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1.
Curr Protoc ; 1(9): e237, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34499805

RESUMO

Eukaryotic cells contain numerous membrane-bound and membraneless organelles that provide spatiotemporal control for diverse biological processes. The liquid-liquid phase separation of proteins has been proposed as the driving force behind the formation of membraneless organelles. Here, we describe a method to determine the phase separation activities of proteins in plants. This basic method includes protocols for an in vivo fluorescence recovery after photobleaching assay in Nicotiana benthamiana using transient expression, an in vitro liquid droplet reconstitution assay using purified recombinant proteins, and an in vivo fluorescence recovery after photobleaching assay in Arabidopsis thaliana using stable transgenic plants. With these assays, the phase separation characteristics of a protein can be determined. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Detection of protein phase separation activities in N. benthamiana Support Protocol: Fluorescence recovery after photobleaching assay Basic Protocol 2: Detection of protein phase separation in vitro Basic Protocol 3: Detection of protein phase separation in stable transgenic Arabidopsis thaliana plants.


Assuntos
Fracionamento Químico/métodos , Organelas , Proteínas de Plantas , Fenômenos Biofísicos , Membranas
2.
Ultrason Sonochem ; 77: 105680, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34365154

RESUMO

The present study optimised the ultrasound-assisted extraction (UAE) of bioactive compounds from Amaranthus hypochondriacus var. Nutrisol. Influence of temperature (25.86-54.14 °C) and ultrasonic power densities (UPD) (76.01-273.99 mW/mL) on total betalains (BT), betacyanins (BC), betaxanthins (BX), total polyphenols (TP), antioxidant activity (AA), colour parameters (L*, a*, and b*), amaranthine (A), and isoamaranthine (IA) were evaluated using response surface methodology. Moreover, betalain extraction kinetics and mass transfer coefficients (KLa) were determined for each experimental condition. BT, BC, BX, TP, AA, b*, KLa, and A were significantly affected (p < 0.05) by temperature extraction and UPD, whereas L*, a*, and IA were only affected (p < 0.05) by temperature. All response models were significantly validated with regression coefficients (R2) ranging from 87.46 to 99.29%. BT, A, IA, and KLa in UAE were 1.38, 1.65, 1.50, and 29.93 times higher than determined using conventional extraction, respectively. Optimal UAE conditions were obtained at 41.80 °C and 188.84 mW/mL using the desired function methodology. Under these conditions, the experimental values for BC, BX, BT, TP, AA, L*, a*, b*, KLa, A, and IA were closely related to the predicted values, indicating the suitability of the developed quadratic models. This study proposes a simple and efficient UAE method to obtain betalains and polyphenols with high antioxidant activity, which can be used in several applications within the food industry.


Assuntos
Amaranthus/química , Antioxidantes/isolamento & purificação , Betalaínas/isolamento & purificação , Fracionamento Químico/métodos , Polifenóis/isolamento & purificação , Ondas Ultrassônicas , Fatores de Tempo
3.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443623

RESUMO

Phenolic compounds have long been of great importance in the pharmaceutical, food, and cosmetic industries. Unfortunately, conventional extraction procedures have a high cost and are time consuming, and the solvents used can represent a safety risk for operators, consumers, and the environment. Deep eutectic solvents (DESs) are green alternatives for extraction processes, given their low or non-toxicity, biodegradability, and reusability. This review discusses the latest research (in the last two years) employing DESs for phenolic extraction, solvent components, extraction yields, extraction method characteristics, and reviewing the phenolic sources (natural products, by-products, wastes, etc.). This work also analyzes and discusses the most relevant DES-based studies for phenolic extraction from natural sources, their extraction strategies using DESs, their molecular mechanisms, and potential applications.


