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1.
Braz. j. biol ; 83: e245202, 2023. tab, graf
Artigo em Inglês | MEDLINE, LILACS, VETINDEX | ID: biblio-1285622

RESUMO

Abstract Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 μg/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 μg/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 μg/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.


Resumo Embora a própolis tenha sido relatada por ter atividade anti-inflamatória, seus efeitos no sistema complemento, uma parte do sistema imunológico inato, não foram muito estudados. Esta pesquisa foi conduzida para descobrir os efeitos da própolis da Indonésia nos níveis de expressão de C3, C1r/s, Bf, MBL e C6 em larvas de peixe-zebra induzidas por lipopolissacarídeo (LPS). Foram realizadas contagens de macrófagos que migram para o saco vitelino e histologia do fígado. As larvas foram divididas em quatro grupos: CON (cultivadas apenas em meio E3), LPS (cultivadas em meio contendo 0,5 μg/L de LPS), LPSIBU (cultivadas em meio contendo LPS e, em seguida, tratadas com 100 μg/L de ibuprofeno por 24 horas) e LPSPRO (cultivado em meio contendo LPS, e então imerso em própolis 14,000 μg/L por 24 horas). Os resultados mostraram que a expressão do gene do complemento em larvas dos grupos LPSIBU e LPSPRO foi geralmente menor que em larvas do grupo LPS. O número de migrações de macrófagos para a gema no grupo LPSPRO também foi menor que no grupo LPS. A estrutura histológica do fígado em todos os grupos foi considerada normal. Este estudo mostra que a própolis indonésia tem potencial para ser utilizada como alternativa na substituição dos AINEs (anti-inflamatórios não esteroides).


Assuntos
Animais , Própole/farmacologia , Peixe-Zebra/genética , Regulação para Baixo , Lipopolissacarídeos/farmacologia , Indonésia , Larva/genética
2.
Methods Mol Biol ; 2489: 115-127, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524048

RESUMO

Fungal natural products have extensive biological activities, and thus have been largely commercialized in the pharmaceutical, agricultural, and food industries. Recently, heterologous expression has become an irreplaceable technique to functionalize fungal biosynthetic gene clusters and synthesize fungal natural products in various chassis organisms. This chapter describes the general method of using Pichia pastoris as a chassis host to investigate fungal biosynthetic pathways.


Assuntos
Produtos Biológicos , Saccharomycetales , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Proteínas Fúngicas/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
3.
Methods Mol Biol ; 2477: 129-147, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524116

RESUMO

Mapping the epigenome is key to describe the relationship between chromatin landscapes and the control of DNA-based cellular processes such as transcription. Cleavage under targets and release using nuclease (CUT&RUN) is an in situ chromatin profiling strategy in which controlled cleavage by antibody-targeted Micrococcal Nuclease solubilizes specific protein-DNA complexes for paired-end DNA sequencing. When applied to budding yeast, CUT&RUN profiling yields precise genome-wide maps of histone modifications, histone variants, transcription factors, and ATP-dependent chromatin remodelers, while avoiding cross-linking and solubilization issues associated with the most commonly used chromatin profiling technique Chromatin Immunoprecipitation (ChIP). Furthermore, targeted chromatin complexes cleanly released by CUT&RUN can be used as input for a subsequent native immunoprecipitation step (CUT&RUN.ChIP) to simultaneously map two epitopes in single molecules genome-wide. The intrinsically low background and high resolution of CUT&RUN and CUT&RUN.ChIP allows for identification of transient genomic features such as dynamic nucleosome-remodeling intermediates. Starting from cells, one can perform CUT&RUN or CUT&RUN.ChIP and obtain purified DNA for sequencing library preparation in 2 days.


