Basal Respiration Research Articles

Non-native and native tree species plantations and seasonality could have substantial impacts on the diversity of indigenous soil fauna in a semi-arid forest ecosystem.

The temporal and spatial dynamics of soil fauna in many terrestrial ecosystems are still not fully understood, while soil fauna is one of the most critical characteristics in assessing soil quality. Therefore, the effects of native [Quercus brantii (QP) and Amygdalus scoparia (AMP)] and non-native [Cupressus arizonica (CUP) and Pinus eldarica (PIN)] plantations and natural trees [Quercus brantii coppice trees (QNC), standard (QNS), and Amygdalus scoparia (AMN)] on diversity and abundance of macro- and mesofauna were done in the semi-arid forest of Zagros, Iran. Samples were collected beneath the canopy of woody species and the outer edge of the canopy in spring and summer seasons. For this purpose, soil samples [(7 samples per woody species + control) × 2 seasons × 3 replicates] were taken from 0 to 20 cm depths. Each soil sample was a mix of three soil cores. For the macrofauna, 15 species belonging to four families (in spring) and 17 species in nine families (in summer) were collected and identified. For the soil mesofauna, 14 species belonging to 14 families (in spring) and 13 species in 13 different families (in summer) were identified, respectively. The fauna diversity indices under the canopy of studied species were higher in summer season than in spring. The result showed that the macrofauna diversity was affected by tree species, while mesofauna was affected by seasonal changes. Macrofauna biodiversity was higher under the canopy of PIN and CUP than other trees. Principle component analysis showed that the diversity of the macrofauna was higher under the canopy of PIN and CUP, and influenced by soil characteristic properties, soil properties did not influence them. Yet the diversity of the mesofauna was affected by soil characteristics and was higher in areas with higher organic carbon, nitrogen, substrate-induced respiration, basal respiration, microbial carbon biomass, and alkaline phosphatase. In addition, mesofauna biodiversity had a significant positive correlation with the soil quality index (SQI). SQI was higher under the canopy of natural stands, especially the QNS. Conservation of native species (QNS, QNC, and AMN) and plantation with native deciduous species (QP and AMP) seem to moderate environmental conditions and increase soil macro- and mesofauna diversity and SQI.

Paraoxonase 2 Deficiency Causes Mitochondrial Dysfunction in Retinal Pigment Epithelial Cells and Retinal Degeneration in Mice.

Although AMD is a complex disease, oxidative stress is a crucial contributor to its development, especially in view of the higher oxygen demand of the retina. Paraoxonase 2 (PON2) is a ubiquitously and constitutively expressed antioxidant protein that is found intracellularly associated with mitochondrial membranes and modulates mitochondrial ROS production and function. The contribution of PON2 to AMD has not been studied to date. In this study, we examined the role of PON2 in AMD utilizing both in vitro and in vivo models of AMD with emphasis on mitochondrial function. Mitochondrial localization and regulation of PON2 following oxidative stress were determined in human primary cultured retinal pigment epithelium (hRPE) cells. PON2 was knocked down in RPE cells using siRNA and mitochondrial bioenergetics were measured. To investigate the function of PON2 in the retina, WT and PON2-deficient mice were administered NaIO3 (20 mg/kg) intravenously; fundus imaging, optical coherence tomography (OCT), electroretinography (ERG) were conducted; and retinal thickness and cell death were measured and quantified. In hRPE, mitochondrial localization of PON2 increased markedly with stress. Moreover, a time-dependent regulation of PON2 was observed following oxidative stress, with an initial significant increase in expression followed by a significant decrease. Mitochondrial bioenergetic parameters (basal respiration, ATP production, spare respiratory capacity, and maximal respiration) showed a significant decrease with oxidative stress, which was further exacerbated in the absence of PON2. NaIO3 treatment caused significant retinal degeneration, retinal thinning, and reduced rod and cone function in PON2-deficient mice when compared to WT mice. The apoptotic cells and active caspase 3 significantly increased in PON2-deficient mice treated with NaIO3, when compared to WT mice. Our investigation demonstrates that deficiency of PON2 results in RPE mitochondrial dysfunction and a decline in retinal function. These findings imply that PON2 may have a beneficial role in retinal pathophysiology and is worthy of further investigation.

Increased platelet mitochondrial function correlates with clot strength in a rodent fracture model.

