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1.
Proc Natl Acad Sci U S A ; 120(18): e2221097120, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37094155

RESUMO

Western dietary patterns have been unfavorably linked with mental health. However, the long-term effects of habitual fried food consumption on anxiety and depression and underlying mechanisms remain unclear. Our population-based study with 140,728 people revealed that frequent fried food consumption, especially fried potato consumption, is strongly associated with 12% and 7% higher risk of anxiety and depression, respectively. The associations were more pronounced among male and younger consumers. Consistently, long-term exposure to acrylamide, a representative food processing contaminant in fried products, exacerbates scototaxis and thigmotaxis, and further impairs exploration ability and sociality of adult zebrafish, showing anxiety- and depressive-like behaviors. Moreover, treatment with acrylamide significantly down-regulates the gene expression of tjp2a related to the permeability of blood-brain barrier. Multiomics analysis showed that chronic exposure to acrylamide induces cerebral lipid metabolism disturbance and neuroinflammation. PPAR signaling pathway mediates acrylamide-induced lipid metabolism disorder in the brain of zebrafish. Especially, chronic exposure to acrylamide dysregulates sphingolipid and phospholipid metabolism, which plays important roles in the development of anxiety and depression symptoms. In addition, acrylamide promotes lipid peroxidation and oxidation stress, which participate in cerebral neuroinflammation. Acrylamide dramatically increases the markers of lipid peroxidation, including (±)5-HETE, 11(S)-HETE, 5-oxoETE, and up-regulates the expression of proinflammatory lipid mediators such as (±)12-HETE and 14(S)-HDHA, indicating elevated cerebral inflammatory status after chronic exposure to acrylamide. Together, these results both epidemiologically and mechanistically provide strong evidence to unravel the mechanism of acrylamide-triggered anxiety and depression, and highlight the significance of reducing fried food consumption for mental health.


Assuntos
Metabolismo dos Lipídeos , Peixe-Zebra , Masculino , Animais , Depressão , Doenças Neuroinflamatórias , Acrilamida , Ansiedade , Contaminação de Alimentos/análise
2.
Trends Genet ; 38(3): 218-221, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34702578

RESUMO

Implementations and improvements of genome editing techniques used in plant science have increased exponentially. For some crops, such as potato, the use of transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) has moved to the next step of trait development and field trials, and should soon be applied to commercial cultivation.


Assuntos
Edição de Genes , Solanum tuberosum , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Produtos Agrícolas/genética , Edição de Genes/métodos , Genoma de Planta/genética , Solanum tuberosum/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética
3.
J Biol Chem ; 299(8): 105002, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37394003

RESUMO

Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)guanine (dfG), a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis polymerases (Polη, Polι, Polκ, and Polζ) and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-KO cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.


Assuntos
Adutos de DNA , Mutagênicos , Humanos , Acrilamidas , Desoxiguanosina , DNA , Dano ao DNA , Replicação do DNA , Mutagênese , Mutagênicos/toxicidade , Contaminação de Alimentos
4.
Small ; 20(22): e2308775, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38126895

RESUMO

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.


Assuntos
Fibrose Pulmonar , RNA Interferente Pequeno , Espermina , Espermina/química , Espermina/farmacologia , Humanos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Fibrose Pulmonar/terapia , Animais , Pulmão/patologia , Pulmão/metabolismo , Polímeros/química , Acrilamidas/química
5.
Toxicol Appl Pharmacol ; 489: 117011, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38906510

