An RNA-immunoprecipitation via CRISPR/dCas13 reveals an interaction between the SARS-CoV-2 5'UTR RNA and the process of human lipid metabolism.

We herein elucidate the function of SARS-CoV-2derived 5'UTR in the human cells. 5'UTR bound host cellular RNAs were immunoprecipitated by gRNA-dCas13 (targeting luciferase RNA fused to SARS-CoV-2 5'UTR) in HEK293T and A549 cells. The 5'UTR bound RNA extractions were predominantly enriched for regulating lipid metabolism. Overexpression of SARS-CoV-2 5'UTR RNA altered the expression of factors involved in the process of the human Mevalonate pathway. In addition, we found that HMG-CoA reductase inhibitors were shown to suppress SARS-CoV-2 5'UTR-mediated translation activities. In conclusion, we deduce the array of host RNAs interacting with SARS-CoV-2 5'UTR that drives SARS-CoV-2 translation and influences host metabolic pathways.

Journey Toward Improved Long-Term Outcomes After Norwood-Sano Procedure: Focus on the Aortic Arch Reconstruction.

The disadvantage of right ventricle-to-pulmonary artery (RV-PA) shunt is the need for more unplanned interventions to address stenosis in the shunt or branch pulmonary arteries, as compared to the modified Blalock-Taussig shunt group. Ring-enforced RV-PA PTFE conduit and dunk technique minimized these complications and right ventricle (RV) damage. Aortic arch obstruction increases afterload and leads to ventricular dysfunction and tricuspid regurgitation; therefore, most surgeons prefer to use homograft, autologous pericardium, or bovine pericardium to reconstruct the neoaorta. Artificial materials decrease the elastic properties, increase wall stiffness, and decrease the distensibility of the aorta; and as a result, RV function gradually deteriorates. This inelastic reconstructed aorta may be one of the reasons why long-term outcomes after the Fontan procedure are worse in hypoplastic left heart syndrome (HLHS) patients, in comparison to non-HLHS. Reconstruction of the neoaorta without any patch materials, or at least techniques that largely minimize the use of non-autologous materials, will offer a further refinement of our ability to optimize ventriculoarterial coupling and thereby long-term RV function.

Intrinsic activation of cardiosphere-derived cells enhances myocardial repair.

OBJECTIVE: Permanent loss of cardiomyocytes after myocardial infarction results in irreversible damage to cardiac function. The present study aims to enhance the cardiomyogenic efficiency of cardiosphere-derived cells (CDCs) to develop into large populations of cardiomyocytes by intrinsic activation of cardio-specific differentiation factors (Gata4, Mef2c, Nkx2-5, Hand2, and Tnnt2) by a CRISPR/dCas9 assisted transcriptional enhancement system. METHODS: Exhaustive screening was performed to identify the specific sequences in endogenous regulatory regions (enhancers and promoters) responsible for transcriptional activation of the target genes, via a CRISPR/dCas9 system fused with transcriptional activator VP64 (CRISPR-dCas9-VP64). In a rat model of acute myocardial infarction, we compared the regenerative potential and functional benefits of CDCs with or without transcriptional activation. RESULTS: We identified a panel of specific CRISPR RNA targeting the enhancers and promoters, which demonstrated significantly higher expression of differentiation factors of Gata4, Hand2, and Tnnt2. The group of CDCs with transcriptional activator VP64 (CDC with VP64) showed significant improvement in the left ventricular ejection fraction (61.9% vs 52.5% and 44.1% in the CDC without transcriptional activation group and control) and decreased scar area in the heart. CONCLUSIONS: We have identified endogenous regulatory regions responsible for an intrinsic activation of cardio-specific differentiation factors assisted via a CRISPR/dCas9 gene transcriptional system. The CRISPR/dCas9 system may provide an efficient and effective means of regulating Tnnt2 gene activation within stem cells. Subsequently, this system can be used to enhance transplanted CDCs differentiation potential within ischemic myocardia to better therapeutic outcomes of patients with ischemic heart disease.

Stem Cell Therapy in Heart Disease: Limitations and Future Possibilities.

