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1.
Talanta ; 238(Pt 1): 122985, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34857320

RESUMO

Numerous chemicals of unknown inhalational toxicity have been measured in electronic cigarette, or vaping, products (EVPs). In addition, little is known about the liquid-to-aerosol transmission and deliveries of these chemicals, including oil-like terpenes such as squalene (SQE) and squalane (SQA). To provide information on the aerosol deliveries of these compounds from EVPs, we developed and validated a quantitative method to measure squalene and squalane in EVP aerosol emissions. Validation parameters include measurement repeatability (SQA and SQE %RSD <6%), intermediate precision (SQA: %RSD 11%, SQE: %RSD 17%), accuracy (SQA: 86-107%, SQE: 104-113%), matrix effects, method robustness, and analyte stability. Limits of detection were 6.06 ng/mL puffed air volume for both squalene and squalane. The method was used to measure squalene and squalane in aerosol emissions of 153 EVPs associated with case patients from a recent outbreak of e-cigarette, or vaping, product use associated lung injury (EVALI). The EVPs analyzed were organized into nicotine, cannabidiol, and tetrahydrocannabinol products by the percentage of nicotine, cannabidiol, and tetrahydrocannabinol in total particulate matter after vaping. In case-associated tetrahydrocannabinol products the detection rates and mean concentrations were 82.4% and 33.0 ng/mL puffed air for squalene and 4.41% and 7.80 ng/mL puffed air for squalane.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Vaping , Aerossóis , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Esqualeno/análogos & derivados
2.
J Agric Food Chem ; 70(1): 229-237, 2022 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-34955018

RESUMO

The study aims to enhance ß-amyrin production in Saccharomyces cerevisiae by peroxisome compartmentalization. First, overaccumulated squalene was determined as a key limiting factor for the production of ß-amyrin since it could inhibit the activity of ß-amyrin synthase GgbAs1. Second, to mitigate the inhibition effect, the enhanced squalene synthesis pathway was compartmentalized into peroxisomes to insulate overaccumulated squalene from GgbAs1, and thus the specific titer of ß-amyrin reached 57.8 mg/g dry cell weight (DCW), which was 2.6-fold higher than that of the cytosol engineering strain. Third, by combining peroxisome compartmentalization with the "push-pull-restrain" strategy (ERG1 and GgbAs1 overexpression and ERG7 weakening), the production of ß-amyrin was further increased to 81.0 mg/g DCW (347.0 mg/L). Finally, through fed-batch fermentation in a 5 L fermenter, the titer of ß-amyrin reached 2.6 g/L, which is the highest reported to date. The study provides a new perspective to engineering yeasts as a platform for triterpene production.


Assuntos
Saccharomyces cerevisiae , Esqualeno , Transferases Intramoleculares , Engenharia Metabólica , Ácido Oleanólico/análogos & derivados , Saccharomyces cerevisiae/genética
3.
J Med Virol ; 94(1): 119-130, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34403141

RESUMO

This study investigates the effect of the nanostructure of squalene in the form of microemulsion on COVID-19 patients. In this blinded clinical trial, a comparison was made between the efficacy of squalene treatment and controls. A total of 30 COVID-19 patients admitted to the emergency department, and the infection ward was equally allocated to case (n = 15) and control (n = 15) groups according to their age and underlying diseases. The baseline characteristics of subjects, including age, gender, time of treatment onset, underlying condition, white blood cells count, and lymphocyte count were similar (p < 0.05). Baseline laboratory tests and computed tomography (CT) scans were performed for the study groups. The treatment group received 5 mg of intravenous squalene twice a day and standard treatment for 6 days, while controls received only standard treatment. After 6 days of treatment, clinical and CT scan changes were evaluated and compared in intervention and control groups. The need for oxygen therapy (p = 0.020), 2 days of no fever (p = 0.025), cough alleviation (p = 0.010), and lung high-resolution computed tomography improvement (p = 0.033) were significantly different between cases and controls within 7 days of admission. No adverse effects were observed in the treatment group. Our data suggest that squalene could be considered as a potential treatment for COVID-19, and further studies are required to confirm the results.