Assuntos
Epigenoma , Saccharomycetales , Cromatina , Imunoprecipitação da Cromatina , DNA/genética , Endonucleases/genética , Nuclease do Micrococo , Nucleossomos/genética , Saccharomycetales/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-35507404

RESUMO

The species Blastobotrys navarrensis Sesma and Ramirez was delineated based on the description of the single strain CBS 139.77T. Based on its phenotypic similarities to Blastobotrys proliferans, B. navarrensis CBS 139.77T was later considered a synonym of B. proliferans. In the present study, we isolated the yeast strain IST 508 (=PYCC 8784=CBS 16671) from the soil surrounding an olive tree in Ferreira do Alentejo, Portugal. The phylogenetic analysis of D1/D2 domain and ITS sequences from strain IST 508 indicates that is closely related to B. navarrensis and B. proliferans. Although strain IST 508 differs from B. navarrensis CBS 139.77T by 14 substitutions and 20 indels (6.6 % divergence) in the ITS sequence, no divergence was detected at the level of D1/D2 domain, mitochondrial small subunit rDNA, and cytochrome oxidase II sequences. On the other hand, strains IST 508 and CBS 139.77 differ from B. proliferans NRRL Y-17577T by eight substitutions (1.4 % divergence) in the D1/D2 domain sequence, by 16 substitutions (2.7 % divergence) in the cytochrome oxidase II sequence, and by 16 substitutions (3.7 % divergence) in the mitochondrial small subunit rDNA sequence. Due to the high number of variable phenotypic tests in B. proliferans and B. navarrensis, strains from the two species are difficult to distinguish. Contrasting with what is described for other Blastobotrys species, no differences were detected at the level of micromorphology between the two species. Nevertheless, based on the molecular differences between the two strains, CBS 139.77 and IST 508, and B. proliferans NRRL Y-17577T and their phylogenetic analysis, strains CBS 139.77 and IST 508 are from B. navarrensis and this species should be considered as an independent species and not a later synonym of B. proliferans. We propose an emended description of B. navarrensis.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Saccharomycetales , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , DNA Fúngico/genética , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Ácidos Graxos/química , Técnicas de Tipagem Micológica , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
5.
Vopr Pitan ; 91(2): 72-80, 2022.
Artigo em Russo | MEDLINE | ID: mdl-35596637

RESUMO

Whey and hen egg white proteins are characterized by high nutritional value, but possess antigenic properties, which limit their use in the production of dietary products. Enzymatic hydrolysis decreases significantly the allergenicity of proteins. The efficiency of hydrolysis depends on the specificity of the proteases used. The aim of this work was to determine the effectiveness of EP-96 enzyme preparation obtained from Bacillus subtilis-96 culture liquid in the hydrolysis of whey and egg white proteins in comparison with commercial bacterial proteases preparations - Alcalase, Neutrase, and Protosubtilin. Material and methods. Whey and egg white protein concentrates were used as substrates. Commercial enzyme preparations Alcalase, Neutrase, and Protosubtilin, and an experimental sample of EP-96 preparation obtained from Bacillus subtilis-96 culture liquid were used for hydrolysis. Hydrolysis was carried out at a substrate concentration of 100 g/L for 3 h at 55 °C or for 24 h at 50 °C. After hydrolysis, the reaction mixture was incubated at 90 °C for 15 min to inactivate the enzymes. The content of peptides with a molecular weight of less than 10 kDa was determined in the obtained hydrolysates. The hydrolysis of the main allergenic proteins was assessed by the disappearance of the corresponding protein bands on the hydrolysate supernatants electrophoregrams. Results and discussion. All the studied preparations showed high efficiency in the hydrolysis of whey proteins and provided the yield of low molecular weight peptides at the level of 18.8-22.8% after 3 h of hydrolysis and 39.4-41.6% after 24 h. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a residual amount of protein with a molecular weight of about 14 kDa, corresponding to α-lactoalbumin, after 3 h of hydrolysis when using Neutrase. The preparations containing serine protease, including EP-96, provided more intensive hydrolysis of whey proteins. In the hydrolysis of egg white protein, Neutrase showed the greatest efficiency. The efficiency of EP-96 was comparable to Neutrase both in the yield of low molecular weight peptides and in the intensity of cleavage of the main allergenic proteins. The effectiveness of preparations with predominant content of serine proteases - Alcalase and Protosubtilin was significantly lower. Conclusion. The optimal ratio of neutral and serine proteases in the EP-96, obtained on the basis of the B. subtilis-96 strain, provided the high efficiency and its versatility in the hydrolysis of the main allergenic proteins of whey and egg white. The parameters of the hydrolysis technology using EP-96 are recommended, which provide intensive conversion of the main immunogenic proteins of whey and egg white to soluble and low molecular weight fractions (duration 3 h at a temperature of 55 °C and the proteolytic activity of the preparation is not less than 2 units per g of substrate) and an increase of subsequent ultrafiltration efficiency in the production of protein hydrolysates for foods for special dietary uses.


