A modified high-resolution melting-based assay (HRM) to identify the SARS-CoV-2 N501Y variant.

High-resolution melting (HRM) analysis is a PCR-based method that can be used as a screening assay to identify SARS-CoV-2 variants. However, conventional HRM assays hardly detect slight melting temperature differences at the A-T to T-A transversion. As the N501Y substitution results from A-T to T-A transversion in A23063, few or no studies have shown that a conventional HRM assay can identify N501Y variants. This study successfully developed an HRM assay for identifying the N501Y mutation. Two HRM assays were used in the N501 site because the discrimination results were affected by the virus copy numbers. One is a conventional HRM assay (detectable at 103-106 copies/mL) and the other is a modified HRM assay by adding the wild-type fragment (detectable at 105-1010 copies/mL). Using viral RNAs from cultured variants (Alpha, Beta, and Gamma), a modified HRM assay correctly identified three N501Y variants because of high-copy-number RNAs in those viral samples. The sensitivity and specificity of the N501Y assay were 93.3% and 100%, respectively, based on 209 clinical samples (105 for N501; 104 for N501Y). These results suggest that our HRM-based assay is a powerful tool for rapidly identifying various SARS-CoV-2 variants.

Rapid Identification of SARS-CoV-2 Omicron BA.5 Spike Mutation F486V in Clinical Specimens Using a High-Resolution Melting-Based Assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.5 emerged as of February 2022 and replaced the earlier Omicron subvariants BA.1 and BA.2. COVID-19 genomic surveillance should be continued as new variants seem to subsequently appear, including post-BA.5 subvariants. A rapid assay is needed to differentiate between the currently dominant BA.5 variant and other variants. This study successfully developed a high-resolution melting (HRM)-based assay for BA.4/5-characteristic spike mutation F486V detection and demonstrated that our assay could discriminate between BA.1, BA.2, and BA.5 subvariants in clinical specimens. The mutational spectra at two regions (G446/L452 and F486) for the variant-selective HRM analysis was the focus of our assay. The mutational spectra used as the basis to identify each Omicron subvariant were as follows: BA.1 (G446S/L452/F486), BA.2 (G446/L452/F486), and BA.4/5 (G446/L452R/F486V). Upon mutation-coding RNA fragment analysis, the wild-type fragments melting curves were distinct from those of the mutant fragments. Based on the analysis of 120 clinical samples (40 each of subvariants BA.1, BA.2, and BA.5), this method's sensitivity and specificity were determined to be more than 95% and 100%, respectively. These results clearly demonstrate that this HRM-based assay is a simple screening method for monitoring Omicron subvariant evolution.

Strain selection and scale-up fermentation for FR901379 acylase production by Streptomyces sp. no. 6907.

Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5'-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.

Clinical benefit of statin pretreatment in patients undergoing percutaneous coronary intervention: a collaborative patient-level meta-analysis of 13 randomized studies.

BACKGROUND: Previous studies suggested that statin pretreatment reduces cardiac events in patients undergoing percutaneous coronary intervention. However, most data were observational, and single randomized trials included limited numbers of patients. METHODS AND RESULTS: We performed a collaborative meta-analysis using individual patient data from 13 randomized studies in which 3341 patients received either high-dose statin (n=1692) or no statin/low-dose statin (n=1649) before percutaneous coronary intervention, with all patients receiving statin therapy after intervention. Occurrence of periprocedural myocardial infarction, defined as postintervention creatine kinase-MB increase ≥3 times the upper limit of normal, and 30-day major adverse cardiac events (death, myocardial infarction, target-vessel revascularization) was evaluated. Incidence of periprocedural myocardial infarction was 7.0% in the high-dose statin versus 11.9% in the control group, which corresponds to a 44% risk reduction in the active-treatment arm (odds ratio by fixed-effects model 0.56, 95% confidence interval, 0.44 to 0.71, P<0.00001). The rate of major adverse cardiac events at 30 days was significantly lower in the high-dose statin group (7.4% versus 12.6%, a 44% risk reduction; P<0.00001), and 1-month major adverse cardiac events, excluding periprocedural events, were also reduced (0.6% versus 1.4%; P=0.05). The benefit of high-dose statins was realized irrespective of clinical presentation (P for interaction=0.43) and was maintained across various subgroups but appeared greater in the subgroup with elevated baseline C-reactive protein levels (n=734; 68% risk reduction for periprocedural myocardial infarction versus 31% in those 1861 patients with normal CRP; P for quantitative interaction=0.025). CONCLUSIONS: High-dose statin pretreatment leads to a significant reduction in periprocedural myocardial infarction and 30-day adverse events in patients undergoing percutaneous coronary intervention. This strategy should be considered in all patients with planned percutaneous coronary intervention.

Implantable cardioverter defibrillator therapy for congenital long QT syndrome: a single-center experience.

