Search Results (37)

Background: Amiodarone is a widely used class III antiarrhythmic agent, but its use can lead to peripheral neuropathy mediated by mitochondrial dysfunction, oxidative stress, and neuroinflammatory injury, while effective preventive options remain limited. Agents that support mitochondrial energy metabolism, sustain redox balance, and modulate inflammation, including adenosine triphosphate (ATP), melatonin, and thiamine pyrophosphate (TPP), may counteract these mechanisms; however, their relative neuroprotective potential in amiodarone-induced neuropathy remains unclear. This study aimed to comparatively evaluate the effects of ATP, melatonin, and TPP on amiodarone-induced peripheral neuropathy and neuropathic pain in rats. Methods: Thirty male albino Wistar rats were assigned to five groups: healthy; amiodarone (50 mg/kg/orally); amiodarone + ATP (5 mg/kg/intraperitoneally); amiodarone + melatonin (10 mg/kg/orally); or amiodarone + TPP (20 mg/kg/intraperitoneally). Treatments were given once daily for 14 days. Oxidative stress indices (malondialdehyde (MDA), total glutathione (tGSH), superoxide dismutase (SOD), catalase (CAT)) and proinflammatory cytokines (tumor necrosis factor-alpha (TNF-α), interleukin-1 Beta (IL-1β), interleukin-6 (IL-6)) were quantified in sciatic nerve by Enzyme-Linked Immunosorbent Assay (ELISA). Paw withdrawal thresholds were measured with the Randall-Selitto test before and after treatment. Histopathology was performed using Hematoxylin-eosin staining. Results: Amiodarone exposure resulted in pronounced elevations in MDA and proinflammatory cytokine levels, accompanied by significant reductions in tGSH, SOD, CAT activities, and paw withdrawal thresholds. ATP, melatonin and TPP ameliorated these alterations to varying degrees. Among them, TPP provided the most robust antioxidant and anti-inflammatory effects, followed by ATP and melatonin. Histopathological examination confirmed most severe axonal degeneration, interstitial edema and Schwann cell proliferation in the amiodarone group, with substantial amelioration in the TPP-treated rats. Conclusions: Amiodarone induces neuropathic pain through oxidative and inflammatory injury to peripheral nerves. TPP exhibited superior neuroprotective efficacy compared with ATP and melatonin, highlighting its potential as a candidate therapeutic agent for amiodarone-related neuropathy. Further clinical research is warranted to support translational application of these findings. Full article

Background: Asarum heterotropoides, a prominent medicinal plant in China, is well known for producing an abundance of monoterpenes and sesquiterpenes, which constitute the primary components of its essential oil and serve as the principal active compounds of the species. However, the biosynthetic pathways for these terpenoids remain largely unelucidated. Methods: Gas chromatography–mass spectrometry analysis, in vitro enzyme assay, subcellular localization experiment and molecular docking were used to characterize the function of terpene synthase from A. heterotropoides. Results: In this study, we isolated and characterized two monoterpene synthases and one sesquiterpene synthase from A. heterotropoides. These enzymes exhibit promiscuous activities, accepting geranyl pyrophosphate and farnesyl pyrophosphate as substrates to yield a variety of monoterpene and sesquiterpene products in in vitro enzymatic assays. All three enzymes possess a conserved RRx8W motif, a hallmark typically associated with TPS-b and TPS-d monoterpene synthases involved in cyclic monoterpene formation. However, these two monoterpene synthases yield linear instead of cyclic products. The sesquiterpene synthase (AhTPS3) is a second example of TPS-a terpene synthase harboring such motif. Conclusions: Our findings significantly expand our understanding of terpene biosynthesis, especially the role of RRx8W motif. Full article