Assuntos
Bacillus subtilis , Soro do Leite , Bacillus subtilis/metabolismo , Proteínas do Ovo/análise , Hidrólise , Peptídeos/análise , Peptídeos/química , Hidrolisados de Proteína/análise , Subtilisinas/análise , Subtilisinas/metabolismo , Soro do Leite/química , Soro do Leite/metabolismo , Proteínas do Soro do Leite/análise
6.
Probiotics Antimicrob Proteins ; 14(3): 595-601, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35451745

RESUMO

Mycocins are substances that have the potential to affect other sensitive yeasts or microorganisms. Wickerhamomyces anomalus is a yeast that produces mycocins that have great biotechnological potential, being highly competitive in many habitats, as it is adaptable to a wide range of environmental conditions. Thus, they are targets for studies in different areas, including the environment, industry, and medical sciences. Yeasts of the genus Candida are of great importance due to the high frequency with which they colonize and infect the human host. Yeast infections are often difficult to treat due to the acquisition of resistance against antifungals, leading to studies focusing in new treatment alternatives. This work aims to verify the inhibition of Candida albicans isolated from vaginal secretion by mycocins produced by Wickerhamomyces anomalus. Tests were carried out in solid medium and microdilution tests, where mycocins proved to be efficient in inhibiting the growth of C. albicans, hemolysis, and irritation in an organotypic model, which showed that the mycocins produced by W. anomalus are safe and non-irritating. Thus, the results of this work can provide scientific evidence for the application of mycocins in the production of new antifungal alternatives.


Assuntos
Candida albicans , Saccharomycetales , Antifúngicos/farmacologia , Candida , Feminino , Humanos , Leveduras
7.
PLoS One ; 17(4): e0266035, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35421110

RESUMO

In meiosis, cells undergo two sequential rounds of cell division, termed meiosis I and meiosis II. Textbook models of the meiosis I substage called pachytene show that nuclei have conspicuous 100-nm-wide, ladder-like synaptonemal complexes and ordered chromatin loops. It remains unknown if these cells have any other large, meiosis-related intranuclear structures. Here we present cryo-ET analysis of frozen-hydrated budding yeast cells before, during, and after pachytene. We found no cryo-ET densities that resemble dense ladder-like structures or ordered chromatin loops. Instead, we found large numbers of 12-nm-wide triple-helices that pack into ordered bundles. These structures, herein called meiotic triple helices (MTHs), are present in meiotic cells, but not in interphase cells. MTHs are enriched in the nucleus but not enriched in the cytoplasm. Bundles of MTHs form at the same timeframe as synaptonemal complexes (SCs) in wild-type cells and in mutant cells that are unable to form SCs. These results suggest that in yeast, SCs coexist with previously unreported large, ordered assemblies.


Assuntos
Saccharomycetales , Cromatina , Meiose , Saccharomyces cerevisiae , Complexo Sinaptonêmico
8.
Appl Environ Microbiol ; 88(9): e0029622, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35435711

RESUMO

Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were EPX1, PAS_chr3_0030, SCW10, and UTH1, suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the PAS_chr3_0030 gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the PAS_chr3_0030 gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris. IMPORTANCE Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the PAS_chr3_0030 gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the PAS_chr3_0030 gene product may facilitate the application of P. pastoris in industrial biocatalysis.


Assuntos
Sinais Direcionadores de Proteínas , Saccharomycetales , Pichia/genética , Pichia/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
9.
World J Microbiol Biotechnol ; 38(6): 99, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35482161

RESUMO

The halophilic yeast Debaryomyces hansenii has been studied for several decades, serving as eukaryotic model for understanding salt and osmotic tolerance. Nevertheless, lack of consensus among different studies is found and, sometimes, contradictory information derived from studies performed in very diverse conditions. These two factors hampered its establishment as the key biotechnological player that was called to be in the past decade. On top of that, very limited (often deficient) engineering tools are available for this yeast. Fortunately Debaryomyces is again gaining momentum and recent advances using highly instrumented lab scale bioreactors, together with advanced -omics and HT-robotics, have revealed a new set of interesting results. Those forecast a very promising future for D. hansenii in the era of the so-called green biotechnology. Moreover, novel genetic tools enabling precise gene editing on this yeast are now available. In this review, we highlight the most recent developments, which include the identification of a novel gene implicated in salt tolerance, a newly proposed survival mechanism for D. hansenii at very high salt and limiting nutrient concentrations, and its utilization as production host in biotechnological processes.