Assuntos
Produtos Biológicos/química , Fracionamento Químico/métodos , Fenóis/isolamento & purificação , Solventes/química
4.
Molecules ; 26(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34443587

RESUMO

Panax spp. (Araliaceae family) are widely used medicinal plants and they mainly include Panax ginseng C.A. Meyer, Panax quinquefolium L. (American ginseng), and Panax notoginseng (notoginseng). Polysaccharides are the main active ingredients in these plants and have demonstrated diverse pharmacological functions, but comparisons of isolation methods, structural features, and bioactivities of these polysaccharides have not yet been reported. This review summarizes recent advances associated with 112 polysaccharides from ginseng, 25 polysaccharides from American ginseng, and 36 polysaccharides from notoginseng and it compares the differences in extraction, purification, structural features, and bioactivities. Most studies focus on ginseng polysaccharides and comparisons are typically made with the polysaccharides from American ginseng and notoginseng. For the extraction, purification, and structural analysis, the processes are similar for the polysaccharides from the three Panax species. Previous studies determined that 55 polysaccharides from ginseng, 18 polysaccharides from American ginseng, and 9 polysaccharides from notoginseng exhibited anti-tumor activity, immunoregulatory effects, anti-oxidant activity, and other pharmacological functions, which are mediated by multiple signaling pathways, including mitogen-activated protein kinase, nuclear factor kappa B, or redox balance pathways. This review can provide new insights into the similarities and differences among the polysaccharides from the three Panax species, which can facilitate and guide further studies to explore the medicinal properties of the Araliaceae family used in traditional Chinese medicine.


Assuntos
Fracionamento Químico/métodos , Panax/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Humanos , Polissacarídeos/isolamento & purificação
5.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443475

RESUMO

Different parts of a plant (seeds, fruits, flower, leaves, stem, and roots) contain numerous biologically active compounds called "phytoconstituents" that consist of phenolics, minerals, amino acids, and vitamins. The conventional techniques applied to extract these phytoconstituents have several drawbacks including poor performance, low yields, more solvent use, long processing time, and thermally degrading by-products. In contrast, modern and advanced extraction nonthermal technologies such as pulsed electric field (PEF) assist in easier and efficient identification, characterization, and analysis of bioactive ingredients. Other advantages of PEF include cost-efficacy, less time, and solvent consumption with improved yields. This review covers the applications of PEF to obtain bioactive components, essential oils, proteins, pectin, and other important materials from various parts of the plant. Numerous studies compiled in the current evaluation concluded PEF as the best solution to extract phytoconstituents used in the food and pharmaceutical industries. PEF-assisted extraction leads to a higher yield, utilizes less solvents and energy, and it saves a lot of time compared to traditional extraction methods. PEF extraction design should be safe and efficient enough to prevent the degradation of phytoconstituents and oils.


Assuntos
Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Compostos Fitoquímicos , Indústria de Processamento de Alimentos , Solventes/química , Tecnologia Farmacêutica
6.
Ultrason Sonochem ; 77: 105696, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34358883

RESUMO

A novel, green and eco-friendly, cost-effective, fast, and reliable high energy ultrasonication (US) extraction with UHPLC-MSMS (Ultrahigh performance liquid chromatography with mass spectrometry) quantification of Glycyrrhizic acid (GZA) is reported herein for the first time. The study provides useful insights regarding the effect of US-factors with statistical analysis and mechanisms, involved in GZA-extraction and analysis. An US-extraction method (US-MD) was developed using three levels of US factors: solvents (AC (acetone), EtOH (ethanol), H2O (water)), time (1, 2, 3 min), amplitudes (30, 40, 50%), pulse (10/0.5, 20/0.5, 30/0.5 sec), particle sizes (0.5, 1, 1.4 mm), and temperatures (20, 30, 40 °C). The US-MD was further validated with high accuracy 98.96 ± 6.82 and r2 = 0.995 whereas, an in-house analytical method (UHPLC-MSMS) was developed and validated to quantify the GZAamount. UHPLCMSMS-MD resulted in a retention time of 0.31 min with MSMS (821.400 > 351.200) in a 1 min run time whereas, UHPLCMSMS-MV showed high accuracy and precision with r2 = 0.998 for GZA. Statistical analysis of K-mean clustering finalized US-set-of-factors showing optimum extract yield (mg/1mg) of 0.48 with sum (2.41 ± 014) and mean (0.27) along with a high GZA-amount (µg/mg) of 8.23 with sum (43.31 ± 2.07) and mean (4.81) for H2O in 3 min at 40 °C using particle size (1.4 mm), amplitude (50%), and pulse (30/0.5). Large scale application of US-UHPLCMSMS confirmed the evaluation power of the method showing the order for GZA amount; Egypt > Pakistan > Syria > India > Palestine > America > Georgia > Morocco. A significant effect for US factors Vs extract yield and GZA amount was observed however, solvent*GZA-amount and extract yield*particle size were more significantly correlated compared to time*temperature*amplitude*pulse analyzed via PCA, GLM-UniANOVA, K-mean, and Pearson's correlation (P ≤ 0.05). A combined mechanism of shear stress, macroturbulence due to acoustic cavitation and implosions, sonochemical, and sonocapillary effect were noted for the US technique producing higher extract yield and GZA amount from licorice.