RESUMO

The critical developmental stages of the embryo are strongly influenced by the dietary composition of the mother. Acrylamide is a food contaminant that can form in carbohydrate-rich foods that are heat-treated. The aim of this study was to investigate the toxicity of a relatively low dose of acrylamide on the development of the neural tube in the early stage chick embryos. Specific pathogen-free fertilized eggs (n = 100) were treated with acrylamide (0.1, 0.5, 2.5, 12.5 mg/kg) between 28-30th hours of incubation and dissected at 48th hours. In addition to morphological and histopathological examinations, proliferating cell nuclear antigen (PCNA) and caspase 3 were analyzed immunohistochemically. The brain and reproductive expression gene (BRE) was analyzed by RT-PCR. Acrylamide exposure had a negative effect on neural tube status even at a very low dose (0.1 mg/kg) (p < 0.05). Doses of 0.5 mg/kg and above caused a delay in neural tube development (p < 0.05). Crown-rump length and somite count decreased dose-dependently, while this decrease was not significant in the very low dose group (p > 0.05), which was most pronounced at doses of 2.5 and 12.5 mg/kg (p < 0.001). Acrylamide exposure dose-dependently decreased PCNA and increased caspase 3, with this change being significant at doses of 0.5 mg/kg and above (p < 0.001). BRE was downregulated at all acrylamide exposures except in the very low dose group (0.1 mg/kg). In conclusion, we find that acrylamide exposure (at 0.5 mg/kg and above) in post-gastrulation delays neural tube closure in chicken embryos by suppressing proliferation and apoptosis induction and downregulating BRE gene expression.


Assuntos
Acrilamida , Relação Dose-Resposta a Droga , Desenvolvimento Embrionário , Antígeno Nuclear de Célula em Proliferação , Animais , Embrião de Galinha , Acrilamida/toxicidade , Antígeno Nuclear de Célula em Proliferação/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Tubo Neural/efeitos dos fármacos , Tubo Neural/embriologia , Caspase 3/metabolismo , Caspase 3/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos
6.
Biopolymers ; : e23620, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39109846

RESUMO

Welan gum (WG) has a wide range of applications, but it is not yet suitable for applications such as oil recovery profile control that have complex requirements for viscosity, gelation properties, and so forth. Grafting modification is an important strategy for improving the property of WG, but there are few reports on controllable modification of WG to customize it for specific application. Acrylamide (AM) dosage was identified as the key factor affecting the grafting ratio of AM onto WG by a uniform experimental design. The grafting ratio can be directly adjusted between 99% and 378% based on the positive correlation with dosage of AM, and viscosity can be adjusted between 206 and 327 mPa s based on the negative correlation with grafting ratio. The 50% weight loss temperature of W11 with a grafting ratio of 110% raised from 314 to 336°C after grafting. The viscosity of the hydrogel formed with WG11 reached 15,654 mPa s, nearly nine times higher than that of unmodified WG. In addition, the gelation time can be controlled within 5 days, so that it can be injected to the optimal area in oilfield profile, avoiding pipeline blockage. This study enables adjusting viscosity of WG grafted with AM by controlling the grafting rate, and enhances gelation performance and thermal stability of WG, which will expand the application of WG in oil recovery and other fields.

7.
Chemphyschem ; 25(4): e202300812, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38227375

RESUMO

Two series of hydrogels based on acrylamide and its copolymers with acrylonitrile and acrylic acid were synthesized by two cross-linking methods - chemical (using N,N'-methylene bis-acrylamide) and physical (using montmorillonite (MMT)) ones. The structure of the gels was characterized by Fourier Transform Infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The swelling and sorption properties were analyzed as a function of both the monomer composition and the cross-linking method. The shift of the band corresponding to Si-O (995-1030 cm-1 ) confirmed the formation of intercalation structures for MMT-cross-linked gels. Moreover, physically cross-linked gels demonstrated a non-monotonic dependence of the swelling degree on the MMT concentration, and acrylamide-acrylic acid copolymer MMT-cross-linked gels showed pH sensitivity and the highest swelling degree of 150 g/g. The highest sorption capacity towards cadmium(II) ions was demonstrated by acrylamide-acrylic acid copolymer gels, both covalently cross-linked (30 mg/g) and MMT-cross-linked (8.9 mg/g).

