Fibronectin-bound fluorescence on both surfaces of the aortic conduits was persistent for at least 8 weeks. Cellular repopulation was examined by histology and immunofluorescence (n = 24). Luminal endothelialization was significantly accelerated in group FN (p = 0.006 after 8 weeks), however, local myofibroblast hyperplasia with significantly increased ratio of intima-to-media thickness occurred (p = 0.0002 after 8 weeks). Originating from the adventitial surface, alpha-smooth muscle actin and desmin positive cell invasion into the media of fibronectin-coated conduits was significantly increased as compared to group C (p < 0.0001). In these medial areas, in situ zymography revealed enhanced matrix metalloproteinase activity. In both groups, inflammatory cell markers (CD3 and CD68) and signs of thrombosis proved negative. With regard to several markers of cell adhesion, inflammation and calcification, quantitative real-time PCR (n = 12) revealed no significant inter-group differences. Fibronectin surface coating of decellularized cardiovascular implants proved feasible and persistent for at least 8 weeks in the systemic circulation. Biofunctional protein coating accelerated the autologous in vivo endothelialization and induced a significantly increased medial recellularization. Therefore, this strategy may contribute to the improvement of current clinically applied bioprostheses.

Background Transgene expression from plasmid DNA is dependent on the expression efficiency per plasmid and the amount of intranuclear plasmid. In the present study, intranuclear dispositions of two types of plasmid DNAs (i.e. the pCpGfree and pLIVE plasmids) that maintain transgene expression in mouse liver were analyzed. In addition, the relationship between transgene expression and plasmid stability in the nucleus was examined. Methods First, the pCpGfree and pLIVE plasmid DNAs, bearing the mouse secreted alkaline phosphatase (Seap) gene, were administered into mouse liver by the hydrodynamics-based method. Next, various Seap-plasmid DNAs containing different promoters, upstream and downstream sequences, and backbones were injected into mice, and both SEAP expression and plasmid DNA amounts were monitored for 28 days. Results At the 14- and 28-day time points, the amount of the pCpGfree plasmid DNA was one order of magnitude less than that of the pLIVE plasmid. Meanwhile, the expression efficiency per plasmid was one order of magnitude more efficient for the pCpGfree plasmid DNA. Moreover, the administration of various Seap-plasmid DNAs revealed that negative correlations exist between plasmid stability and SEAP expression level. Conclusions The results obtained suggest that the pCpGfree plasmid is unstable from the viewpoint of quantity and maintains transgene expression by its high expression efficiency and also that transgene expression negatively affects the stability of plasmid DNA. Copyright © 2013 John Wiley & Sons, Ltd.

In the last decade, cardiovascular tissue engineering has made great progress developing new strategies for regenerative medicine applications. However, while tissue engineered heart valves are already entering the clinical routine, tissue engineered myocardial substitutes are still restrained to experimental approaches. In contrast to the heart valves, tissue engineered myocardium cannot be repopulated in vivo because of its biological complexity, requiring elaborate cultivation conditions ex vivo. Although new promising approaches-like the whole-heart decellularization concept-have entered the myocardial tissue engineering field, bioreactor technology needed for the generation of functional myocardial tissue still lags behind in the sense of user-friendly, flexible and low cost systems. Here, we present a novel customizable modular bioreactor system that can be used for whole-heart cultivation. Out of a commercially obtainable original equipment manufacturer platform we constructed a modular bioreactor system specifically aimed at the cultivation of decellularized whole-hearts through perfusion and controlled 3D biomechanical stimulation with a simple but highly flexible operation platform based on LabVIEW. The modular setup not only allows a wide range of variance regarding medium conditioning under controlled 3D myocardial stretching but can also easily be upgraded for e.g. electrophysiological monitoring or stimulation, allowing for a tailor-made low-cost myocardial bioreactor system.