Assuntos
Debaryomyces , Saccharomycetales , Biotecnologia , Debaryomyces/genética , Amigos , Humanos , Saccharomyces cerevisiae , Saccharomycetales/genética
10.
Gene ; 830: 146500, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35472624

RESUMO

Yolk formation in liver is an important process for egg production in hens. The correlations between egg laying rate decline and liver function changes in Guangxi Ma chickens remain unclear. In this study, a total of 21,750 genes and 76,288 transcripts were identified in the RNA expression profiles isolated from liver tissues of 5 groups of Guangxi Ma chickens divided according to the age and egg laying rate. Numerous differential genes (DEGs) were identified after pairwise comparison among samples, and time series analysis categorization (age-related factors) revealed that down-regulated DEGs with aging were predominantly involved in lipid transportation and metabolic processes in the low egg laying rate groups. Notably, functional enrichment analysis confirmed that DGAT2, LIPG, PNPLA2, LPL, CEL, LIPC, DGKD, AGPAT2, AGPAT1 and AGPAT3 were highlighted as hub genes in glycerolipid metabolism pathway, which may be an essential non-age related factors of egg laying rate by regulating the synthesis of triacylglycerol (TAG) in liver. Finally, we categorized DEGs in Guangxi Ma chickens with different egg laying rate caused by age-related factors and found that DEGs with different expression patterns performing different biological functions. The analysis of DEGs with lower egg laying rate caused by non-age related factors and showed that the transportation of TAG was suppressed. Furthermore, critical genes and pathways involved in the synthesis of TAG in livers were identified, which dynamically regulated the formation of yolk precursors. Our results expanded the knowledge of the molecular mechanisms of the yolk precursor synthesis in chicken livers. The results will be helpful to explore the factors that affect egg laying rate from the perspective of yolk synthesis and provide a theoretical basis for improving the egg production of Guangxi Ma chickens.


Assuntos
Galinhas , Oviposição , Ração Animal/análise , Animais , Galinhas/genética , Galinhas/metabolismo , China , Dieta , Gema de Ovo/metabolismo , Feminino , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Triglicerídeos/metabolismo
11.
Commun Biol ; 5(1): 292, 2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35361876

RESUMO

Microbial drug resistance is an emerging global challenge. Current drug resistance assays tend to be simplistic, ignoring complexities of resistance manifestations and mechanisms, such as multicellularity. Here, we characterize multicellular and molecular sources of drug resistance upon deleting the AMN1 gene responsible for clumping multicellularity in a budding yeast strain, causing it to become unicellular. Computational analysis of growth curve changes upon drug treatment indicates that the unicellular strain is more sensitive to four common antifungals. Quantitative models uncover entwined multicellular and molecular processes underlying these differences in sensitivity and suggest AMN1 as an antifungal target in clumping pathogenic yeasts. Similar experimental and mathematical modeling pipelines could reveal multicellular and molecular drug resistance mechanisms, leading to more effective treatments against various microbial infections and possibly even cancers.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
12.
Food Chem ; 387: 132850, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35381417

RESUMO

This study investigated the effect of dry heat on the aggregation characteristics of egg white protein (EWP) solutions. Turbidity and size-exclusion chromatography (SEC) results showed that dry heat hindered the subsequent bulk aggregation of dry heated EWP (DEWP) in aqueous solutions. A similar trend was also found in the particle size distribution of DEWP aggregates, where longer dry heating times resulted in smaller size distributions. Transmission electron microscopy (TEM) observations showed that native EWP tended to form bulk aggregates when heated in aqueous solutions, while DEWP aggregates presented linear and chain-like morphology. The α-helices of DEWP aggregates were converted to ß-sheets, and more chromogenic amino acids and hydrophobic groups were exposed owing to dry heat. Heat-induced aggregation of DEWP in water is more intense than that induced only by dry heat, but the hydrothermal aggregates with longer dry heating times were smaller in size and molecular weight.


