Search Results (65)

Background: Including Chronic Myeloid Leukemia (CML) patients with deep molecular responses (MR4.5) and those with suboptimal responses provides valuable insights into treatment-associated metabolic changes. This study aimed to characterize the metabolomic alterations associated with CML and identify potential biomarkers for treatment response, particularly in patients achieving a deeper molecular response versus those with poorer responses. Methods: Plasma samples were collected from 51 chronic-phase CML patients and 24 healthy controls. CML patients were classified into two groups based on molecular responses: T1 (BCR-ABL1 IS ≤ 0.0032%) and T2 (BCR-ABL1 IS > 0.0032%, <1%). Metabolomic profiling was conducted using quadrupole time-of-flight liquid chromatography/mass spectrometry. The data analysis involved a partial least squares discriminant analysis, variable importance in projection (VIP) scores, and a pathway enrichment analysis. Significant metabolites were identified. Results: The PLS-DA revealed distinct metabolomic profiles between CML patients and healthy controls as well as between the T1 and T2 groups. Key differentiating metabolites with VIP scores > 1.5 included glutamate, hypoxanthine, and D-galactonic acid. In the T2 group, significant increases in malate and 5-aminoimidazole-4-carboxamide ribonucleotide were observed, reflecting disruptions in purine metabolism, the tricarboxylic acid cycle, and amino acid metabolism. The pathway enrichment analysis highlighted significant alterations in CML energy metabolism, nucleotide synthesis, and amino acid biosynthesis. Conclusions: CML patients exhibit pronounced metabolic changes, particularly in energy and nucleotide metabolism, which are linked to treatment response. These findings provide novel insights into CML biology and suggest potential biomarkers for monitoring treatment efficacy and predicting outcomes and therapeutic targets for improving treatment outcomes and overcoming tyrosine kinase inhibitor resistance. Full article

Methotrexate (MTX) is the conventional synthetic disease-modifying anti-rheumatic drug (csDMARD) recommended as the first-choice anti-rheumatic drug for rheumatoid arthritis (RA). However, responses to MTX may be influenced by genetic variants. We aim to evaluate the association of the rs2372536, rs4673990, and rs4673993 genetic variants of the ATIC gene with therapeutic failure of MTX in patients with RA. A case–control study was performed. Disease activity was measured using the disease activity score based on erythrocyte sedimentation rate (DAS28-ESR). RA patients were classified into two groups: (a) responders (DAS28-ESR ≤ 3.2), which is the group of patients who did respond to methotrexate, and (b) non-responders (DAS28-ESR > 3.2), which is the group of patients who did not respond to methotrexate. Serum levels of the 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) enzyme and Interleukin-6 (IL-6) were quantified using an enzyme-linked immunosorbent assay (ELISA). Genotyping of ATIC genetic variants was performed with quantitative polymerase chain reaction (qPCR) using TaqMan probes. A total of 260 patients with RA were included. In total, 142 (54.6%) were non-responders to MTX. IL-6 levels were increased in the non-responder group (p = 0.002), while no statistical differences were observed in the AICAR levels. The variables associated with non-response were higher HAQ-Di, weekly MTX dose, glucocorticoid use, erythrocyte sedimentation rate, and carriers of the polymorphic homozygous variant of rs4673993 (OR = 4.5, 95% CI: 1.04–19.34; p = 0.04). The use of sulfazaline offered protective effects. Our findings indicate that the polymorphism rs4673993 gene variant of the AICAR protein may significantly influence MTX resistance. Therefore, these results support the importance of the pathway generating extracellular adenosine and its effects on promoting the immune regulation for the mechanism of MTX therapy of RA. Full article

