ATP Concentration Research Articles

Effects of sources and routes of administration of vitamins A, D and copper on postnatal status of these micronutrients in piglets.

Twenty-six nulliparous sows were fed conventional gestation and lactation diets supplemented (N = 13) or not (N = 13) with extra daily supplements of 25-hydroxy-cholecalciferol (25-OH-D3; 4 ĸIU), β-carotene (24 ĸIU), and copper (Cu)-proteinate (45 mg) from day 90 of gestation to 21 d of lactation (L21). In each litter, 10 piglets were divided into 5 pairs received, at 2 (L2) and 8 d (L8) of age, one of the five combinations of micronutrient sources and routes of administration (N = 260 piglets total). These neonatal treatments (N = 26 pairs or 52 piglets each) consisted of oral vitamin D3, retinol acetate and CuSO4 (T1); oral 25-OH-D3, β-carotene, and Cu proteinate (T2); exposure to ultraviolet light (UVB), oral retinol palmitate and Cu gluconate (T3); intramuscular vitamin D3 and retinyl propionate and oral Cu acetate (T4); oral saline (CTRL). Oral or intramuscular provisions corresponded to 12 mg of Cu and 70 and 12 ĸIU of vitamins A and D, respectively. Blood samples were collected from all piglets at L2, L8, and L21 for determination of serum Cu, retinol, and 25-OH-D3. Body weight was measured at birth, L2, L8, and L21. Piglets were weaned at L21, and liver and blood samples were collected 2 d later to evaluate oxidative enzymes in blood and liver and hepatic ATP concentrations and expression of genes associated with antioxidant status. Sow treatments had marginal or no impacts on Cu, retinol, 25-OH-D3, or antioxidant status in piglet blood serum and liver. However, when supplements were given to piglets, hepatic Cu was 38% greater in for all treated piglets compared to CTRL (P < 0.01), hepatic retinol was 3 times higher in T1 than in CTRL (P < 0.01) and intermediate for other treatments whereas serum 25-OH-D3 was markedly increased with T2 and T3 at L8 and L21, respectively, compared to CTRL (Piglet treatment × Age interaction, P < 0.01). Concerning antioxidant activities, glutathione peroxidase, and superoxide dismutase were increased (P < 0.03) in plasma of T2 piglets whereas the highest values (P < 0.03) for indicators of oxidative damage to proteins were observed in T4 piglets. The study revealed that oral Cu proteinate from T2, oral retinol acetate from T1, oral 25-hydroxy-cholecalciferol from T2, and UVB light exposure from T3 were the most efficient ways of increasing the postnatal status of these micronutrients in suckling piglets and this may have some impacts on their peri-weaning antioxidant status.

Tandem mass tag-based quantitative proteomics elucidates the inactivation mechanisms of high-power pulsed microwave treatment on Pseudomonas aeruginosa PAO1

This study explored the effect of HPPM treatment (200 Hz for 3, 5, 7, 9, and 11 min) on P. aeruginosa PAO1 inactivation using tandem mass tag-based quantitative proteomics. HPPM demonstrated marked nucleic acid and protein constituents (p < 0.05) and inhibited ATPase activity and intercellular ATP concentration time-dependently. Hence, 9 min was selected for proteomic profiling to avoid any potential thermal effect. A total of 452 proteins (309 = down-regulated; 143 = up-regulated) were identified as differentially accumulated proteins (DAPs). The DAPs were primarily related to energy production and conversion; DNA replication, transcription, and translation, cell membrane composition; amino acid (AA) transport and metabolism, and biofilm formation and quorum sensing (QS). HPPM significantly disrupted cell membrane composition, altered AA metabolism, impeded cell energy generation, and drastically inhibited biofilm formation in P. aeruginosa. This study provided insights into the underlying mechanisms of HPPM against P. aeruginosa at the proteomic level. Industrial relevancePseudomonas aeruginosa is a key spoiler implicated in food contamination. High-power pulsed microwave (HPPM) is a potential non-thermal inactivation technology. However, the underlying mechanism to validate its efficacy remains unclear. This study provides the industry with the inactivation efficacy of HPPM against P. aeruginosa, highlighting insights into the molecular and biochemical changes after treatment and its remarkable potential to control cross-contamination in food processing, food services, and clinical environments.

Assembly-mediated activation of the SIR2-HerA supramolecular complex for anti-phage defense

SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.

