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1.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077399

RESUMO

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.


Assuntos
Escherichia coli , DNA de Plantas/genética , Escherichia coli/genética , Organismos Geneticamente Modificados , Fosfotransferases (Aceptor do Grupo Álcool) , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes
2.
Int J Mol Sci ; 23(3)2022 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-35162991

RESUMO

Malaria parasites require multiple phosphorylation and dephosphorylation steps to drive signaling pathways for proper differentiation and transformation. Several protein phosphatases, including protein phosphatase 1 (PP1), one of the main dephosphorylation enzymes, have been shown to be indispensable for the Plasmodium life cycle. The catalytic subunit of PP1 (PP1c) participates in cellular processes via dynamic interactions with a vast number of binding partners that contribute to its diversity of action. In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins. Our findings confirm previously known physical interactions of PP1c and reveal enrichment of common biological processes linked to cellular component assembly in both schizonts and gametocytes to biosynthetic processes/translation in schizonts and to protein transport exclusively in gametocytes. Further, our analysis of PP1c and I2 interactomes revealed that nuclear export mediator factor and peptidyl-prolyl cis-trans isomerase, suggested to be essential in P. falciparum, could be potential targets of the complex PP1c/I2 in both asexual and sexual stages. Our study emphasizes the adaptability of Plasmodium PP1 and provides a fundamental study of the protein interaction landscapes involved in a myriad of events in Plasmodium, suggesting why it is crucial to the parasite and a source for alternative therapeutic strategies.


Assuntos
Malária/parasitologia , Plasmodium berghei/fisiologia , Proteína Fosfatase 1/metabolismo , Proteínas/metabolismo , Proteômica/métodos , Animais , Sítios de Ligação , Cromatografia Líquida , Estágios do Ciclo de Vida , Masculino , Camundongos , Organismos Geneticamente Modificados , Plasmodium berghei/patogenicidade , Domínios Proteicos , Mapas de Interação de Proteínas , Proteína Fosfatase 1/genética , Proteínas/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espectrometria de Massas em Tandem
3.
Sci Rep ; 12(1): 2480, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35169205

RESUMO

Algal lipids are expected to become a basis for sustainable fuels because of the highly efficient lipid production by photosynthesis accompanied by carbon dioxide assimilation. Molecular breeding of microalgae has been studied to improve algal lipid production, but the resultant gene-modified algae containing transgenes are rarely used for outdoor culture because the use of genetically modified organisms (GMOs) is strictly restricted under biocontainment regulations. Recently, it was reported that plasmids containing yeast centromere and autonomous replication sequence (CEN/ARS) behaved as episomes in Nannochloropsis species. We previously reported that the Platinum TALEN (PtTALEN) system exhibited high activity in Nannochloropsis oceanica. Therefore, we attempted to develop a genome editing system in which the expression vectors for PtTALEN can be removed from host cells after introduction of mutations. Using all-in-one PtTALEN plasmids containing CEN/ARS, targeted mutations and removal of all-in-one vectors were observed in N. oceanica, suggesting that our all-in-one PtTALEN vectors enable the construction of mutated N. oceanica without any transgenes. This system will be a feasible method for constructing non-GMO high-performance algae.


Assuntos
Centrômero/genética , Replicação do DNA/genética , Edição de Genes/métodos , Vetores Genéticos , Microalgas/genética , Microalgas/metabolismo , Análise de Sequência de DNA/métodos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Dióxido de Carbono/metabolismo , Estudos de Viabilidade , Metabolismo dos Lipídeos/genética , Organismos Geneticamente Modificados , Plasmídeos , Transgenes
4.
Molecules ; 27(3)2022 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35164011

RESUMO

Acetaldehyde dehydrogenases are potential enzyme preparations that can be used to detoxify acetaldehyde and other exogenous aldehydes from pharmaceuticals, food, and biofuel production. In this study, we enhanced the expression of acetaldehyde dehydrogenase sourced from Issatchenkia terricola (istALDH) in Bacillus subtilis using a combinatorial strategy for the optimization of signal peptides, promoters, and growth conditions. First, a library of various signal peptides was constructed to identify the optimal signal peptides for efficient istALDH secretion. The signal peptide yqzG achieved the highest extracellular istALDH activity (204.85 ± 3.31 U/mL). Second, the aprE promoter was replaced by a constitutive promoter (i.e., P43) and an inducible promoter (i.e., Pglv), resulting in 12.40% and 19.97% enhanced istALDH, respectively. Furthermore, the tandem promoter P43-Pglv provided a better performance, resulting in 30.96% enhanced istALDH activity. Third, the production of istALDH was optimized by testing one factor at a time. Physical parameters were optimized including the inducer (e.g., maltose) concentrations, incubation temperatures, and inoculation amounts, and the results were 2.0%, 35 ∘C, and 2.0%, respectively. The optimized medium results were 2.0% glucose, 1.5% peptone, 2.5% yeast extract, 1% NaCl, and 0.5% (NH4)2SO4. The extracellular istALDH activity was 331.19 ± 4.19 U/mL, yielding the highest production reported in the literature to date.


