Spare Capacity Research Articles

Relationships between matrix mineralization, oxidative metabolism, and mitochondrial structure during ATDC5 murine chondroprogenitor cell line differentiation.

The mechanistic relationships betweenthe progression of growth chondrocyte differentiation, matrix mineralization, oxidative metabolism, and mitochondria content and structure were examined in the ATDC5 murine chondroprogenitor cell line. The progression of chondrocyte differentiation was associated with a statistically significant (p ≤ 0.05)~2-fold increase in oxidative phosphorylation. However, as matrix mineralization progressed, oxidative metabolism decreased. In the absence of mineralization, cartilage extracellular matrix mRNA expression for Col2a1, Aggrecan, and Col10a1 were statistically (p ≤ 0.05)~2-3-fold greater than observed in mineralizing cultures. In contrast, BSP and Phex that are associated with promoting matrix mineralization showed statistically (p ≤ 0.05) higher ~2-4 expression, while FGF23 phosphate regulatory factor was significantly lower (~50%) in mineralizing cultures. Cultures induced to differentiate under both nonmineralizing and mineralizing media conditionsshowed statistically greater basal oxidative metabolism and ATP production. Maximal respiration and spare oxidative capacity were significantly elevated (p ≤ 0.05) in differentiated nonmineralizing cultures compared to those that mineralized. Increased oxidative metabolism was associated with both an increase in mitochondria volume per cell and mitochondria fusion, while mineralization diminishedmitochondrial volume and appeared to be associated with fission. Undifferentiated and mineralized cells showed increased mitochondrial co-localization with the actin cytoskeletal. Examination of proteins associated with mitochondria fission and apoptosis and mitophagy, respectively, showed levels of immunological expression consistent with the increasing fission and apoptosis in mineralizing cultures. These results suggest that chondrocyte differentiation is associated with intracellular structural reorganization, promoting increased mitochondria content and fusion that enables increased oxidative metabolism. Mineralization, however, does not need energy derived from oxidative metabolism; rather, during mineralization, mitochondria appear to undergofission and mitophagy. In summary, these studies show that as chondrocytes underwent hypertrophic differentiation, they increased oxidative metabolism, but as mineralization proceeds, metabolism decreased. Mitochondria structure also underwent a structural reorganization that was further supportive of their oxidative capacity as the chondrocytes progressed through their differentiation. Thus, the mitochondria first underwent fusion to support increased oxidative metabolism, then underwent fission during mineralization, facilitating their programed death.

The Value of Flexible Vaccine Manufacturing Capacity: Value Drivers, Estimation Methods, and Approaches to Value Recognition in Health Technology Assessment.

Expanding flexible vaccine manufacturing capacity (FVMC) for routine vaccines could facilitate more timely access to novel vaccines during future pandemics. Vaccine manufacturing capacity is 'flexible' if it is built on a technology platform that allows rapid adaption to new infectious agents. The added value of routine vaccines produced using a flexible platform for pandemic preparedness is not currently recognised in conventional health technology assessment (HTA) methods. We start by examining the current state of play of incentives for FVMC and exploring the relation between flexible and spare capacity. We then establish the key factors for estimating FVMC and draw from established frameworks to identify relevant value drivers. The role of FVMC as a countermeasure against pandemic risks is deemed an additional value attribute that should be recognised. Next, we address the gap in the vaccine-valuation literature between the conceptual understanding of the value of additional FVMC and the availability of accurate and reliable tools for its estimation to facilitate integration into HTA. Three practical approaches for estimating the value of additional FVMC are discussed: stated and revealed preference studies, macroeconomic modelling, and benefit-cost analysis. Lastly, we review how value recognition of additional FVMC can be realised within the HTA process for routine vaccines manufactured on flexible platforms. We argue that, while the value of additional FVMC is uncertain and further research is needed to help to better estimate it, the value of increased pandemic preparedness is likely to be too large to be ignored.

Integrative multi-omics analysis reveals ortho-topolin riboside exhibits anticancer activity by regulating metabolic pathways in radio-resistant triple negative breast cancer cells

