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1.
Res Vet Sci ; 172: 105249, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38579633

RESUMO

The effect of salinomycin sodium alone and in combination with functional oils on performance and microbiota of broiler infected Eimeria were evaluated. 512 broilers were randomly assigned to 4 treatments (8 replicates, 16 birds/pen): a Control group (any additives); Ionophore group: salinomycin supplementation at 66 ppm (SS66); Ionophore +0.075% Functional oil (FO) group (SS66 + FO supplementation at 750 ppm); and Ionophore +0.10% FO group (SS66 + FO supplementation at 1000 ppm). At 14 days of age, birds were gavaged with 1 mL of a saline solution containing sporulated oocysts of E. tenella, E. acervulina and E. maxima. Performance indices were measured weekly. At 28 days, intestinal content was collected for microbiota analysis. Broilers of Control group presented the worst performance indices. Broilers of Ionophore + FO (0.075% and 0.10%) groups exhibited a higher BW at 28 days of age. The supplementation of Ionophore +0.075% FO resulted in a higher relative proportion of Firmicutes and a lower proportion of Actinobacteria in the ileum-jejunum. Lactobacillaceae was the dominant family in the jejunal, and ileal microbiotas of broilers fed diets supplemented with Ionophore, Ionophore +0.075% FO and Ionophore +0.10% FO. The supplementation of ionophore yielded higher numbers of Lactobacillaceae, Enterobactereaceae and Cloritridiaceae in the cecal. Ionophore associated with FO controlled the Lactobacillaceae, Enterobactereaceae and Cloritridiaceae families present in the cecum. Therefore, the combination of salinomycin with functional oil showed synergistic effect on performance and modulation of intestinal microbiota of broilers challenged with Eimeria.


Assuntos
Ração Animal , Galinhas , Coccidiose , Dieta , Suplementos Nutricionais , Eimeria , Microbioma Gastrointestinal , Policetídeos de Poliéter , Doenças das Aves Domésticas , Piranos , Animais , Galinhas/crescimento & desenvolvimento , Piranos/farmacologia , Piranos/administração & dosagem , Coccidiose/veterinária , Coccidiose/tratamento farmacológico , Coccidiose/parasitologia , Microbioma Gastrointestinal/efeitos dos fármacos , Eimeria/efeitos dos fármacos , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/tratamento farmacológico , Ração Animal/análise , Dieta/veterinária , Distribuição Aleatória , Ionóforos/farmacologia , Ionóforos/administração & dosagem , Coccidiostáticos/farmacologia , Coccidiostáticos/administração & dosagem , Masculino
2.
Int J Mol Sci ; 25(8)2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38673887

RESUMO

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.


Assuntos
Antígenos CD28 , Complexo CD3 , Diferenciação Celular , Interferon gama , Ativação Linfocitária , Zinco , Humanos , Zinco/metabolismo , Zinco/farmacologia , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Diferenciação Celular/efeitos dos fármacos , Interferon gama/metabolismo , Ionóforos/farmacologia , Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Cálcio/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/efeitos dos fármacos
3.
J Agric Food Chem ; 72(18): 10640-10654, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38661066

RESUMO

Coronaviruses have consistently posed a major global concern in the field of livestock industry and public health. However, there is currently a lack of efficient drugs with broad-spectrum antiviral activity to address the challenges presented by emerging mutated strains or drug resistance. Additionally, the method for identifying multitarget drugs is also insufficient. Aminopeptidase N (APN) and 3C-like proteinase (3CLpro) represent promising targets for host-directed and virus-directed strategies, respectively, in the development of effective drugs against various coronaviruses. In this study, maduramycin ammonium demonstrated a broad-spectrum antiviral effect by targeting both of the proteins. The binding domains 4 Å from the ligand of both target proteins shared a structural similarity, suggesting that screening and designing drugs based on these domains might exhibit broad-spectrum and highly effective antiviral activity. Furthermore, it was identified that the polyether ionophores' ability to carry zinc ion might be one of the reasons why they were able to target APN and exhibit antiviral effect. The findings of this experiment provide novel perspectives for future drug screening and design, while also offering valuable references for the utilization of polyether ionophores in the management of livestock health.


