Antegrade persufflation of porcine kidneys improves renal function after warm ischemia.

Introduction: Transplantation of kidneys from expanded criteria donors (ECD), including after circulatory death (DCD), is associated with a higher risk of adverse events compared to kidneys from standard criteria donors. In previous studies, improvements in renal transplant outcomes have been seen when kidneys were perfused with gaseous oxygen during preservation (persufflation, PSF). In the present study, we assessed ex-vivo renal function from a Diffusion Contrast Enhanced (DCE)-MRI estimation of glomerular filtration rate (eGFR); and metabolic sufficiency from whole-organ oxygen consumption (WOOCR) and lactate production rates. Methods: Using a porcine model of DCD, we assigned one kidney to antegrade PSF, and the contralateral kidney to static cold storage (SCS), both maintained for 24 h at 4°C. Post-preservation organ quality assessments, including eGFR, WOOCR and lactate production, were measured under cold perfusion conditions, and biopsies were subsequently taken for histopathological analysis. Results: A significantly higher eGFR (36.6 ± 12.1 vs. 11.8 ± 4.3 ml/min, p < 0.05), WOOCR (182 ± 33 vs. 132 ± 21 nmol/min*g, p < 0.05), and lower rates of lactate production were observed in persufflated kidneys. No overt morphological differences were observed between the two preservation methods. Conclusion: These data suggest that antegrade PSF is more effective in preserving renal function than conventional SCS. Further studies in large animal models of transplantation are required to investigate whether integration with PSF of WOOCR, eGFR or lactate production measurements before transplantation are predictive of post-transplantation renal function and clinical outcomes.

The Impact of the Angiotensin-Converting Enzyme Inhibitor Lisinopril on Metabolic Rate in Drosophila melanogaster.

Evidence suggests that angiotensin-converting enzyme inhibitors (ACEIs) may increase metabolic rate by promoting thermogenesis, potentially through enhanced fat oxidation and improved insulin. More research is, however, needed to understand this intricate process. In this study, we used 22 lines from the Drosophila Genetic Reference Panel to assess the metabolic rate of virgin female and male flies that were either fed a standard medium or received lisinopril for one week or five weeks. We demonstrated that lisinopril affects the whole-body metabolic rate in Drosophila melanogaster in a genotype-dependent manner. However, the effects of genotypes are highly context-dependent, being influenced by sex and age. Our findings also suggest that lisinopril may increase the Drosophila metabolic rate via the accumulation of a bradykinin-like peptide, which, in turn, enhances cold tolerance by upregulating Ucp4b and Ucp4c genes. Finally, we showed that knocking down Ance, the ortholog of mammalian ACE in Malpighian/renal tubules and the nervous system, leads to opposite changes in metabolic rate, and that the effect of lisinopril depends on Ance in these systems, but in a sex- and age-specific manner. In conclusion, our results regarding D. melanogaster support existing evidence of a connection between ACEI drugs and metabolic rate while offering new insights into this relationship.

Alterations in the molecular regulation of mitochondrial metabolism in human alveolar epithelial cells in response to cigarette- and heated tobacco product emissions.

Mitochondrial abnormalities in lung epithelial cells have been associated with chronic obstructive pulmonary disease (COPD) pathogenesis. Cigarette smoke (CS) can induce alterations in the molecular pathways regulating mitochondrial function in lung epithelial cells. Recently, heated tobacco products (HTPs) have been marketed as harm reduction products compared with regular cigarettes. However, the effects of HTP emissions on human alveolar epithelial cell metabolism and on the molecular mechanisms regulating mitochondrial content and function are unclear. In this study, human alveolar epithelial cells (A549) were exposed to cigarette or HTP emissions in the form of liquid extracts. The oxygen consumption rate of differently exposed cells was measured, and mRNA and protein abundancy of key molecules involved in the molecular regulation of mitochondrial metabolism were assessed. Furthermore, we used a mitophagy detection probe to visualize mitochondrial breakdown over time in response to the extracts. Both types of extracts induced increases in basal-, maximal- and spare respiratory capacity, as well as in cellular ATP production. Moreover, we observed alterations in the abundancy of regulatory molecules controlling mitochondrial biogenesis and mitophagy. Mitophagy was not significantly altered in response to the extracts, as no significant differences compared to vehicle-treated cells were observed.