Radio-resistant triple negative breast cancer (TNBC) is resistant to conventional drugs and radiation therapy. ortho-topolin riboside (oTR) has been evaluated for its anticancer activity in several types of cancer cells. However, its anti-proliferative activity in radio-resistant TNBC cells has not yet been reported. Therefore, we investigated the anti-proliferative activity of oTR in radio-resistant TNBC cells, and performed metabolome, lipidome, transcriptome, and proteome profiling to reveal the mechanisms of the anticancer activity of oTR. oTR showed cytotoxicity against radio-resistant TNBC cells with an inhibitory concentration (IC50) value of 7.78 μM. Significantly decreased (p value < 0.05) basal and compensatory glycolysis were observed in the oTR-treated group than untreated group. Mitochondrial spare respiratory capacity, which is relevant to cell fitness and flexibility, was significantly decreased (p value < 0.05) in the oTR-treated group. The major metabolic pathways significantly altered by oTR according to metabolome, transcriptome, and proteome profiles were the glycerolipid/glycerophospholipid pathway (log2(FC) of MGLL = −0.13, log2(FC) of acylglycerol lipase = −1.35, log2(FC) of glycerol = −0.81), glycolysis (log2(FC) of EGLN1 = 0.16, log2(FC) of EGLN1 = 0.62, log2(FC) of glucose = −0.76, log2(FC) of lactate = −0.81), and kynurenine pathway (log2(FC) of KYNU = 0.29, log2(FC) of kynureninase = 0.55, log2(FC) of alanine = 0.72). Additionally, proline metabolism (log2(FC) of PYCR1 = −0.17, log2(FC) of proline = −0.73) was significantly altered in the metabolomic and transcriptomic profiles. The MAPK signaling pathway (log2(FC) of CCN1 = −0.15, log2(FC) of CCN family member 1 = −1.02) and Rap 1 signaling pathway (log2(FC) of PARD6B = −0.28, log2(FC) of PAR6B = −3.13) were also significantly altered in transcriptomic and proteomic profiles. The findings of this study revealed that oTR has anticancer activity in radio-resistant TNBC cells by affecting various metabolic pathways, suggesting the potential of oTR as a novel anticancer agent for radio-resistant TNBC patients.

Mitochondrial function of dairy calf lymphocytes from birth to immunologic maturity

The inability of dairy calves to fully respond to immune stimuli until they reach maturity at 6 mo of age severely limits the use of parenteral vaccines to protect calves against disease. Immune responses are metabolically demanding, and immune cells rely on mitochondrial metabolites for their functionality. Due to the essential role of mitochondria in driving T-cell responses necessary for vaccine efficacy, we hypothesized that the mitochondrial function of dairy calf lymphocytes changes with age, from birth to immunologic maturity. In this cross-sectional study, groups of dairy calves (n = 4/group) were blood sampled at birth before colostrum intake and at 1, 2, 3, 4, 6, 8, 16, and 24 wk of age. Mid-lactation adult cows (n = 4) were also sampled to reference fully mature immune cell populations. B, CD4+, CD8+, and γδ T lymphocytes were enriched using magnetic-activated cell sorting, and their mitochondrial function was assessed with an extracellular flux analyzer. Non-mitochondrial oxygen consumption, basal respiration, maximal respiration, spare respiratory capacity, proton leak, and the oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) ratio were reported. Results were compared among groups using a Kruskal-Wallis test. The OCR to ECAR ratio is an indicator of the relative proportions of oxidative phosphorylation and aerobic glycolysis which is associated with effector functions in lymphocytes. The ratio was lower in 0 wk than adults in CD4+ T-cells. For CD8+ T-cells, the OCR to ECAR ratio for the 2 wk group was lower than the 3 wk group. A lower OCR to ECAR ratio indicates more reliance on glycolytic metabolism than oxidative phosphorylation. Maximal respiration is an indication of mitochondrial efficiency and is often associated with mitochondrial mass. For γδ T-cells, the 3 wk group had higher maximal respiration than the 16 wk group, whereas for B cells maximal respiration was higher in the 1 wk compared with the 16 wk group. Basal respiration indicates all cell functions that require oxygen and was lower in the 0 wk group than the 1 wk and 3 wk groups for CD4+ T-cells. γδ T-cells exhibited lower basal respiration in the 2 wk group than the 24 wk one. Although we found minimal differences in the mitochondrial outcomes reported from non-stimulated lymphocytes from birth through 6 mo of age and mid-lactation adults who served as mature immune cell populations, these results align with previous reports from weaning aged calf and adult CD4+ T-cells. In conclusion, there was insufficient evidence to suggest that the mitochondria in the lymphocytes of dairy calves from birth through immunologic maturity had functional changes associated with age. In conclusion, the capacity of unstimulated calf mitochondria to perform oxidative phosphorylation is not associated with age.

Alterations in oxidative phosphorylation complexes in arrhythmogenic cardiomyopathy using desmosomal KO cell model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III (Fondo Investigación Sanitaria FIS) Introduction Arrhythmogenic cardiomyopathy (ACM) is an inherited disease that predominantly affects the right ventricle characterized by progressive cardiomyocyte loss and the replacement of the myocardium by fibro-fatty tissue, which can lead to sudden cardiac death (1). The main genetic causes of ACM are variants in desmosomal genes (PKP2, DSP, DSG2, DSC2 and JUP), representing up to 50% of patients (2). We lack sufficient understanding at molecular level of the pathophysiological mechanisms that may trigger ACM and determine its progression. Recent studies have proposed a deficit in transcripts encoding for proteins from the electron transport chain, which may be a potential ACM molecular signature in samples from patients with ACM carrying mutations in PKP2 (3). Purpose Our study aims to deepen into the knowledge of this proposed mechanism in PKP2 deficiency using edited cell model and elucidating whether these mechanisms are also triggered in DSG2 and DSC2 deficiency, being a potential common ACM pathomechanism, or they are gene specific. Methods HL-1 edited cell lines for desmosomal genes previously generated by CRISPR/Cas9 (PKP2-KO, DSC2-KO and DSG2-KO) in our research group were used to perform our analysis (4). Expression profiling was performed by RNA-seq analysis. To experimentally validate the RNA-seq analysis results, we performed western blot assays using total protein lysate to test protein levels of the five oxidative phosphorylation (OXPHOS) complexes for the PKP2-KO, DSC2-KO and DSG2-KO groups. To test if these differences have an impact at functional level, XFp cell Mito Stress Test (Agilent Technologies) was performed. Results RNA-seq analysis results showed numerous downregulated pathways related to aerobic respiration, the electron transport chain, oxidative phosphorylation and ATP synthesis in all three desmosomal KO lines. Our experimental results showed a significant reduction of the OXPHOS complexes of the mitochondrial respiration chain at protein level in all PKP2-KO, DSC2-KO and DSG2-KO (Fig. 1). At functional level, our preliminary results of Mito Stress Test suggest differences in basal and maximal respiration, ATP production, non-mitochondrial oxygen consumption and spare respiratory capacity between experimental groups. Conclusions Our results support previously published studies describing oxidative phosphorylation as a pathomechanism for PKP2 deficiency and also suggest that these mechanisms may be a common feature for desmosomal mutations in ACM.Total expression of OXPHOS complexes