Exploring the metabolic and antioxidant potential of solergy: Implications for enhanced animal production

Cell models are indispensable tools in biotechnology when investigating the functional properties of organic compounds. The emergence of various additives designed to enhance animal production has introduced the need for in-depth evaluations, which are often hindered by the complexities of in vivo testing. In this study, we harnessed cell-based models to scrutinize the impact of Solergy as a regulator of cellular metabolism with a particular focus on its modulation of glycogen and antioxidant effects. Our experiment was designed to include assessments of the influence of Solergy on the viability of both terrestrial and aquatic vertebrate cell models, which revealed the benign nature of Solergy and its lack of adverse effects. Furthermore, we examined the capacity of Solergy to modulate intracellular ATP concentrations and enhance glycogen accumulation. Notably, the antioxidant potential of Solergy and its ability to mitigate cellular aging were evaluated within the same cellular frameworks. The outcomes of our investigation suggest that Solergy is a potent metabolic regulator that elevates cellular activity while exerting an antioxidant effect. Importantly, our study demonstrates that Solergy does not induce changes in membrane oxidation. These findings indicate the potential of using Solergy to regulate glycogen synthesis, intracellular ATP concentrations, and oxidative stress in production animals. The multifaceted effects of this additive, which acts as both a metabolism enhancer and an antioxidant, open doors to the creation of custom diets tailored to meet specific production needs while maintaining stable production parameters.

Shikonin reverses cancer-associated fibroblast-induced gemcitabine resistance in pancreatic cancer cells by suppressing monocarboxylate transporter 4-mediated reverse Warburg effect

BackgroundGemcitabine is a first-line chemotherapeutic agent for pancreatic cancer (PC); however, most patients who receive adjuvant gemcitabine rapidly develop resistance and recurrence. Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor stroma that contribute to gemcitabine-resistance. There is thus an urgent need to find a novel therapeutic strategy to improve the efficacy of gemcitabine in PC cells under CAF-stimulation. PurposeTo investigate if shikonin potentiates the therapeutic effects of gemcitabine in PC cells with CAF-induced drug resistance. MethodsPC cell-stimulated fibroblasts or primary CAFs derived from PC tissue were co-cultured with PC cells to evaluate the ability of shikonin to improve the chemotherapeutic effects of gemcitabine in vitro and in vivo. Glucose uptake assay, ATP content analysis, lactate measurement, real-time PCR, immunofluorescence staining, western blot, and plasmid transfection were used to investigate the underlying mechanism. ResultsCAFs were innately resistant to gemcitabine, but shikonin suppressed the PC cell-induced transactivation and proliferation of CAFs, reversed CAF-induced resistance, and restored the therapeutic efficacy of gemcitabine in the co-culture system. In addition, CAFs underwent a reverse Warburg effect when co-cultured with PC cells, represented by enhanced aerobic glycolytic metabolism, while shikonin reduced aerobic glycolysis in CAFs by reducing their glucose uptake, ATP concentration, lactate production and secretion, and glycolytic protein expression. Regarding the mechanism underlying these sensitizing effects, shikonin suppressed monocarboxylate transporter 4 (MCT4) expression and cellular membrane translocation to inhibit aerobic glycolysis in CAFs. Overexpression of MCT4 accordingly reversed the inhibitory effects of shikonin on PC cell-induced transactivation and aerobic glycolysis in CAFs, and reduced its sensitizing effects. Furthermore, shikonin promoted the effects of gemcitabine in reducing the growth of tumors derived from PC cells and CAF co-inoculation in BALB/C mice, with no significant systemic toxicity. ConclusionThese results indicate that shikonin reduced MCT4 expression and activation, resulting in inhibition of aerobic glycolysis in CAFs and overcoming CAF-induced gemcitabine resistance in PC. Shikonin is a promising chemosensitizing phytochemical agent when used in combination with gemcitabine for PC treatment. The results suggest that disrupting the metabolic coupling between cancer cells and stromal cells might provide an attractive strategy for improving gemcitabine efficacy.