Assuntos
Antivirais , Antígenos CD13 , Ionóforos , Gado , Animais , Antivirais/farmacologia , Antivirais/química , Ionóforos/farmacologia , Ionóforos/química , Antígenos CD13/metabolismo , Antígenos CD13/química , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/antagonistas & inibidores , Drogas Veterinárias/farmacologia , Drogas Veterinárias/química , Coronavirus/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Policetídeos de Poliéter
4.
ACS Chem Neurosci ; 15(9): 1755-1769, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38602894

RESUMO

Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu2+ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu2+ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu2+ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Cobre , Fator de Crescimento Neural , Peptídeos Cíclicos , Fator A de Crescimento do Endotélio Vascular , Células PC12 , Animais , Ratos , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cobre/metabolismo , Cobre/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ionóforos/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Receptor trkA/metabolismo
5.
Sci Adv ; 10(12): eadl4018, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38517966

RESUMO

In a phenotypical screen of 56 acute myeloid leukemia (AML) patient samples and using a library of 10,000 compounds, we identified a hit with increased sensitivity toward SF3B1-mutated and adverse risk AMLs. Through structure-activity relationship studies, this hit was optimized into a potent, specific, and nongenotoxic molecule called UM4118. We demonstrated that UM4118 acts as a copper ionophore that initiates a mitochondrial-based noncanonical form of cell death known as cuproptosis. CRISPR-Cas9 loss-of-function screen further revealed that iron-sulfur cluster (ISC) deficiency enhances copper-mediated cell death. Specifically, we found that loss of the mitochondrial ISC transporter ABCB7 is synthetic lethal to UM4118. ABCB7 is misspliced and down-regulated in SF3B1-mutated leukemia, creating a vulnerability to copper ionophores. Accordingly, ABCB7 overexpression partially rescued SF3B1-mutated cells to copper overload. Together, our work provides mechanistic insights that link ISC deficiency to cuproptosis, as exemplified by the high sensitivity of SF3B1-mutated AMLs. We thus propose SF3B1 mutations as a biomarker for future copper ionophore-based therapies.


Assuntos
Cobre , Leucemia Mieloide Aguda , Humanos , Cobre/metabolismo , Fatores de Processamento de RNA/genética , Mutação , Leucemia Mieloide Aguda/genética , Ionóforos/farmacologia , Fosfoproteínas/metabolismo
6.
Mol Biol Cell ; 35(5): ar70, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38536415

RESUMO

Lysosome turnover and biogenesis are induced in response to treatment of cells with agents that cause membrane rupture, but whether other stress conditions engage similar homeostatic mechanisms is not well understood. Recently we described a form of selective turnover of lysosomes that is induced by metabolic stress or by treatment of cells with ionophores or lysosomotropic agents, involving the formation of intraluminal vesicles within intact organelles through microautophagy. Selective turnover involves noncanonical autophagy and the lipidation of LC3 onto lysosomal membranes, as well as the autophagy gene-dependent formation of intraluminal vesicles. Here, we find a form of microautophagy induction that requires activity of the lipid kinase PIKfyve and is associated with the nuclear translocation of TFEB, a known mediator of lysosome biogenesis. We show that LC3 undergoes turnover during this process, and that PIKfyve is required for the formation of intraluminal vesicles and LC3 turnover, but not for LC3 lipidation onto lysosomal membranes, demonstrating that microautophagy is regulated by PIKfyve downstream of noncanonical autophagy. We further show that TFEB activation requires noncanonical autophagy but not PIKfyve, distinguishing the regulation of biogenesis from microautophagy occurring in response to agents that induce lysosomal stress.


Assuntos
Lisossomos , Microautofagia , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Membranas Intracelulares/metabolismo , Ionóforos , Lisossomos/metabolismo , Humanos , Linhagem Celular Tumoral
7.
Free Radic Biol Med ; 216: 33-45, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38479632