Assuntos
COVID-19/tratamento farmacológico , Esqualeno/uso terapêutico , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Antivirais/química , Antivirais/uso terapêutico , Emulsões , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óleos Vegetais/química , Esqualeno/administração & dosagem , Esqualeno/efeitos adversos , Esqualeno/química , Resultado do Tratamento
4.
Food Chem ; 368: 130819, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34411865

RESUMO

Squalene and ß-sitosterol are health-benefit compounds due to their nutritional and medicinal properties. It has been reported that the content of these bioactive compounds is relatively high in Torreya grandis nuts. However, it is not yet known what changes in squalene and ß-sitosterol accumulation occur during the special post-ripening process of T. grandis nuts and the effect of the well-known ripening hormone ethylene on the regulatory mechanism of their biosynthetic pathways. Thus, we performed transcriptome and metabolite analyses. The results showed that ethylene not only promoted the post-ripening process but also enhanced the accumulation of squalene by inducing gene expression in the mevalonate pathway. At the same time, ethylene treatment also promoted the accumulation of other sterols but inhibited gene expression in the ß-sitosterol biosynthesis pathway. In addition, co-expression and correlation analysis suggested a framework for the transcriptional regulation of squalene and ß-sitosterol biosynthesis genes under ethylene treatment.


Assuntos
Nozes , Taxaceae , Etilenos , Frutas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Sitosteroides , Esqualeno
5.
AAPS PharmSciTech ; 22(8): 270, 2021 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-34766215

RESUMO

Poor immune responses to inactivated influenza vaccine can be improved by effective and safe adjuvants to increase antibody titers and cellular protective response. In our study, AddaVax and PolyI:C combined adjuvant (AP adjuvant) were used for influenza vaccine development. After immunizing BALB/c mice and Wistar rats intramuscularly, Split inactivated H3N2 vaccine adjuvanted with AP elicited higher serum hemagglutination-inhibition antibodies and IgG titers. We demonstrated that AP induced a transient innate immune cytokines production at the injection site, induced H3N2 uptake by DCs, increased recruitment of monocytes and DCs in LNs, and promoted H3N2 vaccine migration; AP facilitated vaccines to induce a vigorous adaptive immune response. Besides, AP showed good safety as shown by lymph nodes (LNs) size, spleens index of BALB/c mice, and weight changes and C-reaction protein level of BALB/c mice and Wistar rats after repeated administration of high-dose vaccine with or without adjuvant. These findings indicate that AP is a potential novel adjuvant and can be used as a safe and effective adjuvant for MDCK-based influenza inactivated vaccine to induce cellular and antibody protective response.


Assuntos
Vacinas contra Influenza , Infecções por Orthomyxoviridae , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais , Imunidade , Vírus da Influenza A Subtipo H3N2 , Camundongos , Camundongos Endogâmicos BALB C , Polissorbatos , Ratos , Ratos Wistar , Esqualeno
6.
J Agric Food Chem ; 69(42): 12474-12484, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34662105

RESUMO

Transcriptional downregulation is widely used for metabolic flux control. Here, marO, a cis-element of Escherichia coli mar operator, was explored to engineer promoters of Saccharomyces cerevisiae for downregulation. First, the ADH1 promoter (PADH1) and its enhanced variant PUADH1 were engineered by insertion of marO into different sites, which resulted in decrease in both gfp5 transcription and GFP fluorescence intensity to various degrees. Then, marO was applied to engineer the native ERG1 and ERG11 promoters due to their importance for accumulation of value-added intermediates squalene and lanosterol. Elevated squalene content (4.9-fold) or lanosterol content (4.8-fold) and 91 or 28% decrease in ergosterol content resulted from the marO-engineered promoter PERG1(M5) or PERG11(M3), respectively, indicating the validity of the marO-engineered promoters in metabolic flux control. Furthermore, squalene production of 3.53 g/L from cane molasses, a cheap and bulk substrate, suggested the cost-effective and promising potential for squalene production.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação para Baixo , Ergosterol , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esqualeno
7.
Molecules ; 26(19)2021 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-34641552