Cellular Iron Deficiency Disrupts Thyroid Hormone Regulated Gene Expression in Developing Hippocampal Neurons

BackgroundDeveloping neurons have high thyroid hormone and iron requirements to support their metabolically demanding growth. Early-life iron and thyroid-hormone deficiencies are prevalent and often coexist, and each independently increases risk of permanently impaired neurobehavioral function in children. Early-life dietary iron deficiency reduces thyroid-hormone concentrations and impairs thyroid hormone-responsive gene expression in the neonatal rat brain, but it is unclear whether the effect is cell-intrinsic. ObjectivesThis study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. MethodsIron deficiency was induced in primary mouse embryonic hippocampal neuron cultures with the iron chelator deferoxamine (DFO) beginning at 3 d in vitro (DIV). At 11DIV and 18DIV, thyroid hormone-regulated gene messenger ribonucleic acid (mRNA)concentrations indexing thyroid hormone homeostasis (Hairless, mu-crystallin, Type II deiodinase, solute carrier family member 1c1, and solute carrier family member 16a2) and neurodevelopment (neurogranin, Parvalbumin, and Krüppel-like factor 9) were quantified. To assess the effect of iron repletion, DFO was removed at 14DIV from a subset of DFO-treated cultures, and gene expression and adenosine 5'-triphosphate (ATP) concentrations were quantified at 21DIV. ResultsAt 11DIV and 18DIV, neuronal iron deficiency decreased neurogranin, Parvalbumin, and mu-crystallin, and by 18DIV, solute carrier family member 16a2, solute carrier family member 1c1, Type II deiodinase, and Hairless were increased, suggesting cellular sensing of a functionally abnormal thyroid hormone state. Dimensionality reduction with Principal component analysis reveals that thyroid hormone homeostatic genes strongly correlate with and predict iron status. Iron repletion from 14–21DIV did not restore ATP concentration, and Principal component analysis suggests that, after iron repletion, cultures maintain a gene expression signature indicative of previous iron deficiency. ConclusionsThese novel findings suggest there is an intracellular mechanism coordinating cellular iron/thyroid hormone activities. We speculate this is a part of the homeostatic response to acutely match neuronal energy production and growth signaling. However, the adaptation to iron deficiency may cause permanent deficits in thyroid hormone-dependent neurodevelopmental processes even after recovery from iron deficiency.

IMMU-19. MUTANT ISOCITRATE DEHYDROGENASE 1 IN GLIOMA CAUSES A REDUCTION IN ADENOSINERGIC PATHWAY SIGNALING WITHIN THE TUMOR MICROENVIRONMENT

Abstract Glioma is one of the most aggressive cancers and represents approximately 80% of malignant brain tumors. The majority of CNS World Health Organization (WHO) grades 2-3 gliomas and a subset of high-grade gliomas (CNS WHO grade 4) harbor mutated isocitrate dehydrogenase 1 (IDH1R132H; mIDH1). Mutant IDH1 results in the accumulation of 2-hydroxyglutarate (2HG) which elicits profound changes in the glioma transcriptome/biology. Consequently, mIDH1 glioma patients have a significantly increased median survival compared to wildtype IDH1 patients. Recent data from our lab showed that mIDH1 gliomas exhibit increased immune-reactivity of tumor-infiltrating immune cells. A critical pathway that mediates reduction of anti-tumor immune cell reactivity is the adenosinergic pathway (AP), which converts ATP into adenosine via the enzymes CD39 and CD73. Adenosine binds to adenosine receptors, A2AR and A2BR, on immune cells resulting in a decreased immunoreactivity. Although mIDH1 has been implicated in altering the AP, the extent to which the AP is affected by mIDH1 in gliomas is largely unknown. Here we used human and mouse mIDH1 glioma cells in vitro along with in vivo mIDH1 glioma mouse models to examine changes in the AP. Our results show reduced levels of the enzyme, CD73, on the surface of mIDH1 glioma cells, and the ATP concentration in the mIDH1 glioma cell growth media is lower than from wildtype IDH1 glioma cells. Additionally, we discovered that mIDH1 glioma-infiltrating macrophages have decreased expression of CD39 and A2AR compared to their wildtype IDH1 glioma counterparts. Taken together, these data suggest a reduction of the AP mediated-immune suppression in mIDH1 gliomas. Uncovering these differences in the AP provides novel insights on the use of AP inhibitors for glioma treatment, revealing whether the AP is a preferred target in specific glioma types based on the IDH1 mutation status.