Heart diseases are one of the major causes of morbidity and mortality worldwide. Despite major advances in drug and interventional therapies, surgical procedures, and organ transplantation, further research into new therapeutic options is still necessary. Stem cell therapy has emerged as one option for the treatment of a variety of heart diseases. Although a large number of clinical trials have shown stem cell therapy to be a promising therapeutic approach, the results obtained from these clinical studies are inconsistent, and stem cell-based improvements of heart performance and cardiac remodeling were found to be quite limited. Since the precise mechanisms underlying the therapeutic actions of stem cells are still under debate, researchers have developed a variety of strategies to improve and boost the potency of stem cells in repair. In this Reviews, we summarize both the current therapeutic strategies using stem cells and future directions for enhancing stem cell potency.

An overview of stem cell therapy for paediatric heart failure.

Significant achievements in paediatric cardiology, surgical treatment and intensive care of congenital heart disease have drastically changed clinical outcomes for paediatric patients. Nevertheless, late-onset heart failure in children after staged surgeries still remains a serious concern in the medical community. Heart transplantation is an option for treatment; however, the shortage of available organs is a persistent problem in many developed countries. In order to resolve these issues, advanced technologies, such as innovative mechanical circulatory support devices and regenerative therapies, are strongly desired. Accumulated evidence regarding cell-based cardiac regenerative therapies has suggested their safety and efficacy in treating adult heart failure. Given that young children seem to have a higher regenerative capacity than adults, stem cell-based therapies appear a promising treatment option for paediatric heart failure as well. Based on the findings from past trials and studies, we present the potential of various different types of stem cells, ranging from bone marrow mononuclear cells to cardiosphere-derived stem cells for use in paediatric cell-based therapies. Here, we assess both the current challenges associated with cell-based therapies and novel strategies that may be implemented in the future to advance stem cell therapy in the paediatric population.

Impact of Cardiac Progenitor Cells on Heart Failure and Survival in Single Ventricle Congenital Heart Disease.

RATIONALE: Intracoronary administration of cardiosphere-derived cells (CDCs) in patients with single ventricles resulted in a short-term improvement in cardiac function. OBJECTIVE: To test the hypothesis that CDC infusion is associated with improved cardiac function and reduced mortality in patients with heart failure. METHODS AND RESULTS: We evaluated the effectiveness of CDCs using an integrated cohort study in 101 patients with single ventricles, including 41 patients who received CDC infusion and 60 controls treated with staged palliation alone. Heart failure with preserved ejection fraction (EF) or reduced EF was stratified by the cardiac function after surgical reconstruction. The main outcome measure was to evaluate the magnitude of improvement in cardiac function and all-cause mortality at 2 years. Animal studies were conducted to clarify the underlying mechanisms of heart failure with preserved EF and heart failure with reduced EF phenotypes. At 2 years, CDC infusion increased ventricular function (stage 2: +8.4±10.0% versus +1.6±6.4%, P=0.03; stage 3: +7.9±7.5% versus -1.1±5.5%, P<0.001) compared with controls. In all available follow-up data, survival did not differ between the 2 groups (log-rank P=0.225), whereas overall patients treated by CDCs had lower incidences of late failure (P=0.022), adverse events (P=0.013), and catheter intervention (P=0.005) compared with controls. CDC infusion was associated with a lower risk of adverse events (hazard ratio, 0.411; 95% CI, 0.179-0.942; P=0.036). Notably, CDC infusion reduced mortality (P=0.038) and late complications (P<0.05) in patients with heart failure with reduced EF but not with heart failure with preserved EF. CDC-treated rats significantly reversed myocardial fibrosis with differential collagen deposition and inflammatory responses between the heart failure phenotypes. CONCLUSIONS: CDC administration in patients with single ventricles showed favorable effects on ventricular function and was associated with reduced late complications except for all-cause mortality after staged procedures. Patients with heart failure with reduced EF but not heart failure with preserved EF treated by CDCs resulted in significant improvement in clinical outcome. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01273857 and NCT01829750.

10-year results of On-X bileaflet mechanical heart valve in the aortic position: low target INR regimen in Japanese.