Assuntos
Aldeído Oxirredutases/metabolismo , Bacillus subtilis/metabolismo , Pichia/enzimologia , Proteínas Recombinantes/metabolismo , Acetaldeído/metabolismo , Aldeído Oxirredutases/genética , Bacillus subtilis/genética , Clonagem Molecular/métodos , Engenharia Metabólica/métodos , Organismos Geneticamente Modificados , Pichia/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/genética , Via Secretória/genética
5.
Sci Rep ; 12(1): 1745, 2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-35110640

RESUMO

Superoxide dismutases are important group of antioxidant metallozyme and play important role in ROS homeostasis in salinity stress. The present study reports the biochemical properties of a salt-tolerant Cu, Zn-superoxide from Avicennia marina (Am_SOD). Am_SOD was purified from the leaf and identified by mass-spectrometry. Recombinant Am_SOD cDNA was bacterially expressed as a homodimeric protein. Enzyme kinetics revealed a high substrate affinity and specific activity of Am_SOD as compared to many earlier reported SODs. An electronic transition in 360-400 nm spectra of Am_SOD is indicative of Cu2+-binding. Am_SOD activity was potentially inhibited by diethyldithiocarbamate and H2O2, a characteristic of Cu, Zn-SOD. Am_SOD exhibited conformational and functional stability at high NaCl concentration as well in alkaline pH. Introgression of Am_SOD in E. coli conferred tolerance to oxidative stress under highly saline condition. Am_SOD was moderately thermostable and retained functional activity at ~ 60 °C. In-silico analyses revealed 5 solvent-accessible N-terminal residues of Am_SOD that were less hydrophobic than those at similar positions of non-halophilic SODs. Substituting these 5 residues with non-halophilic counterparts resulted in > 50% reduction in salt-tolerance of Am_SOD. This indicates a cumulative role of these residues in maintaining low surface hydrophobicity of Am_SOD and consequently high salt tolerance. The molecular information on antioxidant activity and salt-tolerance of Am_SOD may have potential application in biotechnology research. To our knowledge, this is the first report on salt-tolerant SOD from mangrove.


Assuntos
Avicennia , Tolerância ao Sal/fisiologia , Superóxido Dismutase , Avicennia/genética , Avicennia/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Espectrometria de Massas , Organismos Geneticamente Modificados , Estresse Oxidativo/fisiologia , Folhas de Planta/metabolismo , Estresse Salino/fisiologia , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo
6.
GM Crops Food ; 13(1): 26-37, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-35094657

RESUMO

This study explores how the type of information search and information channel can influence the objective knowledge of consumers on genetically modified organisms. We divided the types of information search on genetically modified organisms into active and passive seekers, and then examined how their knowledge differed depending on preferred the information channel (i.e., government, portals, non-government organization (NGO) sites). An online survey was conducted with Korean men and women aged 19 or older. The main and interaction effects of the type of information search, and government, portal, and NGO sites were statistically significant. The results showed that active information seekers who prefer government, portal, and NGO sites have lower scores of knowledge on genetically modified organisms than that of passive information seekers, given the confusion of competing and sometimes inaccurate information sources.


Assuntos
Conhecimento , Organismos Geneticamente Modificados , Adulto , Feminino , Humanos , Masculino , República da Coreia , Inquéritos e Questionários
7.
Public Underst Sci ; 31(6): 732-750, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35086388

RESUMO

We examine stakeholder participation in the online debate on genetically modified organisms in China and assess how the debate has changed over time. Therefore, we compare messages posted between 2013 and 2020 on the Chinese microblog website Weibo by using discourse network analysis. Our findings reveal strong opposition to genetically modified crops, along with the existence of two competing coalitions of supporters and opponents. We further observe an increasing number of posts supporting genetically modified organisms by the public in recent years. Consequently, there is an indication that the positions of stakeholders have changed over time. We discuss the policy implications for China and draw conclusions for other countries.


Assuntos
Produtos Agrícolas , Dissidências e Disputas , China , Organismos Geneticamente Modificados , Plantas Geneticamente Modificadas
8.
Nat Commun ; 13(1): 101, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013170

RESUMO

A Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated.