Paclitaxel-resistance facilitates glycolytic metabolism via Hexokinase-2-regulated ABC and SLC transporter genes in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) frequently develops resistance to platinum-based therapies, which is regarded as an aggressive subtype. However, metabolic changes in paclitaxel resistance remain unclear. Herein, we present the metabolic alternations of paclitaxel resistance in bioenergetic profiling in OCCC. Paclitaxel-resistant OCCC cells were developed and metabolically active with oxygen consumption rates (OCR) compared to parental cells. Metabolite profiling analysis revealed that paclitaxel-resistant OCCC cells reduced intracellular ATP and GTP influx rates, increasing the NADH/NAD+ ratio. We further demonstrated that paclitaxel-resistant OCCC cells led to characteristic alternations of metabolite levels in energy-requiring and energy-releasing steps of glycolysis and their corresponding glycolytic enzymes. Copy number alterations and RNA sequencing analysis demonstrated that ATP-binding cassette (ABC) transporters and solute carrier (SLC) transporter genes involved in glycolysis metabolism and molecular transport were enriched in paclitaxel-resistant OCCC cells. We first identified that Hexokinase 2 (HK2) expression is upregulated in paclitaxel-resistant OCCC cells to determine the quantity of glucose entering glycolysis. Utilizing proteolysis-targeting chimera (PROTAC) HK2 degraders, we also found that paclitaxel sensitivity, viability, and oxygen consumption rates under paclitaxel treatment were restored by HK2 degraders treatment, and decreased downstream expression of the ABC and SLC transporters was shown in OCCC cells. Taken together, these findings highlight the paclitaxel resistance in OCCC elucidates metabolic alternation, including ABC- and SLC- drug transporters, thereby affecting glycolysis metabolism in response to paclitaxel resistance, and HK2 may become a novel potential therapeutic target for paclitaxel resistance.

Potential (co-)contamination of dairy milk with AFM1 and MC-LR and their synergistic interaction in inducing mitochondrial dysfunction in HepG2 cells.

Several toxic metabolites, such as aflatoxin M1 (AFM1), are known to contaminate dairy milk. However, as mentioned in an external EFSA report, there is a knowledge gap regarding the carry-over of certain emerging toxins such as microcystin-LR (MC-LR). Therefore, this work aimed to develop an LC-MS/MS method for MC-LR quantification in dairy milk. Also, the method included AFM1 as a common fungal metabolite and applied to analyze 113 dairy milk samples collected directly after the end of the summer peak. Both toxins were below their LODs, keeping the question on MC-LR carry-over still unanswered. Moreover, an in silico analysis, using a 3D molecular modeling was performed, pointing to a possible interaction between MC-LR and milk proteins, especially ß-lactoglobulin. Since AFM1 and MC-LR are hepatotoxic, their interaction in inducing mitochondrial dysfunction in HepG2 cells was investigated at low (subcytotoxic) concentrations. Live cell imaging-based assays showed an inhibition in cell viability, without involvement of caspase-3/7, and a hyperpolarization in the mitochondrial membrane potential after the exposure to a mixture of 100 ng mL-1 AFM1 and 1000 ng mL-1 MC-LR for 48h. Extracellular flux analysis revealed inhibitions of several key parameters of mitochondrial function (basal respiration, ATP-linked respiration, and spare respiratory capacity).

MitoTracker Red for isolation of zone-specific hepatocytes and characterization of hepatic sublobular metabolism.

BACKGROUND: The liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics. AIM: Our goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones. METHODS: Mice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed. RESULTS: Multiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes. CONCLUSIONS: MTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

Differential regulation of mitochondrial uncoupling protein 2 in cancer cells.

