Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

Mitochondrial membrane potential (ΔΨm) is a global indicator of mitochondrial function. Previous reports on heterogeneity of ΔΨm were qualitative or semiquantitative. Here, we quantified intercellular differences in ΔΨm in unsynchronized human cancer cells, cells synchronized in G1, S, and G2, and human fibroblasts. We assessed ΔΨm using a two-pronged microscopy approach to measure relative fluorescence of tetramethylrhodamine methyl ester (TMRM) and absolute values of ΔΨm. We showed that ΔΨm is more heterogeneous in cancer cells compared to fibroblasts, and it is maintained throughout the cell cycle. The effect of chemical inhibition of the respiratory chain and ATP synthesis differed between basal, low and high ΔΨm cells. Overall, our results showed that intercellular heterogeneity of ΔΨm is mainly modulated by intramitochondrial factors, it is independent of the ΔΨm indicator and it is not correlated with intercellular heterogeneity of plasma membrane potential or the phases of the cell cycle.

Voltage-Dependent Anion Channels Influence Cytotoxicity of ME-344, a Therapeutic Isoflavone.

ME-344 is a second-generation cytotoxic isoflavone with anticancer activity promulgated through interference with mitochondrial functions. Using a click chemistry version of the drug together with affinity-enriched mass spectrometry, voltage-dependent anion channels (VDACs) 1 and 2 were identified as drug targets. To determine the importance of VDAC1 or 2 to cytotoxicity, we used lung cancer cells that were either sensitive (H460) or intrinsically resistant (H596) to the drug. In H460 cells, depletion of VDAC1 and VDAC2 by small interfering RNA impacted ME-344 effects by diminishing generation of reactive oxygen species (ROS), preventing mitochondrial membrane potential dissipation, and moderating ME-344-induced cytotoxicity and mitochondrial-mediated apoptosis. Mechanistically, VDAC1 and VDAC2 knockdown prevented ME-344-induced apoptosis by inhibiting Bax mitochondrial translocation and cytochrome c release as well as apoptosis in these H460 cells. We conclude that VDAC1 and 2, as mediators of the response to oxidative stress, have roles in modulating ROS generation, Bax translocation, and cytochrome c release during mitochondrial-mediated apoptosis caused by ME-344. SIGNIFICANCE STATEMENT: Dissecting preclinical drug mechanisms are of significance in development of a drug toward eventual Food and Drug Administration approval.

Statin-dependent modulation of mitochondrial metabolism in cancer cells is independent of cholesterol content.

Statins, widely used to treat hypercholesterolemia, inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of de novo cholesterol (Chol) synthesis. Statins have been also reported to slow tumor progression. In cancer cells, ATP is generated both by glycolysis and oxidative phosphorylation. Mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, is sustained by the oxidation of respiratory substrates in the Krebs cycle to generate NADH and flavin adenine dinucleotide, which are further oxidized by the respiratory chain. Here, we studied the short-term effects of statins (3-24 h) on mitochondrial metabolism on cancer cells. Lovastatin (LOV) and simvastatin (SIM) increased ΔΨ in HepG2 and Huh7 human hepatocarcinoma cells and HCC4006 human lung adenocarcinoma cells. Mitochondrial hyperpolarization after LOV and SIM was dose and time dependent. Maximal increase in ΔΨ occurred at 10 µM and 24 h for both statins. The structurally unrelated atorvastatin also hyperpolarized mitochondria in HepG2 cells. Cellular and mitochondrial Chol remained unchanged after SIM. Both LOV and SIM decreased basal respiration, ATP-linked respiration, and ATP production. LOV and SIM did not change the rate of lactic acid production. In summary, statins modulate mitochondrial metabolism in cancer cells independently of the Chol content in cellular membranes without affecting glycolysis.-Christie, C. F., Fang, D., Hunt, E. G., Morris, M. E., Rovini, A., Heslop, K. A., Beeson, G. C., Beeson, C. C., Maldonado, E. N. Statin-dependent modulation of mitochondrial metabolism in cancer cells is independent of cholesterol content.