Die optimale Therapie der herzinsuffizienzbegleitenden Mitralinsuffizienz bleibt umstritten. Ein fehlender Konsens zwischen Kardiologen und Kardiochirurgen – aufgrund eingeschrankter Studien- und heterogener Datenlage – hat in der Vergangenheit zu vage definierten Leitlinien und unterschiedlicher Handhabung in der klinischen Routine gefuhrt. Fortschritte im operativen Management mit exzellenten Ergebnissen einzelner erfahrener Zentren legen allerdings nahe, dass eine Vielzahl von Patienten von einer chirurgischen Therapie zu profitieren scheint. Die Evaluation hinsichtlich des Nutzens einer chirurgischen Therapie sollte hierbei fur jeden Patienten individuell vorgenommen werden und erfordert aufgrund der komplexen Pathophysiologie und der anspruchsvollen Diagnostik einen interdisziplinaren Ansatz („Heart-Team“).

BACKGROUND The objective of this study was to describe a small animal aortic conduit model that could analyze long-term conduit valve (CV) function by echocardiography. METHODS AND RESULTS Recipient Wistar rats (200-250g, n=20) underwent aortic leaflet injury of their native aortic valve under echocardiographic control. After 2 weeks, U-shaped decellularized CVs obtained from other rats were implanted onto the abdominal aorta. Implanted CVs were analyzed via pulsed-wave echocardiography at day 0, 4 and 12 weeks. CV stenosis was assessed as systolic flow velocity (post-pre CV)/flow velocity in the ascending aorta. CV regurgitation was assessed as the ratio of the amount of reversed diastolic flow to forward systolic flow in post-pre CV. The endpoint was set at 12 weeks. Three rats died immediately after aortic valve injury and all surviving rats received CV implantation (n=17, 85%). The survival rate after conduit implantation was 100% at 4 weeks and 88% (15/17) at 12 weeks. Regarding the CV function at 0, 4 and 12 weeks, the average observed value of CV stenosis was 3.8±7.9%, 3.1±4.1% and 14±10% (P

We report on the unique clinical course of a 44-year-old male HIV-positive heart transplant recipient, who was bridged by mechanical circulatory support (MCS). The patient was admitted with acute ischemic heart failure due to severe myocardial infarction. After emergency coronary artery bypass grafting and nine days of extracorporeal life support, we implanted a left ventricular assist device. As HIV infection was effectively treated and other contraindications were not present, we decided to perform a heart transplantation (HTX). At the current time, 34 months after unremarkable HTX, rejection or opportunistic infections have not occurred.

The first description of aortic valve replacement (AVR) as minimally invasive cardiac surgery (MICS) was given by Rao et al. in 1993. Recently, the number of MICS-AVR has increased and this procedure has been established as the routine standard operative technique in some institutes. MICS-AVR can be performed with two different approaches. One is an upper partial sternotomy and the other is a right small anterior minithoracotomy. MICS-AVR via upper partial sternotomy can be performed without any special instruments and the technical hurdles are relatively few, but the cosmetic benefit is limited. MICS-AVR via right anterior minithoracotomy offers maximal cosmetic benefit, but the technical hurdles are fairly high. For MICS-AVR, longer aortic clamping time and cardiopulmonary bypass time are needed, but this disadvantage can be overcome with sutureless aortic valve repair, and therefore this procedure may become a standard open surgical procedure in the era of transcatheter aortic valve implantation.

A 61-year-old man presented with lower leg edema, left-sided parasternal systolic heart murmur, and electrocardiographic abnormalities. Transthoracic echocardiography revealed myocardial hypertrophy and severe obstruction of the outflow tract of the right ventricle (RV) with a gradient of 134 mmHg. Cardiac catheterization confirmed the presence of double chambered right ventricle, with a pressure gradient of 84 mmHg between RV inlet and outlet. Chest computed tomography showed his aortic root enlarged to 55 mm. Holter electrocardiogram revealed nonsustained ventricular tachycardia originated from RV outlet. He underwent resection of the muscular band via right ventriculotomy, aortic valve sparing reimplantation using a straight graft in 28 mm diameter, and cryoablation of RV outflow tract under cardiopulmonary bypass. His postoperative course was uneventful. Postoperative examination showed removal of the obstruction of RV outflow tract.