Assuntos
Proteínas do Ovo , Temperatura Alta , Proteínas do Ovo/química , Calefação , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Água
13.
J Food Sci ; 87(5): 2009-2017, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35411557

RESUMO

Herein, the water and lipid migration of salted duck eggs during storage were systematically explored in three different packaging conditions of long-term salting, no packaging, and vacuum packaging. Bound water, multilayer bound water, lipid, and bulk water were observed in the whole duck egg by low-field nuclear magnetic resonance (LF-NMR) relaxation. Five weeks of salting process led to the redistribution of water and lipid due to the watery state of egg white and the gelation of egg yolk due to the permeation of salt, and boiling mainly caused an obvious decrease in the mobility of bulk water due to the gelation of egg white. Among these three conditions, long-term salting with 6 months storage caused the most serious redistribution of water and lipid as well as the rupture of the vitelline membrane, but could prevent the oxidation of egg yolk. Vacuum packaging had the least influence on the water and lipid distribution, mass change, and water content but led to lipid oxidation with high degree in egg yolk. However, the most obvious mass loss was observed in the salted duck eggs during the storage without packaging. In addition, principal component analysis of Carr-Purcell-Meiboom-Gill data suggested that LF-NMR could distinguish the salted duck eggs with different storage times during the early stage of the storage. Practical Application Water and lipid migration of salted duck eggs during storage with three packaging conditions were explored by using low-field nuclear magnetic resonance and magnetic resonance imaging. Understanding the impacts of packaging conditions on water and lipid migration of salted duck eggs during storage could provide a new method for the quality identification.


Assuntos
Patos , Água , Animais , Gema de Ovo/química , Ovos/análise , Lipídeos/análise , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Cloreto de Sódio/análise , Água/análise
14.
Cells ; 11(8)2022 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-35455932

RESUMO

Precise DNA replication is pivotal for ensuring the accurate inheritance of genetic information. To avoid genetic instability, each DNA fragment needs to be amplified only once per cell cycle. DNA replication in eukaryotes starts with the binding of the origin recognition complex (ORC) to the origins of DNA replication. The genes encoding ORC subunits have been conserved across eukaryotic evolution and are essential for the initiation of DNA replication. In this study, we conducted an extensive physiological and aging-dependent analysis of heterozygous cells lacking one copy of ORC genes in the BY4743 background. Cells with only one copy of the ORC genes showed a significant decrease in the level of ORC mRNA, a delay in the G1 phase of the cell cycle, and an extended doubling time. Here, we also show that the reducing the levels of Orc1-6 proteins significantly extends both the budding and average chronological lifespans. Heterozygous ORC/orcΔ and wild-type diploid cells easily undergo haploidization during chronological aging. This ploidy shift might be related to nutrient starvation or the inability to survive under stress conditions. A Raman spectroscopy analysis helped us to strengthen the hypothesis of the importance of lipid metabolism and homeostasis in aging.


Assuntos
Complexo de Reconhecimento de Origem , Saccharomycetales , Cromossomos/metabolismo , Replicação do DNA/genética , Eucariotos/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
15.
STAR Protoc ; 3(2): 101298, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35463467

RESUMO

Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1-5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).


Assuntos
Aquaporinas , Saccharomycetales , Aquaporinas/genética , Humanos , Pichia/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
16.
Microb Cell Fact ; 21(1): 70, 2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468837

RESUMO

BACKGROUND: The yeast genus Komagataella currently consists of seven methylotrophic species isolated from tree environments. Well-characterized strains of K. phaffii and K. pastoris are important hosts for biotechnological applications, but the potential of other species from the genus remains largely unexplored. In this study, we characterized 25 natural isolates from all seven described Komagataella species to identify interesting traits and provide a comprehensive overview of the genotypic and phenotypic diversity available within this genus. RESULTS: Growth tests on different carbon sources and in the presence of stressors at two different temperatures allowed us to identify strains with differences in tolerance to high pH, high temperature, and growth on xylose. As Komagataella species are generally not considered xylose-utilizing yeasts, xylose assimilation was characterized in detail. Growth assays, enzyme activity measurements and 13C labeling confirmed the ability of K. phaffii to utilize D-xylose via the oxidoreductase pathway. In addition, we performed long-read whole-genome sequencing to generate genome assemblies of all Komagataella species type strains and additional K. phaffii and K. pastoris isolates for comparative analysis. All sequenced genomes have a similar size and share 83-99% average sequence identity. Genome structure analysis showed that K. pastoris and K. ulmi share the same rearrangements in difference to K. phaffii, while the genome structure of K. kurtzmanii is similar to K. phaffii. The genomes of the other, more distant species showed a larger number of structural differences. Moreover, we used the newly assembled genomes to identify putative orthologs of important xylose-related genes in the different Komagataella species. CONCLUSIONS: By characterizing the phenotypes of 25 natural Komagataella isolates, we could identify strains with improved growth on different relevant carbon sources and stress conditions. Our data on the phenotypic and genotypic diversity will provide the basis for the use of so-far neglected Komagataella strains with interesting characteristics and the elucidation of the genetic determinants of improved growth and stress tolerance for targeted strain improvement.