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in both Type 1 (T1D) and Type 2 (T2D). While there are no specific medications to prevent or treat DPN, certain strategies can help halt its progression. In T1D, maintaining tight glycemic control through insulin therapy can effectively prevent or delay the onset of DPN. However, in T2D, overall glucose control may only have a moderate impact on DPN, although exercise is clearly beneficial. Unfortunately, optimal exercise may not be feasible for many patients with DPN because of neuropathic foot pain and poor balance. Exercise has several favorable effects on health parameters, including body weight, glycemic control, lipid profile, and blood pressure. We investigated the impact of an exercise mimetic, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), on DPN. AICAR treatment prevented or reversed experimental DPN in mouse models of both T2D and T1D. AICAR in high-fat diet (HFD-fed) mice increased the phosphorylation of AMPK in DRG neuronal extracts, and the ratio of phosphorylated AMPK to total AMPK increased by 3-fold (HFD vs. HFD+AICAR; p < 0.001). Phospho AMP increased the levels of dynamin-related protein 1 (DRP1, a mitochondrial fission marker), increased phosphorylated autophagy activating kinase 1 (ULK1) at Serine-555, and increased microtubule-associated protein light chain 3-II (LC3-II, a marker for autophagosome assembly) by 2-fold. Mitochondria isolated from DRG neurons of HFD-fed had a decrease in ADP-stimulated state 3 respiration (120 ± 20 nmol O₂/min in HFD vs. 220 ± 20 nmol O₂/min in control diet (CD); p < 0.001. Mitochondria isolated from HFD+AICAR-treated mice had increased state 3 respiration (240 ± 30 nmol O₂/min in HFD+AICAR). However, AICAR’s protection in DPN in T2D mice was also mediated by its effects on insulin sensitivity, glucose metabolism, and lipid metabolism. Drugs that enhance AMPK phosphorylation may be beneficial in the treatment of DPN. Full article

The impact of aging on body composition and glucose metabolism is not well established in C57BL/6J mice, despite being a common pre-clinical model for aging and metabolic research. The purpose of this study was to examine the effect of advancing age on body composition, in vivo glucose metabolism, and skeletal muscle AKT expression in young (Y: 4 months old, n = 7), old (O: 17–18 months old, n = 10), and very old (VO: 26–27 month old, n = 9) male C57BL/6J mice. Body composition analysis, assessed by nuclear magnetic resonance, demonstrated O mice had a significantly greater fat mass and body fat percentage when compared to Y and VO mice. Furthermore, VO mice had a significantly greater lean body mass than both O and Y mice. We also found that the VO mice had greater AKT protein levels in skeletal muscle compared to O mice, an observation that explains a portion of the increased lean body mass in VO mice. During glucose tolerance (GT) testing, blood glucose values were significantly lower in the VO mice when compared to the Y and O mice. No age-related differences were observed in insulin tolerance (IT). We also assessed the glucose response to AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). The change in blood glucose following AICAR administration was significantly reduced in VO mice compared to Y and AG mice. Our findings indicate that lean body mass and AKT2 protein expression in muscle are significantly increased in VO mice compared to O mice. The increase in AKT2 likely plays a role in the greater lean body mass observed in the oldest of old mice. Finally, despite the increased GT, VO mice appear to be resistant to AMPK-mediated glucose uptake. Full article

As a result of screening a panel of marine organisms to identify lead molecules for the stimulation of endochondral bone formation, the calcareous sponge Pericharax heteroraphis was identified to exhibit significant activity during endochondral differentiation. On further molecular networking analysis, dereplication and chemical fractionation yielded the known clathridine A-related metabolites 3–6 and the homodimeric complex (clathridine A)₂ Zn²⁺ (9), together with the new unstable heterodimeric complex (clathridine A–clathridimine)Zn²⁺ (10). With the presence of the zinc complexes annotated through the LC-MS analysis of the crude extract changing due to the instability of some metabolites and complexes constituting the mixture, we combined the isolation of the predicted molecules with their synthesis in order to confirm their structure and to understand their reactivity. Interestingly, we also found a large quantity of the contaminant benzotriazoles BTZ (7) and its semi-dimer (BTZ)₂CH₂ (8), which are known to form complexes with transition metals and are used for preventing corrosion in water. All isolated 2-aminoimidazole derivatives and complexes were synthesized not only for structural confirmation and chemical understanding but to further study their bioactivity during endochondral differentiation, particularly the positively screened imidazolone derivatives. Compounds leucettamine B, clathridine A and clathridimine were found to increase type X collagen transcription and stimulate endochondral ossification in the ATDC5 micromass model. Full article

