Mitochondrial Sequencing Identifies Long Non-Coding RNA Features that Promote Binding to PNPase.

Extranuclear localization of long non-coding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where Polynucleotide Phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and crosslinked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. LncRNA sequence and structure were evaluated through supervised (Classification and Regression Trees (CART) and Support Vector Machines, (SVM)) machine learning algorithms. In HL-1 cells, qPCR of PNPase CLIP knockout mutants (KH and S1) were performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. 112 (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most highly expressed. Most non-coding RNAs binding PNPase were lncRNAs, including Malat1. LncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. Binding of lncRNAs to PNPase was decreased through knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs to bind to PNPase and undergo import into the mitochondrion.

A pilot study of mitochondrial response to an in vivo prosthetic joint Staphylococcus aureus infection model.

Prosthetic joint infections (PJI) are associated with orthopaedic morbidity and mortality. Mitochondria, the "cell's powerhouses," are thought to play crucial roles in infection response and in increased risk of sepsis mortality. No current research discusses PJI's effect on mitochondrial function and a lack of understanding of immune-infection interactions potentially hinders patient care. The purpose of this pilot study was to evaluate the impact of simulated PJI on local tissue mitochondrial function. Using an established prosthetic implant-associated in vivo model, tissues were harvested from the surgical limb of a methicillin-sensitive Staphylococcus aureus implant-associated infection group (n = 6) and compared to a noninfected group (n = 6) at postoperative day (POD) 21. Using mitochondrial coupling assays, oxygen consumption rate and extracellular acidification rate were assessed in each group. Electron flow through mitochondrial complexes reflected group activity. Electron Paramagnetic Resonance (EPR) spectrometry measured the oxidizing potential of serum samples from infected versus noninfected groups. On POD21, colony-forming units per gram of tissue showed 5 × 109 in the infected group and 101 in the noninfected group (p < 0.0001). Maximal respiration and oxygen consumption due to adenosine triphosphate synthesis were significantly lower in isolated mitochondria from infected limbs (p = 0.04). Both groups had similar complex I, III, IV, and V activity (p > 0.1). Infected group EPR signal intensity reflecting reactive oxygen species levels was 1.31 ± 0.30 compared to 1.16 ± 0.28 (p = 0.73) in the noninfected group. This study highlights PJI's role in mammalian cell mitochondrial dysfunction and oxidative tissue damage, which can help develop interventions to combat PJI.

A nitroalkene derivative of salicylate alleviates diet-induced obesity by activating creatine metabolism and non-shivering thermogenesis.

Obesity-related type II diabetes (diabesity) has increased global morbidity and mortality dramatically. Previously, the ancient drug salicylate demonstrated promise for the treatment of type II diabetes, but its clinical use was precluded due to high dose requirements. In this study, we present a nitroalkene derivative of salicylate, 5-(2-nitroethenyl)salicylic acid (SANA), a molecule with unprecedented beneficial effects in diet-induced obesity (DIO). SANA reduces DIO, liver steatosis and insulin resistance at doses up to 40 times lower than salicylate. Mechanistically, SANA stimulated mitochondrial respiration and increased creatine-dependent energy expenditure in adipose tissue. Indeed, depletion of creatine resulted in the loss of SANA action. Moreover, we found that SANA binds to creatine kinases CKMT1/2, and downregulation CKMT1 interferes with the effect of SANA in vivo. Together, these data demonstrate that SANA is a first-in-class activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue and emerges as a candidate for the treatment of diabesity.

Machine learning for spatial stratification of progressive cardiovascular dysfunction in a murine model of type 2 diabetes mellitus.