RESUMO

NADPH oxidase is a target of hyperglycemia in type 2 diabetes mellitus (T2DM), which causes dysregulation of enzyme. Alterations in regulation of NADPH oxidase activity mediated receptor and non-receptor signaling in bone marrow granulocytes of mice with obesity-induced T2DM were studied. The animals fed high fat diet (516 kcal/100 g) for 16 weeks. NADPH oxidase-related generation of reactive species (RS) at normo- and hyperthermia was estimated using chemiluminescent analysis. The redox status of the cells was assessed by Redox Sensor Red CC-1. Baseline biochemical indicators in blood (glucose, cholesterol, HDL and LDL levels) were significant higher in T2DM mice versus controls. Using specific inhibitors, signaling mediated by formyl peptide receptors (FPRs) to NADPH oxidase was shown to involve PLC, PKC, cytochrome p450 in both control and T2DM groups and PLA2 in controls. In T2DM regulation of NADPH oxidase activity via mFpr1, a high-affinity receptors, occurred with a significant increase of the role of PKC isoforms and suppression of PLA2 participation. Significant differences between this regulation via mFpr2, low-affinity receptors, were not found. Non-receptor activation of NADPH oxidase with ionomycin (Ca2+ ionophore) or phorbol ester (direct activator of PKC isoforms) did not revealed differences in the kinetic parameters between groups at 37 °C and 40 °C. When these agents were used together (synergistic effect), lower sensitivity of cells to ionophore was observed in T2DM at both temperatures. Redox status in responses to opsonized zymosan was higher in T2DM mice at 37 °C and similar to control levels at 40 °C. ROC-analysis identified Tmax, RS production and effect of opsonized zymosan as the most significant predictors for discriminating between groups. It was concluded that Ca2+-dependent/PKC-mediated regulation of NADPH oxidase activity was altered in BM granulocytes from diabetic mice.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Camundongos , Animais , Zimosan/farmacologia , Granulócitos , NADPH Oxidases/genética , Isoformas de Proteínas , Ionóforos/farmacologia , Fosfolipases A2 , Obesidade/complicações , Espécies Reativas de Oxigênio/farmacologia
8.
Biochem Pharmacol ; 222: 116092, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38408679

RESUMO

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is an antimicrobial agent whose actions as a zinc or copper ionophore and an iron chelator revived the interest in similar compounds for the treatment of fungal and bacterial infections, neurodegeneration and cancer. Recently, we reported zinc ionophores, including clioquinol, cause vasorelaxation in isolated arteries through mechanisms that involve sensory nerves, endothelium and vascular smooth muscle. Here, we report that clioquinol also uniquely acts as a competitive alpha-1 (α1) adrenoceptor antagonist. We employed ex vivo functional vascular contraction and pharmacological techniques in rat isolated mesenteric arteries, receptor binding assays using stabilized solubilized α1 receptor variants, or wild-type human α1-adrenoceptors transfected in COS-7 cells (African green monkey kidney fibroblast-like cells), and molecular dynamics homology modelling based on the recently published α1A adrenoceptor cryo-EM and α1B crystal structures. At higher concentrations, all ionophores including clioquinol cause a non-competitive antagonism of agonist-mediated contraction due to intracellular zinc delivery, as reported previously. However, at lower concentration ranges, clioquinol has an additional mechanism of competitively inhibiting α1-adrenoceptors that contributes to decreasing vascular contractility. Molecular dynamic simulation showed that clioquinol binds stably to the orthosteric binding site (Asp106) of the receptor, confirming the structural basis for competitive α1-adrenoceptor antagonism by clioquinol.


Assuntos
Clioquinol , Ratos , Humanos , Animais , Chlorocebus aethiops , Clioquinol/farmacologia , Oxiquinolina , Receptores Adrenérgicos alfa 1/metabolismo , Ionóforos , Zinco
9.
Methods Mol Biol ; 2772: 273-283, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38411821

RESUMO

Single-particle tracking (SPT) of biomolecules in the plant endoplasmic reticulum has the potential to inform on the formation of protein-protein complexes, metabolons, and the transport of molecules through both the ER membrane and lumen. Plant cells are particularly challenging for observing and tracking single molecules due to their unique structure, size, and considerable autofluorescence. However, by using variable-angle or highly inclined epifluorescence microscopy (VAEM) and transient expression in tobacco, it is possible to observe single-particle dynamics in the ER. Selecting the appropriate fluorophore, and ensuring the correct fluorophore density in the ER, is essential for successful SPT. By using tuneable fluorophores, which can be photoconverted and photoactivated, it is possible to vary the density of visible fluorophores in the ER dynamically. Here we describe methods to prepare plant samples for VAEM and two methods for determining and analyzing single-particle tracks from VAEM time series.


