Assessment of Mitochondrial Respiration During Hypothermic Storage of Liver Biopsies Following Normothermic Machine Perfusion.

Organ quality can be assessed prior to transplantation, during normothermic machine perfusion (NMP) of the liver. Evaluation of mitochondrial function by high-resolution respirometry (HRR) may serve as a viability assessment concept in this setting. Freshly collected tissue is considered as optimal sample for HRR, but due to technical and personnel requirements, more flexible and schedulable measurements are needed. However, the impact of cold storage following NMP before processing biopsy samples for mitochondrial analysis remains unknown. We aimed at establishing an appropriate storage protocol of liver biopsies for HRR. Wedge biopsies of 5 human livers during NMP were obtained and assessed by HRR. Analysis was performed after 0, 4, 8, and 12 h of hypothermic storage (HTS) in HTK organ preservation solution at 4°C. With HTS up to 4 h, mitochondrial performance did not decrease in HTS samples compared with 0 h (OXPHOS, 44.62 [34.75-60.15] pmol·s-1·mg wet mass-1 vs. 43.73 [40.69-57.71], median [IQR], p > 0.999). However, at HTS beyond 4 h, mitochondrial respiration decreased. We conclude that HTS can be safely applied for extending the biopsy measurement window for up to 4 h to determine organ quality, but also that human liver respiration degrades beyond 4 h HTS following NMP.

Assessment of neuro-pulmonary crosstalk in asthmatic mice: effects of DiNP exposure on cellular respiration, mitochondrial oxidative status and apoptotic signaling.

Human health is becoming concerned about exposure to endocrine disrupting chemicals (EDCs) emanating from plastic, such as phthalates, which are industrially employed as plasticizers in the manufacturing of plastic products. Due to some toxicity concerns, di(2-ethylhexyl) phthalate (DEHP) was replaced by diisononyl phthalate (DiNP). Recent data, however, highlights the potential of DiNP to interfere with the endocrine system and influence allergic responses. Asthma affects brain function through hypoxia, systemic inflammation, oxidative stress, and sleep disturbances and its effective management is crucial for maintaining respiratory and brain health. Therefore, in DiNP-induced asthmatic mice, this study investigated possible crosstalk between the lungs and the brain inducing perturbations in neural mitochondrial antioxidant status, inflammation biomarkers, energy metabolizing enzymes, and apoptotic indicators. To achieve this, twelve (n = 12, 20-30 g) male BALB/c mice were divided into two (2) experimental groups, each with five (6) mice. Mice in group II were subjected to 50 mg/kg body weight (BW) DiNP (Intraperitoneal and intranasal), while group I served as the control group for 24 days. The effects of DiNP on neural energy metabolizing enzymes (Hexokinase, Aldolase, NADase, Lactate dehydrogenase, Complex I, II, II & IV), biomarkers of inflammation (Nitric oxide, Myeloperoxidase), oxidative stress (malondialdehyde), antioxidants (catalase, glutathione-S-transferase, and reduced glutathione), oncogenic and apoptotic factors (p53, K-ras, Bcl, etc.), and brain histopathology were investigated. DiNP-induced asthmatic mice have significantly (p < 0.05) altered neural energy metabolizing capacities due to disruption of activities of enzymes of glycolytic and oxidative phosphorylation. Other responses include significant inflammation, oxidative distress, decreased antioxidant status, altered oncogenic-apoptotic factors level and neural degeneration (as shown in hematoxylin and eosin-stained brain sections) relative to control. Current findings suggest that neural histoarchitecture, energy metabolizing potentials, inflammation, oncogenic and apoptotic factors, and mitochondrial antioxidant status may be impaired and altered in DiNP-induced asthmatic mice suggesting a pivotal crosstalk between the two intricate organs (lungs and brain).

The Determination of Mitochondrial Mass Is a Prerequisite for Accurate Assessment of Peripheral Blood Mononuclear Cells' Oxidative Metabolism.