Assuntos
Fracionamento Químico/métodos , Cromatografia Líquida de Alta Pressão , Glycyrrhiza/química , Ácido Glicirrízico/análise , Ácido Glicirrízico/isolamento & purificação , Espectrometria de Massas em Tandem , Ondas Ultrassônicas , Química Verde , Fatores de Tempo
7.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361739

RESUMO

We developed an alternative whipping cream fat using shea butter but with low saturation. Enriched stearic-oleic-stearic (SOS) solid fat was obtained from shea butter via solvent fractionation. Acyl migration reactant, which mainly contains asymmetric SSO triacylglycerol (TAG), was prepared through enzymatic acyl migration to obtain the creaming quality derived from the ß'-crystal form. Through enzymatic acyl migration, we obtained a 3.4-fold higher content of saturated-saturated-unsaturated (SSU) TAG than saturated-unsaturated-saturated (SUS) TAG. The acyl migration reactant was refined to obtain refined acyl migration reactant (RAMR). An alternative fat product was prepared by blending RAMR and hydrogenated palm kernel oil (HPKO) at a ratio of 4:6 (w/w). The melting points, solid fat index (SFI), and melting curves of the alternative products were similar to those of commercial whipping cream fat. The alternative fat had a content of total unsaturated fatty acids 20% higher than that of HPKO. The atherogenic index (AI) of alternative fat was 3.61, much lower than those of whipping cream fat (14.59) and HPKO (1220.3), because of its low atherogenic fatty acid content and high total unsaturated fatty acids. The polymorphic crystal form determined by X-ray diffraction spectroscopy showed that the ß'-crystal form was predominant. Therefore, the alternative fat is comparable with whipping cream that requires creaming quality, and has a reduced saturated fat content.


Assuntos
Gorduras na Dieta/análise , Ácidos Graxos Insaturados/química , Tecnologia de Alimentos/métodos , Ácidos Oleicos/química , Óleos Vegetais/química , Fracionamento Químico , Cristalização , Ácidos Graxos Insaturados/análise , Humanos , Ácido Oleico/análise , Ácido Oleico/química , Ácidos Oleicos/análise , Óleo de Palmeira/análise , Óleo de Palmeira/química , Óleos Vegetais/análise , Ácidos Esteáricos/análise , Ácidos Esteáricos/química , Triglicerídeos/análise , Triglicerídeos/química
8.
Water Res ; 203: 117519, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34391022

RESUMO

1H-benzotriazole is part of a larger family of benzotriazoles, which are widely used as lubricants, polymer stabilizers, corrosion inhibitors, and anti-icing fluid components. It is frequently detected in urban runoff, wastewater, and receiving aquatic environments. 1H-benzotriazole is typically resistant to biodegradation and hydrolysis, but can be transformed via direct photolysis and photoinduced mechanisms. In this study, the phototransformation mechanisms of 1H-benzotriazole were characterized using multi-element compound-specific isotope analysis (CSIA). The kinetics, transformation products, and isotope fractionation results altogether revealed that 1H-benzotriazole direct photolysis and indirect photolysis induced by OH radicals involved two alternative pathways. In indirect photolysis, aromatic hydroxylation dominated and was associated with small carbon (εC = -0.65 ± 0.03‰), moderate hydrogen (εH = -21.6‰), and negligible nitrogen isotope enrichment factors and led to hydroxylated forms of benzotriazole. In direct photolysis of 1H-benzotriazole, significant nitrogen (εN = -8.4 ± 0.4 to -4.2 ± 0.3‰) and carbon (εC = -4.3 ± 0.2 to -1.64 ± 0.04‰) isotope enrichment factors indicated an initial N-N bond cleavage followed by nitrogen elimination with a C-N bond cleavage. The results of this study highlight the potential for multi-element CSIA application to track 1H-benzotriazole degradation in aquatic environments.