8.
Neurochem Res ; 49(3): 617-635, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37989894

RESUMO

Acrylamide (ACR) is a colorless, odorless, and water-soluble solid molecule. In addition to being an important industrial material, ACR is found in fried and baked carbohydrate-rich foods. ACR is regarded as a typical axonal neurotoxin that induces neuropathy. The brain is protected from oxidative damage by vitamin E, which is regarded as the most powerful fat-soluble antioxidant vitamin. This study aimed to reveal the toxic effect of ACR on the development of myelin in the brain at the molecular level and to examine whether Vitamin E has a neuroprotective effect on the harmful effect of ACR. The study was started by dividing 40 pregnant rats into 4 groups and after lactation, the study was continued with offspring rats (females and males offspring rats) from each group. Offspring rats were equally divided into Control, Vitamin E, ACR, ACR + Vitamin E groups. Following the ACR administration, the Water Maze test was applied to evaluate cognitive function. To evaluate the level of demyelination and remyelination, MBP, MAG, and MOG proteins and mRNA levels were performed. In addition, the degeneration of myelin and glial cells was examined by immunohistochemistry and electron microscopic analysis. Analysis results showed that ACR administration decreased gene and protein levels of myelin-related proteins MBP, MAG, and MOG. The findings were confirmed by histopathological, immunohistochemical, and microscopic examinations. The application of vitamin E improved this negative effect of ACR. It has been observed that ACR may play a role in the pathogenesis of myelin-related neurodegenerative diseases by causing demyelination during gestation, lactation, and post-lactation. In addition, it has been understood that vitamin E supports myelination as a strong neuroprotective vitamin against the toxicity caused by ACR. Our research results suggest that acrylamide may play a role in the etiopathogenesis of demyelinating diseases such as multiple sclerosis in humans since fast-food-type nutrition is very common today and people are chronically exposed to acrylamide.


Assuntos
Acrilamida , Doenças Desmielinizantes , Humanos , Masculino , Gravidez , Feminino , Ratos , Animais , Acrilamida/toxicidade , Bainha de Mielina , Vitamina E/farmacologia , Lactação , Vitaminas/farmacologia
9.
Int Microbiol ; 2024 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-38519776

RESUMO

The L-asparaginase (ASPN) enzyme has received recognition in various applications including acrylamide degradation in the food industry. The synthesis and application of thermostable ASPN enzymes is required for its use in the food sector, where thermostable enzymes can withstand high temperatures. To achieve this goal, the bacterium Bacillus subtilis was isolated from the hot springs of Tapovan for screening the production of thermostable ASPN enzyme. Thus, ASPN with a maximal specific enzymatic activity of 0.896 U/mg and a molecular weight of 66 kDa was produced from the isolated bacteria. The kinetic study of the enzyme yielded a Km value of 1.579 mM and a Vmax of 5.009 µM/min with thermostability up to 100 min at 75 °C. This may have had a positive indication for employing the enzyme to stop polyacrylamide from being produced. The current study has also been extended to investigate the interaction of native and mutated ASPN enzymes with acrylamide. This concluded that the M10 (with 10 mutations) has the highest protein and thermal stability compared to the wild-type ASPN protein sequence. Therefore, in comparison to a normal ASPN and all other mutant ASPNs, M10 is the most favorable mutation. This research has also demonstrated the usage of ASPN in food industrial applications.

10.
Extremophiles ; 28(3): 44, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39313567

RESUMO

L-asparaginase (ASNase, E.C. 3.5.1.1) catalyzes the deamination of L-asparagine to L-aspartic acid and ammonia and is widely used in medicine to treat acute lymphocytic leukemia. It also has significant applications in the food industry by inhibiting acrylamide formation. In this study, we characterized a thermostable ASNase from the hyper thermophilic strain, Pyrococcus yayanosii CH1. The recombinant enzyme (PyASNase) exhibited maximal activity at pH 8.0 and 85 °C. Moreover, PyASNase demonstrated promising thermostability across temperatures ranging from 70 to 95 °C. The kinetic parameters of PyASNase for L-asparagine were a Km of 6.3 mM, a kcat of 1989s-1, and a kcat/Km of 315.7 mM-1 s-1. Treating potato samples with 10 U/mL of PyASNase at 85 °C for merely 10 min reduced the acrylamide content in the final product by 82.5%, demonstrating a high efficiency and significant advantage of PyASNase in acrylamide inhibition.