Mitochondria are responsible for ATP synthesis through oxidative phosphorylation in cells. However, there are limited data on the influence of mitochondrial mass (MM) in the adequate assessment of cellular stress assay (CSA) results in human peripheral blood mononuclear cells (PBMCs). Therefore, the aim of this study was to determine MM in PBMCS and assess its influence on the results of CSA measurements. Blood samples were collected and sent to the laboratory for MM and CSA measurements during different seasons of the year. The mitochondrial mass was determined based on the mtDNA:nDNA ratio in PBMCs using quantitative real-time PCR (qRT-PCR). CSA was measured using Seahorse technology. The MM was significantly lower during summer and autumn compared to winter and spring (p < 0.0001). On the contrary, we found that the maximal respiration per mitochondrion (MP) was significantly higher in summer and autumn compared to winter and spring (p < 0.0001). The estimated effect of MM on mitochondrial performance was -0.002 pmol/min/mitochondrion (p < 0.0001) and a correlation coefficient (r) of -0.612. Similarly, MM was negatively correlated with maximal respiration (r = -0.12) and spare capacity (in % r = -0.05, in pmol/min r = -0.11). In conclusion, this study reveals that MM changes significantly with seasons and is negatively correlated with CSA parameters and MP. Our findings indicate that the mitochondrial mass is a key parameter for determination of mitochondrial fitness. Therefore, we recommend the determination of MM during the measurement of CSA parameters for the correct interpretation and assessment of mitochondrial function.

Mechanical Permeabilization as a New Method for Assessment of Mitochondrial Function in Insect Tissues.

Respirometry analysis is an effective technique to assess mitochondrial physiology. Insects are valuable biochemical models to understand metabolism and human diseases. Insect flight muscle and brain have been extensively used to explore mitochondrial function due to dissection feasibility and the low sample effort to allow oxygen consumption measurements. However, adequate plasma membrane permeabilization is required for substrates/modulators to reach mitochondria. Here, we describe a new method for study of mitochondrial physiology in insect tissues based on mechanical permeabilization as a fast and reliable method that do not require the use of detergents for chemical permeabilization of plasma membrane, while preserves mitochondrial integrity.

Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments.

Changes in mitochondrial function are central to many forms of kidney disease, including acute injury, diabetic nephropathy, hypertension, and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close to in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared with in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here, we report the development and validation of a new approach for the rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular fragments using Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models: the C57BL/6J mouse, the diabetic db/db mouse and matching db/+ control mouse, and the Dahl salt-sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for the rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator's choice. The isolation methods presented here ensure viable and functional proximal tubular fragments and glomeruli, with a preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.

Unimolecular Photodynamic O2-Economizer To Overcome Hypoxia Resistance in Phototherapeutics.

Tumor hypoxia has proven to be the major bottleneck of photodynamic therapy (PDT) to clinical transformation. Different from traditional O2 delivery approaches, here we describe an innovative binary photodynamic O2-economizer (PDOE) tactic to reverse hypoxia-driven resistance by designing a superoxide radical (O2•-) generator targeting mitochondria respiration, termed SORgenTAM. This PDOE system is able to block intracellular O2 consumption and down-regulate HIF-1α expression, which successfully rescues cancer cells from becoming hypoxic and relieves the intrinsic hypoxia burden of tumors in vivo, thereby sparing sufficient endogenous O2 for the PDT process. Photosensitization mechanism studies demonstrate that SORgenTAM has an ideal intersystem crossing rate and triplet excited state lifetime for generating O2•- through type-I photochemistry, and the generated O2•- can further trigger a biocascade to reduce the PDT's demand for O2 in an O2-recycble manner. Furthermore, SORgenTAM also serves to activate the AMPK metabolism signaling pathway to inhibit cell repair and promote cell death. Consequently, using this two-step O2-economical strategy, under relatively low light dose irradiation, excellent therapeutic responses toward hypoxic tumors are achieved. This study offers a conceptual while practical paradigm for overcoming the pitfalls of phototherapeutics.

High-Resolution Respirometry in Assessment of Mitochondrial Function in Neuroblastoma SH-SY5Y Intact Cells.