Assuntos
Carbono , Hidrogênio , Biodegradação Ambiental , Isótopos de Carbono/análise , Fracionamento Químico , Nitrogênio , Isótopos de Nitrogênio , Triazóis
9.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445303

RESUMO

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.


Assuntos
Transição de Fase/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Água/farmacologia , Motivos de Aminoácidos/efeitos dos fármacos , Fracionamento Químico/métodos , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Cinética , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Teoria Quântica , Viscosidade , Água/química
10.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199500

RESUMO

In the search of new alternatives for weed control, spices appear as an option with great potential. They are rich in bioactive natural products and edible, which might minimize toxicity hazard. Marjoram (Origanum majorana L.) is an aromatic herb that has been widely employed as a seasoning herb in Mediterranean countries. Although marjoram boasts a plethora of therapeutic properties (painkiller, antibiotic, treatment for intestinal disorders, etc.), the potential for its extracts for weed control is still to be more thoroughly explored. In order to determine their phytotoxic potential, marjoram leaves were subjected to different bioguided extraction processes, using water, ethyl acetate, acetone or methanol. The most active extract (acetone) was sequentially fractionated to identify its most active compounds. This fractionation led to the isolation and identification of 25 compounds that were classified as monoterpenes, diterpenes or flavonoids. Among them, a new compound named majoradiol and several compounds are described in marjoram for the first time. The phytotoxicity of the major compounds to etiolated wheat coleoptiles was compared against that of the commercial herbicide (Logran®), with similar or higher activity in some cases. These results confirm the extraordinary potential of the extracts from this edible plant to develop safer and more environmentally friendly herbicides.


Assuntos
Herbicidas/farmacologia , Origanum/química , Compostos Fitoquímicos/farmacologia , Fracionamento Químico , Flavonoides/química , Flavonoides/farmacologia , Herbicidas/química , Região do Mediterrâneo , Estrutura Molecular , Compostos Fitoquímicos/química , Folhas de Planta/química , Compostos de Sulfonilureia/farmacologia , Terpenos/química , Terpenos/farmacologia , Controle de Plantas Daninhas
11.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204452

RESUMO

Intercellular communication governs multicellular interactions in complex organisms. A variety of mechanisms exist through which cells can communicate, e.g., cell-cell contact, the release of paracrine/autocrine soluble molecules, or the transfer of extracellular vesicles (EVs). EVs are membrane-surrounded structures released by almost all cell types, acting both nearby and distant from their tissue/organ of origin. In the kidney, EVs are potent intercellular messengers released by all urinary system cells and are involved in cell crosstalk, contributing to physiology and pathogenesis. Moreover, urine is a reservoir of EVs coming from the circulation after crossing the glomerular filtration barrier-or originating in the kidney. Thus, urine represents an alternative source for biomarkers in kidney-related diseases, potentially replacing standard diagnostic techniques, including kidney biopsy. This review will present an overview of EV biogenesis and classification and the leading procedures for isolating EVs from body fluids. Furthermore, their role in intra-nephron communication and their use as a diagnostic tool for precision medicine in kidney-related disorders will be discussed.


Assuntos
Biomarcadores/urina , Vesículas Extracelulares/metabolismo , Nefropatias/metabolismo , Animais , Comunicação Celular , Micropartículas Derivadas de Células/metabolismo , Fracionamento Químico , Gerenciamento Clínico , Suscetibilidade a Doenças , Exossomos/metabolismo , Humanos , Nefropatias/diagnóstico , Nefropatias/etiologia , Nefropatias/urina , Biópsia Líquida/métodos , Medicina de Precisão/métodos , Urinálise/métodos
12.
Environ Sci Technol ; 55(14): 10164-10174, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34213316