Speckle tracking echocardiography (STE) has been utilized to evaluate independent spatial alterations in the diabetic heart, but the progressive manifestation of regional and segmental cardiac dysfunction in the type 2 diabetic (T2DM) heart remains understudied. Therefore, the objective of this study was to elucidate if machine learning could be utilized to reliably describe patterns of the progressive regional and segmental dysfunction that are associated with the development of cardiac contractile dysfunction in the T2DM heart. Non-invasive conventional echocardiography and STE datasets were utilized to segregate mice into two pre-determined groups, wild-type and Db/Db, at 5, 12, 20, and 25 weeks. A support vector machine model, which classifies data using a single line, or hyperplane, that best separates each class, and a ReliefF algorithm, which ranks features by how well each feature lends to the classification of data, were used to identify and rank cardiac regions, segments, and features by their ability to identify cardiac dysfunction. STE features more accurately segregated animals as diabetic or non-diabetic when compared with conventional echocardiography, and the ReliefF algorithm efficiently ranked STE features by their ability to identify cardiac dysfunction. The Septal region, and the AntSeptum segment, best identified cardiac dysfunction at 5, 20, and 25 weeks, with the AntSeptum also containing the greatest number of features which differed between diabetic and non-diabetic mice. Cardiac dysfunction manifests in a spatial and temporal fashion, and is defined by patterns of regional and segmental dysfunction in the T2DM heart which are identifiable using machine learning methodologies. Further, machine learning identified the Septal region and AntSeptum segment as locales of interest for therapeutic interventions aimed at ameliorating cardiac dysfunction in T2DM, suggesting that machine learning may provide a more thorough approach to managing contractile data with the intention of identifying experimental and therapeutic targets.

Chemotherapeutic Activity of Pitavastatin in Vincristine Resistant B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (ALL) is derived from an accumulation of malignant, immature B cells in the bone marrow and blood. Relapse due, in part, to the emergence of tumor cells that are resistant to front line standard chemotherapy is associated with poor patient outcomes. This challenge highlights the need for new treatment strategies to eliminate residual chemoresistant tumor cells. Based on the use of pitavastatin in acute myeloid leukemia (AML), we evaluated its efficacy in an REH ALL cell line derived to be resistant to vincristine. We found that pitavastatin inhibited the proliferation of both parental and vincristine-resistant REH tumor cells at an IC50 of 449 nM and 217 nM, respectively. Mitochondrial bioenergetic assays demonstrated that neither vincristine resistance nor pitavastatin treatment affected cellular oxidative phosphorylation, beta-oxidation, or glycolytic metabolism in ALL cells. In a co-culture model of ALL cells with bone marrow stromal cells, pitavastatin significantly decreased cell viability more robustly in the vincristine-resistant ALL cells compared with their parental controls. Subsequently, NSG mice were used to develop an in vivo model of B-cell ALL using both parental and vincristine-resistant ALL cells. Pitavastatin (10 mg/kg i.p.) significantly reduced the number of human CD45+ REH ALL cells in the bone marrow of mice after 4 weeks of treatment. Mechanistic studies showed that pitavastatin treatment in the vincristine-resistant cells led to apoptosis, with increased levels of cleaved PARP and protein-signaling changes for AMP-activated protein kinase/FoxO3a/Puma. Our data suggest the possible repurposing of pitavastatin as a chemotherapeutic agent in a model of vincristine-resistant B-cell ALL.

NUDT7 regulates total hepatic CoA levels and the composition of the intestinal bile acid pool in male mice fed a Western diet.

Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the ß-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.

N6-methyladenosine (M6A) in fetal offspring modifies mitochondrial gene expression following gestational nano-TiO2 inhalation exposure.

N6-methyladenosine (m6A) is the most prominent epitranscriptomic modification to RNA in eukaryotes, but it's role in adaptive changes within the gestational environment are poorly understood. We propose that gestational exposure to nano titanium dioxide (TiO2) contributes to cardiac m6A methylation in fetal offspring and influences mitochondrial gene expression. 10-week-old pregnant female FVB/NJ wild-type mice underwent 6 nonconsecutive days of whole-body inhalation exposure beginning on gestational day (GD) 5. Mice were exposed to filtered room air or nano-TiO2 with a target aerosol mass concentration of 12 mg/m3. At GD 15 mice were humanely killed and cardiac RNA and mitochondrial proteins extracted. Immunoprecipitation with m6A antibodies was performed followed by sequencing of immunoprecipitant (m6A) and input (mRNA) on the Illumina NextSeq 2000. Protein extraction, preparation, and LC-MS/MS were used for mitochondrial protein quantification. There were no differences in maternal or fetal pup weights, number of pups, or pup heart weights between exposure and control groups. Transcriptomic sequencing revealed 3648 differentially expressed mRNA in nano-TiO2 exposed mice (Padj ≤ 0.05). Transcripts involved in mitochondrial bioenergetics were significantly downregulated (83 of 85 genes). 921 transcripts revealed significant m6A methylation sites (Padj ≤ 0.10). 311 of the 921 mRNA were identified to have both 1) significantly altered expression and 2) differentially methylated sites. Mitochondrial proteomics revealed decreased expression of ATP Synthase subunits in the exposed group (P ≤ 0.05). The lack of m6A modifications to mitochondrial transcripts suggests a mechanism for decreased transcript stability and reduced protein expression due to gestational nano-TiO2 inhalation exposure.