Impact of cardiac health status on bone marrow cell secretome and myocardial repair

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): University of Fribourg Graduate assistant Background Restoring the damaged myocardium remains a significant clinical challenge. Administration of bioactive factors, exosomes, or cell secretomes has shown promising results in promoting angiogenesis, tissue repair, immunomodulation, and extracellular matrix remodelling, particularly for cardiovascular disease treatments. Bone marrow (BM) cells are an essential source of secretome-based therapies for myocardial infarction (MI). Harnessing the potential of the cellular secretome for therapeutic applications requires a comprehensive understanding of its composition, functions, and mechanisms of action. MI affects the entire hematopoietic hierarchy, from stem cells to myeloid progenitors in BM. The study investigates how the cardiac status healthy versus infarcted affects the BM cell compositions and the properties of their secretomes. Methods Bone marrow cells (BMC) were isolated from healthy male Lewis rats (hBMC) and infarcted one (iBMC) obtained 2 weeks post MI induced by coronary artery ligation and cultured for 2 weeks. Cells composition, particularly macrophages, was characterised using multiplex flow cytometry Cytek Aurora. (CD90, CD29, CD45. CD68, CD206, CD163, Arg1, CD80, CCR7, iNOS). Cell proliferation (Real-time cell analyser and EdU incorporation), metabolic assays (seahorse assays), PCR, MS proteomic, and potency assays for immunomodulatory properties were performed. Results Multiplex flow cytometry showed distinctions in the percentage of cells: CD34+ stem cells and hematopoietic CD45+ cells were significantly upregulated in hBMCs, while CD68+ cells were higher in iBMCs. Cell proliferation assay demonstrated that iBMCs had significantly lower proliferation rates compared to hBMCs. Cell Metabolism was also impacted, iBMCs showed increased basal respiration, ATP production, and maximal respiration compared to hBMCs. An increase in respiratory spare capacity in iBMCs suggests an increased potential for ATP production under increased energy demands. Relative to hBMC, iBMC significantly regulated the expression of genes involved in immune response, inflammation, angiogenesis, and ECM remodelling gm-csf, il-17, vcam1, stat6, mmp9, cam, arg1, mhcII, arg2 were downregulated and tlr2, cxcl10, il-6, timp1, rantes, mcp-1, vegf, il-1ra, il-1beta, mip-1alpha, tlr4, b2m, and mmp3 upregulated. Secreted proteins also varied. Finally, the secretome of iBMC showed an enhanced capacity to induce a phenotype switch of macrophages toward an anti-inflammatory profile. Conclusion Our study shows that MI significantly alters the properties of bone marrow cells, impacting their composition, proliferation, metabolism, gene expression and the immunomodulatory potential of the secretome. These findings highlight the importance of considering donor health status in developing restoring secretome-based therapies for cardiac repair and emphasise the need to incorporate potency assay of the biologics for a standardised therapy and quality control.

Effects of sodium glucose co-transporter-2 inhibitor on atrial tachyarrhythmia burden in patients with cardiovascular implantable electronic device

Abstract Introduction Previous studies showed that sodium glucose co-transporter-2 inhibitors (SGLT2i) were associated with a lower atrial fibrillation (AF) incidence. Purpose Since mitochondrial dysfunction has been linked with AF, we aimed to examine the effects of SGLT2i on AF burden and mitochondrial function in patients with cardiovascular implantable electronic device (CIED). Methods Patients with CIED were randomly assigned to receive either dapagliflozin 10 mg daily or placebo for 3 months. The AF burden was measured as atrial high-rate episodes (AHRE) via CIED interrogations, and data were collected at baseline and 3-months post-treatment. Oxidative stress and mitochondrial function were determined in peripheral blood mononuclear cells (PBMCs). Results The study enrolled 54 CIED patients. There were 9 (16.7%) patients with diabetes mellitus and 36 (66.7%) patients with history of clinical AF. The changes of AHRE burden after 3-month treatments did not differ between dapagliflozin and placebo groups. However, in those with clinical AF, dapagliflozin significantly decreased the number of AHRE per month as compared to placebo (-2.2 vs. +0.6 episodes per month, p=0.048) (Table 1). Mitochondrial spare respiratory capacity (SRC) percentage was significantly increased after treatment with dapagliflozin (162.1% to 203.2%, p=0.003), whereas this was not found in the placebo group (Figure 1). In the subgroup analysis of patients with clinical AF, dapagliflozin showed significant improvement in both the SRC percentage (165.7 to 201.7%, p=0.016) and a reduction in cellular oxidative stress (26,840.5 to 18,164.0 arbitrary unit, p=0.049). Conclusions Dapagliflozin significantly reduced the number of AHRE per month in patients with history of clinical AF. The improvement in PBMC’s mitochondrial function and reduced cellular oxidative stress among dapagliflozin users could be the mechanisms responsible for its anti-atrial arrhythmic effect.Table 1Figure 1

