Cycle Enzymes Research Articles

Period3 modulates the NAD+-SIRT3 axis to alleviate depression-like behaviour by enhancing NAMPT activity in mice.

PER3 deficiency is associated with depression-like behaviors, but the underlying mechanisms remain unclear. This study aims to elucidate the role and mechanism of PER3 in regulating depression-like behaviors in mice. Depression-like behaviors were assessed using the sucrose preference test, tail suspension test, and forced swimming test. Metabolomic analysis was conducted on hippocampal tissues from Per3 knockout mice using chromatography-mass spectrometry. The regulatory role of PER3 on the expression of nicotinamide phosphoribosyltransferase (Nampt) was investigated through co-immunoprecipitation and chromatin immunoprecipitation assays. Metabolomic analysis revealed that Per3 deficiency disrupts mitochondrial function, as evidenced by reduced activities of key tricarboxylic acid (TCA) cycle enzymes (succinate dehydrogenase, citrate synthase, and α-ketoglutarate dehydrogenase), diminished expression of mitochondrial respiratory chain complexes I-V, and decreased nicotinamide adenine dinucleotide (NAD)+ levels in Per3 knockout mice. Supplementation with the NAD+ precursor nicotinamide (NAM) rescued mitochondrial function and alleviated depression-like behaviors in Per3 knockout mice. Similar effects were observed with intraperitoneal administration of the NAMPT activator P7C3-A20, while these effects were abolished by the NAMPT inhibitor FK866. Mechanistically, PER3 was found to regulate Nampt expression by binding to E-box elements within its intronic regions in conjunction with BMAL1. This interaction enhanced NAD+ production, activating SIRT3 to mitigate mitochondrial dysfunction in Per3 knockout mice. These findings uncover a novel mechanism by which PER3 ameliorates depressive behaviors through the regulation of NAMPT-controlled NAD+ levels and mitochondrial function, underscoring the critical role of PER3 in depression-related pathophysiology.

The chloroplast RNA–binding protein CP29A supports rbcL expression during cold acclimation

The chloroplast genome encodes key components of the photosynthetic light reaction machinery as well as the large subunit of the enzyme central for carbon fixation, Ribulose-1,5-bisphosphat-carboxylase/-oxygenase (RuBisCo). Its expression is predominantly regulated posttranscriptionally, with nuclear-encoded RNA-binding proteins (RBPs) playing a key role. Mutants of chloroplast gene expression factors often exhibit impaired chloroplast biogenesis, especially in cold conditions. Low temperatures pose a challenge for plants as this leads to electron imbalances and oxidative damage. A well-known response of plants to this problem is to increase the production of RuBisCo and other Calvin Cycle enzymes in the cold, but how this is achieved is unclear. The chloroplast RBP CP29A has been shown to be essential for cold resistance in growing leaf tissue of Arabidopsis thaliana. Here, we examined CP29A-RNA interaction sites at nucleotide resolution. We found that CP29A preferentially binds to the 5'-untranslated region of rbcL, downstream of the binding site of the pentatricopeptide repeat protein MATURATION OF RBCL 1 (MRL1). MRL1 is an RBP known to be necessary for the accumulation of rbcL. In Arabidopsis mutants lacking CP29A, we were unable to observe significant effects on rbcL, possibly due to CP29A's restricted role in a limited number of cells at the base of leaves. In contrast, CRISPR/Cas9-induced mutants of tobacco NtCP29A exhibit cold-dependent photosynthetic deficiencies throughout the entire leaf blade. This is associated with a parallel reduction in rbcL mRNA and RbcL protein accumulation. Our work indicates that a chloroplast RNA-binding protein contributes to cold acclimation of RbcL production.

