Changes In Oxygen Consumption Rate Research Articles

Rv0547c, a functional oxidoreductase, supports Mycobacterium tuberculosis persistence by reprogramming host mitochondrial fatty acid metabolism

Mycobacterium tuberculosis (Mtb) successfully thrives in the host by adjusting its metabolism and manipulating the host environment. In this study, we investigated the role of Rv0547c, a protein that carries mitochondria-targeting sequence (MTS), in mycobacterial persistence. We show that Rv0547c is a functional oxidoreductase that targets host-cell mitochondria. Interestingly, the localization of Rv0547c to mitochondria was independent of the predicted MTS but depended on specific arginine residues at the N- and C-terminals. As compared to the mitochondria-localization defective mutant, Rv0547c-2SDM, wild-type Rv0547c increased mitochondrial membrane fluidity and spare respiratory capacity. To comprehend the possible reason, comparative lipidomics was performed that revealed a reduced variability of long-chain and very long-chain fatty acids as well as altered levels of phosphatidylcholine and phosphatidylinositol class of lipids upon expression of Rv0547c, explaining the increased membrane fluidity. Additionally, the over representation of propionate metabolism and β-oxidation intermediates in Rv0547c-targeted mitochondrial fractions indicated altered fatty acid metabolism, which corroborated with changes in oxygen consumption rate (OCR) upon etomoxir treatment in HEK293T cells transiently expressing Rv0547c, resulting in enhanced mitochondrial fatty acid oxidation capacity. Furthermore, Mycobacterium smegmatis over expressing Rv0547c showed increased persistence during infection of THP-1 macrophages, which correlated with its increased expression in Mtb during oxidative and nutrient starvation stresses. This study identified for the first time an Mtb protein that alters mitochondrial metabolism and aids in survival in host macrophages by altering fatty acid metabolism to its benefit and, at the same time increases mitochondrial spare respiratory capacity to mitigate infection stresses and maintain cell viability.

Comprehensive characterization of mitochondrial bioenergetics at different larval stages reveals novel insights about the developmental metabolism of Caenorhabditis elegans.

Mitochondrial bioenergetic processes are fundamental to development, stress responses, and health. Caenorhabditis elegans is widely used to study developmental biology, mitochondrial disease, and mitochondrial toxicity. Oxidative phosphorylation generally increases during development in many species, and genetic and environmental factors may alter this normal trajectory. Altered mitochondrial function during development can lead to both drastic, short-term responses including arrested development and death, and subtle consequences that may persist throughout life and into subsequent generations. Understanding normal and altered developmental mitochondrial biology in C. elegans is currently constrained by incomplete and conflicting reports on how mitochondrial bioenergetic parameters change during development in this species. We used a Seahorse XFe24 Extracellular Flux (XF) Analyzer to carry out a comprehensive analysis of mitochondrial and non-mitochondrial oxygen consumption rates (OCR) throughout larval development in C. elegans . We optimized and describe conditions for analysis of basal OCR, basal mitochondrial OCR, ATP-linked OCR, spare and maximal respiratory capacity, proton leak, and non-mitochondrial OCR. A key consideration is normalization, and we present and discuss results as normalized per individual worm, protein content, worm volume, mitochondrial DNA (mtDNA) count, nuclear DNA (ncDNA) count, and mtDNA:ncDNA ratio. Which normalization process is best depends on the question being asked, and differences in normalization explain some of the discrepancies in previously reported developmental changes in OCR in C. elegans . Broadly, when normalized to worm number, our results agree with previous reports in showing dramatic increases in OCR throughout development. However, when normalized to total protein, worm volume, or ncDNA or mtDNA count, after a significant 2-3-fold increase from L1 to L2 stages, we found small or no changes in most OCR parameters from the L2 to the L4 stage, other than a marginal increase at L3 in spare and maximal respiratory capacity. Overall, our results indicate an earlier cellular shift to oxidative metabolism than suggested in most previous literature.