The persistent growth of cancer cells is underscored by complex metabolic reprogramming, with mitochondria playing a key role in the transition to aerobic glycolysis and representing new therapeutic targets. Mitochondrial uncoupling protein 2 (UCP2) has attracted interest because of its abundance in rapidly proliferating cells, including cancer cells, and its involvement in cellular metabolism. However, the specific contributions of UCP2 to cancer biology remain poorly defined. Our investigation of UCP2 expression in various human and mouse cancer cell lines aimed to elucidate its links to metabolic states, proliferation, and adaptation to environmental stresses such as hypoxia and nutrient deprivation. We observed significant variability in UCP2 expression across cancer types, with no direct correlation to their metabolic activity or proliferation rates. UCP2 abundance was also differentially affected by nutrient availability in different cancer cells, but UCP2 was generally downregulated under hypoxia. These findings challenge the notion that UCP2 is a marker of malignant potential and suggest its more complex involvement in the metabolic landscape of cancer.

Role of mitochondrial ribosomal protein L7/L12 (MRPL12) in diabetic ischemic heart disease.

BACKGROUND: Myocardial infarction (MI) is a significant cause of death in diabetic patients. Growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. METHODS: We examined MRPL12 levels in right atrial appendage tissues from diabetic patients undergoing coronary artery bypass graft (CABG) surgery. Using AC-16 cells overexpressing MRPL12 under normal and hyperglycemic conditions we performed mitochondrial functional assays OXPHOS, bioenergetics, mitochondrial membrane potential, ATP production and cell death. RESULTS: We observed elevated MRPL12 levels in heart tissue samples from diabetic patients with ischemic heart disease compared to non-diabetic patients. Overexpression of MRPL12 under hyperglycemic conditions did not affect oxidative phosphorylation (OXPHOS) levels, cellular ATP levels, or cardiomyocyte cell death. However, notable impairment in mitochondrial membrane potential (MMP) was observed under hyperglycemic conditions, along with alterations in both basal respiration oxygen consumption rate (OCR) and maximal respiratory capacity OCR. CONCLUSIONS: Overall, our results suggest that MRPL12 may have a compensatory role in the diabetic myocardium with ischemic heart disease, suggesting that MRPL12 may implicate in the pathophysiology of MI in diabetes.

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.

1H and 31P Magnetic Resonance Spectroscopic Metabolomic Imaging: Assessing Mitogen-Activated Protein Kinase Inhibition in Melanoma.

The MAPK signaling pathway with BRAF mutations has been shown to drive the pathogenesis of 40-60% of melanomas. Inhibitors of this pathway's BRAF and MEK components are currently used to treat these malignancies. However, responses to these treatments are not always successful. Therefore, identifying noninvasive biomarkers to predict treatment responses is essential for personalized medicine in melanoma. Using noninvasive 1H magnetic resonance spectroscopy (1H MRS), we previously showed that BRAF inhibition reduces lactate and alanine tumor levels in the early stages of effective therapy and could be considered as metabolic imaging biomarkers for drug response. The present work demonstrates that these metabolic changes observed by 1H MRS and those assessed by 31P MRS are also found in preclinical human melanoma models treated with MEK inhibitors. Apart from 1H and 31P MRS, additional supporting in vitro biochemical analyses are described. Our results indicate significant early metabolic correlations with response levels to MEK inhibition in the melanoma models and are consistent with our previous study of BRAF inhibition. Given these results, our study supports the potential clinical utility of noninvasive MRS to objectively image metabolic biomarkers for the early prediction of melanoma's response to MEK inhibition.

Proliferation of SH-SY5Y neuroblastoma cells on confined spaces.