Assuntos
Acrilamida , Asparaginase , Estabilidade Enzimática , Pyrococcus , Asparaginase/química , Asparaginase/metabolismo , Asparaginase/genética , Acrilamida/química , Acrilamida/metabolismo , Pyrococcus/enzimologia , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Temperatura Alta
11.
Fish Shellfish Immunol ; 146: 109411, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38301813

RESUMO

This study looked at the toxic impacts of water-born acrylamide (ACR) on Nile tilapia (Oreochromis niloticus) in terms of behaviors, growth, immune/antioxidant parameters and their regulating genes, biochemical indices, tissue architecture, and resistance to Aeromonas hydrophila. As well as the probable ameliorative effect of Chlorella vulgaris (CV) microalgae as a feed additive against ACR exposure was studied. The 96-h lethal concentration 50 of ACR was investigated and found to be 34.67 mg/L for O. niloticus. For the chronic exposure study, a total of 180 healthy O. niloticus (24.33 ± 0.03 g) were allocated into four groups in tri-replicates (15 fish/replicate), C (control) and ACR groups were fed a basal diet and exposed to 0 and 1/10 of 96-h LC50 of ACR (3.46 mg/L), respectively. ACR+ CV5 and ACR+ CV10 groups were fed basal diets with 5 % and 10 % CV supplements, respectively and exposed to 1/10 of 96-h LC50 of ACR for 60 days. After the exposure trial (60 days) the experimental groups were challenged with A. hydrophila. The findings demonstrated that ACR exposure induced growth retardation (P˂0.01) (lower final body weight, body weight gain, specific growth rate, feed intake, protein efficiency ratio, final body length, and condition factor as well as higher feed conversion ratio). A substantial decrease in the immune/antioxidant parameters (P˂0.05) (lysozyme, serum bactericidal activity %, superoxide dismutase, and reduced glutathione) and neurotransmitter (acetylcholine esterase) (P˂0.01) was noticed with ACR exposure. A substantial increase (P˂0.01) in the serum levels of hepato-renal indicators, lipid peroxidation biomarker, and cortisol was noticed as a result of ACR exposure. ACR exposure resulted in up-regulation (P˂0.05) of the pro-inflammatory cytokines and down-regulation (P˂0.05) of the antioxidant-related gene expression. Furthermore, the hepatic, renal, brain, and splenic tissues were badly affected by ACR exposure. ACR-exposed fish were more sensitive to A. hydrophila infection and recorded the lowest survival rate (P˂0.01). Feeding the ACR-exposed fish with CV diets significantly improved the growth and immune/antioxidant status, as well as modulating the hepatorenal functions, stress, and neurotransmitter level compared to the exposed-non fed fish. In addition, modulation of the pro-inflammatory and antioxidant-related gene expression was noticed by CV supplementation. Dietary CV improved the tissue architecture and increased the resistance to A. hydrophila challenge in the ACR-exposed fish. Noteworthy, the inclusion of 10 % CV produced better results than 5 %. Overall, CV diets could be added as a feed supplement in the O. niloticus diet to boost the fish's health, productivity, and resistance to A. hydrophila challenge during ACR exposure.


Assuntos
Chlorella vulgaris , Ciclídeos , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Animais , Antioxidantes/metabolismo , Resistência à Doença , Dieta/veterinária , Suplementos Nutricionais , Neurotransmissores/metabolismo , Peso Corporal , Transtornos do Crescimento , Acrilamidas , Ração Animal/análise , Doenças dos Peixes/induzido quimicamente , Infecções por Bactérias Gram-Negativas/veterinária
12.
J Comput Aided Mol Des ; 38(1): 21, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38693331