BACKGROUND: Long QT syndrome's (LQTS) marked heterogeneity necessitates both evidence-based and individualized therapeutic approaches. OBJECTIVE: This study sought to analyze a single LQTS specialty center's experience regarding the relationship between risk factors and appropriate ventricular fibrillation (VF)-terminating therapies among LQTS patients treated with an implantable cardioverter-defibrillator (ICD). METHODS: An internal review board-approved, retrospective analysis of the electronic medical records of 459 patients with genetically confirmed LQTS including the 51 patients (14 LQT1, 22 LQT2, and 15 LQT3) who received an ICD from 2000 to 2010 was performed. RESULTS: Twelve patients (24%, 4 LQT1, 8 LQT2) experienced an appropriate, VF-terminating therapy with an average follow-up of 7.3 years, including 7 of 17 LQT2 female patients but none of the 15 LQT3 patients. Conversely, 15 (29%) patients (8 LQT3) have experienced an inappropriate shock. Secondary prevention indications (P = .008), non-LQT3 genotype (P = .02), QTc ≥ 500 ms (P = .0008), documented syncope (P = .05), documented torsades de pointes (P = .003), and a negative family history (P = .0001) were most predictive of an appropriate therapy. Importantly, no LQT-related deaths have occurred among the 408 non-ICD-treated patients. CONCLUSION: The vast majority of LQTS patients can be treated effectively without an ICD. Potentially life-saving therapies were rendered at a 5% to 6% per year rate among those selected for ICD therapy; similar inappropriate shock frequencies were also noted. Secondary prevention, genotype, and QTc predicted those most likely to receive appropriate therapy. Although the ICD implant frequency is greatest among LQT3 patients, the greatest "save" rate has occurred among LQT2 women, who were assessed to be at high risk.

Creating frog heart as an organ: in vitro-induced heart functions as a circulatory organ in vivo.

Cardiomyocytes have been induced from various pluripotent cells, such as embryonic stem cells and myeloid stem cells; however, the generation of cardiac tissues beyond two-dimensional cell-sheets has not been reported. Creating higher order, three-dimensional structures that are unique to heart is the long-awaited next step in realizing cardiac regenerative medicine. We have previously shown that cardiomyocytes can be induced in vitro from undifferentiated cells (animal caps) excised from Xenopus embryos. Cardiomyocytes were induced by first dissociating the animal caps and then reaggregating them following treatment with activin. Here, we describe an interesting method for creating a complete ectopic heart in vivo, involving the introduction of in vitro-created tissue during early embryogenesis. Thus, animal cap reaggregates were transplanted into the abdomen of late-neurula-stage embryos, resulting in two-chambered hearts being formed. The dual-heart larvae matured into adult animals with transplanted hearts intact. Involvement of transplanted hearts in systemic circulation was demonstrated. Moreover, the ectopic hearts possessed higher order structures such as atrium and ventricle, and were morphologically, histologically, and electrophysiologically identical to original hearts. This system should facilitate the study of heart organogenesis and may promote a shift from tissue to organ engineering for clinical applications.

Role of smoking in the recurrence of atrial arrhythmias after cardioversion.

We aimed to determine whether smoking status affects the recurrence of atrial fibrillation or atrial flutter in patients after cardioversion. The clinical data of patients undergoing cardioversion for atrial flutter from January 1, 2000 to December 31, 2005 were prospectively collected. Arrhythmia recurrences were detected by retrospective review of comprehensive medical records and were determined using electrocardiography. The smoking history was prospectively collected through a standardized clinical form and subsequently categorized as lifetime nonsmoker, exsmoker, or current smoker. Univariate and multivariate associations with end points for clinical and lifestyle variables were assessed with Cox proportional hazards models. Women who were current smokers at cardioversion had a greater risk of atrial arrhythmia recurrence than did nonsmokers (hazard ratio 1.71, 95% confidence interval 1.10 to 2.67, p = 0.02). The increased risk of arrhythmia recurrence in female smokers was not seen in male smokers. Compared to lifetime nonsmokers, the mortality hazard ratio among men was 1.18 (95% confidence interval 0.88 to 1.58; p = 0.28) in exsmokers and 1.93 (95% confidence interval 1.20 to 3.11; p = 0.007) in current smokers. The risk of death after cardioversion was not increased in women. In conclusion, smoking is an independent predictor of atrial arrhythmia recurrence after cardioversion in women; however, an increased mortality risk, but not arrhythmia recurrence risk, was seen in men.

Percutaneous epicardial left atrial appendage closure: preliminary results of an electrogram guided approach.