RESUMO

Mass-independent fractionation (MIF) of stable even mass number mercury (Hg) isotopes is observed in rainfall and gaseous elemental Hg0 globally and is used to quantify atmospheric Hg deposition pathways. The chemical reaction and underlying even-Hg MIF mechanism are unknown however and speculated to be caused by Hg photo-oxidation on aerosols at the tropopause. Here, we investigate the Hg isotope composition of free tropospheric Hg0 and oxidized HgII forms at the high-altitude Pic du Midi Observatory. We find that gaseous oxidized Hg has positive Δ199Hg, Δ201Hg, and Δ200Hg and negative Δ204Hg signatures, similar to rainfall Hg, and we document rainfall Hg Δ196Hg to be near zero. Cloud water and rainfall Hg show an enhanced odd-Hg MIF of 0.3‰ compared to gaseous oxidized HgII, potentially indicating the occurrence of in-cloud aqueous HgII photoreduction. Diurnal MIF observations of free tropospheric Hg0 show how net Hg0 oxidation in high-altitude air masses leads to opposite even- and odd-MIF in Hg0 and oxidized HgII. We speculate that even-Hg MIF takes place by a molecular magnetic isotope effect during HgII photoreduction on aerosols that involves magnetic halogen nuclei. A Δ200Hg mass balance suggests that global Hg deposition pathways in models are likely biased toward HgII deposition. We propose that Hg cycling models could accommodate the Hg-isotope constraints on emission and deposition fluxes.


Assuntos
Mercúrio , Fracionamento Químico , Monitoramento Ambiental , Isótopos , Mercúrio/análise , Isótopos de Mercúrio/análise , Oxirredução
13.
Molecules ; 26(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34299398

RESUMO

Rice straw hydrotropic lignin was extracted from p-Toluene sulfonic acid (p-TsOH) fractionation with a different combined delignification factor (CDF). Hydrotropic lignin characterization was systematically investigated, and alkaline lignin was also studied for the contrast. Results showed that the hydrotropic rice straw lignin particle was in nanometer scopes. Compared with alkaline lignin, the hydrotropic lignin had greater molecular weight. NMR analysis showed that ß-aryl ether linkage was well preserved at low severities, and the unsaturation in the side chain of hydrotropic lignin was high. H units and G units were preferentially degraded and subsequently condensed at high severity. High severity also resulted in the cleavage of part ß-aryl ether linkage. 31P-NMR showed the decrease in aliphatic hydroxyl groups and the increasing carboxyl group content at high severity. The maximum weight loss temperature of the hydrotropic lignin was in the range of 330-350 °C, higher than the alkaline lignin, and the glass conversion temperature (Tg) of the hydrotropic lignin was in the range of 107-125 °C, lower than that of the alkaline lignin. The hydrotropic lignin has high ß-aryl ether linkage content, high activity, nanoscale particle size, and low Tg, which is beneficial for its further valorization.


Assuntos
Lignina/química , Lignina/isolamento & purificação , Oryza/química , Água/química , Biomassa , Fracionamento Químico , Hidrólise , Lignina/análise
14.
J Hazard Mater ; 418: 126342, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34329001

RESUMO

In this study, chromium (Cr) was used as an example of the most toxic heavy metals that threaten human health, and Cr(VI) bioremediation was implemented by using a new type of aerobic granular sludge (AGS), i.e., algal-bacterial AGS. Results showed that the total Cr removal efficiency by active algal-bacterial AGS was 85.1 ± 0.6% after 6 h biosorption at pH 6 and room temperature, which could be further improved to 93.8 ± 0.4% with external electron donor (glucose) supply. However, inactivation dramatically decreased the total Cr removal efficiency to 29.6 ± 3.5%, and no effect was noticed when external electron donor was provided. With an antibiotic (levofloxacin) or metabolic inhibitor (NaN3) addition, the total Cr removal efficiency of bacterial AGS was inhibited by 16.0% or 10.1%, but this efficiency was maintained in the case of algal-bacterial AGS. Analysis of extracellular polymeric substances (EPS) composition revealed that under Cr(VI) exposure, more loosely bound EPS were secreted by algal-bacterial AGS, favoring Cr(VI) reduction. Results from chemical fractionation indicated that 90.5 ± 4.2% of the loaded Cr on algal-bacterial AGS was in an immobile form, reflecting the low environmental risk of Cr-loaded algal-bacterial AGS after biosorption of hazardous heavy metals from wastewater.