RESUMO

Covalent inhibition offers many advantages over non-covalent inhibition, but covalent warhead reactivity must be carefully balanced to maintain potency while avoiding unwanted side effects. While warhead reactivities are commonly measured with assays, a computational model to predict warhead reactivities could be useful for several aspects of the covalent inhibitor design process. Studies have shown correlations between covalent warhead reactivities and quantum mechanic (QM) properties that describe important aspects of the covalent reaction mechanism. However, the models from these studies are often linear regression equations and can have limitations associated with their usage. Applications of machine learning (ML) models to predict covalent warhead reactivities with QM descriptors are not extensively seen in the literature. This study uses QM descriptors, calculated at different levels of theory, to train ML models to predict reactivities of covalent acrylamide warheads. The QM/ML models are compared with linear regression models built upon the same QM descriptors and with ML models trained on structure-based features like Morgan fingerprints and RDKit descriptors. Experiments show that the QM/ML models outperform the linear regression models and the structure-based ML models, and literature test sets demonstrate the power of the QM/ML models to predict reactivities of unseen acrylamide warhead scaffolds. Ultimately, these QM/ML models are effective, computationally feasible tools that can expedite the design of new covalent inhibitors.


Assuntos
Cisteína , Desenho de Fármacos , Aprendizado de Máquina , Teoria Quântica , Cisteína/química , Acrilamida/química , Humanos , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Modelos Lineares , Estrutura Molecular
13.
Bioorg Med Chem ; 112: 117902, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39236467

RESUMO

In the development of covalent inhibitors, acrylamides warhead is one of the most popular classes of covalent warheads. In recent years, researchers have made different structural modifications to acrylamides warheads, resulting in the creation of fluorinated acrylamide warheads and cyano acrylamide warheads. These new warheads exhibit superior selectivity, intracellular accumulation, and pharmacokinetic properties. Additionally, although ketoamide warheads have been applied in the design of covalent inhibitors for viral proteins, it has not received sufficient attention. Combined with the studies in kinase inhibitors and antiviral drugs, this review presents the structural features and the progression of acrylamides warheads, offering a perspective on future research and development in this field.


Assuntos
Acrilamidas , Desenho de Fármacos , Inibidores de Proteínas Quinases , Humanos , Acrilamidas/química , Acrilamidas/síntese química , Acrilamidas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Antivirais/química , Antivirais/farmacologia , Antivirais/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Estrutura Molecular , Relação Estrutura-Atividade
14.
Mol Biol Rep ; 51(1): 739, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874886

RESUMO

Acrylamide (ACR) is an industrial chemical used to produce polyacrylamide, a synthetic polymer with a wide range of applications. Depending on the dosage, its presence in occupational and environmental sources poses potential health risks to humans and animals. ACR can be formed in starchy foods cooked at high temperatures. Its effects on human sperm are not well understood. Animal studies indicate that ACR induces toxicity in the male reproductive system through oxidative stress mechanisms. Exposure to ACR alters the normal structure of testicular tubules, leading to congestion, interstitial edema, degeneration of spermatogenic cells, formation of abnormal spermatid giant cells, and necrosis and apoptosis. It also disrupts the balance of important biomarkers such as malondialdehyde, nitric oxide, superoxide dismutase, catalase, and glutathione. ACR has a negative impact on mitochondrial function, antioxidant enzymes, ATP production, and sperm membrane integrity, resulting in decreased sperm quality. Furthermore, it interferes with the expression of steroidogenic genes associated with testosterone biosynthesis. This review explores the detrimental effects of ACR on sperm and testicular function and discusses the potential role of antioxidants in mitigating the adverse effects of ACR on male reproduction.


Assuntos
Acrilamida , Estresse Oxidativo , Espermatozoides , Testículo , Masculino , Acrilamida/toxicidade , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Humanos , Testículo/efeitos dos fármacos , Testículo/metabolismo , Animais , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Antioxidantes/farmacologia
15.
Mol Biol Rep ; 51(1): 817, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39012451

RESUMO

BACKGROUND: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and ß-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. METHODS AND RESULTS: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of ß-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of ß-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. CONCLUSION: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.