Transcriptomic and enzymatic analysis of peroxidase families at the early growth stage of halophyte ice plant (Mesembryanthemum crystallinum L.) under salt stress

Ice plant (Mesembryanthemum crystallinum L.) is a halophyte and an inducible CAM plant. Ice plant seedlings display moderate salt tolerance, with root growth unaffected by 200 mM NaCl treatments, though hypocotyl elongation is hindered in salt-stressed etiolated seedlings. Superoxide anion accumulation was prominent in cotyledons and primary leaves but decreased in root tissues over time, with no significant effect from salt treatment. Hydrogen peroxide levels initially surged in both control and salt-treated seedlings, with higher and more persistent accumulation in the salt-treated seedlings. The activities of H2O2-scavenging ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase, and dehydroascorbate reductase increased, while guaiacol-dependent peroxidase activity decreased and catalase activity showed no change, indicating APX activity as the primary response to salt stress. Salt-induced APX activities were detected mainly in the microsomal fraction for light-grown seedlings and the cytosolic fraction for etiolated seedlings, highlighting plastids as the primary site of ROS accumulation under salt stress. An RNA-seq analysis of etiolated seedlings revealed about 8% unigenes showing more than a four-fold change in expression after a 6-h 200 mM NaCl treatment. GO enrichment analysis indicated that differentially expressed genes (DEGs) with increased transcript abundance were associated with ion transport, antioxidant activity, and stress responses, while DEGs with decreased transcript abundance were linked to metabolic and biosynthesis processes such as ribosomal protein synthesis and cell wall formation. This indicates that salt stress hinders growth but enhances ion homeostasis and stress response mechanisms. The expression of all eight APX genes were induced by a 48-hour salt treatment, with varying expression patterns. For class III peroxidase family, 14 out of 53 identified unigenes qualified as DEGs. The time-course expression patterns revealed that the transcript levels of McPrx4.1, McPrx12.1, and McPrx12.3 increased, while McPrx60.3 decreased. These findings highlight the distinct roles of class III peroxidases in balancing plant growth and stress responses, advancing our understanding of the mechanisms behind salt tolerance in halophytes. This study comprehensively analyzed changes in gene expression, antioxidant enzyme activity, and ROS accumulation in ice plant seedlings. Unveiling these responses will advance our understanding of the growth–stress balance in the intrinsic salt tolerance in halophytes.

Long-term cultivation of retinal pigment epithelium cells on nanofiber scaffolds.

The retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age-related macular degeneration (AMD) and other retinal degenerative diseases. The introduction of healthy RPE cell cultures into the subretinal space offers a potential treatment strategy. The aim of this study was the long-term culture and characterisation of RPE cells on nanofiber scaffolds. Nanofiber scaffolds consisting of polycaprolactone (PCL) and collagen were prepared by electrospinning. Porcine RPE cell cultures were maintained on PCL scaffolds, PCL-collagen scaffolds, and controls at the bottom of 24-well plates. Cell culture analysis was performed by immunohistochemistry, while the release of inflammatory cytokines IL-6, IL-8, TNF-α, and PDGF-β was measured by ELISA and multiplex assays. Ultrastructural features were examined by transmission electron microscopy. The observation period averaged 42.7 weeks for controls, 38.7 weeks for PCL scaffold cultures, and 36.1 weeks for PCL-collagen scaffold cultures, with cell number and morphology remaining stable. TNF-α levels in the supernatants were minimal, IL-6 levels were consistently low, and IL-8 levels decreased from initially high to lower levels over time. RPE cells were stably cultured on nanofiber scaffolds for extended periods of time. The long-term physiological properties of RPE cells, including phagocytic ability and visual cycle enzyme activity, need to be further investigated before clinical application. In addition, controlling the expression of inflammatory mediators is a major challenge. Despite these hurdles, overcoming them is critical given the increasing prevalence of retinal degenerative diseases.

Resource recovery from wastewater by directing microbial metabolism toward production of value-added biochemicals.

Dynamic oxygen fluctuations in activated sludge were investigated to enhance valuable biochemical production during wastewater treatment. Batch experiments compared constant aeration with rapid cycling between oxygen-rich and oxygen-poor states. Fluctuating oxygen concentrations (0-2 mg/L) significantly increased production of valuable biochemicals compared to constant oxygen concentration (2 mg/L). Continuous oxygen perturbations increased free amino acids by 35.7 ± 7.6 % and free fatty acids by 76.4 ± 13.0 %, while intermittent perturbations with anoxic periods enhanced free amino acids by 42.4 ± 8.1 % and free fatty acids by 39.3 ± 7.7 %. Fourteen standard amino acids showed significant increases, and most fatty acids had carbon chain lengths between C12-C22. Mechanistically, oxygen perturbations activated FNR and ArcA regulons, resulting in lower relative abundances of TCA cycle enzymes and higher abundances of amino acid and fatty acid biosynthetic enzymes. These findings demonstrate that controlled oxygen fluctuations in wastewater treatment can enhance the biochemical value of activated sludge with minimal process modifications, facilitating resource recovery.