Mitochondria are organelles with significant cellular functions, especially cellular bioenergetics and apoptosis. They are structural and functional elements of cell respiration with the electron transport system (ETS), whose role is to provide adenosine triphosphate (ATP), used as a source of chemical energy. The Krebs cycle and fatty acid oxidation take place within mitochondria. Other metabolic pathways and cycles include some steps inside and outside the mitochondria (e.g., the urea cycle, steroid biosynthesis, heme biosynthesis, and cardiolipin synthesis). Dysfunction of mitochondria plays a critical role in the pathophysiology of a variety of diseases including degenerative diseases, aging, and cancer, etc. Nowadays the interest of the mitochondrial respiratory function is still increasing due to their importance in the physiology and pathophysiology of an organism. Neuroblastoma cell line SH-SY5Y is widely used as an in vitro model in neurodegenerative diseases, where mitochondrial dysfunction is considered as a key mechanism in pathophysiology of neurodegenerative disorders. This paper gives first insight into the mitochondrial respiration and characterization of SH-SY5Y cells, with basic information of respiration in different coupling control states including ROUTINE, LEAK, and maximal electron transport (ET) capacity.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study.

A physiologically relevant in vitro human-based model could be the 'gold standard' to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D.

Integration of demand and supply sides in the ATP energy economics of cells.

The central aspects of the energy economics of living cells revolve around the synthesis and utilization of molecules of adenosine triphosphate (ATP). Current descriptions of cell metabolism and its regulation in most textbooks of biochemistry assume that enzymes and transporters behave in the same way in isolation and in a cell. Calculations of the mechanistic or maximal P/O ratios in oxidative phosphorylation by mammalian cells generally consider only the supply side of the problem without linking to ATP-demand processes. The purpose of this article is to calculate the mechanistic P/O ratio by integration of the supply and demand sides of ATP reactions. The mechanistic stoichiometry calculated from an integrated approach is compared with that obtained from the standard model that considers only ATP supply. After accounting for leaks, slips, and other losses, the actual or operative P/O calculated by the integrated method is found to be in good agreement with the experimental values of the P/O ratio determined in mitochondria for both succinate and NADH-linked respiratory substrates. The thermodynamic consequences of these results and the biological implications are discussed. An integrated model of oxidative phosphorylation that goes beyond the chemiosmotic theory is presented, and a solution to the longstanding fundamental problem of respiratory control is found.

A yield-cost tradeoff governs Escherichia coli's decision between fermentation and respiration in carbon-limited growth.

Living cells react to changes in growth conditions by re-shaping their proteome. This accounts for different stress-response strategies, both specific (i.e., aimed at increasing the availability of stress-mitigating proteins) and systemic (such as large-scale changes in the use of metabolic pathways aimed at a more efficient exploitation of resources). Proteome re-allocation can, however, imply significant biosynthetic costs. Whether and how such costs impact the growth performance are largely open problems. Focusing on carbon-limited E. coli growth, we integrate genome-scale modeling and proteomic data to address these questions at quantitative level. After deriving a simple formula linking growth rate, carbon intake, and biosynthetic costs, we show that optimal growth results from the tradeoff between yield maximization and protein burden minimization. Empirical data confirm that E. coli growth is indeed close to Pareto-optimal over a broad range of growth rates. Moreover, we establish that, while most of the intaken carbon is diverted into biomass precursors, the efficiency of ATP synthesis is the key driver of the yield-cost tradeoff. These findings provide a quantitative perspective on carbon overflow, the origin of growth laws and the multidimensional optimality of E. coli metabolism.

Assessment of platelet respiration as emerging biomarker of disease.

Mitochondrial dysfunction is currently acknowledged as a central pathomechanism of most common diseases of the 21(st) century. Recently, the assessment of the bioenergetic profile of human peripheral blood cells has emerged as a novel research field with potential applications in the development of disease biomarkers. In particular, platelets have been successfully used for the ex vivo analysis of mitochondrial respiratory function in several acute and chronic pathologies. An increasing number of studies support the idea that evaluation of the bioenergetic function in circulating platelets may represent the peripheral signature of mitochondrial dysfunction in metabolically active tissues (brain, heart, liver, skeletal muscle). Accordingly, impairment of mitochondrial respiration in peripheral platelets might have potential clinical applicability as a diagnostic and prognostic tool as well as a biomarker in treatment monitoring. The aim of this minireview is to summarize current information in the field of platelet mitochondrial dysfunction in both acute and chronic diseases.