Thromboelastographic measures of clot strength increase early after injury, portending higher risks for thromboembolic complications during recovery. Understanding the specific role of platelets is challenging because of a lack of clinically relevant measures of platelet function. Platelet mitochondrial respirometry may provide insight to global platelet function but has not yet been correlated with functional coagulation studies. Wistar rats underwent anesthesia and either immediate sacrifice for baseline values (n = 6) or (1) bilateral hindlimb orthopedic injury (n = 12), versus (2) sham anesthesia (n = 12) with terminal phlebotomy/hepatectomy after 24 hours. High-resolution respirometry was used to measure basal respiration, mitochondrial leak, maximal oxidative phosphorylation, and Complex IV activity in intact platelets; Complex I- and Complex II-driven respiration was measured in isolated liver mitochondria. Results were normalized to platelet number and protein mass, respectively. Citrated native thromboelastography (TEG) was performed in triplicate. Citrated native TEG maximal amplitude was significantly higher (81.0 ± 3.0 vs. 73.3 ± 3.5 mm, p < 0.001) in trauma compared with sham rats 24 hours after injury. Intact platelets from injured rats had higher basal oxygen consumption (17.7 ± 2.5 vs. 15.1 ± 3.2 pmol O 2 /[s × 10 8 cells], p = 0.045), with similar trends in mitochondrial leak rate ( p = 0.19) when compared with sham animals. Overall, platelet basal respiration significantly correlated with TEG maximal amplitude ( r = 0.44, p = 0.034). As a control for sex-dependent systemic mitochondrial differences, females displayed higher liver mitochondria Complex I-driven respiration (895.6 ± 123.7 vs. 622.1 ± 48.7 mmol e - /min/mg protein, p = 0.02); as a control for systemic mitochondrial effects of injury, no liver mitochondrial respiration differences were seen. Platelet mitochondrial basal respiration is increased after injury and correlates with clot strength in this rodent hindlimb fracture model. Several mitochondrial-targeted therapeutics exist in common use that are underexplored but hold promise as potential antithrombotic adjuncts that can be sensitively evaluated in this preclinical model.

Highly Selective MIF Ketonase Inhibitor KRP-6 Diminishes M1 Macrophage Polarization and Metabolic Reprogramming.

Macrophage polarization is highly involved in autoimmunity. M1 polarized macrophages drive inflammation and undergo metabolic reprogramming, involving downregulation of mitochondrial energy production and acceleration of glycolysis. Macrophage migration inhibitory factor (MIF), an enigmatic tautomerase (ketonase and enolase), was discovered to regulate M1 polarization. Here, we reveal that KRP-6, a potent and highly selective MIF ketonase inhibitor, reduces MIF-induced human blood eosinophil and neutrophil migration similarly to ISO-1, the most investigated tautomerase inhibitor. We equally discovered that KRP-6 prevents M1 macrophage polarization and reduces ROS production in IFN-γ-treated cells. During metabolic reprogramming, KRP-6 improved mitochondrial bioenergetics by ameliorating basal respiration, ATP production, coupling efficiency and maximal respiration in LPS+IFN-γ-treated cells. KRP-6 also reduced glycolytic flux in M1 macrophages. Moreover, the selective MIF ketonase inhibitor attenuated LPS+IFN-γ-induced downregulation of PARP-1 and PARP-2 mRNA expression. We conclude that KRP-6 represents a promising novel therapeutic compound for autoimmune diseases, which strongly involves M1 macrophage polarization.

Impact of anthropogenic land uses on soil microbiological activity in a peri-urban landscape.

Land use and land cover patterns impact soil properties and negatively affect soil microbial community and related processes. However, the information regarding the influence of urban land use on soil microbial composition and functioning is limited. Here, we investigated the impact of urban land use patterns on soil microbiological parameters by comparing five contrasting anthropogenic land use classes, i.e. agriculture, park, roadside plantation, street green, and bare land. Soil physicochemical properties, basal respiration (BR), microbial biomass carbon (MBC), and enzyme activities were estimated and correlated. The results revealed that soil physicochemical and microbiological properties greatly varied across the five land use classes. Among all the land use types, the roadside plantation had the highest nutrient content, i.e. soil organic carbon (SOC), total nitrogen (TN), and mineral nitrogen (MN) (1.33%, 0.13%, 84.0mgkg-1, respectively), while the soil functional capacities measured in terms of BR, MBC, microbial quotient (QCO2), soil microbial activity (SMA), and dehydrogenase activity (DHA) (9.90 C µg g-1h-1, 300µgg-1, 0.045µgh-1/ µg MBC, 9.0µgml-1, 1.30 TPF g-1h-1, respectively) were highest in the park. Disturbed street greens were markedly nutrient depleted and apparently exhibited lower microbial activity. Variations in soil BR, MBC, and enzyme activity were revealed to be primarily influenced by soil moisture, available phosphorus, and SOC content. We concluded that the negative impacts of anthropogenic land use soil quality and microbiological functioning can be managed by integrating proper management approaches for various land use classes in urban systems.