RESUMO

This work is a continuation of efforts to establish the nutritional profile of virgin olive oil (VOO) from cv. Chondrolia Chalkidikis and Chalkidiki and to strengthen its positioning in the global VOO landscape. VOOs produced at an industrial scale in different olive mills of the Chalkidiki (Greece) regional unit as well as VOOs obtained at the laboratory scale from drupes of different maturity stages for four consecutive harvesting years were examined for their squalene (SQ) content using both HPLC and GC procedures. The mean values of SQ were found to be 4228 (HPLC) and 4865 (GC) mg/kg oil (n = 15) and were of the same magnitude as that in VOOs from cv Koroneiki (4134 mg/kg, n = 23). Storage of VOOs in the dark at room temperature for 18 months indicated an insignificant mean SQ content loss (~2%) in comparison to a mean loss of 26% for alpha-tocopherol content. This finding strengthens our view that SQ does not act as a radical scavenger that donates hydrogen atoms to the latter. The four consecutive harvest years studied indicated a clear declining trend in VOO SQ concentration upon olive ripening. To our knowledge, this is the first systematic work concerning the SQ content of Chondrolia Chalkidikis and Chalkidiki VOOs.


Assuntos
Azeite de Oliva/química , Esqualeno/análise , Grécia , Valor Nutritivo , Olea/química , Olea/classificação
8.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638551

RESUMO

Different approaches have been reported to enhance penetration of small drugs through physiological barriers; among them is the self-assembly drug conjugates preparation that shows to be a promising approach to improve activity and penetration, as well as to reduce side effects. In recent years, the use of drug-conjugates, usually obtained by covalent coupling of a drug with biocompatible lipid moieties to form nanoparticles, has gained considerable attention. Natural products isolated from plants have been a successful source of potential drug leads with unique structural diversity. In the present work three molecules derived from natural products were employed as lead molecules for the synthesis of self-assembled nanoparticles. The first molecule is the cytotoxic royleanone 7α-acetoxy-6ß-hydroxyroyleanone (Roy, 1) that has been isolated from hairy coleus (Plectranthus hadiensis (Forssk.) Schweinf). ex Sprenger leaves in a large amount. This royleanone, its hemisynthetic derivative 7α-acetoxy-6ß-hydroxy-12-benzoyloxyroyleanone (12BzRoy, 2) and 6,7-dehydroroyleanone (DHR, 3), isolated from the essential oil of thicket coleus (P. madagascariensis (Pers.) Benth.) were employed in this study. The royleanones were conjugated with squalene (sq), oleic acid (OA), and/or 1-bromododecane (BD) self-assembly inducers. Roy-OA, DHR-sq, and 12BzRoy-sq conjugates were successfully synthesized and characterized. The cytotoxic effect of DHR-sq was previously assessed on three human cell lines: NCI-H460 (IC50 74.0 ± 2.2 µM), NCI-H460/R (IC50 147.3 ± 3.7 µM), and MRC-5 (IC50 127.3 ± 7.3 µM), and in this work Roy-OA NPs was assayed against Vero-E6 cells at different concentrations (0.05, 0.1, and 0.2 mg/mL). The cytotoxicity of DHR-sq NPs was lower when compared with DHR alone in these cell lines: NCI-H460 (IC50 10.3 ± 0.5 µM), NCI-H460/R (IC50 10.6 ± 0.4 µM), and MRC-5 (IC5016.9 ± 0.5 µM). The same results were observed with Roy-OA NPs against Vero-E6 cells as was found to be less cytotoxic than Roy alone in all the concentrations tested. From the obtained DLS results, 12BzRoy-sq assemblies were not in the nano range, although Roy-OA NP assemblies show a promising size (509.33 nm), Pdl (0.249), zeta potential (-46.2 mV), and spherical morphology from SEM. In addition, these NPs had a low release of Roy at physiological pH 7.4 after 24 h. These results suggest the nano assemblies can act as prodrugs for the release of cytotoxic lead molecules.