The Open Symposium of the Japanese Environmental Mutagen Society (JEMS), entitled “Life Style and Cancer”, was held at the Shiba-Kyoritsu campus of Keio University, Tokyo, on May 30, 2015. The major aim of this symposium was to provide information about the relationships between lifestyle habits and cancers and an opportunity to inform a wide range of people about the JEMS activities.

Guanine oxidation occurs in both DNA and the cellular nucleotide pool, and one of the major products is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). The mutagenic potentials of this oxidized base have been examined in various experimental systems. In this review, we summarize the mutagenicity of the base in mammalian cells. We also describe the effects of specialized DNA polymerases, DNA repair proteins, and nucleotide pool sanitization enzymes.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease (CKD) and the optimal glycemic control is key to delay the progression of the disease and prevent major complications. Dipeptidyl peptidase-4 (DPP-4) inhibitors have emerged as a promising therapeutic option. However, the benefits and harms of the treatment have yet to be clarified for diabetic patients with CKD.Type 2 diabetic patients with moderate to severe CKD including end-stage renal disease were eligible and randomized controlled trials comparing DPP-4 inhibitors with no treatment or placebo or other antihyperglycemic agents were included. A systematic electronic search was conducted through the Medline Ovid, EMBASE, Cochrane Central Register of Controlled Trials and ClinicalTrials.gov.Six placebo/open and 2 active controlled trials (1747 participants) were included. The adjusted mean difference of hemoglobin ADPP-4 inhibitors demonstrated beneficial effects on the glycemic control for diabetic patients with CKD without causing any additional adverse effects. However, a definitive conclusion has yet to be drawn due to serious methodological problems and a small number of studies.

Takemura T, Akashi T, Kamiya H, Ikushima S, Ando T, Oritsu M, Sawahata M & Ogura T (2012) Histopathology Pathological differentiation of chronic hypersensitivity pneumonitis from idiopathic pulmonary fibrosis/usual interstitial pneumonia Aims: To evaluate the histological characteristics differentiating chronic hypersensitivity pneumonitis (chronic HP) with a usual interstitial pneumonia (UIP)-like pattern from idiopathic pulmonary fibrosis (IPF)/UIP. Methods and results: Surgical lung biopsy specimens from 22 patients with chronic HP diagnosed as having a UIP-like pattern upon histological examination and 13 patients with IPF/UIP were examined and the incidences of bronchiolitis, perilobular fibrosis, centrilobular fibrosis, bridging fibrosis, organizing pneumonia, fibroblastic foci, honeycombing, granulomas, giant cells, lymphocytic alveolitis and lymphoid follicles were compared. Bronchiolitis, centrilobular fibrosis, bridging fibrosis, organizing pneumonia, granulomas, giant cells and lymphocytic alveolitis were significantly more frequent among patients with chronic HP than among patients with IPF (all P

The formation of 8-oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA and in the nucleotide pool results in G:C→T:A and A:T→C:G substitution mutations, respectively, since G(O) can pair with both C and A. In this study, the role of DNA polymerase κ in the mutagenicity of G(O) was investigated, using a supF shuttle plasmid propagated in human U2OS cells. This translesion synthesis DNA polymerase was knocked down by siRNA, and plasmid DNAs containing G(O):C and G(O):A pairs were transfected into the knock-down cells. The supF plasmid DNAs replicated in the cells were then introduced into Escherichia coli. Mutation analyses indicated that the knock-down of DNA polymerase κ by siRNA decreased the frequency of G:C→T:A mutation caused by G(O):C, although no effects of the DNA polymerase κ reduction were observed for the A:T→C:G substitution induced by G(O):A. These results suggested that DNA polymerase κ is involved in the mutagenic bypass of G(O) in living human cells, when the damaged base is generated by direct DNA oxidation.