Pedobiological properties of a lignite spoil heap in the Provence coal mine basin (south-east of France)

The aim of this study was to investigate the bio-physico-chemical properties of soils developed on dumps originating from lignite extraction and to decipher the respective roles of the geomorphology and the presence of lignite on the biogeochemical characteristics of these soils. Soils from the Armand spoil heap, located in Peypin (Bouches-du-Rhône, France), and natural neighboring Mediterranean soils without lignite were sampled and compared. Lignite presence was assessed by 13C CPMAS NMR spectroscopy allowing for the quantification of C pools such as aromatic C groups, which are characteristic signature of lignite. Physico-chemical (texture, pH, organic C, carbonate, total N, total S, CEC), mineralogical and biological (enzyme activities: β-glucosidase, arylsulfatase, acid phosphatase, arylamidase, lipase and fluorescein diacetate hydrolase; Oxitop basal respiration and Biolog® catabolic profile) parameters were analyzed in the two types of soil. Microbial activities were higher in the natural soils than in the spoil heap soils, while spoil heap samples exhibited enriched aromatic-C pools. Some variables exhibited a gradient along the slope: carbonate and sand contents were larger at the bottom of the heap while clay content, CEC, organic C and C/N ratio were larger at the top of the heap. On the contrary, microbiological activities did not follow any structured pattern according to the slope and appeared distributed as patches. Variance partitioning combined with multiple linear regressions demonstrated that the 13C NMR quality of soil organic matter was the main structuring factor of microbial properties, which were in most cases reduced by the presence of lignite.

Analysis of oxygen consumption rates in zebrafish reveals differences based on sex, age and physical activity recovery.

Introduction: Mitochondrial dysfunction is linked to a variety of human diseases. Understanding the dynamic alterations in mitochondrial respiration at various stages of development is important to our understanding of disease progression. Zebrafish provide a system for investigating mitochondrial function and alterations during different life stages. The purpose of this study was to investigate our ability to measure mitochondrial oxygen consumption rates in zebrafish embryos, larvae, and adults as an indicator of mitochondrial function. Methods: Basal respiration of entire zebrafish embryos (5dpf), larvae (0.6-0.9cm), young adults (3-month-old), and old adults (12-month-old) was measured using an Oroboros Oxygraph, with a stirrer speed of 26rpm. For embryos and larvae, "leak" respiration (plus oligomycin), maximum respiration (plus uncoupler), non-mitochondrial respiration (plus inhibitors), and complex IV activity were also measured. To induce physical activity in adult fish, the stirrer speed was increased to 200rpm. Results and Discussion: We demonstrate the ability to accurately measure respiration rates in zebrafish at various ages using the Oroboros Oxygraph. When comparing zebrafish embryos to larvae, embryos have a higher maximum respiration. Three-month-old zebrafish males have higher basal respiration than females, while 12-month-old zebrafish females exhibit greater rates of respiration than males and younger females. When the stirrer speed was increased, respiration rates decrease, but with differences depending on sex. This study demonstrates a simple and accessible method to assess zebrafish physiology by mitochondrial oxygen consumption measurements in an unmodified Oroboros Oxygraph. The method should facilitate studies to understand the intricate interplay between mitochondrial function, development, and aging.

Extracellular thiamine concentration influences thermogenic competency of differentiating neck area-derived human adipocytes.