#1492 Metabolic switch in CD8+ T cells may influence immune response in chronic antibody-mediated rejection (CAMR)

Abstract Background and Aims CAMR is the main cause of chronic graft injury and subsequent graft loss, but its pathogenesis is still largely unclear. Our group demonstrated an increase in CD8+ T cells infiltrating biopsies from CAMR patients (PMID: 20595199), but their role remains to be defined. Recently, immunometabolism has emerged as a field connecting cell energy metabolism with the immune cell differentiation and functions. Particularly, the permanent aerobic glycolysis of CD8+ lymphocytes induces their transition from naïve to activated state and effector T cells (PMID: 37315709). Thus, the aim of the study was to investigate the molecular mechanisms underlying the development of CAMR by the analysis of gene expression profiles of CD8+ T lymphocytes in conjunction with their metabolic state. Method We enrolled 14 patients with biopsy-proven CAMR and 12 stable transplant recipients with normal graft histology and function (control group). CD8+ T cells were purified by CD8 MicroBeads (Miltenyi Biotec) then counted (TC20™ Automated Cell Counter). Gene expression profiles of CD8+ T lymphocytes isolated from both groups were assessed using Agilent microarrays. Data obtained were statistically and functionally evaluated (IPA software) and validated by qPCR. Oxygen consumption rate (OCR) in CD8+ and CD8- cells was measured by a Resipher oxygen sensing lid (Lucid Scientific Inc., USA). The contribution of mitochondrial respiration to OCR was investigated in isolated CD8+ T cells from CAMR and controls treated with rotenone/antimycin A (Rot/AA), oligomycin (Olygo) or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to evaluate spare respiratory capacity (SRC), ATP-linked respiration, proton leak and maximal respiration. Results Gene expression profiles of CD8+ T cells showed a characteristic alteration of lipid metabolism in CAMR patients with the triglyceride biosynthesis pathway (p = 2.69E-3) as the most altered pathway and several genes de-regulated all potentially involved in the altered metabolic state of CD8+ T cells. Among others, we validated the increased expression of Nicotinamide Phosphoribosyltransferase (NAMPT) (FC = +2.39), the rate-limiting enzyme of nicotinamide adenine dinucleotide (NAD+) salvage pathway involved in the glycolysis progression, mostly activated in effector T cells. We then decided to evaluate the OCR and the metabolic state of CD8+T cells. Basal OCR was higher in CD8+ rather than CD8- cells, but with different trends among cells isolated from control and CAMR patients. Indeed, CD8+ cells from CAMR patients showed higher OCR compared to control, while OCR was lower in CD8- from CAMR rather than control patients. CD8+ OCR from both CAMR and control patients was mostly dependent on mitochondrial respiration, since it was deeply reduced by Rot/AA treatment. Interestingly, inhibitory response to Oligo treatment by CD8+ cells from CAMR patients was modest, while OCR was noticeably stimulated by FCCP in these cells, suggesting that mitochondrial oxygen uptake was not ATP linked but rather associated with increased uncoupling. These data suggest that CD8+ T cells from CAMR patients are activated effector T cells primarily utilizing aerobic glycolysis. To confirm this hypothesis, we evaluated the gene expression levels of: i) HIF1a, which involves permanent dependence on glycolysis even in the presence of oxygen (Warburg effect); ii) fatty acid transporter CD36, whose levels mimic an energy dependence on the beta oxidation of fatty acids, a process activated in memory T cells. We confirmed an increased expression of HIF1a and a reduced expression of CD36 in CD8+T cells from CAMR patients compared to controls (p&amp;lt;0.05), that confirm the T effector phenotype. Conclusion Our data suggest that CD8+ T cell from CAMR patients showed permanent dependency on glycolysis leading to altered immune phenotype and response, thus indicating novel opportunities to modulate the immune system for therapeutic purposes in kidney transplanted patients and reduce graft rejection.