Taraxacum kok-saghyz Rodin, an important rubber-producing plant, has emerged as a potential alternative crop for the natural rubber industry. Geranyl diphosphate synthase (GDS) catalyzes the condensation of dimethylallyl pyrophosphate and isopentenyl pyrophosphate into geranyl pyrophosphate in the mevalonate pathway in plants. However, its specific functions in natural rubber biosynthesis in T. kok-saghyz remain unclear. Methods: We conducted genome-wide analyses of TkGDS genes, followed by transient transformation assay, expression profiling, natural rubber quantification, and analysis of T. kok-saghyz photosynthesis. Results: Seven TkGDS genes are located on chromosomes A6 and A7 with an uneven distribution. All encoded TkGDS proteins contain FARM and SARM motifs. TkGDS1, TkGDS2, and TkGDS7 possess lspA domains, while TkGDS3, TkGDS4, TkGDS5, and TkGDS6 contain PLN02890 domains; both subgroups share similar domain architecture. TkGDS1, TkGDS2, and TkGDS7 exhibit interspecies collinearity with Arabidopsis thaliana; no intraspecies collinearity was detected. The putative cis-acting elements in promoter region of TkGDS genes mainly comprised abscisic acid responsiveness, anaerobic induction, light responsiveness, and MeJA responsiveness. Transient expression assays confirmed chloroplast localization of all TkGDS proteins. A strong positive correlation was observed between TkGDS1/TkGDS7 expression and natural rubber content, as confirmed by both transcriptome and qPCR analyses in T. kok-saghyz lines. Furthermore, overexpression of TkGDS1 and TkGDS7 improved photosynthetic efficiency and significantly increased natural rubber content (OE-TkGDS1: 6.08 ± 0.16%; OE-TkGDS7: 5.62 ± 0.32%; WT: 4.76 ± 0.28%). Conclusions: Our study elucidates the role of GDS1 and GDS7 in promoting growth and latex content, offering a genetic strategy for enhancing rubber accumulation in T. kok-saghyz. Full article

Uncaria rhynchophylla, a medicinal plant extensively used in traditional Chinese medicine, is an important plant source of terpenoid indole alkaloids (TIAs), but the mechanism of TIA biosynthesis at molecular level remains unclear. Geraniol synthase (GES) serves as a crucial enzyme in catalyzing the formation of geraniol from geranyl pyrophosphate (GPP) in various plants, but the functional characterization of the GES gene in U. rhynchophylla has not been investigated. In this study, a GES was identified and characterized through genome mining and bioinformatic analysis. Functional validation was performed via a protein catalysis experiment, transient expression in Nicotiana benthamiana, and methyl jasmonate (MeJA) induction experiments. The full-length UrGES gene was 1761 bp, encoding a protein product of 586 amino acids with an estimated 67.5 kDa molecular weight. Multiple sequence alignments and phylogenetic analysis placed UrGES within the terpene synthase g (TPS-g) subfamily, showing high similarity to known GESs from other plants. Enzymatic assays confirmed that recombinant UrGES catalyzed GPP conversion to a single product of geraniol. The transient expression of UrGES resulted in geraniol accumulation in N. benthamiana, further confirming its function in vivo. UrGES expression was observed in leaves, stems, and roots, where leaves had the highest transcript levels. Moreover, MeJA treatment significantly upregulated UrGES expression, which positively correlated with an increase in alkaloid content. This study functionally characterizes UrGES as a geraniol synthase in U. rhynchophylla, contributing to the current knowledge of the TIA biosynthetic pathway. These findings may offer insights for future metabolic engineering aiming to enhance TIA yields for pharmaceutical and industrial applications. Full article

Toxicological studies of pesticides in animal models provide critical insights into their mechanisms of action, while adsorption strategies offer potential solutions for decontaminating polluted waters. We evaluated toxicity induced by tetraethyl pyrophosphate (TEPP), an organophosphate pesticide and AChE inhibitor, on zebrafish (Danio rerio) development and behavior, alongside the efficacy of wine cork granules as a natural adsorbent. TEPP exposure reduced embryo viability following an inverted U-shaped dose–response curve, suggesting non-monotonic neurodevelopmental effects, but did not alter developmental timing or morphology in survivors. In juveniles, TEPP increased preference for dark environments (33% vs. controls) and enhanced swimming endurance approximately 3-fold, indicating disrupted phototaxis and stress responses. Most strikingly, water treated with cork granules retained toxicity, increasing mortality, delaying embryogenesis, and altering behavior. This directly contradicts in vitro adsorption studies that suggested cork’s efficacy. These results demonstrate the high sensitivity of zebrafish to TEPP at nanomolar concentrations, which contrasts with in vitro models that require doses approximately 1000 times higher. Our findings not only highlight TEPP’s ecological risks but also reveal unexpected limitations of cork granules for environmental remediation, urging caution in their application. Full article