Brown adipose tissue (BAT) dissipates energy in the form of heat majorly via the mitochondrial uncoupling protein 1 (UCP1). The activation of BAT, which is enriched in the neck area and contains brown and beige adipocytes in humans, was considered as a potential therapeutic target to treat obesity. Therefore, finding novel agents that can stimulate the differentiation and recruitment of brown or beige thermogenic adipocytes are important subjects for investigation. The current study investigated how the availability of extracellular thiamine (vitamin B1), an essential cofactor of mitochondrial enzyme complexes that catalyze key steps in the catabolism of nutrients, affects the expression of thermogenic marker genes and proteins and subsequent functional parameters during ex vivo adipocyte differentiation. We differentiated primary human adipogenic progenitors that were cultivated from subcutaneous (SC) or deep neck (DN) adipose tissues in the presence of gradually increasing thiamine concentrations during their 14-day differentiation program. mRNA and protein expression of thermogenic genes were analyzed by RT-qPCR and western blot, respectively. Cellular respiration including stimulated maximal and proton-leak respiration was measured by Seahorse analysis. Higher thiamine levels resulted in increased expression of thiamine transporter 1 and 2 both at mRNA and protein levels in human neck area-derived adipocytes. Gradually increasing concentrations of thiamine led to increased basal, cAMP-stimulated, and proton-leak respiration along with elevated mitochondrial biogenesis of the differentiated adipocytes. The extracellular thiamine availability during adipogenesis determined the expression levels of UCP1, PGC1a, CKMT2, and other browning-related genes and proteins in primary SC and DN-derived adipocytes in a concentration-dependent manner. Providing abundant amounts of thiamine further increased the thermogenic competency of the adipocytes. Case studies in humans reported that thiamine deficiency was found in patients with type 2 diabetes and obesity. Our study raises the possibility of a novel strategy with long-term thiamine supplementation, which can enhance the thermogenic competency of differentiating neck area-derived adipocytes for preventing or combating obesity.

Microbial attributes as structural quality index for physical health of an Oxisol under compaction levels

Microbial activity is a sensitive indicator of soil structure improvement provided by plant species in the physical recovery of compacted soils. This study aimed to assess the impacts of crop species on soil aggregate classes for soil recovery after compaction and identify which microbial attributes best describe the structural quality index for soil physical health in an Oxisol under no-tillage. The experiment comprised three crop species—maize, ruzigrass, and black oats—cultivated under four degrees of compactness produced by chiseling or machine traffic under long-term no-tillage. Soil aggregates were separated by dry sieving into five diameter classes to assess the basal respiration, microbial biomass carbon, metabolic quotient for CO2 (qCO2), labile carbon, cellulase activity, and total organic carbon. The values of microbial indicators for the bulk soil were estimated as a weighted mean of each attribute using aggregate size distribution. The lowest values for total organic carbon, labile carbon, and microbial biomass carbon were observed in aggregates > 4 mm. The basal respiration values increased from 8.2 µg C-CO2 g d−1 in the aggregates > 4 mm, to 22.1 µg C-CO2 g soil d−1 in the aggregates < 0.5 mm (+170%). Similarly, the cellulase activity increased approximately four times comparing aggregates > 4 mm (38 µg glucose g−1 d−1) to those < 1 mm (181 µg glucose g−1 d−1). The total organic carbon and microbial biomass carbon were found to be little affected by crops. However, the plots cultivated with black oats or ruzigrass exhibited higher cellulase activity, basal respiration, and qCO2 in comparison with maize, primarily due to the greater content of labile carbon. Increasing the mean weight diameter from 3 mm to 8 mm led to a decrease in the labile carbon (−27.4%), microbial biomass carbon (−25.5%), cellulase activity (−70%), and basal respiration (−21.6%), thereby revealing negative effects of soil compaction on microbial attributes. The degree of compactness did not influence the values of total organic carbon and qCO2. The response of the microbial indicators depends on the aggregate diameter classes. The results of this study indicate that cultivation of cover crops decreases the negative impacts of soil structure degradation on soil microbial quality. Cellulase activity and labile carbon were considered as new potential indicators of soil compaction for soil physical health studies—highly sensitive and quickly responsive to managements that induce soil structure changes in an Oxisol.

Brain-derived neurotrophic factor protects neurons by stimulating mitochondrial function through protein kinase A.