The Bayu-Undan liquid CO2 import facilities – finding an effective solution

Carbon capture and storage (CCS) is widely considered to be one of the key enablers to decarbonising the economy. It is currently being implemented in a number of projects in gas, power, chemicals, cement, steel, and other industries. In the Asia–Pacific region, shipping of liquid carbon dioxide (LCO2) is widely seen as a key component of the CCS value chain. Shipping allows the emitters located in the industrial centres of Japan, Korea, and Singapore to access distant CO2 storage locations. The repurposing of the Bayu-Undan (BU) platforms and pipeline from gas production to CO2 injection presents a regional opportunity for the disposal of shipped CO2. Multiple LCO2 development concepts were assessed for unloading and storing the imported LCO2. Unloading can be carrier-to-shore, carrier-to-ship/barge, or carrier-to-platform; storage can be onshore, nearshore, offshore or no buffer storage at all with direct injection from carriers. These concepts required evaluation for technical functionality, maturity, complexity, operability, and availability. Imported LCO2 is too cold and low pressure to be transported or injected at the carrier transport conditions. The pressure needs to be boosted and temperature increased to meet the requirements of the injection system. This requires sources of power and heat. An evaluation of existing facilities, their condition, available space and spare capacity, and their constructability considerations revealed important opportunities and constraints. A study was carried out, which evaluated options for the BU LCO2 import using key evaluation criteria to select the most appropriate concept. This paper explores addressing the challenges involved.

Probucol-Ursodeoxycholic Acid Otic Formulations: Stability and In Vitro Assessments for Hearing Loss Treatment

Targeted drug delivery is an ongoing aspect of scientific research that is expanding through the design of micro- and nanoparticles. In this paper, we focus on spray dried microparticles as carriers for a repurposed lipophilic antioxidant (probucol). We characterise the microparticles and quantify probucol prior to assessing cytotoxicity on both control and cisplatin treated hair cells (known as House Ear Institute-Organ of Corti 1; HEI-OC1). The addition of water-soluble polymers to 2% β-cyclodextrin resulted in a stable probucol formulation. Ursodeoxycholic acid (UDCA) used as formulation excipient increases probucol miscibility and microparticle drug content. Formulation characterisations reveals spray drying results in spherical UDCA-drug microparticles with a mean size distribution of ∼5–12 μm. Probucol microparticles show stable short-term storage conditions accounting for only ∼10% loss over seven days. By mimicking cell culture conditions, both UDCA-probucol (67%) and probucol only (82%) microparticles show drug release in the initial two hours. Furthermore, probucol formulations with or without UDCA preserve cell viability and reduce cisplatin-induced oxidative stress. Mitochondrial bioenergetics results in lower basal respiration and non-mitochondrial respiration, with higher maximal respiration, spare capacity, ATP production and proton leak within cisplatin challenged UDCA-probucol groups. Overall, we present a facile method for incorporating lipophilic antioxidant carriers in polymer-based particles that are tolerated by HEI-OC1 cells and show stable drug release, sufficient in reducing cisplatin-induced reactive oxygen species accumulation.

Cilia-deficient renal tubule cells are primed for injury with mitochondrial defects and aberrant tryptophan metabolism.

The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism.NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI.

A proactive transshipment model for prototype parts logistics in the automotive industry

AbstractIn the logistics of prototype parts in the automotive industry, unique parts are stored in warehouses and provided to assembly service providers to ensure a timely assembly of the associated prototype vehicles. As the allocation of the assembly orders to the assembly service providers may change before the start of the assembly, the shipments are planned at short notice. This article considers the short-term task of planning shipments of individual prototype parts from multiple warehouses to multiple assembly service providers. The current literature on planning shipments focuses mainly on parts available in batch sizes larger than one. However, as prototype parts are individual, storing them in adequate warehouses is crucial. A mixed-integer linear programming model for planning transshipments of parts between the warehouses and shipments to the assembly service providers to minimize overall costs is presented. The model enables the utilization of spare capacities of planned transports for a proactive transshipment of parts between warehouses. To this end, the future shipment costs of parts to assembly service providers are approximated to determine suitable warehouses. A numerical study shows that the proactive approach leads to a considerable cost reduction.

Sample intelligence-based progressive hedging algorithms for the stochastic capacitated reliable facility location problem

Selecting facility locations requires significant investment to anticipate and prepare for disruptive events like earthquakes, floods, or labor strikes. In practice, location choices account for facility capacities, which often cannot change during disruptions. When a facility fails, demand transfers to others only if spare capacity exists. Thus, capacitated reliable facility location problems (CRFLP) under uncertainty are more complex than uncapacitated versions. To manage uncertainty and decide effectively, stochastic programming (SP) methods are often employed. Two commonly used SP methods are approximation methods, i.e., Sample Average Approximation (SAA), and decomposition methods, i.e., Progressive Hedging Algorithm (PHA). SAA needs large sample sizes for performance guarantee and turn into computationally intractable. On the other hand, PHA, as an exact method for convex problems, suffers from the need to iteratively solve numerous sub-problems which are computationally costly. In this paper, we developed two novel algorithms integrating SAA and PHA for solving the CRFLP under uncertainty. The developed methods are innovative in that they blend the complementary aspects of PHA and SAA in terms of exactness and computational efficiency, respectively. Further, the developed methods are practical in that they allow the specialist to adjust the tradeoff between the exactness and speed of attaining a solution. We present the effectiveness of the developed integrated approaches, Sampling Based Progressive Hedging Algorithm (SBPHA) and Discarding SBPHA (d-SBPHA), over the pure strategies (i.e. SAA). The validation of the methods is demonstrated through two-stage stochastic CRFLP. Promising results are attained for CRFLP, and the method has great potential to be generalized for SP problems.