5-HT1F receptor regulates mitochondrial homeostasis and its loss potentiates acute kidney injury and impairs renal recovery.

Our laboratory previously reported that agonists of the 5-hydoxytryptamine 1F (5-HT1F) receptor induce renal mitochondrial biogenesis (MB) and that stimulation of the 5-HT1F receptor following ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) accelerated the recovery of renal function in mice. The goal of this study was to examine the contribution of the 5-HT1F receptor in the regulation of renal mitochondrial homeostasis and renal function in naïve and injured mice. Although 5-HT1F receptor knockout (KO) mice were healthy and fertile, and did not exhibit renal dysfunction, renal mitochondrial DNA copy number and mitochondrial fission gene expression increased at 10 wk of age. The 5-HT1F receptor KO mice exhibited greater proximal tubular injury and diminished renal recovery after I/R-induced AKI compared with wild-type mice. These findings were associated with persistent suppression of renal cortical MB and ATP levels after injury. In summary, the 5-HT1F receptor is a component of physiological MB regulation in the kidney, and its absence potentiates renal injury and impedes recovery.

Antibody response to SARS-CoV-2 vaccination or infection in a prospective cohort of children with neuroinflammatory diseases.

INTRODUCTION: Immune medications affect antibody responses to SARS-CoV-2 vaccination in adults with neuroinflammatory disorders, but little is known about antibody responses in children with neuroinflammation and on immune treatments. Here we measure antibody levels in response to SARS-CoV-2 vaccination in children receiving anti-CD20 monoclonal antibodies, or fingolimod. METHODS: Children under 18 years of age with pediatric-onset neuroinflammatory disorders who received at least two mRNA vaccines were included. Plasma samples were assayed for SARS-CoV-2 antibodies (spike, spike receptor binding domain-RBD, nucleocapsid) and neutralization antibodies. RESULTS: Seventeen participants with pediatric onset neuroinflammatory diseases were included: 12 multiple sclerosis, one neuromyelitis optica spectrum disorder, two MOG-associated disease, and two autoimmune encephalitis. Fourteen were on medications (11 on CD20 monoclonal antibodies-mAbs, one on fingolimod, one on steroids, one on intravenous immunoglobulin) and three were untreated. Nine patients also had pre-vaccination samples available. All participants had seropositivity to spike or spike RBD antibodies except for those receiving CD20 mAbs. However, this proportion was higher in children than in an adult MS patient cohort. The most significant contributor to antibody levels was duration of DMT. CONCLUSION: SARS-CoV-2 antibodies are decreased in children on CD20 monoclonal antibodies than on other treatments. Treatment duration associated with vaccination responses.

Sleep Characteristics in Pediatric Anti-N-methyl-d-aspartate (NMDA) Receptor Encephalitis: A Retrospective Cohort Study.

Background: Rates of sleep problems in children with anti-N-methyl-d-aspartate (NMDA) receptor encephalitis are unknown. Methods: We used a retrospective observational cohort database of children with a diagnosis of NMDA receptor encephalitis at a single freestanding institution. One-year outcomes were assessed with the pediatric modified Rankin Score (mRS), with 0 to 2 as good and 3 or greater as poor outcome. Results: Ninety-five percent (39/41) of children with NMDA receptor encephalitis had sleep dysfunction at onset; 34% (11/32) reported sleep problems at 1 year. Sleep problems at onset and propofol use were not associated with poor outcomes at 1 year. Poor sleep at 1 year correlated with mRS scores (range 2-5) at 1 year. Discussion: High rates of sleep dysfunction occur in children with NMDA receptor encephalitis. Persistent sleep problems at 1 year may correlate with outcomes as assessed by mRS at 1 year. Further studies comparing the relationship of poor sleep with outcomes in NMDA receptor encephalitis are needed.