IL-7 promotes integrated glucose and amino acid sensing during homeostatic CD4+ Tcell proliferation.

Interleukin (IL)-7 promotes Tcell expansion during lymphopenia. We studied the metabolic basis in CD4+ Tcells, observing increased glucose usage for nucleotide synthesis and oxidation in the tricarboxylic acid (TCA) cycle. Unlike other TCA metabolites, glucose-derived citrate does not accumulate upon IL-7 exposure, indicating diversion into other processes. In agreement, IL-7 promotes glucose-dependent histone acetylation and chromatin accessibility, notable at the loci of the amino acid-sensing Ragulator complex. Consistently, the expression of its subunit late endosomal/lysosomal adaptor, MAPK and mTOR activator 5 (LAMTOR5) is promoted by IL-7 in a glucose-dependent manner, and glucose availability determines amino acid-dependent mechanistic target of rapamycin (mTOR) activation, confirming integrated nutrient sensing. LAMTOR5 deletion impairs IL-7-mediated Tcell expansion, establishing that glycolysis in the absence of Ragulator activation is insufficient to support this. Clinically, CD4+ Tcells from stem cell transplant recipients demonstrate coordinated upregulation of glycolytic and TCA cycle enzymes, amino acid-sensing machinery, and mTOR targets, highlighting the potential to therapeutically target this pathway to fine-tune lymphopenia-induced Tcell proliferation.

Vitamin C Ameliorates Potassium Dichromate-Induced Oxidative Stress and Mitochondrial Dysfunction via PGC-1α/Nrf-2/TFAM Pathway.

Exposure to potassium dichromate (K2Cr2O7) is well known for its nephrotoxic effects on humans and animals. This study investigated the protective effects of vitamin C against K2Cr2O7-induced nephrotoxicity, focusing on its impact on altered carbohydrate metabolism, mitochondrial dysfunction, and associated molecular mechanisms in the cortical and medullary kidney segments. Male Wistar rats (n = 8) were divided into four groups: Group I received saline, Group II received a single 250 mg/kg body weight (bwt) intraperitoneal (i.p.) injection of vitamin C, Group III received K2Cr2O7 (15 mg/kg bwt, i.p.), and Group IV received vitamin C 6 h before K2Cr2O7 administration. Vitamin C significantly mitigated K2Cr2O7-induced nephrotoxic effects, restoring normal renal function and histological architecture. It preserved the activities of glycolytic and gluconeogenic enzymes altered by K2Cr2O7. Additionally, vitamin C mitigated K2Cr2O7-induced mitochondrial dysfunction by maintaining tricarboxylic acid (TCA) cycle enzymes, electron transport chain proteins, mitochondrial DNA copy number, and ATP content. It also reduced oxidative stress markers and enhanced antioxidant enzyme activity. The protective mechanism of vitamin C against K2Cr2O7-induced renal damage involved upregulation of the protein expression of peroxisome proliferation-activated receptor-γ coactivator-1α (PGC-1α), which further elevated the protein expression of nuclear factor erythroid 2-related factor-2 (Nrf-2) and transcription factor A, mitochondrial (TFAM), crucial for protecting cells from oxidative stress, enhancing mitochondrial function, and promoting cellular health. Overall, this study highlights the significant protective role of vitamin C against K2Cr2O7-induced renal damage by preserving carbohydrate metabolism and mitigating mitochondrial dysfunction through the PGC-1α/Nrf-2/TFAM pathway, offering valuable insights into its protective mechanisms in nephrotoxicity.