Assuntos
Microscopia , Imagem Individual de Molécula , Nicotiana , Retículo Endoplasmático , Corantes Fluorescentes , Ionóforos
10.
Chembiochem ; 25(7): e202400013, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38329925

RESUMO

Carboxylic polyether ionophores (CPIs) are among the most prevalent agricultural antibiotics (notably in the US) and these compounds have been in use for decades. The potential to reposition CPIs beyond veterinary use, e. g. through chemical modifications to enhance their selectivity window, is an exciting challenge and opportunity, considering their general resilience towards resistance development. Given the very large societal impact of these somewhat controversial compounds, it is surprising that many aspects of their mechanisms and activities in cells remain unclear. Here, we report comparative biological activities of the CPI routiennocin and two stereoisomers, including its enantiomer. We used an efficient convergent synthesis strategy to access the compounds and conducted a broad survey of antibacterial activities against planktonic cells and biofilms as well as the compounds' effects on mammalian cells, the latter assessed both via standard cell viability assays and broad morphological profiling. Interestingly, similar bioactivity of the enantiomeric pair was observed across all assays, strongly suggesting that chiral interactions do not play a decisive role in the mode of action. Overall, our findings are consistent with a mechanistic model involving highly dynamic behaviour of CPIs in biological membranes.


Assuntos
Antibacterianos , Policetídeos de Poliéter , Animais , Antibacterianos/farmacologia , Ionóforos/química , Mamíferos/metabolismo
11.
J Sep Sci ; 47(4): e2300761, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38403454

RESUMO

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.


Assuntos
Coccidiostáticos , Coccidiostáticos/análise , Cromatografia Líquida de Alta Pressão , Ionóforos/análise , Aminoácidos , Monensin/análise
12.
Parasitol Res ; 123(2): 139, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38381180

RESUMO

The flow of calcium ions (Ca2+) is involved in numerous vital activities of Toxoplasma gondii. Calreticulin is a type of Ca2+-binding protein in the endoplasmic reticulum (ER) that is involved in Ca2+ signaling pathway regulation, Ca2+ storage, and protein folding. In this work, the calreticulin (CALR), a protein predicted to possess a conserved domain of calreticulin in T. gondii, was characterized. The CALR localized in the ER. Using reverse genetics, we discovered that CALR is not necessary for the lytic cycle, including invasion and replication. However, depletion of CALR affected microneme secretion triggered by A23187, which is a Ca2+ ionophore used to increase cytoplasmic Ca2+ concentration. Furthermore, we discovered that CALR influences Ca2+ release. Transcriptomic comparison between Δcalr and Δku80 parasites showed that 226 genes in the Δcalr parasites were significantly downregulated (p < 0.05). The cellular biological functions of the downregulated genes were mainly involved in calmodulin-dependent protein kinase pathways. Furthermore, in the absence of CALR, tachyzoites were still able to cause acute infection in mice. These results imply that by influencing ER Ca2+ release content, CALR may further impair the ionophore-induced secretion of the parasite. However, this protein is not required for the completion of the parasite's lytic cycle or for the acute virulence of the parasite.


Assuntos
Toxoplasma , Animais , Camundongos , Toxoplasma/genética , Calreticulina/genética , Micronema , Retículo Endoplasmático , Ionóforos
13.
J Am Chem Soc ; 146(10): 6566-6579, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38422385

RESUMO

Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.


Assuntos
Corantes Fluorescentes , Humanos , Rodaminas , Microscopia de Fluorescência/métodos , Células HeLa , Ionóforos
14.
Chembiochem ; 25(5): e202300857, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38206088

RESUMO

As the research of biological systems becomes increasingly complex, there is a growing demand for fluorophores with a diverse range of wavelengths. In this study, we introduce phosphole-based fluorophores that surpass existing options like dansyl chloride. The reactive S-Cl bond in chlorosulfonylimino-5-phenylphosphole derivatives allows rapid and direct coupling to peptides making the fluorophores easily introducible to peptides. This coupling process occurs under mild conditions, demonstrated for [F7 ,P34 ]-NPY and its shorter analogues. Peptides linked with our fluorophores exhibit similar receptor activation to the control peptide, while maintaining high stability and low toxicity, making them ideal biolabeling reagents. In fluorescence microscopy experiments, they can be easily visualized even at low concentrations, without suffering from the typical issue of bleaching. These phosphole-based fluorophores represent a significant leap forward in the field. Their versatility, ease of modification, superior performance, and applicability in biological labeling make them a promising choice for researchers seeking advanced tools to unravel the details of complex biological systems.