Assuntos
Abietanos/química , Abietanos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Animais , Linhagem Celular , Chlorocebus aethiops , Humanos , Hidrocarbonetos Bromados/química , Ácido Oleico/química , Extratos Vegetais/química , Plectranthus/química , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacologia , Esqualeno/química , Testes de Toxicidade Aguda/métodos , Células Vero
9.
Metab Eng ; 68: 232-245, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34710614

RESUMO

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.


Assuntos
Saccharomyces cerevisiae , Esqualeno , Acetilcoenzima A , Engenharia Metabólica , Mitocôndrias/genética , Saccharomyces cerevisiae/genética
10.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2813-2824, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472299

RESUMO

Squalene is widely used in pharmaceutical, nutraceutical, cosmetics and other fields because of its strong antioxidative, antibacterial and anti-tumor activities. In order to produce squalene, a gene ispA encoding farnesyl pyrophosphate synthase was overexpressed in a previously engineered Escherichia coli strain capable of efficiently producing terpenoids, resulting in a chassis strain that efficiently synthesizes triterpenoids. Through phylogenetic analysis, screening, cloning and expression of squalene synthase derived from different prokaryotes, engineered E. coli strains capable of efficiently producing squalene were obtained. Among them, squalene produced by strains harboring squalene synthase derived from Thermosynechococcus elongatus and Synechococcus lividus reached (16.5±1.4) mg/g DCW ((167.1±14.3) mg/L broth) and (12.0±1.9) mg/g DCW ((121.8±19.5) mg/L broth), respectively. Compared with the first-generation strains harboring the human-derived squalene synthase, the squalene synthase derived from T. elongatus and S. lividus remarkably increased the squalene production by 3.3 times and 2.4 times, respectively, making progress toward the cost-effective heterologous production of squalene.


Assuntos
Esqualeno , Synechococcus , Clonagem Molecular , Escherichia coli/genética , Humanos , Filogenia
11.
Biomater Sci ; 9(19): 6623-6640, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34582532

RESUMO

Biocompatible hydrophilic polyethylene glycol (PEG) is widely used in biomedical applications, such as drug or gene delivery, tissue engineering or as an antifouling component in biomedical devices. Experimental studies have shown that the size of PEG can weaken polycation-polyanion interactions, like those between branched polyethyleneimine (b-PEI) and DNA in gene carriers, but details of its cause and underlying interactions on the atomic scale are still not clear. To better understand the interaction mechanisms in the formation of polyplexes between b-PEI-PEG based carriers and DNA, we have used a combination of in silico tools and experiments on three multicomponent systems differing in PEG MW. Using the PEI-PEG-squalene-dsDNA systems of the same size, both in the all-atom MD simulations and in experimental in-gel electrophoresis measurements, we found that the binding between DNA and the vectors is highly influenced by the size of PEG, with the binding efficiency increasing with a shorter PEG length. The mechanism of how PEG interferes with the binding between PEI and DNA is explained using a two-step MD simulation protocol that showed that the DNA-vector interactions are influenced by the PEG length due to the hydrogen bond formation between PEI and PEG. Although computationally demanding we find it important to study molecular systems of the same size both in silico and in a laboratory and to simulate the behaviour of the carrier prior to the addition of bioactive molecules to understand the molecular mechanisms involved in the formation of the polyplex.