Assuntos
Corpos Nucleares/genética , Splicing de RNA , RNA Mensageiro/genética , Transcrição Genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Células Cultivadas , Regulação da Expressão Gênica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Corpos Nucleares/metabolismo , Organismos Geneticamente Modificados , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Mensageiro/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
10.
Food Chem ; 366: 130595, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34298393

RESUMO

Herein, a surface-enhanced Raman scattering (SERS)-integrated LFS platform was developed for rapid and simultaneous screening of multiple genetically modified organism (GMO) components (promoter, codon, and terminator) in soybean. Research demonstrated that, on the same test line (T line) of single LFS, three different GMP components can be well distinguished with the help of three SERS nano tags. Good linear correlations between SERS signal and concentration of each GMO component were also obtained for quantitative analysis. Of greater importance, whether these multiple analytes coexisted or not, varied in the same concentration trend or not, these multiple GMP components can be rapidly (15 min) and accurately screened with satisfied sensitivity and specificity by decoding the signals on the same T line. We envision that this decoding platform can further improve the potential of LFS and SERS for practical applications and provide a promising alternative for multiple screening of GMO identification in food.


Assuntos
Análise Espectral Raman , Organismos Geneticamente Modificados , Sensibilidade e Especificidade
11.
Nat Rev Genet ; 23(3): 154-168, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34611352

RESUMO

Modern genome-scale methods that identify new genes, such as proteogenomics and ribosome profiling, have revealed, to the surprise of many, that overlap in genes, open reading frames and even coding sequences is widespread and functionally integrated into prokaryotic, eukaryotic and viral genomes. In parallel, the constraints that overlapping regions place on genome sequences and their evolution can be harnessed in bioengineering to build more robust synthetic strains and constructs. With a focus on overlapping protein-coding and RNA-coding genes, this Review examines their discovery, topology and biogenesis in the context of their genome biology. We highlight exciting new uses for sequence overlap to control translation, compress synthetic genetic constructs, and protect against mutation.


Assuntos
Bioengenharia , Homologia de Genes/fisiologia , Genoma/genética , Animais , Bioengenharia/métodos , Bioengenharia/tendências , Mapeamento Cromossômico , Humanos , Organismos Geneticamente Modificados/genética
12.
Neurotoxicology ; 88: 14-24, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34718060

RESUMO

In a previous in vitro study, dihydropyrimidinone-derived selenoesteres demonstrated antioxidant properties, metal chelators and inhibitory acetylcholinesterase (AChE) activity, making these compounds promising candidates for Alzheimer's Disease (AD) treatment. However, these effects have yet to be demonstrated in an in vivo animal model; therefore, this study aimed to evaluate the safety and efficacy of eight selenoester compounds in a Caenorhabditis elegans model using transgenic strains for amyloid-beta peptide (Aß) aggregation. The L1 stage worms were acutely exposed (30 min) to the compounds at concentrations ranging from 5 to 200 µM and after 48 h the maintenance temperature was increased to 25 ° C for Aß expression and aggregation. After 48 h, several parameters related to phenotypic manifestations of Aß toxicity and mechanistic elucidation were analyzed. At the concentrations tested no significant toxicity of the compounds was found. The selenoester compound FA90 significantly reduced the rate of paralyzed worms and increased the number of swimming movements compared to the untreated worms. In addition, FA90 and FA130 improved egg-laying induced by levamisole and positively modulated HSP-6 and HSP-4 expression, thereby increasing reticular and mitochondrial protein folding response in C. elegans, which could attenuate Aß aggregation in early exposure. Therefore, our initial screening using an alternative model demonstrated that FA90, among the eight selenoesters evaluated, was the most promising compound for AD evaluation screening in more complex animals.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Pirimidinonas/farmacologia , Acetilcolinesterase/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Caenorhabditis elegans , Modelos Animais de Doenças , Levamisol/farmacologia , Fármacos Neuroprotetores/efeitos adversos , Organismos Geneticamente Modificados , Compostos Organosselênicos/efeitos adversos , Oviposição/efeitos dos fármacos , Pirimidinonas/efeitos adversos
13.
J AOAC Int ; 105(2): 476-482, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34927696