PSIII-4 Effect of Photobiomodulation Treatment on Atp Concentrations in Bovine Oocytes During Maturation

Abstract Oocyte cytoplasmic maturation is critical in determining the success of subsequent embryo development. It has been proposed that light in the red-to-near infrared optical region (~600–1000 nm) enhances cellular metabolic activity through activation of the mitochondrial respiratory chain, increasing intracellular ATP concentrations, and has the potential to modify the oocyte metabolome. Previous research indicated a greater cleavage rate and similar blastocyst rates for bovine oocytes treated with photobiomodulation at hours 16 and 20 from the start of maturation compared with control. For the present study, two separate experiments were conducted to determine ATP concentration in oocytes during maturation after photobiomodulation treatment. A standardized in vitro maturation (IVM) protocol and commercial media (IVF Bioscience) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse, cumulus oocyte complexes (COCs) were collected by aspiration of small (2 to 6 mm) follicles. For experiment 1, compact COCs were cultured in in vitro maturation media (n = 30/well) and treated with no light (control); light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16) and light for 10 minutes at hour 20 from the start of maturation (L-20). At hour 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye (Sigma-Aldrich SCT045), imaged with Cytation-1 (Agilent Technologies Inc.) in 6 replicates. For experiment 2, COCs were cultured in in vitro maturation media (n = 40/well) and treated with no light (control) and light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16). At hours 18, 20, 22 and 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye, and imaged with Cytation-1 in 4 replicates. For experiment 1, one-way ANOVA and Student’s T-Test were performed (JMP software 16.0); in experiment 2, each well was considered as an experimental unit in a model of repeated measures, main effects of time and treatment, blocked by replicate (Mixed procedure, SAS Institute Inc.). In experiment 1, there was a significant (P &amp;lt; 0.05) effect of treatment; ATP concentrations were greater in L-16 than control, and L-20 was intermediate. ATP concentrations were 19,579.3a ± 596.5, 21,652.0b ± 602.7 and 19,710.9ab ± 570.6 relative fluorescence units (RFU) for control, L-16, and L-20 respectively. In experiment 2, there was a significant (P &amp;lt; 0.05) effect of treatment and no significant effect of time. Overall ATP concentrations were 25,668 ± 894.6 and 28,332 ± 894.6 RFU for control and L-16 respectively. In conclusion, photobiomodulation treatment with LED 660-665nm for 10 minutes at hour 16 from start of oocyte maturation increases intracellular ATP concentrations from hour 18 to 24 of maturation.

Elucidating Events within the Black Box of Enzyme Catalysis in Energy Metabolism: Insights into the Molecular Mechanism of ATP Hydrolysis by F1-ATPase.

Oxygen exchange reactions occurring at β-catalytic sites of the FOF1-ATP synthase/F1-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. This work presents a new theory of oxygen exchange and tests it on oxygen exchange data recorded on ATP hydrolysis by mitochondrial F1-ATPase (MF1). The apparent rate constant of oxygen exchange governing the intermediate Pi-HOH exchange accompanying ATP hydrolysis is determined by kinetic analysis over a ~50,000-fold range of substrate ATP concentration (0.1-5000 μM) and a corresponding ~200-fold range of reaction velocity (3.5-650 [moles of Pi/{moles of F1-ATPase}-1 s-1]). Isotopomer distributions of [18O]Pi species containing 0, 1, 2, and 3 labeled oxygen atoms predicted by the theory have been quantified and shown to be in perfect agreement with the experimental distributions over the entire range of medium ATP concentrations without employing adjustable parameters. A novel molecular mechanism of steady-state multisite ATP hydrolysis by the F1-ATPase has been proposed. Our results show that steady-state ATP hydrolysis by F1-ATPase occurs with all three sites occupied by Mg-nucleotide. The various implications arising from models of energy coupling in ATP synthesis/hydrolysis by the ATP synthase/F1-ATPase have been discussed. Current models of ATP hydrolysis by F1-ATPase, including those postulated from single-molecule data, are shown to be effectively bisite models that contradict the data. The trisite catalysis formulated by Nath's torsional mechanism of energy transduction and ATP synthesis/hydrolysis since its first appearance 25 years ago is shown to be in better accord with the experimental record. The total biochemical information on ATP hydrolysis is integrated into a consistent model by the torsional mechanism of ATP synthesis/hydrolysis and shown to elucidate the elementary chemical and mechanical events within the black box of enzyme catalysis in energy metabolism by F1-ATPase.

Yerba Maté (Ilex paraguariensis) Supplement Exerts Beneficial, Tissue-Specific Effects on Mitochondrial Efficiency and Redox Status in Healthy Adult Mice.