Assuntos
Polietilenoglicóis , Esqualeno , Simulação por Computador , DNA , Tamanho da Partícula , Polietilenoimina , Transfecção
12.
Langmuir ; 37(40): 11900-11908, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34585578

RESUMO

Hopanoids are proposed as sterol surrogates in some bacteria, and it has been proved that some hopanoids are able to induce a liquid-order phase state in lipid membranes. The members of this group of molecules have diverse structures, and not all of them have been studied in detail yet. Here, we study membranes with the hopanoid hopene (hop-22 (29)-ene or diploptene), which is the product of the cycling of squalene by squalene-hopene cyclase, and thus is present in the first step of hopanoid biosynthesis. Hopene is particularly interesting because it lacks a polar head group, which opens the question of how does this molecule accommodate in a lipid membrane, and what are the effects promoted by its presence. In order to get an insight into this, we prepared monolayers and bilayers of a phospholipid with hopene and studied their properties in comparison with pure phospholipid membranes, and with the sterol cholesterol or the hopanoid diplopterol. Film stiffness, shear viscosity, and bending dynamics were very affected by the presence of hopene, while zeta-potential, generalized polarization of Laurdan, and conductivity were affected moderately by this molecule. The results suggest that at very low percentages, hopene locates parallel to the phospholipid molecules, while the excess of the hopene molecules stays between leaflets, as previously proposed using molecular dynamics simulations.


Assuntos
Triterpenos , Bactérias , Membranas , Esqualeno , Esteróis
13.
Int J Pharm ; 609: 121117, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34562556

RESUMO

Small interfering RNAs (siRNA) are attractive and powerful tools to inhibit the expression of a targeted gene. However, their extreme hydrophilicities combined with a negative charge and short plasma half-life counteract their use as therapeutics. Previously, we chemically linked siRNA to squalene (SQ) which self-assembled as nanoparticles (NPs) with pharmacological efficiency in cancers and recently in a hereditary neuropathy. In order to understand the siRNA-SQ NP assembly and fate once intravenously injected, the present study detailed characterization of siRNA-SQ NP structure and its interaction with serum components. From SAXS and SANS analysis, we propose that the siRNA-SQ bioconjugate self-assembled as 11-nm diameter supramolecular assemblies, which are connected one to another to form spherical nanoparticles of around 130-nm diameter. The siRNA-SQ NPs were stable in biological media and interacted with serum components, notably with albumin and LDL. The high specificity of siRNA to decrease or normalize gene expression and the high colloidal stability when encapsulated into squalene nanoparticles offer promising targeted therapy with wide applications for pathologies with gene expression dysregulation.


Assuntos
Nanopartículas , RNA Interferente Pequeno , Espalhamento a Baixo Ângulo , Esqualeno , Difração de Raios X
14.
Front Immunol ; 12: 692151, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335601

RESUMO

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos Virais/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas Combinadas/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Ilhas de CpG , Cães , Feminino , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Esqualeno/administração & dosagem , Vacinas Sintéticas/administração & dosagem
16.
Vaccine ; 39(38): 5351-5357, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34393015

RESUMO

Vaccination is the most effective approach to reduce the substantial morbidity and mortality caused by influenza infection. Vaccine efficacy is highly sensitive to antigenic changes causing differences between circulating and vaccine viruses. Adjuvants such as MF59 increase antibody-mediated cross-reactive immunity and therefore may provide broader seasonal protection. A recent clinical trial showed that an MF59-adjuvanted vaccine was more efficacious than a nonadjuvanted comparator in subjects < 2 years of age, although not in those ≥ 2 years, during influenza seasons in which the predominant circulating virus was an A/H3N2 strain that was antigenically different from the vaccine virus. This finding suggested that the increased efficacy of the adjuvanted vaccine in younger subjects may be mediated by strain cross-reactive antibodies. A subset of the trial population, representing subjects with distinct age and/or immunological history, was tested for antibody responses to the vaccine A/H3N2 strain as well as A/H3N2 drifted strains antigenically matching the viruses circulating during the trial seasons. The neutralizing tests showed that, compared with nonadjuvanted vaccine, the adjuvanted vaccine improved not only the neutralizing antibody response to the vaccine strain but also the cross-reactive antibody response to the drifted strains in subjects with lower preexisting antibody titers, regardless of their age or vaccine history. The results demonstrated an immunological benefit and suggested a potential efficacy benefit by adjuvanted vaccine in subjects with lower preexisting antibody responses.