MRI Features and Their Association With Outcomes in Children With Anti-NMDA Receptor Encephalitis.

OBJECTIVES: How brain MRI lesions associate with outcomes in pediatric anti-NMDA receptor encephalitis (pNMDARE) is unknown. In this study, we correlate T2-hyperintense MRI brain lesions with clinical outcomes in pNMDARE. METHODS: This was a multicenter retrospective cohort study from 11 institutions. Children younger than 18 years with pNMDARE were included. One-year outcomes were assessed by the modified Rankin Score (mRS) with good (mRS ≤2) and poor (mRS ≥3) outcomes. RESULTS: A total of 175 pNMDARE subjects were included, with 1-year mRS available in 142/175 (81%) and 60/175 (34%) had abnormal brain MRIs. The most common T2-hyperintense lesion locations were frontal, temporal, and parietal. MRI features that predicted poor 1-year outcomes included abnormal MRI, particularly T2 lesions in the frontal and occipital lobes. After adjusting for treatment within 4 weeks of onset, improvement within 4 weeks, and intensive care unit admission, MRI features were no longer associated with poor outcomes, but after multiple imputation for missing data, T2 frontal and occipital lesions associated with poor outcomes. DISCUSSION: Abnormal frontal and occipital lesions on MRI may associate with 1-year mRS in pNMDARE. MRI of the brain may be a helpful prognostication tool that should be examined in future studies.

The neutrophil-to-lymphocyte ratio is associated with intubation in pediatric anti-NMDA receptor encephalitis: A retrospective study.

Background The neutrophil-to-lymphocyte ratio (NLR) may predict poor outcomes in adult anti-NMDAR encephalitis (NMDARE). The association of NLR with outcomes in pediatric NMDARE was examined. Methods Pediatric NMDARE patients (N = 36) were retrospectively studied. Results High NLR (>6) had a higher proportion of tumors (43% versus 7%) and higher intubation rates (100% versus 38%, p = 0.008). Multivariate analyses showed that high NLR did not correlate with one-year outcomes, inpatient length of stay (LOS), or with tumor, but was associated with intubation and rehabilitation LOS. Conclusion NLR is associated with intubation and rehabilitation LOS. Further investigation is needed for prognostic biomarkers in NMDARE.

Neurosarcoidosis in children: A systematic review and summary of cases, imaging and management.

Neurosarcoidosis is a rare disorder in children. We identified 30 pediatric NS cases through a systematic review. Twenty-one (70%) had systemic sarcoidosis with 30% having primary neurosarcoidosis. Eyes (37%), lymph nodes (37%) and lungs (30%) were most commonly involved. Isolated neurosarcoidosis were more likely in children (30%) than in adults (6%, p = 0.0005). Seizures and optic neuritis were also more common in children than adults (33% vs 14%, p = 0.002; and 30% versus 6%, p = 0.008, respectively). Evaluation, imaging, laboratory findings, and treatments are discussed. Additional research, including multi-center studies, is needed.

Dynamically optimizing experiment schedules of a laboratory robot system with simulated annealing.

A scheduler has been developed for an integrated laboratory robot system that operates in an always-on mode. The integrated system is designed for imaging plates containing protein crystallization experiments, and it allows crystallographers to enter plates at any time and request that they be imaged at multiple time points in the future. The scheduler must rearrange tasks within the time it takes to image one plate, trading off the quality of the schedule for the speed of the computation. For this reason, the scheduler was based on a simulated annealing algorithm with an objective function that makes use of a linear programming solver. To optimize the scheduler, extensive computational simulations were performed involving a difficult but representative scheduling problem. The simulations explore multiple configurations of the simulated annealing algorithm, including both geometric and adaptive annealing schedules, 3 neighborhood functions, and 20 neighborhood diameters. An optimal configuration was found that produced the best results in less than 60 seconds, well within the window necessary to dynamically reschedule imaging tasks as new plates are entered into the system.