The COVID-19 pandemic has highlighted the urgent need for rapid and accurate diagnostic methods for various infectious diseases, including SARS-CoV-2. Traditional RT-PCR methods, while highly sensitive and specific, require complex equipment and skilled personnel. In response, we developed an integrated RT-LAMP-MS assay, which combines rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) with microscanning (MS) technology for detecting SARS-CoV-2. The assay uses magnesium pyrophosphate formed during LAMP amplification as a visual marker, allowing direct observation via microscopy without the need for additional chemical indicators or probes. For the SARS-CoV-2/IC RT-LAMP-MS assay, the sample-LAMP reagent mixture was added to a microchip with SARS-CoV-2 primers and internal controls, then incubated at 62 °C for 30 min in a heat block, followed by amplification analysis using a microscanner. In clinical tests, the RT-LAMP-MS assay showed 99% sensitivity and 100% specificity, which is identical to the RT-LAMP results and comparable to the commercial Allplex^TM SARS-CoV-2 assay results. Additionally, the limit of detection (LOD) was determined to be 10⁻¹ PFU mL⁻¹ (dynamic range: 10³~10⁻¹ PFU mL⁻¹). The assay delivers results in 30 min, uses low-cost equipment, and demonstrates 100% reproducibility in repeated tests, making it suitable for point-of-care use in resource-limited settings. Full article

Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE is only based on a complex multi-organ phenotypic score and/or genetical analysis. Reduced plasma inorganic pyrophosphate concentration [PPi]p has been linked to PXE. In this study, we used a novel and accurate method to measure [PPi]p in one of the largest cohorts of PXE patients, and we reported the valuable contribution of a cutoff value to PXE diagnosis. Plasma samples and clinical records from two French reference centers for PXE (PXE Consultation Center, Angers, and FAVA-MULTI South Competent Center, Nice) were assessed. Plasma PPi were measured in 153 PXE and 46 non-PXE patients. The PPi concentrations in the plasma samples were determined by a new method combining enzymatic and ion chromatography approaches. The best match between the sensitivity and specificity (Youden index) for diagnosing PXE was determined by ROC analysis. [PPi]p were lower in PXE patients (0.92 ± 0.30 µmol/L) than in non-PXE patients (1.61 ± 0.33 µmol/L, p < 0.0001), corresponding to a mean reduction of 43 ± 19% (SD). The PPi cutoff value for diagnosing PXE in all patients was 1.2 µmol/L, with a sensitivity of 83.3% and a specificity of 91.1% (AUC = 0.93), without sex differences. In patients aged <50 years (i.e., the age period for PXE diagnosis), the cutoff PPi was 1.2 µmol/L (sensitivity, specificity, and AUC of 93%, 96%, and 0.97, respectively). The [PPi]p shows high accuracy for diagnosing PXE; thus, quantifying plasma PPi represents the first blood assay for diagnosing PXE. Full article

This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 3′ RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and β-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future. Full article

Two siblings showed increased galactose and galactose-related metabolites in neonatal screening. Diagnostic workup did not reveal abnormalities in any of the known disease-causing enzymes involved in galactose metabolism. Using whole-exome sequencing, we identified a homozygous missense variant in PPA1 encoding the cytosolic pyrophosphatase 1 (PPA1), c.557C>T (p.Thr186Ile). The enzyme activity of PPA1 was determined using a colorimetric assay, and the protein content was visualized via western blotting in skin fibroblasts from one of the affected individuals. The galactolytic activity of the affected fibroblasts was determined by measuring extracellular acidification with a Seahorse XFe96 analyzer. PPA1 activity decreased to 22% of that of controls in the cytosolic fraction of homogenates from patient fibroblasts. PPA1 protein content decreased by 50% according to western blot analysis, indicating a reduced stability of the variant protein. The extracellular acidification rate was reduced in patient fibroblasts when galactose was used as a substrate. Untargeted metabolomics of blood samples revealed an elevation of other metabolites related to pyrophosphate metabolism. Besides hyperbilirubinemia in the neonatal period in one child, both children were clinically unremarkable at the ages of 3 and 14 years, respectively. We hypothesize that the observed metabolic derangement is a possible mild manifestation of PPA1 deficiency. Unresolved abnormalities in galactosemia screening might result in the identification of more individuals with PPA1 deficiency, a newly discovered inborn metabolic disorder (IMD). Full article