The influence of degradation processes on enzymatic activity and the content of forms of mineral nitrogen in agricultural soils

The purpose of the work was to study the concentration of various forms of mineral nitrogen and nitrogen cycle enzymes in farmland soils. The objectives of the research included studying the content of ammonium and nitrate forms of nitrogen, as well as the activity of urease and nitrate reductase in soils of farmland on the territory of the Donetsk People's Republic (DPR). To study the soils of agricultural lands, model sites located in the southern part of the Shakhtersky district of the DPR were selected and represented by moderately washed-out chernozems with low, low and medium humus. The control was an area with steppe vegetation (ordinary chernozem, thick, with medium humus content). The results of the researches allow us to draw conclusions about the development of degradation processes in a number of model sites and their impact on the content of ammonium and nitrate nitrogen in agricultural soils. Growing crops removing substantial amount of mineral nutrients, the slope of the sites surface, and the duration of cultivation of grain crops in the same sites lead to a significant decrease in the concentration of ammonium and nitrate nitrogen. Thus, the most significant decrease in the concentration of nitrogen of ammonium compounds (by 77–82%) was recorded in model slope areas under maize and sunflower. Analysis of the data showed that exchange ammonium is confined to the underlying genetic horizons. The minimum concentration of nitrates in the arable horizon was recorded under the joint influence of such unfavorable factors as: unreasonable layout of fields along the relief elements; violation of crop rotation conditions; use of precursor crops with a significant level of removal of nutrients (wheat, maize). When studying the activity of urease, it was found that a low number of soil microorganisms associated with the cultivation of maize and sunflower, as well as with the development of degradation processes of various genesis, led to the suppression of the functioning of the urease of soils in model areas. The maximum urease activity was observed in areas under winter wheat and amounted to 68–72% in relation to the control indicators. As a result of studies of nitrate reductase activity, a decrease in its values was also found in areas used to grow crops that form a large phytomass (maize and sunflower).

An erythroid-specific lentiviral vector improves anemia and iron metabolism in a new model of XLSA.

X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in ALAS2, a gene responsible for heme synthesis. Treatments are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients. The absence of a suitable animal model has hindered the development of gene therapy research for this disease. We engineered a conditional Alas2-KO mouse model using tamoxifen administration or treatment with lipid nanoparticles (LNP) carrying Cre-mRNA and conjugated to an anti-CD117 antibody. Alas2-KOBM animals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells (RBC), hemoglobin (Hb), and hematocrit (HCT). In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, characterized by reduced oxidative phosphorylation, mitochondria's function, and activity of key Tricarboxylic Acid (TCA) cycle enzymes. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. The IE was associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the liver, spleen, and bone marrow and the formation of ring sideroblasts. To investigate the potential of a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) to direct ALAS2 expression in erythroid cells. Infusion of BM cells with 0.6-1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice improved CBC levels, tissue iron accumulation, and survival rates. These findings suggest our vector could be curative in XLSA patients.

Folate prevents the autism-related phenotype caused by developmental pyrethroid exposure in prairie voles.

Neurodevelopmental disorders (NDDs) have dramatically increased in prevalence to an alarming one in six children, and yet both causes and preventions remain elusive. Recent human epidemiology and animal studies have implicated developmental exposure to pyrethroid pesticides, one of the most common classes of pesticides in the US, as an environmental risk factor for autism and neurodevelopmental disorders. Our previous research has shown that low-dose chronic developmental pyrethroid exposure (DPE) changes folate metabolites in the adult mouse brain. We hypothesize that DPE acts directly on molecular targets in the folate metabolism pathway, and that high-dose maternal folate supplementation can prevent or reduce the biobehavioral effects of DPE. We exposed pregnant prairie vole dams chronically to vehicle or low-dose deltamethrin (3 mg/kg/3 days) with or without high-dose folate supplementation (methylfolate, 5 mg/kg/3 days). The resulting DPE offspring showed broad deficits in five behavioral domains relevant to neurodevelopmental disorders (including the social domain); increased plasma folate concentrations; and increased neural expression of SHMT1, a folate cycle enzyme. Maternal folate supplementation prevented most of the behavioral phenotypes (except for repetitive behaviors) and caused potentially compensatory changes in neural expression of FOLR1 and MTHFR, two folate-related proteins. We conclude that DPE causes neurodevelopmental disorder-relevant behavioral deficits; DPE directly alters aspects of folate metabolism; and preventative interventions targeting folate metabolism are effective in reducing, but not eliminating, the behavioral effects of DPE.