OBJECTIVES: Sternal dehiscence and mediastinitis are rare but serious complications following cardiac surgery. The aim of this study was to investigate the influence of the number of sternal wires used for chest closure on sternal complications. METHODS: From May 2003 to April 2007, 4714 adult patients received cardiac surgery in our institute. X-ray images of all patients were reviewed and the used wires were counted. Patients who received another material or longitudinal wiring technique according to Robicsek for chest closure were excluded from this analysis; thus 4466 patients were included into the final analysis. Figure-of-eight wiring was counted as two wires. RESULTS: Sternal complications occurred in 2.4%, and hospital mortality with or without sternal complications were 2.8 and 2.7%, respectively (P= 0.60). Mean numbers of sternal wires were 7.8 in both patient groups with or without sternal complications (P= 0.79). Multivariate analysis revealed diabetes mellitus [odds ratio (OR) 1.54, 95% CI 1.01–2.34, P= 0.04], chronic obstructive pulmonary disease (OR 1.85, 95% CI 1.12–2.79, P= 0.01) and renal insufficiency (OR 1.70, 95% CI 1.11–2.59, P= 0.001) as significant risk factors for sternal complications. In high-risk patients, the use of less than eight wires was significantly associated with postoperative sternal complications. CONCLUSIONS: Particularly in high-risk patients, careful haemostasis should be done and eight or more wires should be used to avoid sternal complications.

Most of the proteins carrying the 23-residue MutT-related sequence are capable of hydrolyzing compounds with a general structure of nucleoside diphosphate linked to another moiety X and are called the Nudix hydrolases. Among the 22 human Nudix proteins (identified by the sequence signature), some remain uncharacterized as enzymes without a defined substrate. Here, we reveal that the NUDT18 protein, whose substrate was unknown, can degrade 8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2 (NUDT15), we propose that it should be named MTH3. Although these three human proteins resemble each other in their sequences, their substrate specificities differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2 can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme activity of MTH2 is considerably lower than that of MTH1. On the other hand, MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and 8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the finding that MTH1 and MTH2 show a limited activity against the ribonucleotide counterpart, 8-oxo-GTP. These three enzymes may function together to help maintain the high fidelity of DNA replication and transcription under oxidative stress.

Involvement of the upper gastrointestinal tract in sarcoidosis is rare, and an optimal treatment regimen remains to be determined. Here, we report 3 cases of upper gastrointestinal sarcoidosis in Japanese patients aged 57-70 years with or without epigastric symptoms and describe the clinical features, laboratory data, and upper endoscopy and pathological findings. Upper gastrointestinal sarcoidosis was diagnosed based on the presence of noncaseating epithelioid cell granuloma in the lamina propria in the stomach or duodenum. In conclusion, the possibility of upper gastrointestinal sarcoidosis should be considered in patients with epigastric symptoms and a history of sarcoidosis, even in those with stable disease.

The positive feedback system using a fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the transcription activation domain of herpes simplex virus VP16 (GAL4-VP16), in which GAL4-VP16 promotes its own expression as well as that of a reporter gene product, is useful for efficient transgene expression from plasmid DNA. In this study, the transcription activation domains of endogenous proteins, instead of VP16, were fused to the GAL4 DNA binding domain, and the positive feedback systems employing the novel fusion proteins were examined. Plasmid DNAs encoding the transcription factors were introduced into mouse Hepa 1-6 cells by electroporation and lipofection. Among CREB-binding protein (226-460), sterol regulatory element-binding protein-1 (1-140), p53 (1-70), and Med15 (9-73), the CREB-binding protein functioned efficiently as an activator. These results indicated that the GAL4-CREB-binding protein is useful for enhanced transgene expression by the positive feedback system.