BACKGROUND: Microfluidics offers precise drug delivery and continuous monitoring of cell functions, which is crucial for studying the effects of toxins and drugs. Ensuring proper cell growth in these space-constrained systems is essential for obtaining consistent results comparable to standard Petri dishes. NEW METHOD: We investigated the proliferation of SH-SY5Y cells on circular polycarbonate chambers with varying surface areas. SH-SY5Y cells were chosen for their relevance in neurodegenerative disease research. RESULTS: Our study demonstrates a correlation between the chamber surface area and SH-SY5Y cell growth rates. Cells cultured in chambers larger than 10 mm in diameter exhibited growth comparable to standard 60-mm dishes. In contrast, smaller chambers significantly impeded growth, even at identical seeding densities. Similar patterns were observed for HeLaGFP cells, while 16HBE14σ cells proliferated efficiently regardless of chamber size. Additionally, SH-SY5Y cells were studied in a 12-mm diameter sealed chamber to assess growth under restricted gas exchange conditions. COMPARISON WITH EXISTING METHODS: Our findings underscore the limitations of small chamber sizes in microfluidic systems for SH-SY5Y cells, an issue not typically addressed by conventional methods. CONCLUSIONS: SH-SY5Y cell growth is highly sensitive to spatial constraints, with markedly reduced proliferation in chambers smaller than 10 mm. This highlights the need to carefully consider chamber size in microfluidic experiments to achieve cell growth rates comparable to standard culture dishes. The study also shows that while SH-SY5Y and HeLaGFP cells are affected by chamber size, 16HBE14σ cells are not. These insights are vital for designing effective microfluidic systems for bioengineering research.

Acyl-CoA synthetase 4 modulates mitochondrial function in breast cancer cells.

Mitochondria are dynamic organelles that respond to cellular stress through changes in global mass, interconnection, and subcellular location. As mitochondria play an important role in tumor development and progression, alterations in energy metabolism allow tumor cells to survive and spread even in challenging conditions. Alterations in mitochondrial bioenergetics have been recently proposed as a hallmark of cancer, and positive regulation of lipid metabolism constitutes one of the most common metabolic changes observed in tumor cells. Acyl-CoA synthetase 4 (ACSL4) is an enzyme catalyzing the activation of long chain polyunsaturated fatty acids with a strong substrate preference for arachidonic acid (AA). High ACSL4 expression has been related to aggressive cancer phenotypes, including breast cancer, and its overexpression has been shown to positively regulate the mammalian Target of Rapamycin (mTOR) pathway, involved in the regulation of mitochondrial metabolism genes. However, little is known about the role of ACSL4 in the regulation of mitochondrial function and metabolism in cancer cells. In this context, our objective was to study whether mitochondrial function and metabolism, processes usually altered in tumors, are modulated by ACSL4 in breast cancer cells. Using ACSL4 overexpression in MCF-7 cells, we demonstrate that this enzyme can increase the mRNA and protein levels of essential mitochondrial regulatory proteins such as nuclear respiratory factor 1 (NRF-1), voltage-dependent anion channel 1 (VDAC1) and respiratory chain Complex III. Furthermore, respiratory parameters analysis revealed an increase in oxygen consumption rate (OCR) and in spare respiratory capacity (SRC), among others. ACSL4 knockdown in MDA-MB-231 cells led to the decrease in OCR and in SCR, supporting the role of ACSL4 in the regulation of mitochondrial bioenergetics. Moreover, ACSL4 overexpression induced an increase in glycolytic function, in keeping with an increase in mitochondrial respiratory activity. Finally, there was a decrease in mitochondrial mass detected in cells that overexpressed ACSL4, while the knockdown of ACSL4 expression in MDA-MB-231 cells showed the opposite effect. Altogether, these results unveil the role of ACSL4 in mitochondrial function and metabolism and expand the knowledge of ACSL4 participation in pathological processes such as breast cancer.

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.

Scanning electrochemical microscopy for determining oxygen consumption rates of cells in hydrogel fibers fabricated using an extrusion 3D bioprinter.