Assuntos
Microalgas , Esgotos , Biodegradação Ambiental , Reatores Biológicos , Fracionamento Químico , Cromo/toxicidade , Humanos , Viabilidade Microbiana
15.
Nat Methods ; 18(7): 806-815, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34211188

RESUMO

Co-fractionation mass spectrometry (CF-MS) has emerged as a powerful technique for interactome mapping. However, there is little consensus on optimal strategies for the design of CF-MS experiments or their computational analysis. Here, we reanalyzed a total of 206 CF-MS experiments to generate a uniformly processed resource containing over 11 million measurements of protein abundance. We used this resource to benchmark experimental designs for CF-MS studies and systematically optimize computational approaches to network inference. We then applied this optimized methodology to reconstruct a draft-quality human interactome by CF-MS and predict over 700,000 protein-protein interactions across 27 eukaryotic species or clades. Our work defines new resources to illuminate proteome organization over evolutionary timescales and establishes best practices for the design and analysis of CF-MS studies.


Assuntos
Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteoma/análise , Animais , Evolução Biológica , Fracionamento Químico , Bases de Dados de Proteínas , Humanos , Camundongos , Plasmodium berghei/química , Plasmodium berghei/metabolismo , Proteômica/métodos
16.
Food Environ Virol ; 13(3): 303-315, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34296387

RESUMO

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging public health tool to understand the spread of Coronavirus Disease 2019 (COVID-19) in communities. The performance of different virus concentration methods and PCR methods needs to be evaluated to ascertain their suitability for use in the detection of SARS-CoV-2 in wastewater. We evaluated ultrafiltration and polyethylene glycol (PEG) precipitation methods to concentrate SARS-CoV-2 from sewage in wastewater treatment plants and upstream in the wastewater network (e.g., manholes, lift stations). Recovery of viruses by different concentration methods was determined using Phi6 bacteriophage as a surrogate for enveloped viruses. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and E). Using spiked samples, the Phi6 recoveries were estimated at 2.6-11.6% using ultrafiltration-based methods and 22.2-51.5% using PEG precipitation. There was no significant difference in recovery efficiencies (p < 0.05) between the PEG procedure with and without a 16 h overnight incubation, demonstrating the feasibility of obtaining same day results. The SARS-CoV-2 genetic markers were more often detected by RT-ddPCR than RT-qPCR with higher sensitivity and precision. While all three SARS-CoV-2 genetic markers were detected using RT-ddPCR, the levels of E gene were almost below the limit of detection using RT-qPCR. Collectively, our study suggested PEG precipitation is an effective low-cost procedure which allows a large number of samples to be processed simultaneously in a routine wastewater monitoring for SARS-CoV-2. RT-ddPCR can be implemented for the absolute quantification of SARS-CoV-2 genetic markers in different wastewater matrices.


Assuntos
Fracionamento Químico/métodos , SARS-CoV-2/isolamento & purificação , Ultrafiltração/métodos , Águas Residuárias/química , Águas Residuárias/virologia , Precipitação Química , Monitoramento Ambiental , Polietilenoglicóis/química , Saúde Pública , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Esgotos/química , Esgotos/virologia , Proteínas Virais/genética , Poluição da Água/análise
17.
Methods Mol Biol ; 2341: 1-7, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264454

RESUMO

Many S. aureus strains produce membrane-associated carotenoid pigments, advantageous secondary metabolites that can alter membrane fluidity, resistance to antimicrobial peptides (AMPs) and act as antioxidants, properties that can impact resistance against aspects of the host innate immune system. Several studies have reported connections between mutations in both regulatory (i.e., alternative sigma factor B) and metabolic (purine biosynthesis, oxidative phosphorylation) genes, and noticeable differences in carotenoid pigmentation. This chapter outlines a simple protocol to quantify cellular pigments using a methanol extraction method.


Assuntos
Carotenoides/isolamento & purificação , Metanol/química , Staphylococcus aureus/crescimento & desenvolvimento , Carotenoides/química , Fracionamento Químico , Regulação Bacteriana da Expressão Gênica , Fluidez de Membrana , Espectrofotometria , Staphylococcus aureus/metabolismo
18.
Ultrason Sonochem ; 76: 105638, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34225213