Brain-derived neurotrophic factor (BDNF) stimulates dendrite outgrowth and synaptic plasticity by activating downstream protein kinase A (PKA) signaling. Recently, BDNF has been shown to modulate mitochondrial respiration in isolated brain mitochondria, suggesting that BDNF can modulate mitochondrial physiology. However, the molecular mechanisms by which BDNF stimulates mitochondrial function in neurons remain to be elucidated. In this study, we surmised that BDNF binds to the TrkB receptor and translocates to mitochondria to govern mitochondrial physiology in a PKA-dependent manner. Confocal microscopy and biochemical subcellular fractionation assays confirm the localization of the TrkB receptor in mitochondria. The translocation of the TrkB receptor to mitochondria was significantly enhanced upon treating primary cortical neurons with exogenous BDNF, leading to rapid PKA activation. Showing a direct role of BDNF in regulating mitochondrial structure/function, time-lapse confocal microscopy in primary cortical neurons showed that exogenous BDNF enhances mitochondrial fusion, anterograde mitochondrial trafficking, and mitochondrial content within dendrites, which led to increased basal and ATP-linked mitochondrial respiration and glycolysis as assessed by an XF24e metabolic analyzer. BDNF-mediated regulation of mitochondrial structure/function requires PKA activity as treating primary cortical neurons with a pharmacological inhibitor of PKA or transiently expressing constructs that target an inhibitor peptide of PKA (PKI) to the mitochondrion abrogated BDNF-mediated mitochondrial fusion and trafficking. Mechanistically, western/Phos-tag blots show that BDNF stimulates PKA-mediated phosphorylation of Drp1 and Miro-2 to promote mitochondrial fusion and elevate mitochondrial content in dendrites, respectively. Effects of BDNF on mitochondrial function were associated with increased resistance of neurons to oxidative stress and dendrite retraction induced by rotenone. Overall, this study revealed new mechanisms of BDNF-mediated neuroprotection, which entails enhancing mitochondrial health and function of neurons.

In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles

BackgroundThe capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies.MethodsIn the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile.ResultsWe observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake.ConclusionsOverall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.

P10.36.B ROLE OF AMPK IN GLIOBLASTOMA BIOENERGETICS

Abstract BACKGROUND Glioblastoma is the most prevalent and aggressive primary brain tumor. AMP-activated kinase (AMPK), the main energy sensor of the cell, has been previously described by our group as a key factor in glioblastoma oncogenesis and proliferation and is known for taking part in tumoral metabolic transformation. Our group has also formerly reported how AMPK regulates the expression of lipoprotein receptors and alters lipid reliance of glioblastoma cells, suggesting AMPK orchestrates nutrient dependency and flexibility on glioblastoma. Overall, this evidence positions AMPK inhibition as a promising antitumoral therapy for glioblastoma. MATERIAL AND METHODS To further study the role of AMPK in glioblastoma bioenergetics, we used the human glioblastoma cell lines U87 and U373 and silenced AMPK by transfecting the cells with a siRNA for AMPKα1/2. To assess mitochondrial function (OCR), fuel oxidation, and glycolytic parameters (ECAR), we carried out Seahorse metabolic analyses. This technology provides non-invasive and real-time monitoring of glycolytic and mitochondrial metabolic profiles. The expression of genes related to mitochondrial energetics (CPT1C, PPARGC1A, and PPARA) in these cells was studied using RT-qPCR. We are also currently studying lactate dehydrogenase (LDH) function and expression, as well as glutamine metabolism. RESULTS Our data show that silencing of AMPKα1/2 reduces basal and maximum mitochondrial respiration, as well as decreases the expression of CPT1C, PPARGC1A, and PPARA. Altogether, these results suggest alterations in mitochondrial energetic pathways. Our results do not reveal a significant effect of AMPK on the glycolytic activity of these glioblastoma cell lines. Finally, we found a great capacity of these cells to use glucose, but also fatty acids and glutamine as energy substrates, although with a very high dependency on glucose. CONCLUSION Even though in glioblastoma cells there is an increment in aerobic glycolysis (Warburg effect), these tumoral cells still maintain an elevated mitochondrial activity and a great capacity to use lipids as a fuel. AMPK plays an important role in regulating glioblastoma mitochondrial activity and bioenergetics. The authors would like to acknowledge the Funding for the Consolidation of Competitive Groups of Research (ED431B 2020/26, Xunta de Galicia, 2020-2022).