The Impact of Sex Hormones on Renal Mitochondrial Bioenergetics in Aging

Introduction. Aging is accompanied by changes in multiple factors, such as accumulation of mitochondrial dysfunction and the decline of sex hormone levels throughout life. Females are generally protected from renal diseases until menopause, potentially as a result of the presence of female sex hormones. Sex hormones govern a plethora of crucial renal processes, including metabolism and mitochondrial bioenergetics. However, there remains a gap in knowledge regarding the role of sex hormones in mitochondrial bioenergetics in renal aging. We hypothesized in this study that removal of sex hormones will exacerbate the aging-induced decline in renal mitochondrial function. Methods. Male and female Sprague Dawley (SD) rats were randomly divided into groups, and gonadectomy or sham surgery was performed before 4 wks of age. At 6 and 12 months of age kidneys were perfused to remove blood, then cortical tissues were immediately collected for mitochondrial isolations using differential centrifugation or snap-frozen/fixed for biochemical assays. Spectrofluorimetry was utilized to measure mitochondrial membrane potential (ΔΨm) and H2O2 production using TMRM and Amplex Red. Seahorse assay was done to measure oxygen consumption rate (OCR). OriginPro was used for statistical analysis (two-way ANOVA with Holm-Sidak post hoc). Results. Male OCR parameters (basal, ADP-linked, maximal and spare capacity) were lower than OCR observed in females at both 6 and 12 months of age (p<0.05). Orchiectomy resulted in an increase in male OCR parameters at both ages (p<0.05); ovariectomy did not affect renal cortical OCR (p>0.05 for both ages). Of note, gonadectomy in males increased the OCR up to the female level (p>0.05 for both ages). Cortical mitochondrial ΔΨm displayed similar values at 6 months across all groups. However, at 12 months we report lower ΔΨm in males vs female shams (p<0.001). Of note, the male gonadectomy increased ΔΨm up to the level of female values, while ovariectomy had no effect (p=0.3). Female cortical H2O2 production was higher than in males (p<0.001), whereas ovariectomy lowered H2O2 at both time points (p<0.001), indicative of ΔΨm-independent ROS sources. Interestingly, the effect of testosterone on H2O2 production was dependent on age. At 6 months old, orchiectomy did not alter H2O2 production compared to shams, while at 12 months, orchiectomy led to higher H2O2 production (p<0.001). Conclusions. Renal mitochondrial OCR was found to be independent of age in either sex at 6 and 12 months of age. Gonadectomy data allowed us to speculate that testosterone suppresses renal OCR, and the decreased membrane potential in males is likely driving the OCR changes. The observed ΔΨm-independent sources of H2O2 remain to be explored in future experiments. These data established the impact of sex hormones on mitochondrial bioenergetics and will help elucidate sex-specific targets that can mitigate the effects of aging on kidney function. Funding for this project came from 1U54HL169191. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

Glycolytic reprogramming in macrophages and MSCs during inflammation.

Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs). We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE. Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines. This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

Targeting Mitochondrial IDH2 Enhances Antitumor Activity of Cisplatin in Lung Cancer via ROS-Mediated Mechanism

Mitochondrial isocitrate dehydrogenase 2 (IDH2) is an important metabolic enzyme in the tricarboxylic acid cycle (TCA) cycle. Our previous study showed that high expression of wild-type IDH2 promotes the proliferation of lung cancer cells. This study aims to test the potential of targeting IDH2 as a therapeutic strategy to inhibit lung cancer in vitro and in vivo. First, we analyzed the available data from the databases gene expression omnibus (GEO) database to evaluate the clinical relevance of IDH2 expression in affecting lung cancer patient survival. We then generated a stable IDH2-knockdown lung cancer cell line using a lentivirus-based method for in vitro and in vivo study. Cell growth, apoptosis, cell viability, and colony formation assays were conducted to test the sensitivity of lung cancer cells with different IDH2 expression status to cisplatin or radiation treatment in vitro. For mechanistic study, Cellular oxygen consumption and extracellular acidification rates were measured using a Seahorse metabolic analyzer, and reactive oxygen species (ROS) generation was analyzed using flow cytometry. An animal study using a xenograft tumor model was performed to further evaluate the in vivo therapeutic effect on tumor growth. We found that high IDH2 expression was associated with poor survival in lung cancer patients undergoing chemotherapy. Inhibition of IDH2 significantly enhanced the anticancer activity of cisplatin and also increased the effect of radiation against lung cancer cells. IDH2 was upregulated in cisplatin-resistant lung cancer cells, which could be sensitized by targeted inhibition of IDH2. Mechanistic study showed that abrogation of IDH2 caused only minimal changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in lung cancer cells, but induced a significant increase in ROS, which rendered the cancer cells more sensitive to cisplatin. Pretreatment of lung cancer cells with the ROS scavenger N-acetyl-cysteine could partially rescue cells from the cytotoxic effect of cisplatin and IDH2 inhibition. Importantly, abrogation of IDH2 significantly increased the sensitivity of lung cancer cells to cisplatin in vivo.