O2 Economizer for Inhibiting Cell Respiration To Combat the Hypoxia Obstacle in Tumor Treatments.

Hypoxia, a ubiquitously aberrant phenomenon implicated in tumor growth, causes severe tumor resistance to therapeutic interventions. Instead of the currently prevalent solution through intratumoral oxygen supply, we put forward an "O2-economizer" concept by inhibiting the O2 consumption of cell respiration to spare endogenous O2 and overcome the hypoxia barrier. A nitric oxide (NO) donor responsible for respiration inhibition and a photosensitizer for photodynamic therapy (PDT) are co-loaded into poly(d,l-lactide- co-glycolide) nanovesicles to provide a PDT-specific O2 economizer. Once accumulating in tumors and subsequently responding to the locally reductive environment, the carried NO donor undergoes breakdown to produce NO for inhibiting cellular respiration, allowing more O2 in tumor cells to support the profound enhancement of PDT. Depending on the biochemical reallocation of cellular oxygen resource, this O2-economizer concept offers a way to address the important issue of hypoxia-induced tumor resistance to therapeutic interventions, including but not limited to PDT.

Leaf growth rate and nitrogen content determine respiratory costs during leaf expansion in grapevines.

Respiration processes are well recognized as fundamental for the plant carbon balance, but little attention has been paid to the relationships among respiration rates, environment and genetic variability. This can be of particular interest to understand the differences in net carbon balances in crops as grapevines. Night respiration (Rn ) and its associated growth (Rg ) and maintenance (Rm ) components were evaluated during leaf expansion in two grapevine cultivars (Tempranillo cv. and Garnacha cv.) that differ in their plant growth pattern and carbon balance. Simultaneously, leaf traits as leaf mass area, nitrogen (N) and carbon (C) content were evaluated in order to relate to the respiratory processes and the leaf growth. The results showed the differences in respiration rates associated with the leaf expansion pattern. Tempranillo developed leaves with higher leaf area and lower dry weight per leaf unit than Garnacha. Although differences between cultivars were observed in terms of growth costs in expanding leaves, the maintenance costs were similar for both cultivars. Also, a significant linear regression was found between respiration rates and N content in expanding and mature leaves. The results indicate that differences in structure and nitrogen content of expanding leaves may lead to respiratory differences between cultivars. These results also demonstrate the importance of respiratory cost components in carbon balance calculations in grapevines.

High-throughput assessment of oxidative respiration in fish embryos: Advancing adverse outcome pathways for mitochondrial dysfunction.

Mitochondrial dysfunction is a prevalent molecular event that can result in multiple adverse outcomes. Recently, a novel high throughput method to assess metabolic capacity in fish embryos following exposure to chemicals has been adapted for environmental toxicology. Assessments of oxygen consumption rates using the Seahorse XF(e) 24/96 Extracellular Flux Analyzer (Agilent Technologies) can be used to garner insight into toxicant effects at early stages of development. Here we synthesize the current state of the science using high throughput metabolic profiling in zebrafish embryos, and present considerations for those wishing to adopt high throughput methods for mitochondrial bioenergetics into their research. Chemicals that have been investigated in zebrafish using this metabolic platform include herbicides (e.g. paraquat, diquat), industrial compounds (e.g. benzo-[a]-pyrene, tributyltin), natural products (e.g. quercetin), and anti-bacterial chemicals (i.e. triclosan). Some of these chemicals inhibit mitochondrial endpoints in the µM-mM range, and reduce basal respiration, maximum respiration, and spare capacity. We present a theoretical framework for how one can use mitochondrial performance data in zebrafish to categorize chemicals of concern and prioritize mitochondrial toxicants. Noteworthy is that our studies demonstrate that there can be considerable variation in basal respiration of untreated zebrafish embryos due to clutch-specific effects as well as individual variability, and basal oxygen consumption rates (OCR) can vary on average between 100 and 300 pmol/min/embryo. We also compare OCR between chorionated and dechorionated embryos, as both models are employed to test chemicals. After 24 h, dechorionated embryos remain responsive to mitochondrial toxicants, although they show a blunted response to the uncoupling agent carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP); dechorionated embryos are therefore a viable option for investigations into mitochondrial bioenergetics. We present an adverse outcome pathway framework that incorporates endpoints related to mitochondrial bioenergetics. High throughput bioenergetics assays conducted using whole embryos are expected to support adverse outcome pathways for mitochondrial dysfunction.