Three-dimensional (3D)-cultured cells have attracted the attention of researchers in tissue engineering- and drug screening-related fields. Among them, 3D cellular fibers have attracted significant attention because they can be stacked to prepare more complex tissues and organs. Cellular fibers are widely fabricated using extrusion 3D bioprinters. For these applications, it is necessary to evaluate cellular activities, such as the oxygen consumption rate (OCR), which is one of the major metabolic activities. We previously reported the use of scanning electrochemical microscopy (SECM) to evaluate the OCRs of cell spheroids. However, the SECM approach has not yet been applied to hydrogel fibers prepared using the bioprinters. To the best of our knowledge, this is the first study to evaluate the OCR of cellular fibers printed by extrusion 3D bioprinters. First, the diffusion theory was discussed to address this issue. Next, diffusion models were simulated to compare realistic models with this theory. Finally, the OCRs of MCF-7 cells in the printed hydrogel fibers were evaluated as a proof of concept. Our proposed approach could potentially be used to evaluate the OCRs of tissue-engineered fibers for organ transplantation and drug screening using in-vitro models.

Antimicrobial cetylpyridinium chloride causes functional inhibition of mitochondria as potently as canonical mitotoxicants, nanostructural disruption of mitochondria, and mitochondrial Ca2+ efflux in living rodent and primary human cells.

People are exposed to high concentrations of antibacterial agent cetylpyridinium chloride (CPC) via food and personal care products, despite little published information regarding CPC effects on eukaryotes. Here, we show that low-micromolar CPC exposure, which does not cause cell death, inhibits mitochondrial ATP production in primary human keratinocytes, mouse NIH-3T3 fibroblasts, and rat RBL-2H3 immune mast cells. ATP inhibition via CPC (EC50 1.7 µM) is nearly as potent as that caused by canonical mitotoxicant CCCP (EC50 1.2 µM). CPC inhibition of oxygen consumption rate (OCR) tracks with that of ATP: OCR is halved due to 1.75 µM CPC in RBL-2H3 cells and 1.25 µM in primary human keratinocytes. Mitochondrial [Ca2+] changes can cause mitochondrial dysfunction. Here we show that CPC causes mitochondrial Ca2+ efflux from mast cells via an ATP-inhibition mechanism. Using super-resolution microscopy (fluorescence photoactivation localization) in live cells, we have discovered that CPC causes mitochondrial nanostructural defects in live cells within 60 min, including the formation of spherical structures with donut-like cross section. This work reveals CPC as a mitotoxicant despite widespread use, highlighting the importance of further research into its toxicological safety.

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

Effects of Hypoxia Stress on Survival, Antioxidant and Anaerobic Metabolic Enzymes, and Related Gene Expression of Red Swamp Crayfish Procambarus clarkii.

The red swamp crayfish Procambarus clarkii is the most reared shrimp in China, but it is often affected by hypoxia stress in the process of seedling culture and adult crayfish culture. The oxygen consumption rate and asphyxiation point of juvenile crayfish (1.17 ± 0.03 g) and subadult crayfish (11.68 ± 0.11 g) at different temperatures (20, 22, 24, 26, and 28 °C) were studied. The survival, glycolysis, and expression of antioxidant genes were compared under 24 h acute hypoxia stress (1, 2, and 3 mg/L) and normal dissolved oxygen (7.5 mg/L). The results showed that the oxygen consumption rate and asphyxiation point of juvenile and subadult crayfish increased with increasing temperatures (20-28 °C). At the same temperature, the oxygen consumption rate and asphyxiation point of juvenile crayfish were significantly higher than those of subadult crayfish (p < 0.05). Within 24 h, the three hypoxia stress environments did not lead to the death of crayfish, indicating that P. clarkii has a strong ability to adapt to hypoxia. Hypoxia stress significantly affected the activities of antioxidant and anaerobic metabolic enzymes and gene expression in juvenile and subadult crayfish. The activities of the superoxide dismutase (SOD), catalase (CAT), and lactate dehydrogenase (LDH) and the content of lactic acid (LD) in the hepatopancreas of juvenile and subadult crayfish in the hypoxia stress groups increased significantly. The expression levels of SOD mRNA, CAT mRNA, Hsp70 mRNA, and crustin 4 mRNA in the hepatopancreas of juvenile and subadult crayfish in the hypoxia stress groups were significantly higher than those in the control group (p < 0.05), and the higher the degree of hypoxia stress, the higher the expression of each gene. The results showed that the antioxidant system of juvenile crayfish was more sensitive to hypoxia environments, and hypoxia stress resulted in increased stress levels in juvenile crayfish and subadult crayfish.