AMPK regulates immature boar Sertoli cell proliferation through affecting CDK4/Cyclin D3 pathway and mitochondrial function

Sertoli cell (SC) proliferation plays an important role in sperm production and quality; however, the regulatory mechanism of SC proliferation is not well understood. This study investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in the regulation of immature boar SC activity. Cell counting kit-8, Seahorse XFe96, mitochondrial respiratory enzyme-related assay kits, and transmission electron microscopy were used to detect SC proliferative viability, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and the ultrastructure of primary cultured SCs in vitro from the testes of 21-day-old boars. A dual luciferase reporter assay was performed to determine the miRNA-mRNA target interaction. Western blotting was used to analyze cell proliferation-related protein expression of p38, p21, proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinase 4 (CDK4), Cyclin D3, and phosphorylated retinoblastoma protein (Rb). Each experiment had a completely randomized design, with three replicates in each experiment. The results showed that the AMPK inhibitor (Compound C, 20 μM-24 h) increased cell proliferation viability, ATP production, and maximal respiration of SCs by 0.64-, 0.12-, and 0.08-fold (p < 0.05), respectively; increased the SC protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.13-, 0.09-, 0.88-, and 0.12-fold (p < 0.05), respectively; and decreased the SC protein expression of p38 and p21 by 0.36- and 0.27-fold (p < 0.05), respectively. The AMPK agonist AICAR (2 mM-6 h) significantly inhibited SC ultrastructure, OCR, mitochondrial respiratory enzyme activity, and cell proliferation-related protein levels. AMPK was validated to be a target gene of miR-1285 based on the result in which the miR-1285 mimic inhibited the luciferase activity of wild-type AMPK by 0.54-fold (p < 0.001). MiR-1285 mimic promoted the OCR of SCs, with 0.45-, 0.15-, 0.21-, and 0.30-fold (p < 0.01) increases in ATP production, basal and maximal respiration, and spare capacity, respectively. MiR-1285 mimic increased the mitochondrial respiratory enzyme activity of SCs, with 0.63-, 0.70-, and 0.97-fold (p < 0.01) increases in NADH-Q oxidoreductase, cytochrome c oxidase, and ATP synthase, respectively. Moreover, the miR-1285 mimic increased the protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.24-, 0.30-, 0.22-, and 0.13-fold (p < 0.05), respectively, and reduced the protein expression of p38 and p21 by 0.58- and 0.66-fold (p < 0.001). MiR-1285 inhibitor showed opposite effects on the above indicators and induced numerous autophagosomes and large lipid droplets in SCs. A high dose of estradiol (10 μM-6 h, showed a promotion of AMPK activation in a previous study) significantly inhibited SC ultrastructure, mitochondrial function, and proliferation-related pathways, while these adverse effects were weakened by Compound C treatment or miR-1285 mimic transfection. Our findings suggest that the activation and inhibition of AMPK induced by specific drugs or synthesized targeted miRNA fragments could regulate immature boar SC proliferative activity by influencing the CDK4/Cyclin D3 pathway and mitochondrial function; this helps to provide a basis for the prevention and treatment of male sterility in clinical practice.

A Mitochondrial Targeted Ubiquinol (MitoQ) Improves Immune Cell Bioenergetics in Patients with Heart Failure with Preserved Ejection Fraction

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a proinflammatory syndrome. Mitochondrial dysfunction that is characteristic of HFpEF could alter immunometabolism, attenuating the functional reserve of immune cells. In addition, immune cell mitochondrial respiration is a biomarker of systemic mitochondrial health. The aim of this study was to assess the impact of a chronic oral mitochondrial targeted ubiquinol (MitoQ) on the immune cell bioenergetic profile in patients with HFpEF. We hypothesized that MitoQ administration in patients with HFpEF, would improve mitochondrial respiration in peripheral blood mononuclear cells (PBMC’s). METHODS: In this double-blind, crossover trial 8 patients with a clinical diagnosis of HFpEF (6 female, 2 male; 6 African American/Black, 2 white; mean ± SEM, Age, 58±5 years; BMI, 37±1 kg/m2) received a 4 week oral dose of MitoQ (MTQ, 20 mg/day) and placebo (PLB) in random order separated by a 2 week wash out period. Following completion of each trial arm, a venous blood sample was obtained and PBMCs were isolated using density gradient centrifugation. High resolution respirometry coupled with modulators of cellular respiration was performed to obtain basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity of the immune cells. Paired samples t-tests were used to compare variables between conditions. RESULTS: MitoQ was safe, well tolerated, and compliance was high (MTQ, 99 ± 1%; PLB, 89±4%). Basal respiration (MTQ, 106.4 ± 11.5 pmol/min; PLB, 66.5±5.9 pmol/min; p=0.002), ATP-linked respiration (MTQ, 41.4± 16.9 pmol/min; PLB, 16.3±6.7 pmol/min; p=0.04), maximal respiration (MTQ, 323.9 ± 50.0 pmol/min; PLB, 65.4±23.1 pmol/min; p=0.01), spare respiratory capacity (MTQ, 217.5 ± 39.6 pmol/min; PLB, 149.6 ±18.2 pmol/min; p=0.02), were higher following MTQ compared to PLB. CONCLUSIONS: In patients with HFpEF, administration of a mitochondrial targeted ubiquinol improved the immune cell mitochondrial respiration capacity and effciency. An increase in spare respiratory capacity following MTQ reflects an improvement in functional reserve that would augment the ability to respond to an acute stressor. These findings have positive implications for the ability to target and improve mitochondrial function in patients with HFpEF. Future work should investigate if the observed improvements in immune cell mitochondrial function are mirrored in other tissues. Supported by AHA 19CDA3474002. 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.