Assuntos
Hidroliases , Rhodococcus , Rhodococcus/enzimologia , Rhodococcus/genética , Hidroliases/metabolismo , Hidroliases/genética , Hidroliases/química , Estabilidade Enzimática , Estresse Fisiológico , Acrilamida/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética
16.
Anal Bioanal Chem ; 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740590

RESUMO

Acrylamide determination is important to state its quantity in baked food preventing any potential carcinogenic effects. Matrix solid-phase dispersion (MSPD) extraction is an extraction procedure based on a homogenization phase between a solid sample and a solid dispersing material to break sample increasing analyte extraction yield, often used for acrylamide determination. The addition of a green deep eutectic solvent (DES) during the MSPD homogenization phase improves the analyte extraction, giving the possibility to reduce the amount of organic solvent used. In this work, a miniaturized MSPD extraction assisted by a DES was developed to determine acrylamide in bread, using high-performance liquid chromatography coupled with mass spectrometry detection. The optimized procedure provides 1:1 (w/w) matrix-to-dispersing material ratio, 2 mL of methanol as extraction solvent, and 50 µL of choline chloride-glycerol DES added during the homogenization phase. Method validation ensured good results with minimum recoveries of 90%, high precision with a maximum intra-day error of 4%, and inter-day error of 6%. Limit of detection and limit of quantification resulted to be 16 µg/kg and 35 µg/kg, respectively. This miniaturized extraction procedure represents a good alternative to those reported in the literature, guaranteeing great performance and respecting green chemistry principles.

17.
Anal Bioanal Chem ; 416(7): 1541-1560, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38349534

RESUMO

The development and validation of a simple, comprehensive, and environment-friendly procedure to determine pesticide residues, naturally occurring and processing contaminants in roasted coffee is presented. A solid-liquid extraction of pesticides and mycotoxins with ethyl acetate and the concurrent partition of acrylamide to an aqueous phase follows a parallel analytical strategy that requires a single analytical portion to determine contaminants that are typically analyzed by dedicated single residue methods. The partition rules the lipids out of the aqueous extract before an "in-tube" dispersive solid phase microextraction (dSPME) for acrylamide retention. This is followed by the elution with buffer prior to injection. This extract is independently introduced into the system front end followed by the injection of the compounds from the organic phase, yet all spotted in the same run. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method setup enables the quantification of 186 compounds at 10 µg/kg, 226 at 5 µg/kg, and the acrylamide at 200 µg/kg for a total of 414 molecules, with acceptable recoveries (70-120%) and precision (RSD < 20%) making this strategy significantly faster and cost-effective than the dedicated single residue methods. Even though the presence of chlorpyrifos, acrylamide, and ochratoxin A was confirmed on samples of different origins, the findings were below the limit of quantification. During the storage of raw coffee, no proof of masking of OTA was found; however, condensation with glucose was evidenced during thermal processing experiments with sucrose by using stable isotope labeling (SIL). No detected conjugates were found in roasted nor in commercial sugar-added torrefacto samples, an industrial processing usually carried out above the decomposition temperature of the disaccharide.


Assuntos
Micotoxinas , Praguicidas , Café/química , Espectrometria de Massas em Tandem/métodos , Micotoxinas/análise , Praguicidas/análise , Acrilamida/análise
18.
Anal Bioanal Chem ; 416(5): 1117-1126, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38123751

RESUMO

For the analysis of plant-based meat substitutes and the determination of Maillard reaction products such as acrylamide, 5-hydroxymethylfurfural and furaneol, a novel and effective procedure based on hydrophobic natural deep eutectic solvent and liquid chromatography coupled with tandem mass spectrometry was developed for the first time. The 49 compositions of the deep eutectic solvents were designed and screened to select the most suitable option. The terpenoids eugenol and thymol in a molar ratio of 2:1 were selected as precursors for solvent formation, allowing effective extraction of the target analytes. The developed procedure comprised two main steps: extraction - in which the analytes are isolated from the solid sample due to the salting-out effect and pre-concentrated in the deep eutectic solvent, and back-extraction - in which the analytes are re-extracted into the formic acid solution for subsequent mass spectrometric detection. As the density of the aqueous phases changed during the extraction and back-extraction steps, the phenomenon of inversion of the coalesced organic phase was observed, which simplified the withdrawing of the phases. The linear range was 1-50 ng/mL for acrylamide, 10-1000 ng/mL for 5-hydroxymethylfurfural and 200-1000 ng/mL for furaneol with coefficients of determination above 0.9952. The developed method was fully validated and found recoveries were in the range 83-120%, with CVs not exceeding 4.9%. The method was applied to real sample analysis of pea-based meat substitutes.