The energy budget in C4 photosynthesis: insights from a cell-type-specific electron transport model.

Extra ATP required in C4 photosynthesis for the CO2 -concentrating mechanism probably comes from cyclic electron transport (CET). As metabolic ATP : NADPH requirements in mesophyll (M) and bundle-sheath (BS) cells differ among C4 subtypes, the subtypes may differ in the extent to which CET operates in these cells. We present an analytical model for cell-type-specific CET and linear electron transport. Modelled NADPH and ATP production were compared with requirements. For malic-enzyme (ME) subtypes, c. 50% of electron flux is CET, occurring predominantly in BS cells for standard NADP-ME species, but in a ratio of c. 6 : 4 in BS : M cells for NAD-ME species. Some C4 acids follow a secondary decarboxylation route, which is obligatory, in the form of 'aspartate-malate', for the NADP-ME subtype, but facultative, in the form of phosphoenolpyruvate-carboxykinase (PEP-CK), for the NAD-ME subtype. The percentage for secondary decarboxylation is c. 25% and that for 3-phosphoglycerate reduction in BS cells is c. 40%; but these values vary with species. The 'pure' PEP-CK type is unrealistic because its is impossible to fulfil ATP : NADPH requirements in BS cells. The standard PEP-CK subtype requires negligible CET, and thus has the highest intrinsic quantum yields and deserves further studies in the context of improving canopy productivity.

Respiratory hazard assessment of combined exposure to complete gasoline exhaust and respirable volcanic ash in a multicellular human lung model at the air-liquid interface.

Communities resident in urban areas located near active volcanoes can experience volcanic ash exposures during, and following, an eruption, in addition to sustained exposures to high concentrations of anthropogenic air pollutants (e.g., vehicle exhaust emissions). Inhalation of anthropogenic pollution is known to cause the onset of, or exacerbate, respiratory and cardiovascular diseases. It is further postulated similar exposure to volcanic ash can also affect such disease states. Understanding of the impact of combined exposure of volcanic ash and anthropogenic pollution to human health, however, remains limited. The aim of this study was to assess the biological impact of combined exposure to respirable volcanic ash (from Soufrière Hills volcano (SHV), Montserrat and Chaitén volcano (ChV), Chile; representing different magmatic compositions and eruption styles) and freshly-generated complete exhaust from a gasoline vehicle. A multicellular human lung model (an epithelial cell-layer composed of A549 alveolar type II-like cells complemented with human blood monocyte-derived macrophages and dendritic cells cultured at the air-liquid interface) was exposed to diluted exhaust (1:10) continuously for 6 h, followed by immediate exposure to the ash as a dry powder (0.54 ±â€¯0.19 µg/cm2 and 0.39 ±â€¯0.09 µg/cm2 for SHV and ChV ash, respectively). After an 18 h incubation, cells were exposed again for 6 h to diluted exhaust, and a final 18 h incubation (at 37 °C and 5% CO2). Cell cultures were then assessed for cytotoxic, oxidative stress and (pro-)inflammatory responses. Results indicate that, at all tested (sub-lethal) concentrations, co-exposures with both ash samples induced no significant expression of genes associated with oxidative stress (HMOX1, NQO1) or production of (pro-)inflammatory markers (IL-1ß, IL-8, TNF-α) at the gene and protein levels. In summary, considering the employed experimental conditions, combined exposure of volcanic ash and gasoline vehicle exhaust has a limited short-term biological impact to an advanced lung cell in vitro model.

Interleukin 37 reverses the metabolic cost of inflammation, increases oxidative respiration, and improves exercise tolerance.