Assuntos
Vacinas contra Influenza , Influenza Humana , Anticorpos Antivirais , Formação de Anticorpos , Criança , Pré-Escolar , Humanos , Vírus da Influenza A Subtipo H3N2 , Influenza Humana/prevenção & controle , Polissorbatos , Esqualeno
17.
Int J Pharm ; 607: 121024, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34416331

RESUMO

Vaccination is regarded as the most effective intervention for controlling the coronavirus disease 2019 (COVID-19) pandemic. The objective of this study is to provide comprehensive information on lipid squalene nanoparticle (SQ@NP)-adjuvanted COVID-19 vaccines regarding modulating immune response and enhancing vaccine efficacy. After being adjuvanted with SQ@NP, the SARS-CoV-2 spike (S) subunit protein was intramuscularly (i.m.) administered to mice. Serum samples investigated by ELISA and virus neutralizing assay showed that a single-dose SQ@NP-adjuvanted S-protein vaccine can induce antigen-specific IgG and protective antibodies comparable with those induced by two doses of nonadjuvanted protein vaccine. When the mice received a boosting vaccine injection, anamnestic response was observed in the groups of adjuvanted vaccine. Furthermore, the secretion of cytokines in splenocytes, such as interferon (IFN)-γ, interleukin (IL)-5 and IL-10, was significantly enhanced after adjuvantation of S-protein vaccine with SQ@NP; however, this was not the case for the vaccine adjuvanted with conventional aluminum mineral salts. Histological examination of injection sites showed that the SQ@NP-adjuvanted vaccine was considerably well tolerated following i.m. injection in mice. These results pave the way for the performance tuning of optimal vaccine formulations against COVID-19.


Assuntos
COVID-19 , Nanopartículas , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais , Vacinas contra COVID-19 , Humanos , Lipídeos , Camundongos , SARS-CoV-2 , Esqualeno
18.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200063

RESUMO

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.


Assuntos
Archaea/fisiologia , Membrana Celular/fisiologia , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fluidez de Membrana , Esqualeno/análogos & derivados , Archaea/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Esqualeno/farmacologia , Temperatura
19.
J Biotechnol ; 338: 20-30, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34237394

RESUMO

Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as food-additives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic α-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production. Recently, we have developed modular tools allowing to engineer the biosynthesis of terpene precursors. These tools were now applied to boost the biosynthesis of the diterpene casbene, the triterpene squalene and the tetraterpene ß-carotene in R. capsulatus SB1003. Selected enzymes of the intrinsic isoprenoid pathway and the heterologous mevalonate (MVA) pathway were co-expressed together with the respective terpene synthases in various combinations. Remarkably, co-expression of genes ispA, idi and dxs enhanced the synthesis of casbene and ß-carotene. In contrast, co-expression of precursor biosynthetic genes with the squalene synthase from Arabidopsis thaliana reduced squalene titers. Therefore, we further employed four alternative pro- and eukaryotic squalene synthases. Here, the synthase from Methylococcus capsulatus enabled highest product levels of 90 mg/L squalene upon co-expression with ispA. In summary, we demonstrate the applicability of R. capsulatus for the heterologous production of diverse terpene classes and provide relevant insights for further development of such platforms.


Assuntos
Rhodobacter capsulatus , Triterpenos , Ácido Mevalônico , Rhodobacter capsulatus/genética , Esqualeno , Terpenos
20.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 2105-2115, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227297

RESUMO

Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.


Assuntos
Esqualeno Mono-Oxigenase , Esqualeno , Animais , Escherichia coli/genética , Proteínas de Escherichia coli , NADPH-Ferri-Hemoproteína Redutase , Engenharia de Proteínas , Ratos , Proteínas Repressoras , Esqualeno Mono-Oxigenase/genética
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