In 2019, five million deaths associated with antimicrobial resistance were reported by The Centers for Disease Control and Prevention (CDC). Acinetobacter baumannii, a Gram-negative bacterial pathogen, is among the list of urgent threats. Previously, we reported 2-aminoimidazole (2-AI) adjuvants that potentiate macrolide activity against A. baumannii. In this study, we identify several of these adjuvants that sensitize A. baumannii to aminoglycoside antibiotics. Lead compounds 1 and 7 lower the tobramycin (TOB) minimum inhibitory concentration (MIC) against the TOB-resistant strain AB5075 from 128 μg/mL to 2 μg/mL at 30 μM. In addition, the lead compounds lower the TOB MIC against the TOB-susceptible strain AB19606 from 4 μg/mL to 1 μg/mL and 0.5 μg/mL, respectively, at 30 μM and 15 μM. The evolution of resistance to TOB and 1 in AB5075 revealed mutations in genes related to protein synthesis, the survival of bacteria under environmental stressors, bacteriophages, and proteins containing Ig-like domains. Full article

Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco’s Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium. Full article

Although dry eye disease (DED) is one of the most common ocular surface diseases worldwide, its pathogenesis is incompletely understood, and treatment options are limited. There is growing evidence that complex interactions between the ocular surface microbiome (OSM) and tear fluid constituents, potentially leading to inflammatory processes, are associated with ocular surface diseases such as DED. In this study, we aimed to find unique compositional and functional features of the OSM associated with human and microbial tear proteins in patients with DED. Applying whole-metagenome shotgun sequencing of forty lid and conjunctival swabs, we identified 229 taxa, with Actinobacteria and Proteobacteria being the most abundant phyla and Propionibacterium acnes the dominating species in the cohort. When DED patients were compared to controls, the species Corynebacterium tuberculostearicum was more abundant in conjunctival samples, whereas the family Propionibacteriaceae was more abundant in lid samples. Functional analysis showed that genes of L-lysine biosynthesis, tetrapyrrole biosynthesis, 5-aminoimidazole ribonucleotide biosynthesis, and the super pathway of L-threonine biosynthesis were enriched in conjunctival samples of controls. The relative abundances of Acinetobacter johnsonii correlated with seven human tear proteins, including mucin-16. The three most abundant microbial tear proteins were the chaperone protein DnaK, the arsenical resistance protein ArsH, and helicase. Compositional and functional features of the OSM and the tear proteome are altered in patients with DED. Ultimately, this may help to design novel interventional therapeutics to target DED. Full article

Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N¹⁰-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5′-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function. Full article

A new 2-amino imidazole derivative, 5-(4-chlorophenyl)-N,1-di-o-tolyl-1H-imidazole-2-amine (3), has been synthesized using a green approach. The reaction was conducted in a ChCl (cholinium chloride)/urea eutectic mixture, which is a nature-inspired and environmentally friendly reaction medium. The proposed reaction mechanism involves the preliminary regioselective alkylation of the N_sp2 of guanidine (2), followed by an intramolecular condensation between the carbonyl moiety and the secondary N′_sp3. Finally, a tautomerization/aromatization step furnished the final product (3). Notably, 2-amino imidazole (3) could be isolated in high yield (91%), just by filtration from the DES/water mixture and subsequent crystallization; the remaining ChCl/urea could be recycled, after water removal, for four consecutive reactions without any significant drop in the (3) yield. The product has been fully characterized by ¹H, ¹³C, 2D ¹H-¹³C HSQC, and 2D ¹H-¹³C HMBC NMR; FT-IR spectroscopy; and EI-MS spectrometry. Full article