Yerba maté, a herbal tea derived from Ilex paraguariensis, has previously been reported to be protective against obesity-related and other cardiometabolic disorders. Using high-resolution respirometry and reverse-phase high-performance liquid chromatography, the effects of four weeks of yerba maté consumption on mitochondrial efficiency and cellular redox status in skeletal muscle, adipose, and liver, tissues highly relevant to whole-body metabolism, were explored in healthy adult mice. Yerba maté treatment increased the mitochondrial oxygen consumption in adipose but not in the other examined tissues. Yerba maté increased the ATP concentration in skeletal muscle and decreased the ATP concentration in adipose. Combined with the observed changes in oxygen consumption, these data yielded a significantly higher ATP:O2, a measure of mitochondrial efficiency, in muscle and a significantly lower ATP:O2 in adipose, which was consistent with yerba maté-induced weight loss. Yerba maté treatment also altered the hepatic glutathione (GSH)/glutathione disulfide (GSSG) redox potential to a more reduced redox state, suggesting the treatment's potential protective effects against oxidative stress and for the preservation of cellular function. Together, these data indicate the beneficial, tissue-specific effects of yerba maté supplementation on mitochondrial bioenergetics and redox states in healthy mice that are protective against obesity.

Adenylate kinase 9 is essential for sperm function and male fertility in mammals

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

Accelerated Extracellular Nucleotide Metabolism in Brain Microvascular Endothelial Cells in Experimental Hypercholesterolemia

Hypercholesterolemia affects the neurovascular unit, including the cerebral blood vessel endothelium. Operation of this system, especially in the context of energy metabolism, is controlled by extracellular concentration of purines, regulated by ecto-enzymes, such as e-NTPDase-1/CD39, ecto-5′-NT/CD73, and eADA. We hypothesize that hypercholesterolemia, via modulation of the activity of nucleotide metabolism-regulating ecto-enzymes, deteriorates glycolytic efficiency and energy metabolism of endothelial cells, which may potentially contribute to development of neurodegenerative processes. We aimed to determine the effect of hypercholesterolemia on the concentration of purine nucleotides, glycolytic activity, and activity of ecto-enzymes in the murine brain microvascular endothelial cells (mBMECs). We used 3-month-old male LDLR−/−/Apo E−/− double knockout mice to model hypercholesterolemia and atherosclerosis. The age-matched wild-type C57/BL6 mice were a control group. The intracellular concentration of ATP and NAD and extracellular activity of the ecto-enzymes were measured by HPLC. The glycolytic function of mBMECs was assessed by means of the extracellular acidification rate (ECAR) using the glycolysis stress test. The results showed an increased activity of ecto-5′-NT and eADA in mBMECs of the hypercholesterolemic mice, but no differences in intracellular concentration of ATP, NAD, and ECAR between the hypercholesterolemic and control groups. The changed activity of ecto-5′-NT and eADA leads to increased purine nucleotides turnover and a shift in their concentration balance towards adenosine and inosine in the extracellular space. However, no changes in the energetic metabolism of the mBMECs are reported. Our results confirm the influence of hypercholesterolemia on regulation of purine nucleotides metabolism, which may impair the function of the cerebral vascular endothelium.Graphical abstractThe effect of hypercholesterolemia on the murine brain microvascular endothelial cells (mBMECs). An increased activity of ecto-5′-NT and eADA in mBMECs of the LDLR−/−/Apo E−/− mice leads to a shift in the concentration balance towards adenosine and inosine in the extracellular space with no differences in intracellular concentration of ATP. Figure was created with Biorender.com.

Depth profiles of biological aerated contactors: Characterizing microbial activity treating reduced contaminants

The biological treatment process consisting of an aerated contactor and filter is effective for groundwaters containing elevated ammonia and other reduced contaminants, including iron, manganese, arsenic, and methane. Depth profiles characterizing microbial activity across aerated contactors are lacking. A 1-year pilot study comparing gravel- and ceramic-packed contactors was conducted, and media depth profile samples were collected at the conclusion of the study. Media and water samples also were collected from pilot-scale aerated contactors at 4 other water systems. Water quality, media surface metals concentrations, and a suite of biofilm parameters were analyzed. Media surface metals concentrations were greatest at the influent end. ATP concentrations, extracellular polymeric substances, and extracellular enzyme activities tended to be similar across depth. Bacteria and functional genes involved in contaminant oxidation co-occurred and tended to decrease across depth, but were not correlated to the media metals concentration. Microbial community composition changed with depth, and the diversity either decreased or remained similar. The microbial activity profiles through aerated contactors differed from what is typically reported for groundwater biofilters, suggesting that the different reactor flow and dissolved oxygen profiles impacted the microbial community.