Ultrasonic Texture Features for Assessing Cardiac Remodeling and Dysfunction.

BACKGROUND: Changes in cardiac size, myocardial mass, cardiomyocyte appearance, and, ultimately, the function of the entire organ are interrelated features of cardiac remodeling that profoundly affect patient outcomes. OBJECTIVES: This study proposes that the application of radiomics for extracting cardiac ultrasonic textural features (ultrasomics) can aid rapid, automated assessment of left ventricular (LV) structure and function without requiring manual measurements. METHODS: This study developed machine-learning models using cardiac ultrasound images from 1,915 subjects in 3 clinical cohorts: 1) an expert-annotated cardiac point-of-care-ultrasound (POCUS) registry (n = 943, 80% training/testing and 20% internal validation); 2) a prospective POCUS cohort for external validation (n = 275); and 3) a prospective external validation on high-end ultrasound systems (n = 484). In a type 2 diabetes murine model, echocardiography of wild-type (n = 10) and Leptr-/- (n = 8) mice were assessed longitudinally at 3 and 25 weeks, and ultrasomics features were correlated with histopathological features of hypertrophy. RESULTS: The ultrasomics model predicted LV remodeling in the POCUS and high-end ultrasound external validation studies (area under the curve: 0.78 [95% CI: 0.68-0.88] and 0.79 [95% CI: 0.73-0.86], respectively). Similarly, the ultrasomics model predicted LV remodeling was significantly associated with major adverse cardiovascular events in both cohorts (P < 0.0001 and P = 0.0008, respectively). Moreover, on multivariate analysis, the ultrasomics probability score was an independent echocardiographic predictor of major adverse cardiovascular events in the high-end ultrasound cohort (HR: 8.53; 95% CI: 4.75-32.1; P = 0.0003). In the murine model, cardiomyocyte hypertrophy positively correlated with 2 ultrasomics biomarkers (R2 = 0.57 and 0.52, Q < 0.05). CONCLUSIONS: Cardiac ultrasomics-based biomarkers may aid development of machine-learning models that provide an expert-level assessment of LV structure and function.

Left Ventricular Segmental Strain Identifies Unique Myocardial Deformation Patterns After Intrinsic and Extrinsic Stressors in Mice.

We used segmental strain analysis to evaluate whether intrinsic (diet-induced obesity [DIO]) and extrinsic (unpredictable chronic mild stress [UCMS]) stressors can alter deformational patterns of the left ventricle. Six-week-old male C57BL/6J mice were randomized into the lean or obese group (n = 24/group). Mice underwent 12 wk of DIO with a high-fat diet (HFD). At 18 wk, lean and obese mice were further randomized into UCMS and non-UCMS groups (UCMS, 7 h/d, 5 d/wk, for 8 wk). Echocardiography was performed at baseline (6 wk), post-HFD (18 wk) and post-UCMS (26 wk). Machine learning was applied to the DIO and UCMS groups. There was robust predictive accuracy (area under the receiver operating characteristic curve [AUC] = 0.921) when comparing obese with lean mice, with radial strain changes in the lateral (-64%, p ≤ 0.001) and anterior free (-53%, p < 0.001) walls being most informative. The ability to predict mice that underwent UCMS, irrespective of diet, was assessed (AUC = 0.886), revealing longitudinal strain rate of the anterior midwall and radial strain of the posterior septal wall as the top features. The wall segments indicate a predilection for changes in deformation patterns to the free wall (DIO) and septal wall (UCMS), indicating disease-specific alterations to the myocardium.

Manipulation of the miR-378a/mt-ATP6 regulatory axis rescues ATP synthase in the diabetic heart and offers a novel role for lncRNA Kcnq1ot1.