Multicentric Castleman's Disease (MCD) is a systemic disease characterized by generalized lymphadenopathy and the proliferation of plasma cells. The development of MCD in a patient with preexisting sarcoidosis has not been previously reported. We herein describe a case of MCD developing in a 78-year-old woman with a 17-year history of sarcoidosis. The patient's serum interleukin-6 (IL-6) levels were only slightly elevated; however, the IL-6 levels in the fluid of both pleural effusion and ascites were markedly elevated. The administration of steroid-pulse therapy and prednisolone was ineffective in treating the MCD, although treatment with tocilizumab proved highly effective.

Lactoferrin, a multifunctional protein with antimicrobial activity, is a component of the innate immune system. It may possibly prevent clinical isolates of Pseudomonas aeruginosa from developing biofilm, but this hypothesis is yet to be widely accepted. We evaluated the in vitro effects of lactoferrin on biofilm formation by various clinical isolates of P. aeruginosa using a modified method of the microtiter plate biofilm assay. Lactoferrin significantly inhibited biofilm formation in these isolates. The effect was the most marked at 2 mg/ml, which suggested that an optimal concentration of lactoferrin might exist. Lactoferrin inhibited biofilm formation in eight of nine clinical isolates after 1 day of incubation; however, the inhibitory effects were maintained until 7 days of incubation in only two of those eight strains. Suppression of biofilm formation may be caused by a mechanism that is independent of the bactericidal effects of lactoferrin because the number of viable bacteria was not influenced by lactoferrin under the experimental conditions. Supplementation of lactoferrin to preformed biofilm demonstrated a reduction in biofilm, which suggests that lactoferrin may have a destructive effect on biofilm. Pretreatment with ferric chloride partially restored biofilm formation, suggesting an iron-chelating action may be involved in the inhibitory mechanism of lactoferrin. These results suggest that lactoferrin provides inhibitory effects on biofilm formation in many clinical isolates of P. aeruginosa and that it may also have destructive effects on preformed biofilm, but further research using multiple clinical strains should be undertaken to clarify if those effects are universal.

The intranuclear disposition of plasmid DNA is extremely important for transgene expression. The interactions between the plasmid DNA and the histone proteins are one of the keys for controlling the disposition. In this study, the effects of a left-handedly curved sequence (20-40 repeated A•T tracts) on transgene expression from a plasmid were examined in vivo. A naked luciferase plasmid with the curved sequence was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Interestingly, transgene expression was markedly increased by the addition of the curved sequence. An analysis of the nucleosome positions near the left-handedly curved sequence suggested that the sequence functions as an acceptor of the histone core and allows nucleosome sliding, resulting in transcriptional activation. These results suggested that the designed curved DNA sequences could control transgene expression from plasmid DNAs in vivo.

Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (α and β phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5–ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at Pβ, whereas ADPR is attacked at Pα. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site.

Escherichia coli Orf135 protein is thought to be an enzyme that efficiently hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-hydroxy-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP, thus preventing mutations in cells caused by unfavorable base pairing. Nucleotide pool sanitization by Orf135 is important since organisms are continually subjected to potential damage by reactive oxygen species produced during respiration. It is known that the frequency of spontaneous and H2O2-induced mutations is two to threefold higher in the orf135- strain compared with the wild-type. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, although they recognize various substrates and possess a variety of substrate binding pockets. We are interested in delineating the mechanism by which Orf135 recognizes oxidatively damaged nucleotides. To this end, we are investigating the tertiary structure of Orf135 and its interaction with substrate using NMR. Herein, we report on the 1H, 13C and 15N resonance assignments of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrate.

With the introduction of heart valve prostheses cardiac valvular disease has become much more accessible to therapeutic options. However, currently available prostheses display significant limitations, such as limited long-term durability (biological prostheses) and a long-term necessity for anticoagulation therapy. Hence, alternative prosthesis types have been extensively explored in recent years particularly aiming at the development of vital and regenerative prostheses by means of tissue engineering. In the scientific field, different competing concepts have been introduced, including biological or synthetic scaffolds which can be further enhanced by cellular or extracellular components to promote further in vivo development of the prosthesis after implantation. Nowadays, decellularized donor heart valves are among the most advanced prosthesis types experiencing growing clinical attention and widespread use.