Co-exposure to cadmium and microplastics promotes liver fibrosis through the hemichannels -ATP-P2X7 pathway

Microplastics (MPs) and cadmium (Cd) are important environmental pollutants, that damage the liver. However, the effect and mechanism of combined Cd and MPs exposure on liver fibrosis are still largely unknown. In this study investigated, Cd + MPs exposure increased superoxide anion production and promoted extracellular ATP release compared with exposure to Cd or MPs individually. Cd + MPs increased inflammatory cell infiltration, activated the P2X7-NLRP3 signaling pathway, and promoted inflammatory factor release. Cd + MPs aggravated Cd- or MPs-induced liver fibrosis and induced liver inflammation. In AML12/HSC-T6 cell in vitro poisoning model, exposure of AML12 cells to Cd + MPs increased the opening of connexin hemichannels and promoted extracellular ATP release. Treatment of HSC-T6 cells with the supernatant of AML12 cells exposed to Cd + MPs significantly promoted HSC-T6 cell activation. Treatment of HSC-T6 cells with different concentrations of ATP produced similar results. TAT-Gap19TFA, an inhibitor of connexin hemichannels, significantly inhibited the ATP release and activation of Cd + MPs-treated HSC-T6 cells. Finally, the expression of the ATP receptor P2X7 was silenced in HSC-T6 cells, which significantly inhibited their activation. In conclusion, exposure to Cd + MPs promoted liver fibrosis through the ATP-P2X7 pathway and synergistically affected liver inflammation and fibrosis.

Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling.

Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC50 of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

N-doped carbon quantum dots enhance anaerobic digestion under light condition: The performances and potential mechanisms

The treatment of organic waste (OW) by anaerobic digestion (AD) conforms to the concept of sustainable development. But AD is facing the issue of low conversion rate. In this work, the photo-AD system using visible light (LED lamp) as the source was constructed and the performances and mechanism of N-doped carbon quantum dots (NCQD) were explored in the system for the first time. The results showed that 0.5 g/L NCQD promoted a 23.1 % increase in cumulative CH4 yield in the photo-AD system. Microbial analysis results showed that in photo-AD with NCQD, the dominant strain was Methanosarciniales, with an abundance of 69.0 %. Microbial activity and structural integrity tests showed that the microorganisms were not damaged by free radicals. In addition, NCQD increased the redox peak intensity of the CV curve and increased photocurrent intensity of photo-AD. Furthermore, it promoted an increase of 18.2 % (0.26 ± 0.03 μmol/mL) in ATP concentration. The photoelectrochemical analysis and quantitative analysis of functional genes results indicated that NCQD mainly promoted methanogenesis by providing photoelectrons. This promotion mechanism increased the copynumber (61,652.8 g−1) of EchA in photo-AD, rather than Vht and Hdr related to cytochrome. This work provided new strategies for the enhancement of AD and clarified potential mechanisms.

Sestrin2 protects against hypoxic nerve injury by regulating mitophagy through SESN2/AMPK pathway.

Hypoxia induced by high altitude can lead to severe neurological dysfunction. Mitophagy is known to play a crucial role in hypoxic nerve injury. However, the regulatory mechanism of mitophagy during this injury remains unclear. Recent studies have highlighted the role of Sestrin2 (SESN2), an evolutionarily conserved stress-inducible protein against acute hypoxia. Our study demonstrated that hypoxia treatment increased SESN2 expression and activated mitophagy in PC12 cells. Furthermore, the knock-out of Sesn2 gene led to a significant increase in mitochondrial membrane potential and ATP concentrations, which protected the PC12 cells from hypoxic injury. Although the AMPK/mTOR pathway was significantly altered under hypoxia, it does not seem to participate in mitophagy regulation. Instead, our data suggest that the mitophagy receptor FUNDC1 plays a vital role in hypoxia-induced mitophagy. Moreover, SESN2 may function through synergistic regulation with other pathways, such as SESN2/AMPK, to mediate cellular adaptation to hypoxia, including the regulation of mitophagy in neuron cells. Therefore, SESN2 plays a critical role in regulating neural cell response to hypoxia. These findings offer valuable insights into the underlying molecular mechanisms governing the regulation of mitophagy under hypoxia and further highlight the potential of SESN2 as a promising therapeutic target for hypoxic nerve injury.