Chalcone-thiosemicarbazone Hybrids as Inhibitors of Human Hepatocellular Carcinoma HepG2 Cells Viability and Oxygen Consumption

Background: Chalcones are open-chain flavonoids especially attractive to medicinal chemistry due to their easy synthesis and the possibility of structural modifications. Objective: This study aims to evaluate the in vitro anticancer activity of a series of hybrids chalcones- thiosemicarbazones against the human hepatocellular carcinoma cell line HepG2. Methods: Seven hybrid chalcones-thiosemicarbazones (CTs), 3-(4’-X-phenyl)-1-phenylprop- 2-en-1-one thiosemicarbazone, where X=H (CT-H), CH3 (CT-CH3), NO2 (CT-NO2), Cl (CTCl), CN (CT-CN), F (CT-F), and Br (CT-Br), were synthesized and their effects on cells’ viability and mitochondrial oxygen consumption were assessed. Results: Incubation with CTs caused a decrease in HepG2 cells viability in a time-concentration-dependent manner. The most effective compounds in inhibiting cell viability, after 24 hours of treatment, were CT-Cl and CT-CH3 (IC50 20.9 and 23.63 μM, respectively). In addition, using 10 μM and only 1 hour of pre-incubation, CT-CH3 caused a reduction in basal respiration (-37 %), oxygen consumption coupled with ATP synthesis (-60 %), and maximum oxygen consumption (-54 %). These alterations in respiratory parameters may be involved with the inhibitory effects of CT-CH3 since significant changes in oxygen consumption rates were observed in a condition that anticipates more significant losses of cell viability. The ADME parameters and the no violation of Lipinski Rule of Five showed that all compounds are safe. Conclusion: These results may contribute to the knowledge about the effects of CTs on these cells and the development of new treatments against HCCs.