RESUMO

BACKGROUND: With the commercialization of genetically modified organisms (GMOs) in the market, laboratories have undergone a significantly increased workload. A universal analytical approach was designed to achieve cost-efficient and high-throughput GMOs screening with high specificity and accuracy. The approach provides accurate qualification of authorized and unauthorized GMOs. OBJECTIVE: This article describes the assessment of this analytical approach developed to detect the majority of commercialized GMOs over the world. METHOD: Seven elements and three events were detected by qPCR in a single laboratory to detect 59 commercialized GMOs. Certificated reference materials and food/feed samples from the Chinese market were also evaluated for the specificity, conformity, and robustness of this approach and were challenged in the interlaboratory study. RESULTS: The results showed that elements and events selected can best detect GMO presence with good specificity and sensitivity. The results showed a concordance between 97.5 and 99.56% and the variance between 0.65 and 12.88%, which is in line with the minimum requirement of analytical methods of GMO testing. CONCLUSIONS: The approach validated here can be used to manipulate GMO presence in food and feed and showed the capacity to manipulate GMO trace in the trade and domestic agriculture market in China. HIGHLIGHTS: A universal analytical approach used to track GMO presence was evaluated for its specificity, sensitivity, and robustness.


Assuntos
Agricultura , Alimentos Geneticamente Modificados , China , Laboratórios , Organismos Geneticamente Modificados/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real
14.
Int J Mol Sci ; 22(21)2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34768859

RESUMO

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain's genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fusarium/metabolismo , Tricotecenos/metabolismo , Cultura Axênica , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Organismos Geneticamente Modificados/genética , Tricotecenos/química
15.
Toxins (Basel) ; 13(11)2021 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-34822538

RESUMO

Multicopper oxidases (MCOs) are a diverse group of enzymes that could catalyze the oxidation of different xenobiotic compounds, with simultaneous reduction in oxygen to water. Aside from laccase, one member of the MCO superfamily has shown great potential in the biodegradation of mycotoxins; however, the mycotoxin degradation ability of other MCOs is uncertain. In this study, a novel MCO-encoding gene, StMCO, from Streptomyces thermocarboxydus, was identified, cloned, and heterologously expressed in Escherichia coli. The purified recombinant StMCO exhibited the characteristic blue color and bivalent copper ion-dependent enzyme activity. It was capable of oxidizing the model substrate ABTS, phenolic compound DMP, and azo dye RB5. Notably, StMCO could directly degrade aflatoxin B1 (AFB1) and zearalenone (ZEN) in the absence of mediators. Meanwhile, the presence of various lignin unit-derived natural mediators or ABTS could significantly accelerate the degradation of AFB1 and ZEN by StMCO. Furthermore, the biological toxicities of their corresponding degradation products, AFQ1 and 13-OH-ZEN-quinone, were remarkably decreased. Our findings suggested that efficient degradation of mycotoxins with mediators might be a common feature of the MCOs superfamily. In summary, the unique properties of MCOs make them good candidates for degrading multiple major mycotoxins in contaminated feed and food.


Assuntos
Aflatoxina B1/metabolismo , Proteínas de Bactérias/metabolismo , Oxirredutases/metabolismo , Streptomyces/enzimologia , Zearalenona/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Lacase/metabolismo , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo
16.
ACS Chem Biol ; 16(11): 2641-2650, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34723462

RESUMO

Filamentous soil bacteria are known to produce diverse specialized metabolites. Despite having enormous potential as a source of pharmaceuticals, they often produce bioactive metabolites at low titers. Here, we show that inactivation of the pactamycin, NFAT-133, and conglobatin biosynthetic pathways in Streptomyces pactum ATCC 27456 significantly increases the production of the mitochondrial electron transport inhibitors piericidins. Similarly, inactivation of the pactamycin, NFAT-133, and piericidin pathways significantly increases the production of the heat-shock protein (Hsp) 90 inhibitor conglobatin. In addition, four new conglobatin analogues (B2, B3, F1, and F2) with altered polyketide backbones, together with the known analogue conglobatin B1, were identified in this mutant, indicating that the conglobatin biosynthetic machinery is promiscuous toward different substrates. Among the new conglobatin analogues, conglobatin F2 showed enhanced antitumor activity against HeLa and NCI-H460 cancer cell lines compared to conglobatin. Conglobatin F2 also inhibits colony formation of HeLa cells in a dose-dependent manner. Molecular modeling studies suggest that the new conglobatins bind to human Hsp90 and disrupt Hsp90/Cdc37 chaperone/co-chaperone interactions in the same manner as conglobatin. The study also showed that genes that are involved in piericidin biosynthesis are clustered in two different loci located distantly in the S. pactum genome.