Fosmidomycin (FOS) is a naturally occurring compound active against the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) enzyme in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and using it as a template for lead structure design is an effective strategy to develop new active compounds. In this work, by replacing the hydroxamate unit of FOS with pyrazole, isoxazole and the related heterocycles that also have metal ion binding affinity, while retaining the monophosphonic acid in FOS or replacing it with a bisphosphonic acid group, heterocycle-containing mono- and bisphosphonic acid compounds as FOS analogs were designed. The key steps involved in the facile synthesis of these FOS analogs included the Michael addition of diethyl vinylphosphonate or tetraethyl vinylidenebisphosphonate to β-dicarbonyl compounds and the subsequent cyclic condensation with hydrazine or hydroxylamine. Two additional isoxazolinone-bearing FOS analogs were synthesized via the Michaelis–Becker reaction with diethyl phosphite as a key step. The bioactivity evaluation on model plants demonstrated that several compounds have better herbicidal activities compared to FOS, with the most active compound showing a 3.7-fold inhibitory activity on Arabidopsis thaliana, while on the roots and stalks of Brassica napus L. and Echinochloa crus-galli in a pre-emergence inhibitory activity test, the activities of this compound were found to be 3.2- and 14.3-fold and 5.4- and 9.4-fold, respectively, and in a post-emergency activity test on Amaranthus retroflexus and Echinochloa crus-galli, 2.2- and 2.0-fold inhibition activities were displayed. Despite the significant herbicidal activity, this compound exhibited a DXR inhibitory activity lower than that of FOS but comparable to that of other non-hydroxamate DXR inhibitors, and the dimethylallyl pyrophosphate rescue assay gave no statistical significance, suggesting that a different target might be involved in the inhibiting process. This work demonstrates that using bioisosteric replacement can be considered as a valuable strategy to discover new FOS analogs that may have high herbicidal activities. Full article

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage calcification, loss of articular cartilage, bone changes, pain, and disability. Cartilage calcification is one hallmark of OA and is predominantly caused by basic calcium crystals formed due to an imbalance of the pyrophosphate pathway. Sortilin is a transmembrane protein that contributes to vascular calcification in atherosclerosis by externalizing alkaline phosphatase (ALP)-containing vesicles. Calcification in atherosclerosis and osteoarthritis has been associated with cellular senescence. The aim of this study was to investigate the potential role of sortilin and senescence in osteoarthritis-dependent cartilage calcification. Osteoarthritic cartilage from human knee joints was collected after joint replacement, and samples were analyzed by immunohistochemistry and quantitative RT-PCR analysis. Human chondrocytes were treated with osteogenic medium for up to 21 days to induce calcification. Western blots for sortilin and ALP, as well as an ALP activity assay, were performed. Human chondrocytes were treated with mitomycin C to induce senescence, and sortilin expression was quantified at the protein and gene levels. Sections of knee joints from a murine model of osteoarthritis were stained for sortilin and p16 and analyzed by immunohistochemistry. Treatment of wild-type chondrocytes using an osteogenic medium similar to human chondrocytes was performed. Osteoarthritic cartilage from mouse and human knee joints showed an increased number of sortilin and p16-positive chondrocytes compared to healthy cartilage. This observation was corroborated by increased gene expression of sortilin and p16 in mild and moderate osteoarthritic cartilage samples. To investigate the mechanism of sortilin regulation, human chondrocytes were treated with osteogenic medium to induce calcification. Sortilin protein levels and expression were increased after 7 days of stimulation, whereas ALP levels and activity were upregulated after 21 days of stimulation. Similar observations were made in a murine osteoarthritis model. Mechanistically, senescent chondrocytes induced by mitomycin C showed an upregulation of sortilin and ALP gene expression compared to non-senescent chondrocytes. Our data indicate that sortilin and ALP are upregulated during cartilage calcification, which is associated with chondrocyte senescence and thus might contribute to the pathogenesis of osteoarthritis. Cellular senescence seems to induce sortilin expression. Full article

The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can be used to identify compounds interacting with the enzymes of isoprenoid biosynthesis, an important part of the ergosterol biosynthesis pathway. The method was validated according to the EMEA guideline on bioanalytical method validation. Aspergillus fumigatus hyphae and Saccharomyces cerevisiae cells were lysed mechanically in an aqueous buffer optimized for the enzymatic deconjugation of isoprenoid pyrophosphates. The residual alcohols were extracted, silylated and analyzed by GC-MS. The obtained isoprenoid pattern provides an indication of the inhibited enzyme, due to the accumulation of specific substrates. By analyzing terbinafine-treated A. fumigatus and mutant strains containing tunable gene copies of erg9 or erg1, respectively, the method was verified. Downregulation of erg9 resulted in a high accumulation of intracellular farnesol as well as elevated levels of geranylgeraniol and isoprenol. The decreased expression of erg1 as well as terbinafine treatment led to an increased squalene content. Additional analysis of growth medium revealed high farnesyl pyrophosphate levels extruded during erg9 downregulation. Full article