BACKGROUND: Pharmacologic therapies to prevent stroke in atrial fibrillation (AF) have numerous limitations, prompting the development of device-based therapies. We investigated whether an electrogram-based approach using a novel hollow suture can safely capture and ligate the left atrial appendage (LAA). METHODS AND RESULTS: A novel system for closure of the LAA within the confines of the closed pericardium with a single sheath puncture was tested in 4 dogs. The tool used to grasp the appendage was fitted with electrodes and utilized electrical navigation to identify and confirm LAA capture. A hollow suture preloaded with a mechanical support wire to permit its manipulation and fluoroscopic visualization was advanced over the grasper, and the wire removed after the suture was positioned. The LAA was successfully closed in all dogs. In 2 dogs, after closure, a thoracotomy was performed and the LAA amputated without bleeding, confirming closure integrity. Necropsy confirmed closure in all animals. CONCLUSIONS: Using electrical navigation, percutaneous epicardial LAA ligation with a remotely tightened suture was performed successfully within the confines of the intact pericardial space. This technique may allow decreasing the risk of stroke in AF patients without the need for thoracotomy or an endocardially placed prosthetic device.

Randomized trial of statin administration for myocardial injury: is intensive lipid-lowering more beneficial than moderate lipid-lowering before percutaneous coronary intervention?

BACKGROUND: Minor myocardial damage after percutaneous coronary intervention (PCI) is associated with cardiac risks, which statins seem to reduce. The aim of this study was to examine whether intensive lipid-lowering therapy is more effective in decreasing the risk of cardiac injury after PCI than moderate lipid-lowering therapy. METHODS AND RESULTS: Subjects comprised 42 patients with stable angina without previous statin treatment, randomly assigned to either an intensive lipid-lowering group (Group A: target low-density lipoprotein-cholesterol (LDL-C)<70 mg/dl) or a moderate lipid-lowering group (Group B: target LDL-C<100 mg/dl) 2 weeks before PCI. All patients took statins to reach target LDL-C levels. Incidence of periprocedural myocardial injury was assessed by analyzing levels of creatine kinase myocardial isozyme (CK-MB) and cardiac troponin T (TnT) before and 6, 12 and 24 h after PCI. Minor myocardial damage was defined as TnT elevation to >0.01 ng/ml. Frequency of minor myocardial damage was 14.2% in Group A and 47.6% in Group B (p=0.043). CK-MB was above the upper limit of normal (ULN) in 19% of Group A and 33.3% of Group B (p=0.44), and CK-MB was >3x ULN in 9.5% of Group A and 19% of Group B (p=0.66). CONCLUSIONS: Intensive lipid-lowering therapy before PCI reduces minor myocardial damage during PCI with stenting compared with moderate lipid-lowering therapy.

Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells are a promising source of cardiomyocytes, but clinical application of ES cells has been hindered by the lack of reliable selective differentiation methods. Differentiation into any lineage is partly dependent on the regulatory mechanisms of normal early development. Although several signals, including bone morphogenetic protein (BMP), Wnt and FGF, are involved in heart development, scarce evidence is available about the exact signals that mediate cardiomyocyte differentiation. While investigating the involvement of BMP signaling in early heart formation in the mouse, we found that the BMP antagonist Noggin is transiently but strongly expressed in the heart-forming region during gastrulation and acts at the level of induction of mesendoderm to establish conditions conducive to cardiogenesis. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by inhibition of BMP signaling.

Amphibian in vitro heart induction: a simple and reliable model for the study of vertebrate cardiac development.

Amphibian embryos are an excellent model system for analyzing the mechanisms of vertebrate cardiogenesis. Studies of heart development in Xenopus have, for example, revealed that the inductive interaction of the heart primordia with the adjacent underlying endoderm and dorsal lip starts at the early stages of gastrulation. However, the molecular basis of those early inductive events and the genes expressed during the early phases of heart differentiation remain largely unknown. Amphibian blastula embryos contain pluripotent cells in their ectodermal region, called the "animal cap," which fortunately can be exploited for understanding a variety of organogenesis processes. Despite an enormous potential for analysis, the use of this system in cardiogenesis research has languished due to a lack of information concerning appropriate culture methods. Herein we report conditions for generating an in vitro heart induction system and present evidence from two types of in vivo transplantations, that the cultured heart rudiment can develop and function in the adult organism. It is expected that the fundamental principles established in this model system will provide a versatile research platform for a variety of organ engineering projects, including modifying in vitro organ growth with exogenous components (e.g. various growth factors) and developing methods for preparing tissue for transplantation.

[Regenerative medicine for heart disease].

Recent studies revealed that cardiomyocytes can be regenerated from both embryonic- and adult bone marrow-stem cells in animal experiment. Regenerated cardiomyocytes had fetal ventricular phenotype, expressed alpha 1, beta 1, and beta 2-adrenergic, and M2 cholinergic receptors, and revealed spontaneous beating. These cells could be transplanted into the recipient heart, and survive for long period. Clinical application of these cells had some problems at present. Embryonic stem cells had ethical problems, and had a possibility of making teratoma when undifferentiated cells could not be eliminated. Adult stem cells had a difficulty in isolation and culture expansion, since they were not yet well characterized. However, transplantation of regenerated cardiomyocytes would become a future method for the treatment of severe heart failure.