Na/K‐ATPase Alpha1 Isoform Signaling in Cardiac Metabolism

Understanding cardiac metabolic signaling and its relation to pathological hypertrophy is critical to develop new therapeutic intervention in heart failure (HF). Decreased Na/K‐ATPase (NKA) activity and expression have long been associated with HF in experimental models and in patients. Classically known for its crucial role in transmembrane ion transport, electrical excitability, and contractility, NKA also supports non‐enzymatic receptor functions that differ among major cardiac isoforms (α1 and α2 in rodents). Studies from our group and others have suggested a role of these receptor functions in cardiac myocyte hypertrophy. Using epithelial (LLC‐PK1)‐derived cell lines, we have previously observed a NKA α1‐specific ability to modulate metabolic reserve and capacity. Mechanistically, this property is highly dependent upon NKA α1 ability to regulate the signaling partner Src kinase and is not supported by NKA α2, which lacks the Src binding domain. In this work, our goal was to assess the impact of NKA α1 ablation in the heart and demonstrate the importance of NKA α1 signaling function for proper cardiomyocyte metabolic function. We used a Myh6‐Cre/Lox approach to delete NKA α1 in the mouse heart (cardio α1‐/‐). These animals (3‐month‐old male mice) presented a normal cardiac structure, reduced NKA α1 expression (more than 100‐fold; n = 6‐7; p < 0.01) and increased NKA α2 expression (10‐fold; n = 6‐7; p < 0.01) by Western blot. RNA sequencing followed by gene set enrichment analysis revealed that cardio α1‐/‐ hearts have downregulated pathways related to mitochondrial metabolism, such as cytochrome complex, NADH dehydrogenase complex, respiratory chain, and mitochondrial fatty acid β‐oxidation (n = 3; FDR < 0.05). We next generated human induced pluripotent stem cells (hiPSCs) with a mutant form of the NKA α1 isoform that can pump ions but have blunted Src signaling (A420P mutant) using a CRISPR/Cas9‐mediated knock‐in approach. Genotyping and sequencing confirmed the substitution of alanine to proline in the NaKtide sequence to generate the desired mutation. These cells were differentiated into cardiomyocytes, as confirmed by overexpression of the cardiac markers myosin heavy chain 6 (MYH6) and cardiac muscle troponin T (TNNT2) (more than 10‐fold; n = 3; p < 0.05). Metabolic phenotyping using the Agilent Seahorse Extracellular Flux technology suggested that the A420P mutant hiPSC‐derived cardiomyocytes (A420P iCM) have decreased basal respiration, maximal respiration, spare respiratory capacity, and ATP production compared to the controls (WT iCM) based on changes in oxygen consumption rate (OCR) using the Agilent Seahorse XF Cell Mito Stress Test Kit. In addition, A420P iCM have reduced glycolytic capacity and reserve, based on changes in extracellular acidification rate (ECAR) using the Agilent Seahorse XF Glycolysis Stress Test Kit. Taken together, these results suggest that NKA α1 isoform signaling modulates the cardiomyocyte metabolism. Further studies may reveal a link between NKA/Src signaling, metabolic flexibility, and structural cardiac remodeling in the context of HF and cardiac hypertrophy.

Mitochondrial and metabolic remodelling in human skin fibroblasts in response to glucose availability.

Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine, and the validation of ingredients for cosmetics. Given these cells' short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in normal human dermal fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level [low glucose medium (LGm)], or its complete removal and replacement by galactose [medium that forces oxidative phosphorylation (OXPHOSm)], always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm was it possible to observe the selective inhibition of mitochondrial adenosine triphosphate (ATP) production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate and extracellular acidification rate, oxidation of citric acid cycle substrates, fatty acids, lactate, and other substrates, increased mitochondrial network extension and polarization, the increased protein content of voltage-dependent anion channel (VDAC) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha and changes in several key transcripts related to energy metabolism. LGm did not promote significant metabolic changes in NHDF, although mitochondrial network extension and VDAC protein content were increased compared to HGm-cultured cells. Our results indicate that short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.

Analysis of mitochondrial oxygen consumption and hydrogen peroxide release from cardiac mitochondria using electrochemical multi-sensors

Mitochondria are the primary sites of oxygen (O2) consumption and energy metabolism in most cell types, but also produce reactive oxygen species (ROS) that contribute to a wide array of pathological and physiological processes. Accordingly, simultaneous monitoring of mitochondrial ROS release and oxygen consumption rate (OCR) from cells and mitochondrial preparations is an attractive investigative approach in biological research, particularly when sample quantity is scarce. This paper presents the development of a sensitive multi-sensor device capable of measuring ROS production and OCR from biological samples in a single micro-chamber assay. Sensor sensitivities for O2 and hydrogen peroxide (H2O2; the major ROS species released by mitochondria and cells) are 4.32 nA/μM and 54.89 nA/μM, respectively, with limits of detection of 2.9 μM and 58.36 nM, respectively. Proof-of-concept studies in isolated mitochondria from rat cardiac tissue (5 µg protein) demonstrate an expected 3 – 4 fold increase in H2O2 release over the basal rate following addition of respiratory substrates, with a comparatively small change in OCR. The subsequent addition of adenosine diphosphate (ADP) decreased H2O2 release by 73% (p < 0.01) and increased OCR by 168% (p < 0.01), consistent with established shifts in mitochondrial membrane potential and electron flow from an ADP-limited (State 4) to ADP-stimulated (State 3) respiratory state. These studies validate the results from the use of a novel multi-sensor device capable of monitoring OCR and H2O2 simultaneously in scarce biological samples, with potential utility in the non-destructive integrative study of cellular metabolism and mitochondrial function.