Obesity and nonalcoholic fatty liver disease (NAFLD) are global health concerns, and thus, drugs for the long-term treatment of these diseases are urgently needed. We previously discovered that the inositol pyrophosphate biosynthetic enzyme IP6K1 is a target in diet-induced obesity (DIO), insulin resistance, and NAFLD. Moreover, high-throughput screening (HTS) assays and structure−activity relationship (SAR) studies identified LI-2242 as a potent IP6K inhibitor compound. Here, we tested the efficacy of LI-2242 in DIO WT C57/BL6J mice. LI-2242 (20 mg/kg/BW daily, i.p.) reduced body weight in DIO mice by specifically reducing the accumulation of body fat. It also improved glycemic parameters and reduced hyperinsulinemia. LI-2242-treated mice displayed reduced the weight of various adipose tissue depots and an increased expression of metabolism- and mitochondrial-energy-oxidation-inducing genes in these tissues. LI-2242 also ameliorated hepatic steatosis by reducing the expression of genes that enhance lipid uptake, lipid stabilization, and lipogenesis. Furthermore, LI-2242 enhances the mitochondrial oxygen consumption rate (OCR) and insulin signaling in adipocytes and hepatocytes in vitro. In conclusion, the pharmacologic inhibition of the inositol pyrophosphate pathway by LI-2242 has therapeutic potential in obesity and NAFLD. Full article

Magnesium (Mg) has been explored during the last few decades in the biomedical industry as a biodegradable implant. However, mechanical properties and corrosion resistance are still big concerns for clinical use. Therefore, this study proposes a suitable surface modification of the Mg by plasma electrolytic oxidation (PEO) to improve its corrosion resistance and biological performance. Mg samples were processed in a galvanostatic mode using an electrolytic solution of a phosphate compound supplemented with either potassium pyrophosphate or sodium-potassium tartrate. The obtained coatings were physiochemically characterized by SEM, XRD, EDS, and micro-Raman spectroscopy. The corrosion resistance of the coatings was studied using a hydrogen evolution setup and electrochemical tests. Finally, the biological performance of the material was evaluated by using an indirect test with osteoblasts. Obtained coatings showed a porous morphology with thicknesses ranging from 2 to 3 µm, which was closely dependent on the PEO solution. The corrosion resistance tests improved the degradation rate compared to the raw material. Additionally, an unreported active–passive corrosion behavior was evidence of a protective layer of corrosion products underneath the anodic coating. Indirect in vitro cytotoxicity assays indicated that the coatings improved the biocompatibility of the material. In conclusion, it was found that the produced coatings from this study not only lead to material protection but also improve the biological performance of the material and ensure cell survival, indicating that this could be a potential material used for bone implants. Full article

In the present study, the chemical composition of the volatile oil and methanolic extract from Ducrosia flabellifolia Boiss. was investigated. The antimicrobial, antioxidant, and anticancer activities of the methanolic extract from D. flabellifolia aerial parts were screened using experimental and computational approaches. Results have reported the identification of decanal (28.31%) and dodecanal (16.93%) as major compounds in the essential oil obtained through hydrodistillation. Farnesyl pyrophosphate, Methyl 7-desoxypurpurogallin-7-carboxylate trimethyl ether, Dihydro-Obliquin, Gummiferol, 2-Phenylaminoadenosine, and 2,4,6,8,10-dodecapentaenal, on the other hand, were the dominant compounds in the methanolic extract. Moreover, the tested extract was active against a large collection of bacteria and yeast strains with diameter of growth inhibition ranging from 6.67 ± 0.57 mm to 17.00 ± 1.73 mm, with bacteriostatic and fungicidal activities against almost all tested microorganisms. In addition, D. flabellifolia methanolic extract was dominated by phenolic compounds (33.85 ± 1.63 mg of gallic acid equivalent per gram of extract) and was able to trap DPPH• and ABTS•+ radicals with IC₅₀ about 0.05 ± 0 mg/mL and 0.105 ± 0 mg/mL, respectively. The highest percentages of anticancer activity were recorded at 500 µg/mL for all cancer cell lines with IC₅₀ about 240. 56 µg/mL (A-549), 202.94 µg/mL (HCT-116), and 154.44 µg/mL (MCF-7). The in-silico approach showed that D. flabellifolia identified compounds bound 1HD2, 2XCT, 2QZW, and 3LN1 with high affinities, which together with molecular interactions and the bond network satisfactorily explain the experimental results using antimicrobial, antioxidant, and anticancer assays. The obtained results highlighted the ethnopharmacological properties of the rare desertic D. flabellifolia plant species growing wild in Hail region (Saudi Arabia). Full article