Assuntos
Corantes Fluorescentes , Ácido Hipocloroso , Ionóforos , Microscopia de Fluorescência , Peptídeos
15.
Langmuir ; 40(6): 3004-3014, 2024 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-38294191

RESUMO

Unequivocally, Pb2+ as a harmful substance damaging children's brain and nerve systems, thereby causing behavior and learning disabilities, should be detected much lower than the elevated blood lead for children, 240 nM, endorsed by US CDC considering the unknown neurotoxic effects, yet the ultralow detection limit up to sub-ppb level remains a challenge due to the intrinsically insufficient sensitivity in the current analytical techniques. Here, we present nanoemulsion (NE)-integrated single-entity electrochemistry (NI-SEE) toward ultrasensitive sensing of blood lead using Pb-ion-selective ionophores inside a NE, i.e., Pb2+-selective NE. Through the high thermodynamic selectivity between Pb2+ and Pb-ionophore IV, and the extremely large partition coefficient for the Pb2+-Pb-ionophore complex inside NEs, we modulate the selectivity and sensitivity of NI-SEE for Pb2+ sensing up to an unprecedentedly low detection limit, 20 ppt in aqueous solutions, and lower limit of quantitation, 40 ppb in blood serums. This observation is supported by molecular dynamics simulations, which clearly corroborate intermolecular interactions, e.g., H-bonding and π*-n, between the aromatic rings of Pb-ionophore and lone pair electrons of oxygen in dioctyl sebacate (DOS), plasticizers of NEs, subsequently enhancing the current intensity in NI-SEE. Moreover, the highly sensitive sensing of Pb2+ is enabled by the appropriate suppression of hydroxyl radical formation during NI-SEE under a cathodic potential applied to a Pt electrode. Overall, the experimentally demonstrated NI-SEE approach and the results position our new sensing technology as potential sensors for practical environmental and biomedical applications as well as a platform to interrogate the stoichiometry of target ion-ionophore recognition inside a NE as nanoreactors.


Assuntos
Chumbo , Água , Criança , Humanos , Eletroquímica/métodos , Ionóforos/química , Eletrodos
16.
Sci Rep ; 14(1): 1802, 2024 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-38245618

RESUMO

Artemisinin combination therapy remains effective for the treatment of falciparum malaria. However, Plasmodium falciparum can escape the effects of artemisinin by arresting their growth. The growth-arrested parasites cannot be distinguished from nonviable parasites with standard microscopy techniques due to their morphological similarities. Here, we demonstrated the efficacy of a new laboratory assay that is compatible with the artemisinin susceptibility test. As a result of the differential cell permeabilities of two DNA-binding fluorophores, growth-arrested P. falciparum can be distinguished from parasites killed by artemisinin, since the latter lose cell membrane permeability. This fluorescence-based assay increased the sensitivity and specificity of the ring survival assay in the assessment of artemisinin susceptibility. When combined with a third fluorophore-conjugated anti-human leukocyte antibody, this trio fluorophore assay became more useful in identifying growth-arrested parasites in mock human blood samples. This novel assay is a simple and rapid technique for monitoring artemisinin resistance with greater sensitivity and accuracy compared with morphology-based observations under a light microscope.


Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Humanos , Plasmodium falciparum , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Eritrócitos/parasitologia , Ionóforos/farmacologia , Resistência a Medicamentos
17.
Proc Natl Acad Sci U S A ; 121(2): e2309579121, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38175865

RESUMO

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple nonhematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKα, p38, and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKα knockout, or pharmacologic inhibitors of ZAKα and p38 kinase activities. By surveying a panel of ionophores, we show that electroneutrality of ion movement is essential to activate ZAKα-driven RSR and a greater extent of K+ depletion is necessary to activate ZAKα-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux, and innate immunity.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nigericina/farmacologia , Potássio/metabolismo , Imunidade Inata , Ionóforos , Proteínas NLR
18.
Chem Biodivers ; 21(2): e202301834, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38179845

RESUMO

We discovered a new tetronomycin analog, C-32-OH tetronomycin (2) from the Streptomyces sp. K20-0247 strain, which produces tetronomycin (1). After NMR analysis of 2, we determined the planar structure. Futhermore, the absolute stereochemistry of 2 was deduced based on the biosynthetic pathway of 1 in the K20-0247 strain and a comparison of experimental electronic circular dichroism (ECD) results of 1 with 2. While 2 exihibits potent antibacterial activity aganist Gram-positive baceria including vancomycin-intermediate Staphylococcus aureus (VISA) strains and vancomycin-resistant Enterococci (VRE), the antibacterial activity of 2 shows 16-32-folds weaker than that of 1 suggesting that the C-34 methyl group in 1 is one of the very important functinal group. Moreover, we evaluated the ionophore activity of 1 and 2 and neither compound shows ionophore activity at reasonable concetrations. Our research suggests that 1 and 2 would have different target(s) from an ionophore mechanism in the antibacterial activity and tetronomycins are promising natural products for broad-spectrum antibiotics.


Assuntos
Antibacterianos , Éteres , Antibacterianos/farmacologia , Bactérias Gram-Positivas , Ionóforos , Testes de Sensibilidade Microbiana
19.
Sci Rep ; 14(1): 1921, 2024 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-38253556

RESUMO

Ten patients undergoing surgical resection for spinal tumors were selected. Samples of tumor, muscle, and bone were resected, de-identified by the treating surgeon, and then scanned with the TumorID technology ex vivo. This study investigates whether TumorID technology is able to differentiate three different human clinical fresh tissue specimens: spine tumor, normal muscle, and normal bone. The TumorID technology utilizes a 405 nm excitation laser to target endogenous fluorophores, thereby allowing for the detection of tissue based on emission spectra. Metabolic profiles of tumor and healthy tissue vary, namely NADH (bound and free emission peak, respectively: 487 nm, 501 nm) and FAD (emission peak: 544) are endogenous fluorophores with distinct concentrations in tumor and healthy tissue. Emission spectra analyzed consisted of 74 scans of spine tumor, 150 scans of healthy normal bone, and 111 scans of healthy normal muscle. An excitation wavelength of 405 nm was used to obtain emission spectra from tissue as previously described. Emission spectra consisted of approximately 1400 wavelength intensity pairs between 450 and 750 nm. Kruskal-Wallis tests were conducted comparing AUC distributions for each treatment group, α = 0.05. Spectral signatures varied amongst the three different tissue types. All pairwise comparisons among tissues for Free NADH were statistically significant (Tumor vs. Muscle: p = 0.0006, Tumor vs. Bone: p < 0.0001, Bone vs. Muscle: p = 0.0357). The overall comparison of tissues for FAD (506.5-581.5 nm) was also statistically significant (p < 0.0001), with two pairwise comparisons being statistically significant (Tumor vs. Muscle: p < 0.0001, Tumor vs. Bone: p = 0.0045, Bone vs. Muscle: p = 0.249). These statistically significant differences were maintained when stratifying tumor into metastatic carcinoma (N = 57) and meningioma (N = 17). TumorID differentiates tumor tissue from normal bone and normal muscle providing further clinical evidence of its efficacy as a tissue identification tool. Future studies should evaluate TumorID's ability to serve as an adjunctive tool for intraoperative assessment of surgical margins and surgical decision-making.


Assuntos
Neoplasias Meníngeas , NAD , Humanos , Espectrometria de Fluorescência , Músculos , Corantes Fluorescentes , Ionóforos , Lasers
20.
mBio ; 15(2): e0315523, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38214510

RESUMO

Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.


Assuntos
Monensin , Infecções Estafilocócicas , Humanos , Animais , Camundongos , Monensin/farmacologia , Virulência , Staphylococcus aureus , Proteômica , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/microbiologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Ionóforos/farmacologia , Ionóforos/metabolismo , Purinas
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