RESUMO

The aim of the present research was to develop green and sustainable extraction procedure for ß-carotene recovery from pumpkin. A series of hydrophobic natural deep eutectic solvents (NADESs) based onfatty acids were prepared to establish high extraction efficiency of ß-carotene and to increase stability of extracted carotenoids from the pumpkin. To intensify extraction process, NADES composed of C8 and C10 fatty acids (3:1) was selected and coupled with ultrasound assisted extraction. Response surface methodology and artificial neural network model (ANN) model was adopted to analyze significance of extraction parameters demonstrating high prediction levels of the ß-carotene yield, experimentally confirming the maximum ß-carotene content of 151.41 µg/mL at the optimal process condition. Extracted carotenoids in the optimal NADES extract have shown high stability during the storing period of 180 days. A switchable-hydrophilicity eutectic solvent system has been introduced as a successful way to recover extracted carotenoids from the NADES solvent. It was capable of precipitating 90% of carotenoids present in the extract. The proposed procedure is simple, easily scalable and has minimal impact on operators and the environment.


Assuntos
Fracionamento Químico/métodos , Cucurbita/química , Solventes/química , beta Caroteno/isolamento & purificação
19.
Ultrason Sonochem ; 76: 105658, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34242865

RESUMO

Phenolic compounds are secondary metabolites involved in plant adaptation processes. The development of extraction procedures, quantification, and identification of this compounds in habanero pepper (Capsicum chinense) leaves can provide information about their accumulation and possible biological function. The main objective of this work was to study the effect of the UAE method and the polarity of different extraction solvents on the recovery of phenolic compounds from C. chinense leaves. Quantification of the total phenolic content (TPC), antioxidant activity (AA) by ABTS+ and DPPH radical inhibition methods, and the relation between the dielectric constant (ε) as polarity parameter of the solvents and TPC using Weibull and Gaussian distribution models was analyzed. The major phenolic compounds in C. chinense leaves extracts were identified and quantified by UPLC-PDA-ESI-MS/MS. The highest recovery of TPC (24.39 ± 2.41 mg GAE g-1 dry wt) was obtained using MeOH (50%) by UAE method. Correlations between TPC and AA of 0.89 and 0.91 were found for both radical inhibition methods (ABTS+ and DPPH). The Weibull and Gaussian models showed high regression values (0.93 to 0.95) suggesting that the highest phenolic compounds recovery is obtained using solvents with "ε" values between 35 and 52 by UAE. The major compounds were identified as N-caffeoyl putrescine, apigenin, luteolin and diosmetin derivatives. The models presented are proposed as a useful tool to predict the appropriate solvent composition for the extraction of phenolic compounds from C. chinense leaves by UAE based on the "ε" of the solvents for future metabolomic studies.


Assuntos
Antioxidantes/isolamento & purificação , Capsicum/química , Fracionamento Químico/métodos , Fenóis/isolamento & purificação , Folhas de Planta/química , Solventes/química , Ondas Ultrassônicas , Antioxidantes/química , Cromatografia Líquida de Alta Pressão , Fenóis/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
20.
Ultrason Sonochem ; 76: 105661, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34252684

RESUMO

In this work, extraction of flavonoids from peanut shells has been studied in the presence of ultrasound and the results are compared with Soxhlet and heat reflux extraction for establishing the process intensification benefits. The process optimization for understanding the effects of operating parameters, such as ethanol concentration, particle size, solvent to solid ratio, extraction temperature, ultrasonic power and ultrasonic frequency, on the extraction of flavonoids has been investigated in details. The highest extraction yield (9.263 mg/g) of flavonoids was achieved in 80 min at optimum operating parameters of particle size of 0.285 mm, solvent to solid ratio of 40 ml/g, extraction temperature of 55 °C, ultrasonic power of 120 W and ultrasonic frequency of 45 kHz with 70% ethanol as the solvent. Two kinetic models (i.e. phenomenological model and Peleg's model) have been introduced to describe the extraction kinetic of flavonoids by fitting experimental data and predict kinetic parameters. Good performance with slight loss of goodness of fit of two models was found by comparing their coefficient of determination (R2), root mean square error (RMSE) and/or mean percentage error (MPE) values. This work would provide the reduction of degradation and the economic evaluation for the extraction processes of flavonoids from peanut shells, as well as give a better explanation for the mechanism of ultrasound.


Assuntos
Arachis/química , Fracionamento Químico/métodos , Flavonoides/isolamento & purificação , Ondas Ultrassônicas , Cinética
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