CoenzymeQ10 and Ischemic Preconditioning Potentially Prevent Tourniquet-Induced Ischemia/Reperfusion in Knee Arthroplasty, but Combined Pretreatment Possibly Neutralizes Their Beneficial Effects

Tourniquet (TQ) use during total knee arthroplasty (TKA) induces ischemia/reperfusion (I/R) injury, resulting in mitochondrial dysfunction. This study aims to determine the effects of coenzyme Q10 (CoQ10) and ischemic preconditioning (IPC), either alone or in combination, on I/R-induced mitochondrial respiration alteration in peripheral blood mononuclear cells (PBMCs) and pain following TKA. Forty-four patients were allocated into four groups: control, CoQ10, IPC, and CoQ10 + IPC. CoQ10 dose was 300 mg/day for 28 days. IPC protocol was three cycles of 5/5-min I/R time. Mitochondrial oxygen consumption rates (OCRs) of PBMCs were measured seven times, at baseline and during ischemic/reperfusion phases, with XFe 96 extracellular flux analyzer. Postoperative pain was assessed for 48 h. CoQ10 improved baseline mitochondrial uncoupling state; however, changes in OCRs during the early phase of I/R were not significantly different from the placebo. Compared to ischemic data, IPC transiently increased basal OCR and ATP production at 2 h after reperfusion. Clinically, CoQ10 significantly decreased pain scores and morphine requirements at 24 h. CoQ10 + IPC abolished analgesic effect of CoQ10 and mitochondrial protection of IPC. In TKA with TQ, IPC enhanced mitochondrial function by a transient increase in basal and ATP-linked respiration, and CoQ10 provides postoperative analgesic effect. Surprisingly, CoQ10 + IPC interferes with beneficial effects of each intervention.

Relative Inference for Paired Data: More than Meets the Eye

Relative Inference for Paired Data: More than Meets the Eye

Effects of Siberian fir terpenes extract Abisil on antioxidant activity, autophagy, transcriptome and proteome of human fibroblasts.

Background: Abisil is an extract of Siberian fir terpenes with antimicrobial and wound healing activities. Previous studies revealed that Abisil has geroprotective, anti-tumorigenic, and anti-angiogenic effects. Abisil decreased the expression of cyclin D1, E1, A2, and increased the phosphorylation rate of AMPK.Objective: In the present study, we analyzed the effect of Abisil on autophagy, the mitochondrial potential of embryonic human lung fibroblasts. We evaluated its antioxidant activity and analyzed the transcriptomic and proteomic effects of Abisil treatment.Results: Abisil treatment resulted in activation of autophagy, reversal of rotenone-induced elevation of reactive oxygen species (ROS) levels and several-fold decrease of mitochondrial potential. Lower doses of Abisil (25 μg/ml) showed a better oxidative effect than high doses (50 or 125 μg/ml). Estimation of metabolic changes after treatment with 50 μg/ml has not shown any changes in oxygen consumption rate, but extracellular acidification rate decreased significantly. Abisil treatment (5 and 50 μg/ml) of MRC5-SV40 cells induced a strong transcriptomic shift spanning several thousand genes (predominantly, expression decrease). Among down-regulated genes, we noticed an over-representation of genes involved in cell cycle progression, oxidative phosphorylation, and fatty acid biosynthesis. Additionally, we observed predominant downregulation of genes encoding for kinases. Proteome profiling also revealed that the content of hundreds of proteins is altered after Abisil treatment (mainly, decreased). These proteins were involved in cell cycle regulation, intracellular transport, RNA processing, translation, mitochondrial organization.Conclusions: Abisil demonstrated antioxidant and autophagy stimulating activity. Treatment with Abisil results in the predominant downregulation of genes involved in the cell cycle and oxidative phosphorylation.