OBJECTIVES: This study was designed to establish clinical outcomes after aortic valve replacement (AVR) with On-X bileaflet mechanical heart valve. METHODS: Between 2006 and 2014, AVR was performed to 686 patients. Of them, 78 patients using On-X valve were enrolled. The mean age was 65 ± 11 years (ranged 33-85); 65% were men; and 81% were in sinus rhythm preoperatively. Calcific or degenerative tricuspid aortic valve was present in 73%. Concomitant procedures included coronary artery bypass grafting (22%), Bentall (8%), mitral valve procedure (3%) and other (9%). They postoperatively received lower dose warfarin [international normalized ratio (INR), 1.5-2.0] and 100 mg aspirin daily. The follow-up duration averaged 5 years (386.6 patient-years). The follow-up rate was 97.3%. RESULTS: In-hospital mortality rates were 3.8% (n = 3). Late mortality rates were 2.6% per patient-years (n = 10). Five-year Kaplan-Meier survival rates were 84%. Freedom from major adverse valve-related stroke and cerebral bleeding events was 93.3% (n = 5, 1.29% per patient-years) and 98.6% (n = 1, 0.26% per patient-years, mild subdural hematoma). The incidence of stroke was two patients of transient ischemic attack, two patients of paralytic event, one patient of asymptomatic stroke (self-interruption of anticoagulation). The median INR was 1.92 ± 0.53, ranged from 1.00 to 8.98 (n = 1181) and 51% of all measured INR values were in the therapeutic range of 1.5-2.0. CONCLUSIONS: AVR using On-X valve with low target INR regimen and low-dose aspirin resulted in a significantly low risk of bleeding.

The Risk Factors for Metastasis in Non-Ampullary Duodenal Neuroendocrine Tumors Measuring 20 mm or Less in Diameter.

BACKGROUND/AIMS: The treatment strategy for non-ampullary duodenal neuroendocrine tumors (NAD-NETs) ≤20 mm in diameter has not been established. In this study, we aimed to evaluate the detailed characteristics of NAD-NETs ≤20 mm in diameter to clarify the risk factors of subsequent metastasis. METHODS: The patients with NAD-NETs ≤20 mm in diameter who had been treated at 12 institutions between 1992 and 2013 were enrolled. Clinical records were retrieved, and histopathological findings of all cases were centrally reviewed by 2 pathologists. RESULTS: We studied 49 patients with a mean follow-up period of 66.5 months. Thirty-five patients were initially treated with endoscopic resection (ER), and 14 with surgery. A univariate analysis revealed the ORs and 95% CIs of the risk factors for metastasis were lymphovascular invasion (12.5 [2.01-77.9]), multiple tumors (9.75 [1.46-65.4]), a tumor size of 11-20 mm (6.67 [1.21-36.6]), and World Health Organization grade G2 (7.13 [1.16-43.9]). Five-year overall and disease-specific survival rates were 86.1 and 97.2%, respectively. CONCLUSION: This is the first study to demonstrate the risk factors of metastasis in NAD-NETs ≤20 mm in diameter. These findings may be helpful for determining the appropriate therapeutic approach and the clinical strategy of treatment following ER.

A reoperation of thrombosed On-X valve detected by multidetector-row computed tomography.

A 62-year-old female patient underwent mitral valve replacement with a 31/33-mm On-X valve for ischemic mitral valve regurgitation. Three months later, transthoracic echocardiography incidentally showed a blocked leaflet with 6 mmHg of mean pressure gradient and 2.4 cm(2) of mitral valve orifice area. Transesophageal echocardiography could not detect thrombus. Electrocardiographically gated multidetector-row computed tomography (MDCT) clearly demonstrated a blocked leaflet in the close position and thrombus (2 cm in length, 0.4 cm(2) in area) attached onto the atrial aspect of the leaflet. These findings observed by MDCT were confirmed at reoperation. MDCT was useful diagnostic method for visualizing prosthetic valve thrombosis.

Mid-term results of 17-mm St. Jude Medical Regent prosthetic valves in elder patients with small aortic annuli: comparison with 19-mm bioprosthetic valves.

