Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induces Post-Translational Modifications of AKAP121, DRP1 and OPA1 That Promote Mitochondrial Fission

Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate- treated neonatal rat ventricular cardiomyocytes (NRVCs). Palmitate exposure to NRVCs initially activates mitochondrial respiration, coupled with increased mitochondrial membrane potential and adenosine triphosphate (ATP) synthesis. However, long-term exposure to palmitate (\u3e8h) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of A-kinase anchor protein (AKAP121) leading to reduced phosphorylation of DRP1 at Ser637 and altered proteolytic processing of OPA1. Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy. 38 pp; includes supplemental materials

Advancing a Conceptual Model of Evidence-Based Practice Implementation in Public Service Sectors

Implementation science is a quickly growing discipline. Lessons learned from business and medical settings are being applied but it is unclear how well they translate to settings with different historical origins and customs (e.g., public mental health, social service, alcohol/drug sectors). The purpose of this paper is to propose a multi-level, four phase model of the implementation process (i.e., Exploration, Adoption/Preparation, Implementation, Sustainment), derived from extant literature, and apply it to public sector services. We highlight features of the model likely to be particularly important in each phase, while considering the outer and inner contexts (i.e., levels) of public sector service systems

Characterization of a Novel Close-to-Root Papillomavirus from a Florida Manatee by Using Multiply Primed Rolling-Circle Amplification: Trichechus manatus latirostris Papillomavirus Type 1

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus

Iterative optimization yields Mcl-1–targeting stapled peptides with selective cytotoxicity to Mcl-1–dependent cancer cells

Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1–binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1–dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or “staple” that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation work-flow applicable to a host of challenging biomedical targets. Keywords: stapled peptide; Mcl-1; apoptosis; BH3 mimetic; inhibitorUnited States. Department of Energy (Contract DE-AC02-06CH11357)National Institutes of Health (U.S.) (Grant R01 GM110048

Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induces Post-Translational Modifications of AKAP121, DRP1 and OPA1 That Promote Mitochondrial Fission

Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate- treated neonatal rat ventricular cardiomyocytes (NRVCs). Palmitate exposure to NRVCs initially activates mitochondrial respiration, coupled with increased mitochondrial membrane potential and adenosine triphosphate (ATP) synthesis. However, long-term exposure to palmitate (\u3e8h) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of A-kinase anchor protein (AKAP121) leading to reduced phosphorylation of DRP1 at Ser637 and altered proteolytic processing of OPA1. Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy. 38 pp; includes supplemental materials

Thermodynamic parameters for thermal unfolding of TmPV4 oligonucleotides.

<p>Thermodynamic parameters for thermal unfolding of TmPV4 oligonucleotides.</p

Identification of G-quadruplex forming sequences in three manatee papillomaviruses

<div><p>The Florida manatee (<i>Trichechus manatus latirotris</i>) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.</p></div