Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis.

Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.

Assessment of Risk Factors for Rupture in Breast Reconstruction Patients with Macrotextured Breast Implants.

BACKGROUND: Breast implants (BI) are widely used in plastic surgery, though they are not lifetime devices. Average life before rupture is reported to be around 10-15 years. No consensus exists regarding which factors are involved. OBJECTIVES: Following FDA recommendations, this study aims at identifying potential risk factors by evaluating their effect on BI rupture cases. METHODS: In this observational study, 763 BI patients were operated between 2003 and 2019, with a mean implant indwelling of 12.2 years. Patients that returned for follow-up were administered a questionnaire regarding postoperative lifestyle and habits. Implant rupture rate was 15.1%, while BI lifespan was 10.1 years. We obtained complete data from 191 breast implant patients (288 implants). Twenty-three potential risk factors were evaluated and divided in four categories: patient-related, surgery-related, postoperative complications/symptoms, and postoperative care/lifestyle habits. Odds Ratio (OR) for each factor was calculated. Linear regression analysis was calculated for those with a significant OR. RESULTS: We report 120 patients (195 implants) with intact and 71 (93 implants) with ruptured devices. BIs were macrotextured in 95.1% of cases (86.8% Allergan BIOCELL). OR was significant for underwire bra use (OR: 2.708), car seat belts (OR: 3.066), mammographic imaging (OR: 2.196), weightlifting (OR: 0.407) and carry-on heavy purses and backpacks (OR: 0.347). CONCLUSION: Wearing underwire bras, seat belts and undergoing mammography increases the risk of rupture. Weightlifting and carry heavy bags do not increase that risk. Implant rupture is directly linked with time of indwelling. Postoperative recommendations in BI patients should consider findings from our study, though larger multicenter studies should be encouraged. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Guiding Nipple-Areola Complex Reconstruction: Literature Review and Proposal of a New Decision-Making Algorithm.

BACKGROUND: Nipple-areola complex reconstruction (NAR) most commonly represents the finishing touch to breast reconstruction (BR). Nipple presence is particularly relevant to the patient's psyche, beyond any shadow of doubt. Many reconstructive options have been described in time. Surgery is easy, but final result is often disappointing on the long run. METHODS: The goal of this manuscript is to analyze and classify knowledge concerning NAR techniques and the factors that influence success, and then to elaborate a practical evidence-based algorithm. Out of the 3136 available articles as of August 8th, 2020, we selected 172 manuscripts that met inclusion criteria, which we subdivided into 5 main topics of discussion, being the various NAR techniques; patient factors (including patient selection, timing and ideal position); dressings; potential complications and finally, outcomes/patient satisfaction. RESULTS: We found 92 articles describing NAR techniques, 41 addressing patient factors (out of which 17 discussed patient selection, 14 described ideal NAC location, 10 described appropriate timing), 10 comparing dressings, 7 studying NAR complications, and 22 addressing outcomes and patient satisfaction. We elaborated a comprehensive decision-making algorithm to help narrow down the choice among NAR techniques, and choose the correct strategy according to the various scenarios, and particularly the BR technique and skin envelope. CONCLUSIONS: No single NAR technique provides definitive results, which is why we believe there is no "end-all be-all solution". NAR must be approached as a case-by-case situation. Furthermore, despite NAR being such a widely discussed topic in scientific literature, we still found a lack of clinical trials to allow for more thorough recommendations to be elaborated. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Transcriptional and epigenetic characterization of a new in vitro platform to model the formation of human pharyngeal endoderm.

BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.

Immediate prepectoral breast reconstruction using an ADM with smooth round implants: A prospective observational cohort study.

Prepectoral breast reconstruction using acellular dermal matrices (ADMs) is well established and used in candidates for nipple/skin-sparing mastectomies; it is based on many different matrices and a great variability in breast implant selection. We describe our experience and clinical outcomes using Braxon® ADMs and smooth round breast implants. Females aged 18-80 years who underwent mastectomies with immediate prepectoral reconstruction between April 2019 and April 2021 were prospectively included. Complications were classified as mastectomy-related (hematoma, necrosis) or reconstruction-related (seroma, infection, red breast syndrome). Binary logistic regression analysis was performed to assess correlation between complication rate and selected variables, which were analyzed per breast with Kruskal-Wallis H test. Fifty-eight patients (102 breasts) received 45 bilateral and 12 unilateral procedures. Drains collected 485.9 cc [range: 100-1260] and were removed 15.7 days [range: 6-29] postoperatively. We report 41 complications (40.2%): 33 mastectomy-related, 8 reconstruction-related. Reoperation occurred in 14 patients: 7 wound debridement and revisions under local anesthesia; and 7 explantation. Implant loss rate was 6.8%. Mastectomy and reconstruction complications were not correlated with any variable. In conclusion, we found prepectoral reconstruction with Braxon® ADMs and smooth round implants to be associated with acceptable complication rates that are not influenced by any patient- or surgery-related factors. Drainage volume is comparable to other breast implant reconstructive techniques, but drains are left in place for longer.

Human iPSC modeling of heart disease for drug development.

Human induced pluripotent stem cells (hiPSCs) have emerged as a promising platform for pharmacogenomics and drug development. In cardiology, they make it possible to produce unlimited numbers of patient-specific human cells that reproduce hallmark features of heart disease in the culture dish. Their potential applications include the discovery of mechanism-specific therapeutics, the evaluation of safety and efficacy in a human context before a drug candidate reaches patients, and the stratification of patients for clinical trials. Although this new technology has the potential to revolutionize drug discovery, translational hurdles have hindered its widespread adoption for pharmaceutical development. Here we discuss recent progress in overcoming these hurdles that should facilitate the use of hiPSCs to develop new medicines and individualize therapies for heart disease.

Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes.

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy.

Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20.

Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs.

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.