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

The general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary heart disease

The purpose of the study: to develop a general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary heart disease (CHD). Materials and methods. Examination of 60 patients with CHD: stable angina pectoris of I-III functional classes was performed. According to the standard general clinical methods for verifying the diagnosis were used: ECG, Doppler echocardiography, coronary angiography, lipid profile, complete blood count, coagulogram, renal, hepatic complex. Also the condition of stress-realizing and stress-limiting systems of the functional system of adaptation was deeply examined in patients: determination of the level of personal anxiety by the method of questioning, determination of the concentration of cortisol, insulin, cAMP and cGMP in blood serum by the method of enzyme immunoassay; determination of the endothelial and inducible nitric oxide synthase levels in erythrocyte lysate by the method of enzyme immunoassay; the study of ATP and ADP concentrations in blood serum and erythrocytes, also the study of 2,3-DFG concentration in erythrocytes by spectrophotometric methods. The individual reactivity of blood leukocytes’ mitochondria of patients was estimated in vitro under the influence of metabolic drugs (trimetazidine, meldonium, cytoflavin) by confocal microscopy according to the technique developed and patented by us (mitochondria were stained with pyrene). The materials were processed statistically. Results. Two variants of the leukocyte mitochondrial response in patients with CHD to the introduction of metabolic correctors in vitro were found — in the form of their activation or inhibition, depending on numerous parameters of the initial state of patients, which served as the basis for the development of a general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary heart disease. According to this concept, the individual reactivity of a patient with CHD to the administration of a metabolic drug depends on the initial state of the functional adaptation system, which can be determined by the activity of stress-realizing and stress-limiting systems (cortisol/insulin ratio, serum cAMP/cGMP, eNOS, iNOS of erythrocyte lysate) and by the degree of preservation of the structure and function of effector organs. It is appropriate to prescribe drugs that stimulate energy metabolism in cells only to patients with initial hypoergosis, reduced mitochondrial activity at the phases of activation or resistance of the general adaptation syndrome, while maintaining reserves for energy adaptation, by short courses. Conclusion. A general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary artery disease has been developed, according to which metabolic correctors should be prescribed in short courses, provided that individual reserves for energy adaptation are preserved in patients with initial mitochondrial hypoergosis in the activation or resistance phase of the general adaptation syndrome.

Relaxin (a regulatory-peptide) enhances cryotolerance, fertility potential and plasma reproductive hormones of Nili Ravi buffalo (Bubalus bubalis) during low breeding season

This study investigated the beneficial effects of relaxin on cryotolerance of buffalo spermatozoa and reproductive hormones during low breeding season. Collected semen was diluted in five aliquots with relaxin addition (0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1 mg/mL, and control). After gentle dilution (37°C), cooling (4°C, 2 h), equilibration (4°C, 4 h), and packaging (straws, polyvinyl French, 0.5 mL), frozen (cell freezer), and thawed (37°C, 30 s) for analysis. Blood samples were collected at different time intervals i.e., −60, −30 and 0 min (pre-dose) and 30, 60, 90, 120 and 150 min (post-dose) from a jugular vein. This study manifest that adding relaxin (1 mg/ mL) in freezing medium ameliorates sperm motility, functionality (%), and seminal plasma total antioxidant capacity (TAC, μM/L) than control during low breeding season. Furthermore, we found that relaxin supplementation at 1 mg/mL significantly improves seminal plasma ATP concentrations (nmol/million) than control, 0.25 mg/mL, and 0.50 mg/mL, and fertility (control, and 0.75 mg/mL). Further, relaxin injection significantly improves plasma T, LH and IGF-1 levels (150 and 120 min vs. −60, and − 30), and FSH, KP, and GnRH concentrations (150 min vs. −60), during low breeding season. Taken together, this study revealed that relaxin ameliorates motility, functionality, and fertility of buffalo spermatozoa. Moreover, relaxin injection (1 mg/mL) improves essential reproductive hormones levels in buffalo signifying its importance in the field of reproductive physiology. Further studies are required to determine the exact mechanism of action of relaxin in enhancing semen quality, fertility and reproductive hormones.