This study was designed to compare the mid-term outcomes after aortic valve replacement (AVR) between 17-mm mechanical heart valves (MV) and 19-mm bioprosthetic valves (BV) in elderly patients with small aortic annuli. Between 2000 and 2011, 127 consecutive patients (mean age 79 years; 87 % female) underwent AVR for aortic valve stenosis with a small aortic annulus. 19-mm BV (n = 67) was implanted. When the 19-mm BV did not fit the annulus, 17-mm St. Jude Medical Regent prosthetic mechanical valve (n = 60) was used instead of an aortic root-enlargement procedure. The follow-up rate was 94.0 % in the BV group, and 98.5 % in the MV group. No significant differences in survival rate and valve-related complications were found between the 2 groups. In-hospital mortality rates were 1.5 % (n = 1) in the BV group and 5.0 % (n = 3) in the MV group. Late mortality rates were 3.9 % per patient-years (p-y; n = 8) in the BV group, and 6.0 % per p-y (n = 10) in the MV group. Five-year Kaplan-Meier survival rates were 62 % in the BV group, and 72 % in the MV group (log-rank P = 0.280). Freedom from major adverse valve-related stroke and cerebral bleeding events was 92.5 and 98.5 % in the BV group, and 94.7 and 100 % in the MV group. AVR using 17-mm MV in elder patients with small aortic annuli provided equivalent mid-term clinical results to that with 19-mm BV.

Beyerane derivatives and a sesquiterpene dimer from Japanese Cypress (Chamaecyparis obtusa).

In the course of studies on biological active constituents from woody plants, we previously reported the isolation of many lignan derivatives as neurite outgrowth-promoting compounds from an ethyl acetate soluble fraction of Japanese Cypress (Chamaecyparis obtusa). Further chemical investigation on the residual parts of the ethyl acetate soluble fraction of a methanol extract of Japanese Cypress resulted in the isolation of four new beyerene type derivatives and a novel sesquiterpene dimer formed between cryptomeridiol and hinokiic acid. Their structures were elucidated as 18-O-(Z)-p-coumaroylbeyer-15-ene-18-ol (1), 18-O-(E)-p-coumaroylbeyer-15-ene-18-ol (2), 18-O-(E)-p-coumaroylbeyer-15-ene-11beta,18-diol (3), 18-O-(Z)-p-coumaroylbeyer-15-ene-11beta,18-diol (4) and ent-cryptomeridiol-4-yl-hinokiiate (5) by (1)H-NMR, (13)C-NMR, 2D-NMR, and HR-MS spectral analysis.

Neurite outgrowth-promoting active constituents of the Japanese cypress (Chamaecyparis obtusa).

In the screening of biologically active constituents from woody plants, the methanol extract of leaves of Chamaecyparis obtusa showed potent neurite outgrowth-promoting activity in neuronal PC12 cells. The ethyl acetate-soluble fraction of the methanol extract showed potent activity and was separated by means of various chromatographic methods to give the two new compounds 1 and 2, as well as 11 known lignan and sesquiterpene derivatives. The structures of the new compounds were determined to be 9-O-acetyldihydrosesamin (1) and 9-O-(11-hydroxyeudesman-4-yl)dihydrosesamin (2), respectively, in NMR studies including 2D-NMR experiments. Of the 13 compounds, the known compound hinokinin (5) and the new compound 2 showed potent neurite outgrowth-promoting activity in PC 12 cells.

New diarylheptanoids from Alnus japonica and their antioxidative activity.

In the course of research on the bioactive constituents of woody plants in the Cyugoku area of Japan, a methanol extract of the leaves of Alnus japonica were found to have strong antioxidative activity. Ethyl acetate soluble and n-buthanol soluble fractions of the methanol extract had a potent antioxidative effect. Both fractions were purified by silica gel column chromatography and HPLC using an ODS column to give four new diarylheptanoids along with known diarylheptanoids and flavonoids. These new compounds were elucidated to be 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-(4-hydroxyphenyl)-3-heptanone-5-O-beta-D-xylopyranoside (1), 1-(3,4-dihydroxyphenyl)-5-hydroxy-7-(4-hydroxyphenyl)-3-heptanone-5-O-beta-D-xylopyranoside (2), 1,7-bis-(3,4-dihydroxyphenyl)-5-hydroxy-3-heptanone-5-O-[2-(2-methylbutenoyl)]-beta-D-xylopyranoside (3) and 1,7-bis-(3,4-dihydroxyphenyl)-5-methoxy-3-heptanone (4) using spectral methods and especially 1H-, 13C-NMR and 2D-NMR measurements. The isolated compounds including their main constituent, oregonin (5), were tested for antioxidative activity. Some of these compounds having two catechol structures showed potent antioxidative activity. Compounds having one catechol structure showed moderate antioxidative activity, but a peracetate of 5 having no catechol structure exhibited no antioxidative activity. Thus the catechol structure of the diarylheptanoids is indispensable for antioxidative activity.