Possibilities of Predicting Methotrexate-associated Toxicity in Oncohematology Based on Molecular Genetic Testing Methods

The development of highly effective protocols for the treatment of acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphomas (NHL) followed the path of escalation of doses of cytostatic agents and improvement of supportive care. Methotrexate (MTX), used in high doses (1000–5000 mg/m2), radically changed the results of treatment of ALL and NHL in children, increasing patient survival rates. The downside of the anti-tumor effect of MTX is its organ toxicity, and therefore the development of methods for predicting the development of toxic effects of MTX is an important scientific and practical task. In recent years, the genetic factors of the patient’s organism have been considered as one of the reasons for the individual variability of pharmacokinetic and pharmacodynamic parameters of MTX. Abnormal function of folate cycle enzymes, methotrexate transporter proteins, due to gene polymorphism, may affect the effectiveness and toxicity of the drug. This review summarizes and analyzes the known genetic polymorphisms involved in MTX metabolism. The possibilities of predicting toxicity, as well as the prospects for individualizing therapy, taking into account the results of pharmacogenetic testing, are presented.

Modulation of Urea Transport Attenuates TLR2-Mediated Microglial Activation and Upregulates Microglial Metabolism In Vitro.

Background: Alzheimer's disease (AD) is a neurodegenerative disorder traditionally characterised by the presence of amyloid beta (Aβ) plaques and neurofibrillary tau tangles in the brain. However, emerging research has highlighted additional metabolic hallmarks of AD pathology. These include the metabolic reprogramming of microglia in favour of glycolysis over oxidative phosphorylation. This shift is attributed to an 'M1'-like pro-inflammatory phenotype, which exacerbates neuroinflammation and contributes to neuronal damage. The urea cycle also presents as an altered metabolic pathway in AD, due to elevated urea levels and altered expression of urea cycle enzymes, metabolites, and transporters in the brain. However, to date, these changes remain largely unexplored. Methods: This study focuses on understanding the effects of extracellular urea and urea transporter-B (UT-B) inhibition on inflammatory changes in lipoteichoic acid (LTA)-stimulated BV2 microglia and on the viability of SH-SY5Y neuronal cells under oxidative stress and neurotoxic conditions. Results: In BV2 microglia, UT-B inhibition demonstrated a notable anti-inflammatory effect by reducing the formation of nitric oxide (NO) and the expression of tumour necrosis factor α (TNFα) and CCL2 in response to stimulation with the toll-like receptor (TLR)2 agonist, lipoteichoic acid (LTA). This was accompanied by a reduction in extracellular urea and upregulation of UT-B expression. The application of exogenous urea was also shown to mediate the inflammatory profile of BV2 cells in a similar manner but had only a modest impact on UT-B expression. While exposure to LTA alone did not alter the microglial metabolic profile, inhibition of UT-B upregulated the expression of genes associated with both glycolysis and fatty acid oxidation. Conversely, neither increased extracellular urea nor UT-B inhibition had a significant impact on cell viability or cytotoxicity in SH-SY5Y neurones exposed to oxidative stressors tert-butyl hydroperoxide (t-BHP) and 6-hydroxydopamine (6-OHDA). Conclusions: This study further highlights the involvement of urea transport in regulating the neuroinflammation associated with AD. Moreover, we reveal a novel role for UT-B in maintaining microglial metabolic homeostasis. Taken together, these findings contribute supporting evidence to the regulation of UT-B as a therapeutic target for intervention into neuroinflammatory and neurodegenerative disease.

Soil microbiological assessment on diversified annual cropping systems in China

Recent studies have demonstrated that monocropping of flue-cured tobacco can lead to various issues, including nutrient deficiencies, accumulation of allelopathic substances, and disturbance in soil microbial flora. While diversification in cropping systems has proven effective in alleviating monocropping barriers, however, further exploration is needed to understand the potential microbial mechanisms involved in this process. In our study, we set five cropping systems (RR: rice monocropping; TR: tobacco-rice rotation over 20 years; TRA: tobacco-rice-astragalus rotation; TRW: tobacco-rice-wheat rotation; TRO: tobacco-rice-oilseed rape rotation) to explore the impact on crop yield and quality, soil chemical properties, and microbial diversity. The results showed that the yield and gross margin were significantly decreased. Following diversification in cropping systems, particularly after implementing the TRA treatment, the yield and gross margin increased by 27.35% and 38.67%, respectively, compared to the TR treatment. Additionally, the presence of tobacco in the soil resulted in acidification, reduced soil fertility, and suppression of soil microorganism diversity and metabolite abundance. With diversification in cropping systems, there was an increase in soil pH, carbon and nitrogen cycle enzyme activities, and the relative abundance of beneficial microorganisms (acidobacteria, nitrospirillum, and ascomycota) and soil metabolites. Diversification in cropping systems has the potential to increase crop biomass, soil fertility, and soil microbial environment. Our results suggest a scientific foundation for implementing effective nutrient management practices and rational crop rotation systems.