TRNA derived fragment (tRF)-3009 participates in modulation of IFN-\u03b1-induced CD4+ T cell oxidative phosphorylation in lupus patients

BackgroundAccumulating evidence suggests tRNA-derived fragments (tRFs) play important roles in cellular homeostasis. Here we aimed to explore aberrant expression of tRFs in CD4+ T cells from patients with systemic lupus erythematosus (SLE) and their potential function in the SLE pathogenesis.MethodsFirst, small RNA sequencing was performed on CD4+ T cells from four SLE patients and three healthy controls (HCs). Candidate tRFs were then validated in CD4+ T cells from 97 SLE patients and their relevant disease controls using qRT-PCR. Then sequencing was used to investigate the profiles of HC-derived CD4+ T cells transfected with tRF-3009. Lastly, tRF-3009 siRNA or tRF-3009 mimics were transfected into CD4+ T cells with/without IFN-α. Changes in oxygen consumption rate (OCR), ATP, and ROS production were analyzed.ResultsWe identified 482 differentially expressed tRFs from SLE CD4+ T cells and chose tRF-3009 for further analysis due to its upregulation and the positive correlations between its expression and SLEDAI, active lupus nephritis and serum IFN-α levels. In vitro, tRF-3009 over-expressing CD4+ T cell profiling and putative analysis linked this product to the type I IFN and oxidative phosphorylation (OXPHOS) pathways. Interestingly, IFN-α is capable of inducing ROS and ATP production in CD4+ T cells, while knockdown of tRF-3009 reversed this process. Overexpression of tRF-3009 in CD4+ T cells alone was sufficient to upregulate OCR, ROS, and ATP production.ConclusionsOur study is the first to link aberrant tRF expression and SLE. tRF-3009 may participate in metabolic modulation of IFN-α-induced CD4+ T cell OXPHOS in lupus.

A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.

Dual-emissive, oxygen-sensing boron nanoparticles quantify oxygen consumption rate in breast cancer cells.

.Significance: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex experimental systems.Aim: We present a method for quantifying the OCR of two tumor cell lines using oxygen-sensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay.Approach: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR.Results: Using a Bland–Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation.Conclusions: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland–Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.

6-Amino[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol Derivatives as Efficacious Mitochondrial Uncouplers in STAM Mouse Model of Nonalcoholic Steatohepatitis.

Small molecule mitochondrial uncouplers have recently garnered great interest for their potential in treating nonalcoholic steatohepatitis (NASH). In this study, we report the structure-activity relationship profiling of a 6-amino[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core, which utilizes the hydroxy moiety as the proton transporter across the mitochondrial inner membrane. We demonstrate that a wide array of substituents is tolerated with this novel scaffold that increased cellular metabolic rates in vitro using changes in oxygen consumption rate as a readout. In particular, compound SHS4121705 (12i) displayed an EC50 of 4.3 μM in L6 myoblast cells and excellent oral bioavailability and liver exposure in mice. In the STAM mouse model of NASH, administration of 12i at 25 mg kg-1 day-1 lowered liver triglyceride levels and improved liver markers such as alanine aminotransferase, NAFLD activity score, and fibrosis. Importantly, no changes in body temperature or food intake were observed. As potential treatment of NASH, mitochondrial uncouplers show promise for future development.

Ca2+‐induced Complex I Inactivity: A Model for Early Mitochondrial Dysfunction Following Volumetric Muscle Loss Injury

