Improved Cardiac Function in Postischemic Rats Using an Optimized Cardiac Reprogramming Cocktail Delivered in a Single Novel Adeno-Associated Virus.

BACKGROUND: Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. METHODS: Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. RESULTS: We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. CONCLUSIONS: Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.

Two-dimensional ultrasonography and CEUS of a case with primary biliary cholangitis and vanishing bile duct syndrome.

Vanishing bile duct syndrome (VBDS) is a pathological concept that refers to the gradual reduction, destruction and disappearance of intrahepatic bile ducts caused by drugs, immunity, malignancy, and infections (including HIV and tuberculosis). Its clinical manifestation is cholestasis. The pathological diagnostic criteria for VBDS are the occurrence of intralobular vanishing bile ducts in more than 50% of 10 or more portal areas. At present, the diagnosis of VBDS still relies on liver biopsy. Contrast-enhanced ultrasound has been widely used in the diagnosis of liver-related diseases. The intravenous injection of microbubbles could enhance the observation of tissue microcirculation and significantly expand the possibility of ultrasound hemodynamic research. VBDS is a rare disease, and there are few reports on the early ultrasound and CEUS manifestations. The purpose of this report is to explore the unique performance of ultrasound and CEUS in the diagnosis of VBDS.

Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.

The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.

Epigenomic analysis of multilineage differentiation of human embryonic stem cells.

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Instant spectral assignment for advanced decision tree-driven mass spectrometry.

We have developed and implemented a sequence identification algorithm (inSeq) that processes tandem mass spectra in real-time using the mass spectrometer's (MS) onboard processors. The inSeq algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes ∼16 ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate (i) real-time prediction of peptide elution windows en masse (∼3 min width, 3,000 targets), (ii) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and (iii) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.