Aerobic and anaerobic poise of white swimming muscles of the deep-diving scalloped hammerhead shark: comparison to sympatric coastal and deep-water species

Scalloped hammerhead sharks (Sphyrna lewini) routinely perform rapid dives to forage on mesopelagic prey. These deep dives consist of intensive swimming followed by recovery periods in the surface mixed layer. Swimming muscle temperature profiles suggest that S. lewini suppresses gill function as a means to reduce convective heat loss during dives into cool water. Such intensive swimming behavior coupled with reduced respiration prompted us to test whether the aerobic and anaerobic metabolic capacities of the white swimming muscle tissue of this species are greater than those of other shark species from the same region. The activities of key enzymes used in aerobic and anaerobic metabolism provide an indirect indicator of the metabolic potential (“poise”) of a tissue. Here we measured the maximal activities [international units (µmol substrate converted to product per min, U) per gram of wet tissue mass at 10°C] of the citric acid cycle enzymes citrate synthase (CS) and malate dehydrogenase (MDH) and glycolytic enzymes pyruvate kinase (PK) and lactate dehydrogenase (LDH) from white swimming muscle of S. lewini. Enzyme activities, and ratios of these enzyme activities that indicate relative indexes of aerobic to anaerobic capacity, were compared to those measured in three sympatric coastal carcharhinid sharks and two deep-dwelling species, Echinorhinus cookei and Hexanchus griseus. This is the first report of swimming-muscle enzyme activity for these deep-dwelling species. In comparison to the other species, S. lewini had significantly higher activities of both LDH and MDH in the white muscle, and a higher MDH/CS ratio. The high LDH activities suggest that the white muscle of S. lewini relies on relatively high rates of anaerobic ATP production, with would result in build up of high lactate levels, during deep foraging dives. High MDH activity in S. lewini white muscle suggests the potential for lactate levels to be rapidly reduced when aerobic conditions are restored while in the surface mixed layer between dives. These biochemical characteristics may enable S. lewini to swim rapidly while suppressing gill function during deep dives and thereby exploit a very different ecological niche from sympatric shark species (e.g., coastal carcharhinids) and hunt more rapidly via faster swimming for deep-water prey compared to species that permanently inhabit deep depths.

EXTH-37. ALLOSTERIC MITOCHONDRIAL CLPP AGONIST ONC206 ALTERS STRESS RESPONSE, METABOLIC AND EPIGENETIC PROFILES TO ELICIT ANTI-CANCER EFFICACY IN HIGH-GRADE GLIOMAS

Abstract Dordaviprone (ONC201) is a dopamine receptor D2/3 (DRD2/3) antagonist and allosteric ClpP agonist that has demonstrated durable responses in recurrent H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 with nanomolar potency that also targets DRD2/3 and ClpP, is in Phase I trials for central nervous system tumors. We characterized target relevance, human ClpP binding, in vitro efficacy, downstream signaling and response determinants associated with ONC206 in high-grade gliomas (HGG). Co-crystallization with ClpP that assembles as a tetradecameric complex revealed an allosteric ligand interaction with distinctions in the ONC206-ClpP overall conformation relative to the ONC201-bound or apo complex, including an increase in the resolved pore size and decreased complex height. ONC206 was more potent than ONC201 in cell-free human ClpP casein/peptide degradation assays). Accordingly, HGG cell lines and patient-derived cells exhibited increased sensitivity to ONC206 relative to ONC201 and generally required a shorter duration of exposure. Unlike DRD2, CRISPR-mediated ClpP knockout in HGG cell lines blunted ONC206 sensitivity. Metabolomics revealed elevated α-ketoglutaric acid, α-hydroxyglutaric acid, and glutaric acid. Proteomics indicated inhibition of multiple mitochondrial pathways including OXPHOS and TCA cycle. TCA cycle enzyme α-ketoglutarate dehydrogenase (KGDH) was inhibited in a ClpP-dependent manner. Western blotting showed downregulated ClpX, and upregulated ATF4/CHOP, H3 K27me3 and H3 K36me3. Next-generation sequencing of acquired resistance cell lines, which exhibited cross-resistance to ONC201 and ONC206, revealed diverse ClpP missense mutations distributed across various monomer interfaces. Both intrinsic and well as ONC206-stimulated ClpP proteolysis were mitigated by these mutations, which was congruent with an absence of the tetradecameric assembly. These ClpP mutations recapitulated resistance and, conversely, wildtype ClpP overexpression restored sensitivity. Thus, allosteric ClpP agonism is a key determinant of ONC206 activity in HGG by altering stress response, metabolic, and epigenetic profiles.