Hypoxia Exposure Fine-Tunes Mitochondrial Function in Primary Dermal Fibroblasts Derived from Loggerhead Sea Turtles

Sea turtles are constantly exposed to changes in oxygen tension derived from extended breath-hold diving, but the cellular-level response to such changes is not completely understood. Most studies in hypoxia-tolerant reptiles have focused on the freshwater turtle Trachemys scripta, which responds to anoxia by inducing metabolic rewiring and preserving mitochondrial integrity, facilitating effcient mitochondrial respiration during reoxygenation. Here, we derived primary dermal fibroblasts from loggerhead sea turtles and western fence lizards to study the bioenergetic cellular response to hypoxia exposure in diving and non-diving reptiles. Cells from both species stained positive for the fibroblast markers vimentin and PDGFRb and responded to hypoxia exposure (0.1% O2) by accumulating HIF-1α. We measured oxygen consumption rates (OCR) in intact cells using Seahorse technology. We found that basal, maximal, and spare respiratory capacity was lower in lizard than in loggerhead cells at 26oC under normoxic (room air) conditions (p &lt; 0.0001). Exposure to short (1h) and long-term hypoxia (24h) altered mitochondrial function in both species. Maximal and spare respiratory capacity were lower in loggerhead than in lizard cells (p &lt; 0.01) after 1h hypoxia followed by 1h reoxygenation. In contrast, basal and maximal respiratory capacity was higher in loggerhead compared to lizard cells after 24h hypoxia (p &lt; 0.04). Nonetheless, the overall OCR after 24h hypoxia in both species was lower than that observed after 1h hypoxia. Overall, there was a significant reduction in cellular respiration in both species from short-term to long-term hypoxia exposure. In lizard cells, basal OCR decreased by a factor of three while maximal OCR decreased by a factor of 4.5. In loggerhead cells, both the basal and maximal OCR decreased by a factor of two. Our results demonstrate species-specific regulation of cellular respiration after hypoxia exposure. Remarkably, loggerhead cells show a steady downregulation of cellular respiration in response to hypoxia exposure and faster recovery after extended hypoxia (24h) than lizard cells, aligning with the remarkable hypoxic tolerance exhibited by sea turtles, which can endure up to 7 hours of breath-holding underwater. Understanding the cellular mechanisms that drive hypoxic tolerance in diving animals can provide insights for treating human conditions characterized by hypoxia signaling. Ford Foundation. 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.

Skeletal Muscle Derived Cells from Old Human Donors Show Increased Mitochondrial Lipid Peroxidation and Reduced Mitochondrial Function

Skeletal muscle undergoes atrophy with age progression resulting in decreased mitochondrial function. Similarly, age-related increases in mitochondrial oxidant generation can promote mitochondrial oxidative damage, but the relationship between increased oxidant stress and mitochondrial function is not well understood. Here, we profiled changes in mitochondrial function and mitochondrial lipid peroxidation in intact primary skeletal muscle progenitor cells (myoblasts) derived from the Rectus abdominis muscle of healthy young (19.67 ± 1.70 years) and old (67.0 ± 1.41 years) humans. Using metabolic flux assays, we found that cells derived from young humans showed higher basal and maximal oxygen consumption rates compared to cells derived from old humans (basal: 22.67 ± 6.53 vs. 10.81 ± 5.69; maximal: 41.02 ± 15.20 vs. 17.33 ± 4.84 pmol/min/protein, p &lt; 0.001). Similarly, cells derived from young humans also showed higher ATP production and spare respiratory capacity (ATP: 18.79 ± 5.17 vs. 9.15 ± 4.91; SRC: 18.35 ± 9.62 vs. 6.52 ± 3.42 pmol/min/protein, p &lt; 0.001). In contrast, using live-cell fluorescence microscopy, MitoTracker red and the mitochondrial-target lipid peroxide probe MitoPeDPP, we found higher mitochondrial lipid peroxidation levels in cells derived from old than young humans ( p = 0.0007). We then evaluated whether peroxiredoxin 6 (Prdx6), an enzyme that repairs lipid peroxidation through its phospholipid glutathione peroxidase and phospholipase A2 activities, impacts mitochondrial function using CRISPR/Cas9-mediated knockouts of Prdx6 in C2C12 myoblasts and extracellular flux assays. Spare respiratory capacity was lower in cells with full Prdx6-KO than in cells transfected with non-target gRNAs (NT: 295.10 ± 18.59 vs. Prdx6-KO: 242.20 ± 17.93; p = 0.05). These results show that mitochondrial function decreases with age along with increases in mitochondrial lipid peroxidation in primary muscle cells derived from old humans. Similarly, these results show that the Prdx6 supports mitochondrial function. Overall, our data contributes to our understanding of the mechanistic drivers of age-related skeletal muscle mitochondrial dysfunction. NIGMS-R35GM146951 NIA-R01AG059715. 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.