Evaluation of Mitochondrial Respiratory Function in Murine Splenocytes.

Accumulated evidence has demonstrated a key role of mitochondria in the onset and progression of autoimmune disease. Understanding and modulation of mitochondrial dysfunction could provide new molecular targets for both preventive and therapeutic intervention in disease management. The ability to assess mitochondrial function has enabled rheumatologists to advance the understanding of the contribution of cellular metabolism in cellular physiology and disease pathology and etiology. Direct measurement of oxygen consumption rate using an Agilent Seahorse XF measurement system has been widely used as the gold-standard assay for evaluating mitochondrial function in cells. Using this assay system, measurement of parameters of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and nonmitochondrial respiration can be achieved. An optimized method which works well in mouse splenocytes and a Jurkat cell line is presented in this chapter.

Study on the thermal effect of coal oxidation under different stresses.

The utilization of coal resources has been improved by using the method of narrow coal pillar mining, but this leads to a stress concentration in the coal pillars, which causes differences in the oxidation of coal pillars. To study the effect of stress on the oxidation and spontaneous combustion of coal samples, programmed heating-gas chromatography coupling experiments were carried out on coal samples under different stresses, analyzing the effect rule of stress on the gas derivatives of coal samples in the process of heating and oxidation. Furthermore, the mechanism of stress influence on thermal effect parameters is explored on the basis of that analysis. The results show that the rate of oxygen consumption, CO, CO2 concentration and heat release intensity of coal samples show a changing trend, initially increasing and then decreasing with increasing stress, and these values within coal are at the maximum when the stress is 9 MPa; and with increasing stress, the activation energy shows a "V" type change and reaches the minimum of 26.89 kJ/mol at 6 MPa, which indicates that low stress promotes coal spontaneous combustion (CSC), while high stress inhibits CSC. The thermal conduction coefficient of coal samples shows a negative correlation with temperature at the low-temperature stage, while the thermal conductivity of coal samples shows a positive correlation with temperature at the high-temperature stage, and the thermal conduction coefficient of coal samples reaches a minimum at temperatures of 70 °C and 0 MPa of stress. The porosity within coal decreases, and the thermal conductivity coefficient within coal increases with increasing stress because the increase in stress makes the macromolecules within coal disassemble into small molecules, the structure becomes more compact, and the thermal conductivity increases. The study provides an important theoretical basis for better understanding the effect mechanism of stress on CSC.

High-Resolution Respirometry for the Assessment of Teratogenic Chemicals.

Pesticides are often used in agriculture and residential areas to mitigate pests and weeds. These chemicals can enter aquatic ecosystems via runoff and rain events, exerting negative effects on aquatic species. In rapidly developing fish embryos, metabolic disruption can alter the developmental trajectory and alter ATP levels. Therefore, it is important to quantify mitochondrial integrity in organisms following exposure to pesticides. To achieve this, a high throughput method to assess pesticide effects on oxidative phosphorylation and mitochondria has been optimized for fish embryos. Fish embryos are first exposed to pesticides for 24 or 48 h, and oxygen consumption rates are measured using the Seahorse XFe24/96 Flux Analyzer (formerly Seahorse Biosciences, now Agilent). The assay utilizes a single embryo and precisely measures oxygen consumption and extracellular acidification. Based upon these measurements, characteristics related to mitochondrial bioenergetics are calculated to provide information on mitochondrial integrity. Using this approach, one can identify pesticides affecting the electron transport chain and ultimately ATP production. In this chapter, we describe the mitochondrial stress test to understand mitochondrial dysfunction and metabolic shifts within the fish embryo.