Citations

Abstract Transplantation of pluripotent stem cells has proven beneficial in heart failure, yet the proteomic landscape underlying repair remains largely uncharacterized. In a genetic model of dilated cardiomyopathy elicited by pressure overload in the KCNJ11 (potassium inwardly rectifying channel, subfamily J, member 11) null mutant, proteome-wide profiles were here resolved by means of a systems approach prior to and following disease manifestation in the absence or presence of embryonic stem cell treatment. Comparative two-dimensional gel electrophoresis revealed a unique cardiomyopathic proteome in the absence of therapy, remodeled in response to stem cell treatment. Specifically, linear ion trap quadrupole-Orbitrap mass spectrometry determined the identities of 93 and 109 differentially expressed proteins from treated and untreated cardiomyopathic hearts, respectively. Mapped protein–protein relationships and corresponding neighborhoods incorporated the stem cell-dependent subproteome into a nonstochastic network with divergent composition from the stem cell-independent counterpart. Stem cell intervention produced a distinct proteome signature across a spectrum of biological processes ranging from energetic metabolism, oxidoreductases, and stress-related chaperones to processes supporting protein synthesis/degradation, signaling, and transport regulation, cell structure and scaffolding. In the absence of treatment, bioinformatic interrogation of the disease-only proteome network prioritized adverse cardiac outcomes, ablated or ameliorated following stem cell transplantation. Functional and structural measurements validated improved myocardial contractile performance, reduced ventricular size and decreased cardiac damage in the treated cohort. Unbiased systems assessment unmasked “cardiovascular development” as a prioritized biological function in stem cell-reconstructed cardiomyopathic hearts. Thus, embryonic stem cell treatment transformed the cardiomyopathic proteome to demote disease-associated adverse effects and sustain a procardiogenic developmental response, supplying a regenerative substrate for heart failure repair.

BACKGROUND Next-generation DNA sequencing (NGS) techniques have the potential to revolutionize molecular diagnostics; however, a thorough evaluation of these technologies is necessary to ensure their performance meets or exceeds that of current clinical sequencing methods. METHODS We evaluated the NimbleGen Sequence Capture 385K Human Custom Arrays for enrichment of 22 genes. We sequenced each sample on both the Roche 454 Genome Sequencer FLX (GS-FLX) and the Illumina Genome Analyzer II (GAII) to compare platform performance. RESULTS Although the sequence capture method allowed us to rapidly develop a large number of sequencing assays, we encountered difficulty enriching G+C–rich regions. Although a high proportion of reads consistently mapped outside of the targeted regions, >80% of targeted bases for the GAII and >30% of bases for the GS-FLX were covered by a read depth of ≥20, and >90% of bases for the GAII and >80% of bases for the GS-FLX were covered by a read depth of ≥5. We observed discrepancies among sequence variants identified by the different platforms. CONCLUSIONS Although oligonucleotide arrays are quick and easy to develop, some problematic regions may evade capture, necessitating sequential redesigning for complete optimization. Neither sequencing technology was able to detect every variant identified by Sanger sequencing because of well-known drawbacks of the NGS technologies. The rapidly decreasing error rates and costs of these technologies, however, coupled with advancing bioinformatic capabilities, make them an attractive option for molecular diagnostics in the very near future.

Background: We performed a laboratory-based study of the ankle-hindfoot complex in normal cadaveric ankles with simulated lateral ankle ligament injuries, utilizing an ankle stability testing device. The ankle stability testing device distinguished between intact, injury to anterior talofibular ligament (ATFL) and combined injury to ATFL and calcaneofibular ligament (CFL). Materials and Methods: Ankles were tested in an ankle stability testing device, with a specified inversion torque applied through the range of sagittal plane motion, then internal rotation torque applied through the entire range of sagittal plane motion. Three-dimensional motion of the calcaneus and talus in relation to the tibia were determined with a magnetic tracking system. The specimens were tested in the intact condition, then after sectioning the ATFL, then the CFL. Results: Upon sectioning the ATFL, there was a significant increase in the internal rotation motion at the tibiotalar joint. Subsequent sectioning of the CFL did not significantly increase ankle-hindfoot internal rotation, but it did significantly increase the motion in inversion testing. This increase occurred primarily at the tibiotalar level. Conclusion: The isolated ATFL injury was clearly distinguished from the intact condition by increased talar-tibial internal rotation, but not inversion. The more severe combined ATFL/CFL injury was distinguished from the ATFL injury condition with inversion torque. Clinical Relevance: Inversion torque may disclose combined ATFL/CFL injury, and internal rotation torque may be the key to distinguish isolated ATFL and combined ATFL/CFL injuries.