Diabetes mellitus has been linked to an increase in mitochondrial microRNA-378a (miR-378a) content. Enhanced miR-378a content has been associated with a reduction in mitochondrial genome-encoded mt-ATP6 abundance, supporting the hypothesis that miR-378a inhibition may be a therapeutic option for maintaining ATP synthase functionality during diabetes mellitus. Evidence also suggests that long noncoding RNAs (lncRNAs), including lncRNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (Kcnq1ot1), participate in regulatory axes with microRNAs (miRs). Prediction analyses indicate that Kcnq1ot1 has the potential to bind miR-378a. This study aimed to determine if loss of miR-378a in a genetic mouse model could ameliorate cardiac dysfunction in type 2 diabetes mellitus (T2DM) and to ascertain whether Kcnq1ot1 interacts with miR-378a to impact ATP synthase functionality by preserving mt-ATP6 levels. MiR-378a was significantly higher in patients with T2DM and 25-wk-old Db/Db mouse mitochondria, whereas mt-ATP6 and Kcnq1ot1 levels were significantly reduced when compared with controls. Twenty-five-week-old miR-378a knockout Db/Db mice displayed preserved mt-ATP6 and ATP synthase protein content, ATP synthase activity, and preserved cardiac function, implicating miR-378a as a potential therapeutic target in T2DM. Assessments following overexpression of the 500-bp Kcnq1ot1 fragment in established mouse cardiomyocyte cell line (HL-1) cardiomyocytes overexpressing miR-378a revealed that Kcnq1ot1 may bind and significantly reduce miR-378a levels, and rescue mt-ATP6 and ATP synthase protein content. Together, these data suggest that Kcnq1ot1 and miR-378a may act as constituents in an axis that regulates mt-ATP6 content, and that manipulation of this axis may provide benefit to ATP synthase functionality in type 2 diabetic heart.

Transcriptomics of single dose and repeated carbon black and ozone inhalation co-exposure highlight progressive pulmonary mitochondrial dysfunction.

BACKGROUND: Air pollution is a complex mixture of particles and gases, yet current regulations are based on single toxicant levels failing to consider potential interactive outcomes of co-exposures. We examined transcriptomic changes after inhalation co-exposure to a particulate and a gaseous component of air pollution and hypothesized that co-exposure would induce significantly greater impairments to mitochondrial bioenergetics. A whole-body inhalation exposure to ultrafine carbon black (CB), and ozone (O3) was performed, and the impact of single and multiple exposures was studied at relevant deposition levels. C57BL/6 mice were exposed to CB (10 mg/m3) and/or O3 (2 ppm) for 3 h (either a single exposure or four independent exposures). RNA was isolated from lungs and mRNA sequencing performed using the Illumina HiSeq. Lung pathology was evaluated by histology and immunohistochemistry. Electron transport chain (ETC) activities, electron flow, hydrogen peroxide production, and ATP content were assessed. RESULTS: Compared to individual exposure groups, co-exposure induced significantly greater neutrophils and protein levels in broncho-alveolar lavage fluid as well as a significant increase in mRNA expression of oxidative stress and inflammation related genes. Similarly, a significant increase in hydrogen peroxide production was observed after co-exposure. After single and four exposures, co-exposure revealed a greater number of differentially expressed genes (2251 and 4072, respectively). Of these genes, 1188 (single exposure) and 2061 (four exposures) were uniquely differentially expressed, with 35 mitochondrial ETC mRNA transcripts significantly impacted after four exposures. Both O3 and co-exposure treatment significantly reduced ETC maximal activity for complexes I (- 39.3% and - 36.2%, respectively) and IV (- 55.1% and - 57.1%, respectively). Only co-exposure reduced ATP Synthase activity (- 35.7%) and total ATP content (30%). Further, the ability for ATP Synthase to function is limited by reduced electron flow (- 25%) and translation of subunits, such as ATP5F1, following co-exposure. CONCLUSIONS: CB and O3 co-exposure cause unique transcriptomic changes in the lungs that are characterized by functional deficits to mitochondrial bioenergetics. Alterations to ATP Synthase function and mitochondrial electron flow underly a pathological adaptation to lung injury induced by co-exposure.

Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (ALL) affects both pediatric and adult patients. Chemotherapy resistant tumor cells that contribute to minimal residual disease (MRD) underlie relapse and poor clinical outcomes in a sub-set of patients. Targeting mitochondrial oxidative phosphorylation (OXPHOS) in the treatment of refractory leukemic cells is a potential novel approach to sensitizing tumor cells to existing standard of care therapeutic agents. In the current study, we have expanded our previous investigation of the mitoNEET ligand NL-1 in the treatment of ALL to interrogate the functional role of the mitochondrial outer membrane protein mitoNEET in B-cell ALL. Knockout (KO) of mitoNEET (gene: CISD1) in REH leukemic cells led to changes in mitochondrial ultra-structure and function. REH cells have significantly reduced OXPHOS capacity in the KO cells coincident with reduction in electron flow and increased reactive oxygen species. In addition, we found a decrease in lipid content in KO cells, as compared to the vector control cells was observed. Lastly, the KO of mitoNEET was associated with decreased proliferation as compared to control cells when exposed to the standard of care agent cytarabine (Ara-C). Taken together, these observations suggest that mitoNEET is essential for optimal function of mitochondria in B-cell ALL and may represent a novel anti-leukemic drug target for treatment of minimal residual disease.

Mitochondrial membranes modify mutant huntingtin aggregation.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ tracts are prone to aggregate into oligomers and insoluble fibrils. Mutant htt (mhtt) localizes to variety of organelles, including mitochondria. Specifically, mitochondrial defects, morphological alteration, and dysfunction are observed in HD. Mitochondrial lipids, cardiolipin (CL) in particular, are essential in mitochondria function and have the potential to directly interact with htt, altering its aggregation. Here, the impact of mitochondrial membranes on htt aggregation was investigated using a combination of mitochondrial membrane mimics and tissue-derived mitochondrial-enriched fractions. The impact of exposure of outer and inner mitochondrial membrane mimics (OMM and IMM respectively) to mhtt was explored. OMM and IMM reduced mhtt fibrillization, with IMM having a larger effect. The role of CL in mhtt aggregation was investigated using a simple PC system with varying molar ratios of CL. Lower molar ratios of CL (<5%) promoted fibrillization; however, increased CL content retarded fibrillization. As revealed by in situ AFM, mhtt aggregation and associated membrane morphological changes at the surface of OMM mimics was markedly different compared to IMM mimics. While globular deposits of mhtt with few fibrillar aggregates were observed on OMM, plateau-like domains were observed on IMM. A similar impact on htt aggregation was observed with exposure to purified mitochondrial-enriched fractions. Collectively, these observations suggest mitochondrial membranes heavily influence htt aggregation with implication for HD.

Enhanced antioxidant capacity prevents epitranscriptomic and cardiac alterations in adult offspring gestationally-exposed to ENM.

Maternal engineered nanomaterial (ENM) exposure during gestation has been associated with negative long-term effects on cardiovascular health in progeny. Here, we evaluate an epitranscriptomic mechanism that contributes to these chronic ramifications and whether overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) can preserve cardiovascular function and bioenergetics in offspring following gestational nano-titanium dioxide (TiO2) inhalation exposure. Wild-type (WT) and mPHGPx (Tg) dams were exposed to nano-TiO2 aerosols with a mass concentration of 12.01 ± 0.50 mg/m3 starting from gestational day (GD) 5 for 360 mins/day for 6 nonconsecutive days over 8 days. Echocardiography was performed in pregnant dams, adult (11-week old) and fetal (GD 14) progeny. Mitochondrial function and global N6-methyladenosine (m6A) content were assessed in adult progeny. MPHGPx enzymatic function was further evaluated in adult progeny and m6A-RNA immunoprecipitation (RIP) was combined with RT-qPCR to evaluate m6A content in the 3'-UTR. Following gestational ENM exposure, global longitudinal strain (GLS) was 32% lower in WT adult offspring of WT dams, with preservation in WT offspring of Tg dams. MPHGPx activity was significantly reduced in WT offspring (29%) of WT ENM-exposed dams, but preserved in the progeny of Tg dams. M6A-RIP-qPCR for the SEC insertion sequence region of mPHGPx revealed hypermethylation in WT offspring from ENM-exposed WT dams, which was thwarted in the presence of the maternal transgene. Our findings implicate that m6A hypermethylation of mPHGPx may be culpable for diminished antioxidant capacity and resultant mitochondrial and cardiac deficits that persist into adulthood following gestational ENM inhalation exposure.

The Mitochondrial mitoNEET Ligand NL-1 Is Protective in a Murine Model of Transient Cerebral Ischemic Stroke.

PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC50 of 5.95 µM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

Cardiovascular adaptations to particle inhalation exposure: molecular mechanisms of the toxicology.

Ambient air, occupational settings, and the use and distribution of consumer products all serve as conduits for toxicant exposure through inhalation. While the pulmonary system remains a primary target following inhalation exposure, cardiovascular implications are exceptionally culpable for increased morbidity and mortality. The epidemiological evidence for cardiovascular dysfunction resulting from acute or chronic inhalation exposure to particulate matter has been well documented, but the mechanisms driving the resulting disturbances remain elusive. In the current review, we aim to summarize the cellular and molecular mechanisms that are directly linked to cardiovascular health following exposure to a variety of inhaled toxicants. The purpose of this review is to provide a comprehensive overview of the biochemical changes in the cardiovascular system following particle inhalation exposure and to highlight potential biomarkers that exist across multiple exposure paradigms. We attempt to integrate these molecular signatures in an effort to provide direction for future investigations. This review also characterizes how molecular responses are modified in at-risk populations, specifically the impact of environmental exposure during critical windows of development. Maternal exposure to particulate matter during gestation can lead to fetal epigenetic reprogramming, resulting in long-term deficits to the cardiovascular system. In both direct and indirect (gestational) exposures, connecting the biochemical mechanisms with functional deficits outlines pathways that can be targeted for future therapeutic intervention. Ultimately, future investigations integrating "omics"-based approaches will better elucidate the mechanisms that are altered by xenobiotic inhalation exposure, identify biomarkers, and guide in clinical decision making.

MiR-34a Interacts with Cytochrome c and Shapes Stroke Outcomes.

Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC) in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a-/- mice following stroke reperfusion. We further confirmed CYC is a target of miR-34a by a dural luciferase reporter gene assay in vitro. Our study provides the first description of miR-34a affecting stroke outcomes and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke.

Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

PURPOSE: Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia. METHODS: ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness. RESULTS: PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. CONCLUSION: We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.

Machine-learning to stratify diabetic patients using novel cardiac biomarkers and integrative genomics.

BACKGROUND: Diabetes mellitus is a chronic disease that impacts an increasing percentage of people each year. Among its comorbidities, diabetics are two to four times more likely to develop cardiovascular diseases. While HbA1c remains the primary diagnostic for diabetics, its ability to predict long-term, health outcomes across diverse demographics, ethnic groups, and at a personalized level are limited. The purpose of this study was to provide a model for precision medicine through the implementation of machine-learning algorithms using multiple cardiac biomarkers as a means for predicting diabetes mellitus development. METHODS: Right atrial appendages from 50 patients, 30 non-diabetic and 20 type 2 diabetic, were procured from the WVU Ruby Memorial Hospital. Machine-learning was applied to physiological, biochemical, and sequencing data for each patient. Supervised learning implementing SHapley Additive exPlanations (SHAP) allowed binary (no diabetes or type 2 diabetes) and multiple classification (no diabetes, prediabetes, and type 2 diabetes) of the patient cohort with and without the inclusion of HbA1c levels. Findings were validated through Logistic Regression (LR), Linear Discriminant Analysis (LDA), Gaussian Naïve Bayes (NB), Support Vector Machine (SVM), and Classification and Regression Tree (CART) models with tenfold cross validation. RESULTS: Total nuclear methylation and hydroxymethylation were highly correlated to diabetic status, with nuclear methylation and mitochondrial electron transport chain (ETC) activities achieving superior testing accuracies in the predictive model (~ 84% testing, binary). Mitochondrial DNA SNPs found in the D-Loop region (SNP-73G, -16126C, and -16362C) were highly associated with diabetes mellitus. The CpG island of transcription factor A, mitochondrial (TFAM) revealed CpG24 (chr10:58385262, P = 0.003) and CpG29 (chr10:58385324, P = 0.001) as markers correlating with diabetic progression. When combining the most predictive factors from each set, total nuclear methylation and CpG24 methylation were the best diagnostic measures in both binary and multiple classification sets. CONCLUSIONS: Using machine-learning, we were able to identify novel as well as the most relevant biomarkers associated with type 2 diabetes mellitus by integrating physiological, biochemical, and sequencing datasets. Ultimately, this approach may be used as a guideline for future investigations into disease pathogenesis and novel biomarker discovery.