Effects of deltamethrin on photosynthetic pigments and ascorbate-glutathione (ASA-GSH) cycle in Lemna minor

Deltamethrin is a synthetic pyrethroid insecticide that can cause adverse effects on non-target organisms. This study was designed to investigate the effects of different concentrations (0.001, 0.005 and 0.01 ppm) of deltamethrin on photosynthetic pigments and the ascorbate-glutathione (ASA-GSH) cycle in Lemna minor, a freshwater macrophyte. To assess the effect of deltamethrin on L. minor, photosynthetic pigments, malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels, and the activities of some antioxidant enzymes (SOD, CAT and POD) and enzymatic and non-enzymatic antioxidants associated with the ASA-GSH cycle were measured. The results showed that exposure to deltamethrin decreased chl a, chl b and carotenoid levels and increased MDA and H2O2 levels. In addition, deltamethrin exposure significantly increased SOD, CAT and POD activities. The activities of ASA-GSH cycle enzymes (APX, GR, GPX, MDHAR and DHAR) decreased in L. minor exposed to 0.01 ppm deltamethrin, while GST activity increased. Exposure to low doses of deltamethrin increased ASA and GSH levels, while 0.01 ppm deltamethrin decreased the amounts of ASA and GSH compared to the control. Taken together, the present study revealed that different concentrations of deltamethrin inhibited photosynthetic activity, increased lipid peroxidation and caused oxidative stress and activated the antioxidant defense system of L. minor to eliminate the increased oxidative stress.

Responses of glyoxalase system, ascorbate-glutathione cycle, and antioxidant enzymes in Pontederia cordata to lead stress and its capacity to remove lead

A hydroponic experiment was conducted to investigate the variations in membrane permeabilities, chlorophyll contents, antioxidase activities, the ascorbic acid (AsA)-glutathione (GSH) cycle, and the glyoxalase system in the leaves of Pontederia cordata with 0 ∼ 15.0 mg L−1 lead ion (Pb2+) exposure. The concentrations of Pb2+ accumulated in the plant roots, stems, and leaves were also evaluated. After 7 days of exposure, the plants maintained normal growth, and there was a significant increase in ascorbate peroxidase and dehydroascorbate reductase activities. With 5.0 mg L−1 Pb2+ exposure for 28 days, nearly 66.36% of Pb2+ accumulated in the roots, while excess Pb2+ immobilized in the leaves was not observed. Exposure to 10.0 and 15.0 mg L−1 Pb2+ for 28 days significantly increased Pb2+ contents in the leaves. This led to decrease in chlorophyll a, b, and carotenoid contents, and to increase in the methylglyoxal content in the leaves. With 10 and 15 mg L−1 Pb2+ exposure, NPT and PCs contents in leaves increased. however, the glyoxalase system did not function well in the plant tolerant to Pb2+ at higher concentrations. The AsA-GSH cycle did not cooperate with the glyoxalase system in the plant defense against Pb2+ exposure in the present investigation.

Cephalic ganglia transcriptomics of the American cockroach Periplaneta americana (Blattodea: Blattidae).