Femur head necrosis (FHN), also known as bacterial chondronecrosis with osteomyelitis (BCO), has remained an animal welfare and production concern for modern broilers regardless of efforts to select against it in primary breeder flocks. Characterized by the bacterial infection of weak bone, FHN has been found in birds without clinical lameness and remains only detectable via necropsy. This presents an opportunity to utilize untargeted metabolomics to elucidate potential non-invasive biomarkers and key causative pathways involved in FHN pathology. The current study used ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC–HRMS) and identified a total of 152 metabolites. Mean intensity differences at p < 0.05 were found in 44 metabolites, with 3 significantly down-regulated and 41 up-regulated in FHN-affected bone. Multivariate analysis and a partial least squares discriminant analysis (PLS-DA) scores plot showed the distinct clustering of metabolite profiles from FHN-affected vs. normal bone. Biologically related molecular networks were predicted using an ingenuity pathway analysis (IPA) knowledge base. Using a fold-change cut off of −1.5 and 1.5, top canonical pathways, networks, diseases, molecular functions, and upstream regulators were generated using the 44 differentially abundant metabolites. The results showed the metabolites NAD+, NADP+, and NADH to be downregulated, while 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and histamine were significantly increased in FHN. Ascorbate recycling and purine nucleotides degradation were the top canonical pathways, indicating the potential dysregulation of redox homeostasis and osteogenesis. Lipid metabolism and cellular growth and proliferation were some of the top molecular functions predicted based on the metabolite profile in FHN-affected bone. Network analysis showed significant overlap across metabolites and predicted upstream and downstream complexes, including AMP-activated protein kinase (AMPK), insulin, collagen type IV, mitochondrial complex, c-Jun N-terminal kinase (Jnk), extracellular signal-regulated kinase (ERK), and 3β-hydroxysteroid dehydrogenase (3β HSD). The qPCR analysis of relevant factors showed a significant decrease in AMPKα2 mRNA expression in FHN-affected bone, supporting the predicted downregulation found in the IPA network analysis. Taken as a whole, these results demonstrate a shift in energy production, bone homeostasis, and bone cell differentiation that is distinct in FHN-affected bone, with implications for how metabolites drive the pathology of FHN. Full article

The plant mirid bug Apolygus lucorum is an omnivorous pest that can cause considerable economic damage. The steroid hormone 20-hydroxyecdysone (20E) is mainly responsible for molting and metamorphosis. The adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor regulated by 20E, and its activity is regulated allosterically through phosphorylation. It is unknown whether the 20E-regulated insect’s molting and gene expression depends on the AMPK phosphorylation. Herein, we cloned the full-length cDNA of the AlAMPK gene in A. lucorum. AlAMPK mRNA was detected at all developmental stages, whereas the dominant expression was in the midgut and, to a lesser extent, in the epidermis and fat body. Treatment with 20E and AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AlCAR) or only AlCAR resulted in activation of AlAMPK phosphorylation levels in the fat body, probed with an antibody directed against AMPK phosphorylated at Thr172, enhancing AlAMPK expression, whereas no phosphorylation occurred with compound C. Compared to compound C, 20E and/or AlCAR increased the molting rate, the fifth instar nymphal weight and shortened the development time of A. lucorum in vitro by inducing the expression of EcR-A, EcR-B, USP, and E75-A. Similarly, the knockdown of AlAMPK by RNAi reduced the molting rate of nymphs, the weight of fifth-instar nymphs and blocked the developmental time and the expression of 20E-related genes. Moreover, as observed by TEM, the thickness of the epidermis of the mirid was significantly increased in 20E and/or AlCAR treatments, molting spaces began to form between the cuticle and epidermal cells, and the molting progress of the mirid was significantly improved. These composite data indicated that AlAMPK, as a phosphorylated form in the 20E pathway, plays an important role in hormonal signaling and, in short, regulating insect molting and metamorphosis by switching its phosphorylation status. Full article