Assuntos
Solventes Eutéticos Profundos , Furaldeído/análogos & derivados , Furanos , Microextração em Fase Líquida , Solventes/química , Espectrometria de Massa com Cromatografia Líquida , Cromatografia Líquida , Espectrometria de Massas em Tandem , Acrilamida , Substitutos da Carne , Microextração em Fase Líquida/métodos , Limite de Detecção
19.
J Biochem Mol Toxicol ; 38(8): e23784, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39095945

RESUMO

Acrylamide (AA) is a carcinogenic compound that affects people due to its frequent use in laboratories and industry as well as the high-temperature cooking of foods with high hydrocarbon content. AA is known to cause severe reproductive abnormalities. The main aim of this study is to evaluate the protective effect of rutin (RU), a phytoactive compound, against AA-induced reproductive toxicity in female rats. Initially, rats were exposed to AA (40 mg/kg for 10 days). Therapy of RU was given after AA intoxication consecutively for 3 days. After 24 h of the last treatment, all the animals were sacrificed. The study evaluated reproductive hormones, oxidative stress markers, membrane-bound enzymes, DNA damage, histological findings, and an in silico approach to determine the protective efficacy of RU. The results indicated that RU significantly protected against inflammation, oxidative stress, and DNA damage induced by AA, likely due to its antioxidant properties.


Assuntos
Acrilamida , Dano ao DNA , Inflamação , Estresse Oxidativo , Rutina , Animais , Rutina/farmacologia , Feminino , Estresse Oxidativo/efeitos dos fármacos , Acrilamida/toxicidade , Dano ao DNA/efeitos dos fármacos , Ratos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Ratos Wistar , Simulação por Computador , Antioxidantes/farmacologia , Antioxidantes/metabolismo
20.
J Biochem Mol Toxicol ; 38(2): e23658, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38348719

RESUMO

Vinpocetine (VIN) is a synthetic drug derived from the natural alkaloid vincamine. The antioxidation and anti-inflammation effects of VIN allow it to be used for multiple therapeutic purposes. So, the research aims to discover the possibility of using VIN to improve the nephrotoxicity of acrylamide (ACR). Twenty-four male albino rats were used in the trial: rats in the control group received 0.5 mL of oral saline, rats in the VIN group received an oral dose of VIN (5 mg/kg), rats in the ACR group received an oral dose of ACR (38.27 mg/kg), and rats in the VIN + ACR group received VIN and then ACR 1 h later. Rat blood and kidneys were collected 10 days after the experiment began to assess biochemical parameters and to examine both renal histopathological and immunohistochemistry. The ACR-treated rats showed high levels of serum kidney function biomarkers (creatinine, urea, and uric acid), serum protein biomarkers (total protein, albumin, and globulin), renal kidney injury molecule (KIM)-1, renal malondialdehyde (MDA), and renal caspase-3 immunoexpression. Moreover, ACR lowed both renal superoxide dismutase (SOD) activity and renal glutathione (GSH) level and caused renal histological alterations. While administration of VIN improved serum kidney function biomarkers, serum protein biomarkers, renal KIM-1, renal oxidative stress biomarkers (MDA, SOD, and GSH), renal caspase-3 immunoexpression, and renal histological alterations induced by ACR. The study confirmed the ability of VIN to reduce the nephrotoxic effects of ACR, which was evident through the results of biochemical parameters and histological and immunohistochemical examinations of the kidney tissues.


Assuntos
Acrilamida , Insuficiência Renal , Alcaloides de Vinca , Ratos , Masculino , Animais , Caspase 3/metabolismo , Acrilamida/toxicidade , Rim , Antioxidantes/farmacologia , Estresse Oxidativo , Glutationa/metabolismo , Superóxido Dismutase/metabolismo , Proteínas Sanguíneas/metabolismo , Biomarcadores/metabolismo , Malondialdeído/metabolismo
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