Modulation of Endothelial Cell Mitochondrial Phenotype and Angiogenesis by TRPV4

Transient receptor potential vanilloid type 4 (TRPV4) is a mechanically activated ion channel implicated in the regulation of endothelial cell (EC) proliferation, migration, and angiogenesis. However, the molecular mechanism(s) by which TRPV4 regulates EC functions remains underexplored. Here, we investigated if TRPV4 channels mediate endothelial function via modulation of mitochondria. Confocal microscopy showed a peri-nuclear localization of mitochondria in normal EC (NEC), while they were localized throughout the cell in TRPV4 knockout EC (KOEC). Furthermore, transmission electron microscopy confirmed clear round mitochondria in NEC compared to elongated mitochondria with distinct cristae in KOEC. Importantly, we found increased distribution of mitochondria in KOEC with increasing stiffness, when cultured on extracellular matrix (ECM) gels of varying stiffness that mimic the stiffness of matrix in pathophysiological conditions such as tumor or heart failure (0.2, 8, and 50 kPa). Mechanistically, western blot analysis showed increased fusion/fission protein ratio (Optic Atrophy 1 (OPA1)/mitochondrial fission factor (MFF)), in KOEC compared to NEC. Seahorse flux analyzer analysis demonstrated increased basal oxygen consumption rate (OCR), maximal OCR, ATP-linked OCR, and spare capacity in KOEC compared to NEC, which were significantly attenuated by a small molecule inhibitor of OPA1, MYLS22. Finally, MYLS22 normalized TRPV4-knockout mediated abnormal proliferation, migration, and angiogenesis ex vivo. Taken together, these findings indicate that mechanosensitive TRPV4 channels regulate mitochondrial phenotype and function during angiogenesis through OPA1. This work was supported by the National Institutes of Health (and R01HL148585 and AHA-TPA-971237) to CKT. 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.

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&lt;0.05). Orchiectomy resulted in an increase in male OCR parameters at both ages (p&lt;0.05); ovariectomy did not affect renal cortical OCR (p&gt;0.05 for both ages). Of note, gonadectomy in males increased the OCR up to the female level (p&gt;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&lt;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&lt;0.001), whereas ovariectomy lowered H2O2 at both time points (p&lt;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&lt;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.

Characterization of Sex-Dependent Differences in Brain Microvascular Bioenergetics

Background: Women experience increased incidence of stroke-related deaths and lifetime risk of Alzheimer's disease (AD). Microvascular dysfunction has been implicated in the pathogenesis of ischemic stroke and AD. Our objective is to investigate the sex-dependent differences in bioenergetics of young mouse brain microvessels (BMVs). Methods: BMVs were isolated from young male and female mice (3-4 months), using a combination of gradient centrifugation and filtration (300μm and 40μm filters) methods. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMVs were measured utilizing the Agilent Seahorse XFe 24 analyzer. The levels of proteins and mRNAs of mitochondrial respiratory complexes were measured by western blot and RT-PCR, respectively. Citrate synthase (CS) enzyme activity and ATP levels were determined in lysates of BMVs. Results: Real-Time ATP Rate assay demonstrated a decreased total ATP production rate and ATP contribution from both glycolysis and oxidative phosphorylation (OxPhos) in BMVs from females compared to males. Mitochondrial stress test assay revealed significantly lower values of several mitochondrial respiratory parameters, including basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak in BMVs from females compared to males. Glycolytic Rate assay uncovered reduced basal glycolysis, proton efflux rate (PER) originating from basal glycolysis, and the mitoOCR/glycoPER ratio in BMVs from females compared to males. Interestingly, no sex-dependent differences were observed in basal PER and post-2-DG acidification rates in BMVs. Mito Fuel Flex Test assay showed no sex-dependent difference in the utilization of glucose, glutamine, and long-chain fatty acids as fuel substrates in BMVs. ATP measurements confirmed the reduced ATP levels in BMVs from females compared to males. Measurements of CS activity revealed no sex-dependent difference in the BMVs. Lastly, Western blot and RT-PCR experiments indicated no differences in either protein or mRNA levels of various mitochondrial complex protein levels.Conclusions: Female BMVs showed reduced mitochondrial respiratory and glycolytic parameters compared to male BMVs although relative contribution of various substrates for energy production were not different. These sex-dependent differences involving glycolysis and OxPhos make the microvasculature vulnerable to injury in females. Funding sources: NIH grants AG074489 and NS114286 (PVGK and RM). 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.