P10.33.A TARGETED INHIBITION OF THREE MAJOR PROLIFERATION PATHWAYS IN GLIOBLASTOMA INDUCES SENESCENCE THROUGH A MITOCHONDRIA-DEPENDENT MECHANISM

Abstract BACKGROUND Glioblastoma (GBM) is a fatal brain tumor with a great unmet need for better therapies. Research on the genomic landscape of GBM has revealed that three major proliferation pathways — the PI3K-mTOR-AKT, the MAPK, and the CDK4/6-RB pathways — are overactive in the majority of GBM patients. Based on that knowledge we have developed a multi-target combination therapy (MTCT) by employing inhibitors of these three pathways. When these inhibitors are used in combination, lower, clinically more relevant concentrations of each drug are sufficient for target inhibition and proliferation arrest than with single agents.Excitingly, we find that MTCT not only shuts down proliferation but also causes permanent growth inhibition by the induction of senescence and the secretion of a senescence-associated secretory phenotype (SASP). MATERIAL AND METHODS Senescence is identified in a multimodal way, including morphological characterization, SASP production, beta-galactosidase activity, and colony outgrowth potential. RNAseq together with (phospho)proteomics are also applied to investigate the molecular mechanism in combination with techniques such as DNA damage assays, flow cytometry, western blot, metabolic assays, and fluorescence microscopy. RESULTS MTCT-senescent cells have increased mitochondrial reactive oxygen species (mtROS). However, they do not acquire nuclear DNA damage but only local mitochondrial DNA damage. Addition of antioxidants that deplete mtROS resulted in senescence rescue both in terms of morphology and proliferation arrest. MTCT-senescent cells display an enhancement in total mitochondrial mass, mitochondrial DNA copy numbers, and the transcription of mitochondrial proteins. Despite this enhancement, the basal respiration rate of these cells is comparable to untreated cells, indicating an impaired maximal respiration capacity in MTCT-senescent cells. Concordantly, these cells display striking disturbances of their mitochondrial network.Intriguingly, depletion of functional mitochondria prevents the induction of mtROS and senescence by MTCT but not by classical senescence inducers such as ionizing radiation or high concentrations of CDK4/6 inhibitors. CONCLUSION MTCT induces senescence in GBM cells through a unique nuclear DNA damage-independent mechanism for which the mitochondria are essential.

Oxygen-consumption based quantification of chemogenetic H2O2 production in live human cells.

Reactive Oxygen Species (ROS) in the form of H2O2 can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H2O2 were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2 production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H2O2 formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H2O2. However, a method to directly quantify the amount of H2O2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2 production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, allowing to estimate whether the ensuing level of H2O2 production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTert cells, addition of 5 mM D-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H2O2. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H2O2 production to be able to discriminate the effects of H2O2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.

Uncoupled respiration stability of isolated pancreatic acini as a novel functional test for cell vitality

Background. Assessment of cell viability is crucial in cell studies. Testing plasma membrane integrity is a traditional approach of evaluating cell viability. Mitochondrial functional capacity closely correlates with plasma membrane integrity and overall cell health. This study aimed to investigate whether any aspect of mitochondrial adaptive capacity in isolated pancreatic acini is associated with the quality of isolated pancreatic acini preparations, as determined by the dye exclusion method. Materials and Methods. Experiments were carried out on male Wistar rats weig­hing 250–300 g. A suspension of isolated pancreatic acini was obtained using collagenase. The rate of oxygen consumption of rat isolated pancreatic acini was measured with Clark oxygen electrode. Basal respiration of isolated pancreatic acini was recorded for approximately 2 min. Afterwards, the mitochondrial adaptive capacity was examined using FCCP in concentrations from 0.5 to 2 μM. Uncoupled respiratory stability was calculated as a ratio of respiration rate at high and low FCCP concentrations. Plasma membrane integrity was assessed with trypan blue staining. A total of 74 preparations of isolated pancreatic acini were used in this study. Results. In all experiments, 92–99 % of pancreatic acinar cells exhibited negative trypan blue staining, indicating intact plasma membranes. The basal and maximal uncoupled respiration rates were not affected by the fraction of trypan-negative cells. However, acini preparations with &lt;less than 95 % plasma membrane integrity had significantly lower uncoupled respiration rates when exposed to a high concentration of FCCP (2 µM), indicating reduced stability of uncoupled respiration. Conclusions. Results of the study suggest that the stability of uncoupled respiration can serve as a novel metabolic functional test to complement the existing methods for assessing cell vitality.