The loss of tumor suppressor proteins, p53 and retinoblastoma (Rb), causes genomic instability. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is a major oxidatively damaged base that induces G:C→T:A transversions in cells. In this study, the effects of p53 and Rb reductions on the mutagenicity of GO in DNA were investigated, using a supF shuttle plasmid propagated in human U2OS and HT1080 cells. The p53 and Rb proteins were individually knocked-down by siRNAs, and then the plasmid DNA containing GO was introduced into the knocked-down cells. The knock-downs of p53 and Rb had quite weak effects on mutagenesis by GO in the shuttle plasmid. These results suggested that p53 and Rb minimally affect the GO-induced mutagenic processes in living cells.

The base moieties of DNA precursors in the nucleotide pool are subjected to oxidative damage, and the formation of damaged DNA precursors is an important source of mutagenesis. 8-Hydroxy-2'-deoxyguanosine 5'-triphosphate, also known by the name of its keto-enol tautomer as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate, and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate have been identified as the major products of in vitro oxidation reactions. The mutagenicities of these damaged precursors in living cells will be summarized in this review. In addition, the roles of the nucleotide pool sanitization and DNA repair enzymes, and the translesion synthesis DNA polymerases will be described.

Objective: Successful animal and clinical implantation of decellularised heart valves has been performed in the pulmonary position. Comparable results have not yet been achieved for the aortic position with the high haemodynamic demands of the systemic circulation andthechallengingimplantationprocedure.Methods:Allogenicaorticvalves(n = 10)weredecellularised usingdetergents(decellularised aortic valves (dAoVs)). Five prostheses were analysed for decellularisation quality and scaffold preservation. Five valves were orthotopically implanted in juvenile sheep in a subcoronary technique. After 5 months, echocardiography, immunohistology, histology, electron microscopy and western blot (WB) were used for analysis. Results: All animals survived the follow-up with increased body weight (38.8 2.8 kg vs 56.0 2.6 kg, p < 0.001).Afterimplantation, threedAoVsshowednegligibleandtwoothersminorinsufficiency(I),whichremainedunchangedatexplantation. Effective orifice area increased slightly (1.1 0.2 cm 2 vs 1.6 0.3 cm 2 , p = 0.051). Explanted dAoVs (n = 4) showed excellent macroscopy with minor soft-tissue nodules observed at the free cusp margins of only one dAoV. No valve showed any signs of thrombosis or calcification. On microscopic evaluation, the cusp architecture was preserved with an almost complete endothelial repopulation as confirmed by vimentin + /von Willebrand factor (vWF + )-staining, WB of endothelial markers (eNOS/vWF) and scanning electron microscopy (SEM). Partial interstitial reseeding with vimentin + /alpha-smooth muscle (asm + )-cells was noted. Quantitative measurement of collagen-IV, collagen-I, laminin and elastin (WB) demonstrated preserved scaffold composition as compared to native tissue. Conclusion: The dAoVs showed excellent functional outcome at 5 months in a subcoronary model of juvenile sheep. Advanced endothelial and nascent interstitial repopulation, with preserved structural integrity under the high-shear-stress milieu of the aortic valve, encourage further long-term studies. # 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

To enhance the levels of transgene expression from plasmid-based nonviral vectors, replicating plasmids containing the SV40 origin and the SV40 large T antigen gene, as a model replicating unit, were constructed. The replicating luciferase plasmid DNA produced the luciferase protein more efficiently than the non-replicating luciferase plasmid DNA, as expected. Surprisingly, the introduction of the replicating plasmid DNA containing the Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) gene was highly cytotoxic and caused cell death without nucleoside analogs. Our results confirm that transgenes on a replicating plasmid represent an excellent tool for effective protein production and suggest that efficient production of the Dm-dNK protein in tumor cells could be an attractive cancer therapy.