Calcium (Ca2+) is a secondary messenger, responsible for the regulation of a plethora of metabolic pathways that must be tightly regulated to avoid cytotoxicity 1. During early stage volumetric muscle loss (VML) injuries, damaged muscle fibers are open to the extracellular environment until the myofiber can be sealed, a process that may take hours 2– 3. This allows for free Ca2+ exchange between intra‐ and extracellular environments to concentrations beyond those appropriate for cell homeostasis. The purpose of this study was to model the changes in mitochondrial metabolism of early VML injuries as a response to a drastic increase of intracellular Ca2+.C57BL6 mice underwent unilateral surgery to create a VML defect in the gastrocnemius muscle, as approved by the University of Georgia’s Institutional Animal Care and Use Committee. Animals were sacrificed via CO2 inhalation, 18 hrs. following surgery. Total [Ca2+] per kg of tissue ([Ca2+]TWM) was measured using a BAPTA complexation assay. Gastrocnemius muscles from naïve mice were dissected into individual fiber bundles, permeabilized, and washed in either normal isotonic or Ca2+‐supplemented medium. Mitochondrial metabolic function and mitochondrial membrane potential were measured through oxygen consumption and Safranin‐O fluorescence rates, respectively, in a High‐Resolution Fluoro‐respirometer.[Ca2+]TWM was elevated in VML‐injured tissue, as expected (n=3, p&lt; 0.05). Acute Ca2+ exposure negatively affected Complex I (C‐I) activity of the electron transport chain. Specifically, C‐I activity was targeted by the addition of glutamate and malate (Glu‐Mal) and confirmed by the sequential addition of C‐I inhibitor Rotenone (Rot). In normal fibers, respiration increased after Glu‐Mal addition and subsequently decreased significantly following Rot, while Ca2+‐incubated fibers exhibited no change in oxygen consumption rate. Succinate (Succ) was used to rescue respiratory function by bypassing C‐I. Succ addition significantly increased oxygen consumption in both normal and Ca2+‐incubated fibers (n=3, p&lt;0.01), confirming a C‐I dysfunction in Ca2+‐treated fibers. Safranin fluorescence is sensitive to changes in mitochondrial membrane potential, decreasing with complex activity and increasing upon inhibition. In normal fibers, Safranin signal decreased with Glu‐Mal and Succ additions and increased with Rot, as expected. However, Ca2+‐treated fibers were only sensitive to Succ treatment, further supporting C‐I dysfunction (n=3, p&lt; 0.01).Herein, we show that an acute increase in intracellular Ca2+ negatively affects the process of oxidative phosphorylation within the mitochondria by disturbing the electron potential and inactivating Complex I. This alteration in membrane potential and decrease in complex activity may be the first step in a systematic shut down of mitochondrial metabolic function that leaves the muscle remaining after injury beyond the hope of effective repair.Support or Funding InformationFunded through the Department of Defense Neuromusculoskeletal Injuries Rehabilitation Research Award W81XWH‐18‐1‐0710 to SMG (UMinn) and JAC (UGA)A‐ Quantification of internal [Ca2+] of VML‐injured and control gastrocnemius muscles using BAPTA absorbance (n=3, p &lt; 0.05). B‐ Oxygen consumption rates of control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3 in duplicate, p &lt; 0.05). C‐ Safranin‐O signal fluctuation in control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3, p&lt; 0.05).Figure 1

Mapping mitochondrial respiratory chain deficiencies by respirometry: Beyond the Mito Stress Test

Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that “map” locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.

Evaluation of a Trunk Supporting Exoskeleton for reducing Muscle Fatigue

Although the effect of wearing a back-support exoskeleton during lifting has been demonstrated to reduce overall muscle activation, less is known about how wearing exoskeletons affect muscular fatigue and oxygen consumption. The purpose of this study is to evaluate the effect of wearing a back-support exoskeleton (backX) on muscle fatigue during repetitive lifting by assessing whether wearing backX increases endurance time relative to lifting unassisted. A secondary objective of this study is to quantify changes in oxygen consumption rate while performing a repetitive lifting task with and without backX to address a common industry concern. The UC Ergonomics Lab evaluated backX on twelve male subjects by measuring bilateral muscle activity of the erector spinae and oxygen consumption rate. Summary measures of muscle activity for 50 and 90 percent of the repetitive lifting session were used to characterize peak and mean muscle activity. Oxygen consumption rate was collected continuously during the repetitive lifting session. Compared to the unassisted condition, wearing backX reduced peak lumbar erector spinae activation by 16.5% and 21.8% (p &lt; 0.05). The time subjects could hold a back-straining posture after the repetitive lifting session increased by 52% after wearing backX during the lifting task. The was no significant negative change in oxygen consumption rate. This study confirms that wearing a backX reduces muscle activation in the lower back for this specific dynamic lifting task. Additionally, we find that wearing a backX may reduce the risk of low back injuries by reducing muscle activity and increasing endurance time to fatigue