The American cockroach Periplaneta americana (L.) (Blattodea, Blattidae) has been a model organism for biochemical and physiological study for almost a century, however, its use does not benefit from the genetic tools found in key model species such as Drosophila melanogaster. To facilitate the use of the cockroach as a model system in neuroscience and to serve as a foundation for functional and translational experimentation, a transcriptome of the cephalic ganglia was assembled and annotated, and differential expression profiles between these ganglia were assessed. The transcriptome assembly yielded >400 k transcripts, with >40 k putative coding sequences. Gene ontology and protein domain searches indicate the cerebral and gnathal ganglia (GNG) have distinct genetic expression profiles. The developmental Toll signaling pathway appears to be active in the adult central nervous system (CNS), which may suggest a separate role for this pathway besides innate immune activation or embryonic development. The catabolic glycolytic and citric acid cycle enzymes are well represented in both ganglia, but key enzymes are more highly expressed in the GNG. Both ganglia express gluconeogenic and trehaloneogenic enzymes, suggesting a larger role of the CNS in regulating hemolymph sugar homeostasis than previously appreciated. The annotation and quantification of the cephalic ganglia transcriptome reveal both canonical and novel pathways in signaling and metabolism in an adult insect and lay a foundation for future functional and genetic analysis.

Evidence for interaction of 5,10-methylenetetrahydrofolate reductase (MTHFR) with methylenetetrahydrofolate dehydrogenase (MTHFD1) and general control nonderepressible 1 (GCN1)

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a folate cycle enzyme required for the intracellular synthesis of methionine. MTHFR was previously shown to be partially phosphorylated at 16 residues, which was abrogated by conversion of threonine 34 to alanine (T34A) or truncation of the first 37 amino acids (i.e. expression of amino acids 38–656), and promoted by methionine supplementation. Here, we over-expressed wild-type MTHFR (MTFHRWT), as well as the variants MTHFRT34A and MTHFR38-656 in 293T cells to provide further insights into these mechanisms. We demonstrate that following incubation in high methionine conditions (100–1000 μM) MTHFRWT is almost completely phosphorylated, but in methionine restricted conditions (0–10 μM) phosphorylation is reduced, while MTHFRT34A always remains unphosphorylated. Following affinity purification coupled mass spectrometry of an empty vector, MTHFRWT, MTHFRT34A and MTHFR38-656 in three separate experiments, we identified 134 proteins consistently pulled-down by all three MTHFR protein variants, of which 5 were indicated to be likely true interactors (SAINT prediction threshold of 0.95 and 2 fold-change). Amongst these were the folate cycle enzyme methylenetetrahydrofolate dehydrogenase (MTHFD1) and the amino acid starvation sensor general control nonderepressible 1 (GCN1). Immunoprecipitation-immunoblotting of MTHFRWT replicated interaction with both proteins. An AlphaFold 3 generated model of the MTHFR-MTHFD1 interaction places the MTHFD1 dehydrogenase/cyclohydrolase domain in direct contact with the MTHFR catalytic domain, suggesting their interaction may facilitate direct delivery of methylenetetrahydrofolate. Overall, we confirm methionine availability increases MTHFR phosphorylation, and identified potential interaction of MTHFR with MTHFD1 and GCN1.

Antitrypanosomal Potential of Chalcone‐Based Ursolic Acid Derivatives via Ligand‐Based Virtual Screening, DMPK Analyses, Molecular Dynamics Simulation, and MM/GBSA Binding Energy

AbstractChagas disease, caused by the protozoan parasite Trypanosoma cruzi and transmitted mainly by triatomine insects, represents a significant challenge to public health, especially in impoverished regions. Current treatments, such as benznidazole and nifurtimox, have limitations, including serious side effects and reduced efficacy in the chronic phase. This work aims to evaluate the antitrypanosomal activity of ursolic acid‐derived chalcones (UACD) using a ligand‐based virtual screening approach. To this end, a series of independent molecular docking simulations were carried out (via AutoDock Vina code) with the parasite proliferation cycle enzymes TcGAPDH and cruzain. The most favorable candidates underwent drug metabolism and pharmacokinetics (DMPK) analyses and molecular dynamics (MD) simulations to estimate the pharmacokinetic and pharmacodynamic profile. Molecular docking simulations showed that the UACD derivatives showed better specificity for the TcGAPDH enzyme, emphasizing the ursolic acid and UACD3 derivatives (affinity energy = −9.4 kcal/mol for each). The DMPK prediction showed that the derivatives present viable apparent permeability (Papp) that promotes an excellent absorbed fraction (in 10⁻⁶ cm/s). MD simulations showed that the UACD3 derivative showed better free energy when binding to the TcGAPDH enzyme (−13.27 ± 1.87 kcal/mol) and interacting with the active site residues Cys166 and Thr167. However, both ligands appear to be alternative therapies in treating Chagas disease.