IL-1 family member interleukin 37 (IL-37) has broad antiinflammatory properties and functions as a natural suppressor of innate inflammation. In this study, we demonstrate that treatment with recombinant human IL-37 reverses the decrease in exercise performance observed during systemic inflammation. This effect was associated with a decrease in the levels of plasma and muscle cytokines, comparable in extent to that obtained upon IL-1 receptor blockade. Exogenous administration of IL-37 to healthy mice, not subjected to an inflammatory challenge, also improved exercise performance by 82% compared with vehicle-treated mice (P = 0.01). Treatment with eight daily doses of IL-37 resulted in a further 326% increase in endurance running time compared with the performance level of mice receiving vehicle (P = 0.001). These properties required the engagement of the IL-1 decoy receptor 8 (IL-1R8) and the activation of AMP-activated protein kinase (AMPK), because both inhibition of AMPK and IL-1R8 deficiency abrogated the positive effects of IL-37 on exercise performance. Mechanistically, treatment with IL-37 induced marked metabolic changes with higher levels of muscle AMPK, greater rates of oxygen consumption, and increased oxidative phosphorylation. Metabolomic analyses of plasma and muscles of mice treated with IL-37 revealed an increase in AMP/ATP ratio, reduced levels of proinflammatory mediator succinate and oxidative stress-related metabolites, as well as changes in amino acid and purine metabolism. These effects of IL-37 to limit the metabolic costs of chronic inflammation and to foster exercise tolerance provide a rationale for therapeutic use of IL-37 in the treatment of inflammation-mediated fatigue.

The cost of surviving nitrogen excess: energy and protein demand in the lichen Cladonia portentosa as revealed by proteomic analysis.

MAIN CONCLUSION: Different nitrogen forms affect different metabolic pathways in lichens. In particular, the most relevant changes in protein expression were observed in the fungal partner, with NO 3- mostly affecting the energetic metabolism and NH 4+ affecting transport and regulation of proteins and the energetic metabolism much more than NO 3- did. Excess deposition of reactive nitrogen is a well-known agent of stress for lichens, but which symbiont is most affected and how, remains a mystery. Using proteomics can expand our understanding of stress effects on lichens. We investigated the effects of different doses and forms of reactive nitrogen, with and without supplementary phosphorus and potassium, on the proteome of the lichen Cladonia portentosa growing in a 'real-world' simulation of nitrogen deposition. Protein expression changed with the nitrogen treatments but mostly in the fungal partner, with NO3- mainly affecting the energetic metabolism and NH4+ also affecting the protein synthesis machinery. The photobiont mainly responded overexpressing proteins involved in energy production. This suggests that in response to nitrogen stress, the photobiont mainly supports the defensive mechanisms initiated by the mycobiont with an increased energy production. Such surplus energy is then used by the cell to maintain functionality in the presence of NO3-, while a futile cycle of protein production can be hypothesized to be induced by NH4+ excess. External supply of potassium and phosphorus influenced differently the responses of particular enzymes, likely reflecting the many processes in which potassium exerts a regulatory function.

Toxicity assessment of hydrogen peroxide on Toll-like receptor system, apoptosis, and mitochondrial respiration in piglets and IPEC-J2 cells.

In this study, expressions of toll-like receptors (TLRs) and apoptosis-related genes in piglets and mitochondrial respiration in intestinal porcine epithelial cells were investigated after hydrogen peroxide (H2O2) exposure. The in vivo results showed that H2O2 influenced intestinal expressions of TLRs and apoptosis related genes. H2O2 treatment (5% and 10%) downregulated uncoupling protein 2 (UCP2) expression in the duodenum (P < 0.05), while low dosage of H2O2 significantly increased UCP2 expression in the jejunum (P < 0.05). In IPEC-J2 cells, H2O2 inhibited cell proliferation (P < 0.05) and caused mitochondrial dysfunction via reducing maximal respiration, spare respiratory, non-mitochondrial respiratory, and ATP production (P < 0.05). However, 50 uM H2O2 significantly enhanced mitochondrial proton leak (P < 0.05). In conclusion, H2O2 affected intestinal TLRs system, apoptosis related genes, and mitochondrial dysfunction in vivo and in vitro models. Meanwhile, low dosage of H2O2 might exhibit a feedback regulatory mechanism against oxidative injury via increasing UCP2 expression and mitochondrial proton leak.