Assuntos
Organismos Geneticamente Modificados , Streptomyces/metabolismo , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Genes Bacterianos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oxazóis/química , Oxazóis/metabolismo , Oxazóis/farmacologia , Policetídeo Sintases/metabolismo , Policetídeos/química , Ligação Proteica , Streptomyces/genética , Especificidade por Substrato
17.
Front Immunol ; 12: 711876, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34659202

RESUMO

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1-/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1-/- mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected Ifnar1-/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in PbAAma1OVA-infected Ifnar1-/- mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during PbAAma1OVA-infection in Ifnar1-/- mice and thereby are contributing to the protection from ECM.


Assuntos
Encéfalo/imunologia , Eosinófilos/fisiologia , Malária Cerebral/imunologia , Parasitemia/imunologia , Plasmodium berghei , Linfócitos T/imunologia , Animais , Animais não Endogâmicos , Anopheles/parasitologia , Antígenos de Protozoários/imunologia , Movimento Celular , Quimiocina CCL5/análise , Quimiocina CCL5/fisiologia , Citotoxicidade Imunológica , Feminino , Contagem de Leucócitos , Malária Cerebral/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mosquitos Vetores/parasitologia , Organismos Geneticamente Modificados , Ovalbumina , Parasitemia/parasitologia , Fragmentos de Peptídeos , Plasmodium berghei/genética , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptores CCR5/fisiologia , Baço/química , Baço/imunologia
18.
Nat Commun ; 12(1): 6049, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663809

RESUMO

Microalgae can accumulate various carbon-neutral products, but their real-world applications are hindered by their CO2 susceptibility. Herein, the transcriptomic changes in a model microalga, Chlamydomonas reinhardtii, in a high-CO2 milieu (20%) are evaluated. The primary toxicity mechanism consists of aberrantly low expression of plasma membrane H+-ATPases (PMAs) accompanied by intracellular acidification. Our results demonstrate that the expression of a universally expressible PMA in wild-type strains makes them capable of not only thriving in acidity levels that they usually cannot survive but also exhibiting 3.2-fold increased photoautotrophic production against high CO2 via maintenance of a higher cytoplasmic pH. A proof-of-concept experiment involving cultivation with toxic flue gas (13 vol% CO2, 20 ppm NOX, and 32 ppm SOX) shows that the production of CO2-based bioproducts by the strain is doubled compared with that by the wild-type, implying that this strategy potentially enables the microalgal valorization of CO2 in industrial exhaust.


Assuntos
Dióxido de Carbono/metabolismo , Dióxido de Carbono/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Microalgas/genética , Microalgas/metabolismo , Bombas de Próton/genética , Bombas de Próton/metabolismo , Biodegradação Ambiental , Biocombustíveis , Carbono/metabolismo , Chlamydomonas reinhardtii/metabolismo , Tolerância a Medicamentos , Microalgas/crescimento & desenvolvimento , Organismos Geneticamente Modificados , Transcriptoma , Emissões de Veículos
19.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638966

RESUMO

Bacterial non-coding RNAs (ncRNAs) play important regulatory roles in various physiological metabolic pathways. In this study, a novel ncRNA CsiR (ciprofloxacin stress-induced ncRNA) involved in the regulation of ciprofloxacin resistance in the foodborne multidrug-resistant Proteus vulgaris (P. vulgaris) strain P3M was identified. The survival rate of the CsiR-deficient strain was higher than that of the wild-type strain P3M under the ciprofloxacin treatment condition, indicating that CsiR played a negative regulatory role, and its target gene emrB was identified through further target prediction, quantitative real-time PCR (qRT-PCR), and microscale thermophoresis (MST). Further studies showed that the interaction between CsiR and emrB mRNA affected the stability of the latter at the post-transcriptional level to a large degree, and ultimately affected the ciprofloxacin resistance of P3M. Notably, the base-pairing sites between CsiR and emrB mRNAs were highly conserved in other sequenced P. vulgaris strains, suggesting that this regulatory mechanism may be ubiquitous in this species. To the best of our knowledge, this is the first identification of a novel ncRNA involved in the regulation of ciprofloxacin resistance in P. vulgaris species, which lays a solid foundation for comprehensively expounding the antibiotic resistance mechanism of P. vulgaris.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Membrana/metabolismo , Proteus vulgaris/efeitos dos fármacos , Proteus vulgaris/metabolismo , RNA não Traduzido/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Organismos Geneticamente Modificados , Proteus vulgaris/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
20.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445358

RESUMO

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.


Assuntos
Escherichia coli/genética , Engenharia de Proteínas/métodos , Receptores Dopaminérgicos/genética , Calibragem , Membrana Celular/metabolismo , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Mutação , Organismos Geneticamente Modificados , Engenharia de Proteínas/normas , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Bacteriana , Transgenes
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