The Effect of Mineral Fertilizers on Soil Respiration in Urban Lawns

Application of mineral fertilizers to regulate microbial respiration and carbon dioxide emissions from urban lawn soils was evaluated due to the need to develop technologies for reducing CO2 emissions and for increasing the C-absorption capacity of natural and anthropogenic ecosystems. The studies were performed in the Botanical Garden of Moscow State University on Leninskie Hills in a small-plot experiment with the fractional application of four types of complex fertilizers (NPKS 27 : 6 : 6 : 2, NPKS 21 : 10 : 10 : 2, NPK 15 : 15 : 15 and NPK 18:18:18 + 3 MgO + trace elements (TE)) at the doses of 60 and 120 kg N/ha during the growing season. We studied the basal respiration (BR) of soils, carbon content of microbial biomass (Cmic) by substrate-induced respiration, and the CO2 emission from soils by the method of closed static chambers. Cmic in soil of the control plot in the summer period was 1300–1450 µg/g. Application of NPKS 21 : 10 : 10 : 2 and NPK 18 : 18 : 18 + 3 MgO + TE at a low dose increased Cmic by 12–35% within the first two weeks, and then it dropped. All types of fertilizers applied for a short period of time increased BR of soils and CO2 emission maximum on the sixth day. After two weeks and onwards, their growth decreased or their intensity dropped to the control values (500 mg CO2 m2/h–1 and 1.5 μg C–CO2 g/h, respectively) and lower. The lowest intensity of CO2 emission, a rise in basal respiration, and an increase in microbial biomass were recorded after the application of fertilizer NPKS 21 : 10 : 10 : 2. The change in the functioning of the soil microbial community detected by the maximal qCO2 was the greatest in case of NPKS 27 : 6 : 6 : 2 application. The dynamics of CO2 emission from the soils of the small-plot experiment from April to October correlated with the soil temperature (rS = 0.66, p < 0.05, n = 135). Emissions of CO2 were minimal for the plot with NPKS 21 : 10 : 10 : 2 in all periods of the study.

Bioindication of the Dark Gray Soil State in the Pine Forests of the Krasnoyarsk Forest-Steppe under Anthropogenic Influence

The biological activity of dark gray soil was studied in a 100-year-old pine forb-green-moss forest of the Pogorelsky pine forest of the Krasnoyarsk forest-steppe. In 2017, selective cuttings were carried out in the pine forest, and in May 2022 there was a strong fire. To improve reforestation and increase the biological productivity of the soil, bio-fertilizer based on sawdust-soil substrate with the addition of urea and mycoproduct (SSSU + M) was applied to cut and burned areas. On the experimental plots (Paseka, Volok, Fon), an annual count of self-seeding of pine was carried out. Bioindication of the soil condition was assessed based on the total number and ratio of ecological-trophic groups of microorganisms, enzyme activity, microbial biomass content, intensity of basal respiration and specific respiration of microbial biomass. The application of biofertilizer to the cut areas alkalized the soil by 0.2–0.4 units, retained moisture, increased the content of nitrogen (by 5–14%) and microbial biomass (by 1.2–1.6 times), compared with the control options. The impact of biofertilizers on the germination and growth of self-seeding of pine was noted in the second year after application – in the experimental plots of self-seeding it was 4–6 times greater than in the control ones. The entry of burnt plant residues, coals and ash into the soil in the first week after the fire led to an increase in some microbiological indicators, the activity of urease and invertase, and the activation of carbotrophic microorganisms. However, by the end of the growing season, a decrease in microbiological activity was noted, which indicated a post-pyrogenic depression of microbocenoses. The introduction of biofertilizer on the burnt surface of the plots leveled the effect of pyrogenic effects and stimulated the formation of shoots of scots pine, the number of which was significantly higher than in the control plots. It was found that the universal bioindicators that adequately reflect the state of the soil after all anthropogenic impacts were microbial biomass, specific microbial respiration, enzymatic activity and the total number of microorganisms. A specific bioindication of the soil condition after the fire was an increase in the proportion of bacteria Serratia plymuthica, Bacillus mycoides and fungi of the genera Trichoderma, Penicillium and Mortierela.

The Role of Biochar Co-Pyrolyzed with Sawdust and Zeolite on Soil Microbiological and Physicochemical Attributes, Crop Agronomic, and Ecophysiological Performance

