ALTERED PROFILES OF EXTRACELLULAR MITOCHONDRIAL DNA IN IMMUNOPARALYZED PEDIATRIC PATIENTS AFTER THERMAL INJURY.

ABSTRACT: Background: Thermal injury is a major cause of morbidity and mortality in the pediatric population worldwide with secondary infection being the most common acute complication. Suppression of innate and adaptive immune function is predictive of infection in pediatric burn patients, but little is known about the mechanisms causing these effects. Circulating mitochondrial DNA (mtDNA), which induces a proinflammatory signal, has been described in multiple disease states but has not been studied in pediatric burn injuries. This study examined the quantity of circulating mtDNA and mtDNA mutations in immunocompetent (IC) and immunoparalyzed (IP) pediatric burn patients. Methods: Circulating DNA was isolated from plasma of pediatric burn patients treated at Nationwide Children's Hospital Burn Center at early (1-3 days) and late (4-7 days) time points postinjury. These patients were categorized as IP or IC based on previously established immune function testing and secondary infection. Three mitochondrial genes, D loop, ND1, and ND4, were quantified by multiplexed qPCR to assess both mtDNA quantity and mutation load. Results: At the early time point, there were no differences in plasma mtDNA quantity; however, IC patients had a progressive increase in mtDNA over time when compared with IP patients (change in ND1 copy number over time 3,880 vs. 87 copies/day, P = 0.0004). Conversely, the IP group had an increase in mtDNA mutation burden over time. Conclusion: IC patients experienced a significant increase in circulating mtDNA quantity over time, demonstrating an association between increased mtDNA release and proinflammatory phenotype in the burn patients. IP patients had significant increases in mtDNA mutation load likely representative of degree of oxidative damage. Together, these data provide further insight into the inflammatory and immunological mechanisms after pediatric thermal injury.

Drp1/p53 interaction mediates p53 mitochondrial localization and dysfunction in septic cardiomyopathy.

BACKGROUND: Previous studies have implicated p53-dependent mitochondrial dysfunction in sepsis induced end organ injury, including sepsis-induced myocardial dysfunction (SIMD). However, the mechanisms behind p53 localization to the mitochondria have not been well established. Dynamin-related protein 1 (Drp1), a mediator of mitochondrial fission, may play a role in p53 mitochondrial localization. Here we examined the role of Drp1/p53 interaction in SIMD using in vitro and murine models of sepsis. METHODS: H9c2 cardiomyoblasts and BALB/c mice were exposed to lipopolysaccharide (LPS) to model sepsis phenotype. Pharmacologic inhibitors of Drp1 activation (ψDrp1) and of p53 mitochondrial binding (pifithrin µ, PFTµ) were utilized to assess interaction between Drp1 and p53, and the subsequent downstream impact on mitochondrial morphology and function, cardiomyocyte function, and sepsis phenotype. RESULTS: Both in vitro and murine models demonstrated an increase in physical Drp1/p53 interaction following LPS treatment, which was associated with increased p53 mitochondrial localization, and mitochondrial dysfunction. This Drp1/p53 interaction was inhibited by ΨDrp1, suggesting that this interaction is dependent on Drp1 activation. Treatment of H9c2 cells with either ΨDrp1 or PFTµ inhibited the LPS mediated localization of Drp1/p53 to the mitochondria, decreased oxidative stress, improved cellular respiration and ATP production. Similarly, treatment of BALB/c mice with either ΨDrp1 or PFTµ decreased LPS-mediated mitochondrial localization of p53, mitochondrial ROS in cardiac tissue, and subsequently improved cardiomyocyte contractile function and survival. CONCLUSION: Drp1/p53 interaction and mitochondrial localization is a key prodrome to mitochondrial damage in SIMD and inhibiting this interaction may serve as a therapeutic target.

Synaptic protein interaction networks encode experience by assuming stimulus-specific and brain-region-specific states.

A core network of widely expressed proteins within the glutamatergic post-synapse mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we address the question, how does proteomic composition affect activity-dependent protein-protein interaction networks (PINs) downstream of synaptic activity? Using quantitative multiplex co-immunoprecipitation, we compare the PIN response of in vivo or ex vivo neurons derived from different brain regions to activation by different agonists or different forms of eyeblink conditioning. We report that PINs discriminate between incoming stimuli using differential kinetics of overlapping and non-overlapping PIN parameters. Further, these "molecular logic rules" differ by brain region. We conclude that although the PIN of the glutamatergic post-synapse is expressed widely throughout the brain, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity. This diversity may help explain the challenges in developing molecule-specific drug therapies for neurological disorders.

A Repurposed Drug Screen Identifies Compounds That Inhibit the Binding of the COVID-19 Spike Protein to ACE2.

Repurposed drugs that block the interaction between the SARS-CoV-2 spike protein and its receptor ACE2 could offer a rapid route to novel COVID-19 treatments or prophylactics. Here, we screened 2,701 compounds from a commercial library of drugs approved by international regulatory agencies for their ability to inhibit the binding of recombinant, trimeric SARS-CoV-2 spike protein to recombinant human ACE2. We identified 56 compounds that inhibited binding in a concentration-dependent manner, measured the IC50 of binding inhibition, and computationally modeled the docking of the best inhibitors to the Spike-ACE2 binding interface. The best candidates were Thiostrepton, Oxytocin, Nilotinib, and Hydroxycamptothecin with IC50's in the 4-9 µM range. These results highlight an effective screening approach to identify compounds capable of disrupting the Spike-ACE2 interaction, as well as identify several potential inhibitors of the Spike-ACE2 interaction.

A repurposed drug screen identifies compounds that inhibit the binding of the COVID-19 spike protein to ACE2.

Repurposed drugs that block the interaction between the SARS-CoV-2 spike protein and its receptor ACE2 could offer a rapid route to novel COVID-19 treatments or prophylactics. Here, we screened 2701 compounds from a commercial library of drugs approved by international regulatory agencies for their ability to inhibit the binding of recombinant, trimeric SARS-CoV-2 spike protein to recombinant human ACE2. We identified 56 compounds that inhibited binding by <90%, measured the EC 50 of binding inhibition, and computationally modeled the docking of the best inhibitors to both Spike and ACE2. These results highlight an effective screening approach to identify compounds capable of disrupting the Spike-ACE2 interaction as well as identifying several potential inhibitors that could serve as templates for future drug discovery efforts.