5-Amino-1-β-D-ribofuranosylimidazole-4-carboxamide 5′-monophosphate (ZMP) is a central intermediate in de novo purine nucleotide biosynthesis. Its nucleobase moiety, 5-aminoimidazole-4-carboxamide (Z-base), is considered an ambiguous base that can pair with any canonical base owing to the rotatable nature of its 5-carboxamide group. This idea of ambiguous base pairing due to free rotation of the carboxamide has been applied to designing mutagenic antiviral nucleosides, such as ribavirin and T-705. However, the ambiguous base-pairing ability of Z-base has not been elucidated, because the synthesis of Z-base-containing oligomers is problematic. Herein, we propose a practical method for the synthesis of Z-base-containing DNA oligomers based on the ring-opening reaction of an N¹-dinitrophenylhypoxanthine (Hxa^DNP) base. Thermal denaturation studies of the resulting oligomers revealed that the Z-base behaves physiologically as an A-like nucleobase, preferentially forming pairs with T. We tested the behavior of Z-base-containing DNA oligomers in enzyme-catalyzed reactions: in single nucleotide insertion, Klenow fragment DNA polymerase recognized Z-base as an A-like analog and incorporated dTTP as a complementary nucleotide to Z-base in the DNA template; in PCR amplification, Taq DNA polymerase similarly incorporated dTTP as a complementary nucleotide to Z-base. Our findings will contribute to the development of new mutagenic antiviral nucleoside analogs. Full article

Textiles are important components for the development of lightweight and flexible displays useful in smart materials. In particular, halochromic textiles are fibrous materials with a color-changing ability triggered by pH variations mainly based on pH-sensitive dye molecules. Recently, a novel class of 2-aminoimidazole azo dyes was developed with distinct substituent patterns. In this work, silk fabric was functionalized through exhaustion for the first time with one of these dyes (AzoIz.Pip). The halochromic properties of the dye were assessed in an aqueous solution and after silk functionalization. The solutions and the fabrics were thoroughly analyzed by ultraviolet-visible (UV-vis) spectra, color strength (K/S), color difference (∆E), CIE L*a*b* coordinates, and the ultraviolet protection factor (UPF). The dyeing process was optimized, and the halochromic performance (and reversibility) was assessed in universal Britton–Robinson buffers (ranging from pH 3 to 12) and artificial body fluids (acid and alkaline perspiration, and wound exudate). AzoIz.Pip showed vibrant colors and attractive halochromic properties with a hypsochromic shift from blue (557 nm) to magenta (536 nm) in aqueous buffered solutions. Similarly, the functionalized silk showed a shift in wavelength of the maximum K/S value from 590 nm to 560 nm when pH increases. The silk fabric showed a high affinity to AzoIz.Pip, and promoted additional color stabilization of the dye, avoiding color loss as observed when the dye is in solution at alkaline pH after 24 h. The color reversibility was effective up to the fourth cycle and the fastness tests denoted suitable results, except washing fastness. The cytotoxicity of the silk fabric extracts was assessed, depicting reduced viability of HaCaT cells to <70% only when the dye concentration in the fabric is higher or equal to 64 μg·mL⁻¹. Nevertheless, lower concentrations were also very effective for the halochromic performance in silk. These materials can thus be a helpful tool for developing sensors in several sectors such as biomedicine, packaging, filtration, agriculture, protective apparel, sports, camouflage, architecture, and design. Full article

Non-alcoholic fatty liver disease (NAFLD) is a global health problem characterized by altered lipid and redox homeostasis, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. The AMP-dependent kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been shown to improve the outcome of NAFLD in the context of AMPK activation, yet the underlying molecular mechanism remains obscure. This study investigated the potential mechanism(s) of AICAR to attenuate NAFLD by exploring AICAR’s effects on the HGF/NF-κB/SNARK axis and downstream effectors as well as mitochondrial and ER derangements. High-fat diet (HFD)-fed male Wistar rats were given intraperitoneal AICAR at 0.7 mg/g body weight or left untreated for 8 weeks. In vitro steatosis was also examined. ELISA, Western blotting, immunohistochemistry and RT-PCR were used to explore AICAR’s effects. NAFLD was confirmed by steatosis score, dyslipidemia, altered glycemic, and redox status. HGF/NF-κB/SNARK was downregulated in HFD-fed rats receiving AICAR with improved hepatic steatosis and reduced inflammatory cytokines and oxidative stress. Aside from AMPK dominance, AICAR improved hepatic fatty acid oxidation and alleviated the ER stress response. In addition, it restored mitochondrial homeostasis by modulating Sirtuin 2 and mitochondrial quality gene expression. Our results provide a new mechanistic insight into the prophylactic role of AICAR in the prevention of NAFLD and its complications. Full article