The overuse of synthetic fertilizers has been associated with negative environmental consequences. The use of biochar in this regard has been recommended as a win–win strategy. However, our understanding on the comparative influences of biochar prepared from various feedstocks mixed with other bulking agents on soil health and crop performance remained limited. Therefore, in the present study, three types of biochar produced from sewage sludge, food, and agricultural waste were analyzed and compared for their effects on soil enzymes (dehydrogenase, DHA; β-glucosidase, GLU; phosphatase, PHOS; urease, URE; N-acetyl-β-D-glucosaminidase, NAG; and arylsulphatase, ARS), soil basal, as well as substrate-induced respirations and plant growth and physiology characters. The results revealed that food waste-derived biochar co-pyrolyzed with zeolite and/or sawdust was more effective in improving soil physicochemical properties and carbon and phosphorous cycling enzyme (DHA, GLU, and PHOS) activities in addition to soil basal respiration. While the influence of wastewater sewage sludge-derived biochar was more pronounced on urease, N-acetyl-β-D-glucosaminidase, and arylsulphatase enzymes as well as plant biomass accumulation and physiological attributes. Moreover, agricultural waste-derived biochar was found to be effective in enhancing substrate-induced respirations. This study thus concluded that biochar derived from various feedstocks has the tendency to improve soil health and plant growth attributes which further depend on the type of modification prior to pyrolysis.

THE EFFECT OF MINERAL FERTILIZERS ON SOIL RESPIRATION IN URBAN LAWNS

Due to the need to develop technologies for reducing carbon dioxide emissions and increasing the C-absorption capacity of natural and anthropogenic ecosystems, the possibility of using mineral fertilizers to regulate microbial respiration and CO2 emissions from urban lawn soils was evaluated. The studies were carried out on the territory of the Botanical Garden of Moscow State University on the Lenin Hills in a small-plot experiment with the fractional application of four types of complex fertilizers (NPKS 27:6:6:2, NPKS 21:10:10:2, NPK 15:15:15 and NPK 18:18:18 + 3 MgO + trace elements (TE)) at doses of 60 and 120 kg N ha-1 during the growing season. The carbon content of microbial biomass (Cmic) using substrate-induced respiration was studied, the basal respiration (BR) of soils was estimated, and the emission of CO2 from soils using closed static chambers was determined. Тhe Cmic content in the soil of the control plot in the summer period varied within 1300–1450 μg·g-1. The application of NPKS 21:10:10:2 and NPK 18:18:18 + 3 MgO + TE at a low dose increased the Cmic content by 12–35% for two weeks, then it de-creased. The application of all fertilizers for a short period increased basal respiration of soils and CO2 emission, maximum on the 6th day. Aft er two weeks, the increase in the intensity of BR and CO2 emission decreased or it reached the control values (1,5 μg C-CO2·g-1·h-1 and 500 mg·CO2 ·m-2·h-1 respectively) or less. In the short term, the lowest intensity of CO2 emission and a surge in basal respiration, along with an increase in microbial biomass, were observed with the addition of fertilizer NPKS 21:10:10:2. The greatest change in the functioning of the soil microbial community, revealed by the maximum qCO2, occurred with the introduction of NPKS 27:6:6:2. Th e dy-namics of CO2 emission from the soils of the small-plot experiment from April to October correlated with the soil temperature (rS = 0.66, p&lt;0.05, n=135). In all periods of the study, the minimum CO2 emissions were noted for the plot with NPKS 21:10:10:2 fertilizer.

Noticeable Shifts in Soil Physicochemical and Biological Properties after Contrasting Tillage Management in Crop Rotations of Bean, Maize, and Amaranth in Ecuadorian Highland Soils

Soil biological properties are sensitive indicators of soil quality changes due to perturbations occurred under agricultural management. The effects of contrasting tillage, increasing nitrogen fertilization doses, and crop rotations [e.g., bean, maize, bean (BMB) and bean, amaranth, bean (BAB)] on soil physicochemical and biological properties in an Andean soil from Ecuadorian highlands were evaluated in this study. Acid phosphatase, β-Glucosidase, fluorescein diacetate hydrolysis, microbial biomass carbon (Cmic), soil basal respiration (BR), arbuscular mycorrhizal fungi (AMF) spore density, total glomalin content (TGRSP), and soil physicochemical properties were analyzed. Conventional tillage (CT) and crop rotation showed significant effects on soil physicochemical and biological properties. Towards the final crop rotations, no-tillage (NT) promoted BR, TGRSP, and higher AMF spore density in both crop rotations; the Cmic kept stable along time in BMB and BAB, while BR doubled its value when compared to CT. Results indicated that the AMF spore density increased by 308% at the end of the BMB, and 461% at the end of the BAB, while TGRSP increased by 18% and 32% at the end of BMB and BAB, respectively. Biological traits demonstrated to be strongly associated to the organic matter accumulation originated from crop residues under the NT post-harvest which improved soil moisture, biological activity, and AMF interaction. The conservative soil management system has definitively improved general soil properties when compared to soil conditions under the intensive soil management system in this research.