Assuntos
Saccharomycetales , Xilose , Carbono/metabolismo , Fenótipo , Pichia/metabolismo , Saccharomycetales/genética , Xilose/metabolismo , Leveduras
17.
Biosensors (Basel) ; 12(4)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35448306

RESUMO

The increasing levels of environmental estrogens are causing negative effects on water, soil, wildlife, and human beings; label-free immunosensors with high specificities and sensitivities are being developed to test estrogeneous chemicals in complex environmental conditions. For the first time, highly fluorescent graphene quantum dots (GQDs) were prepared using a visible-Fenton catalysis reaction with graphene oxide (GO) as a precursor. Different microscopy and spectroscopy techniques were employed to characterize the physical and chemical properties of the GQDs. Based on the fluorescence resonance energy transfer (FRET) between amino-functionalized GQDs conjugated with anti-lipovitellin monoclonal antibodies (Anti-Lv-mAb) and reduced graphene oxide (rGO), an ultrasensitive fluorescent "ON-OFF" label-free immunosensor for the detection of lipovitellin (Lv), a sensitive biomarker derived from Paralichthys olivaceus for environmental estrogen, has been established. The immunosensor has a wide linear test range (0.001-1500 ng/mL), a lower limit of detection (LOD, 0.9 pg/mL), excellent sensitivity (26,407.8 CPS/(ng/mL)), and high selectivity and reproducibility for Lv quantification. The results demonstrated that the visible-Fenton is a simple, mild, green, efficient, and general approach to fabricating GQDs, and the fluorescent "ON-OFF" immunosensor is an easy-to-use, time-saving, ultrasensitive, and accurate detection method for weak estrogenic activity.


Assuntos
Técnicas Biossensoriais , Grafite , Pontos Quânticos , Técnicas Biossensoriais/métodos , Proteínas do Ovo , Grafite/química , Humanos , Imunoensaio/métodos , Pontos Quânticos/química , Reprodutibilidade dos Testes
18.
Comput Intell Neurosci ; 2022: 1266332, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35378803

RESUMO

With the rapid development of the computer field in recent years, a series of major breakthroughs have been made in the field of computer vision. The key technologies in image feature recognition, face recognition, image understanding, pattern recognition, and machine learning have been rapidly applied and developed. The research and application of this field provide efficient and convenient means. However, for traditional physical and chemical experimental research, parameter adjustment is time-consuming and costly. In response to the phenomenon, this article starts with the study of the characteristics of the egg white protein thermal gelation image and explores the extraction of external features presented by the optimal parameters of the coagulation image under the thermal coagulation state of the egg white protein, based on the classic PCA and ICA-image feature extraction algorithm and its improved algorithm, respectively. Experiment and simulation research on several image feature extraction algorithms under different egg white solidification states are carried out, and the efficient recognition method and accuracy of the image under the optimal egg white protein thermal gelation state are discussed. It has important reference significance for the research of optimal image feature extraction in the future high-efficiency experimental research.


Assuntos
Interpretação de Imagem Assistida por Computador , Reconhecimento Automatizado de Padrão , Algoritmos , Simulação por Computador , Proteínas do Ovo , Interpretação de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos
19.
Genetics ; 220(4)2022 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-35380658

RESUMO

The Alliance of Genome Resources (the Alliance) is a combined effort of 7 knowledgebase projects: Saccharomyces Genome Database, WormBase, FlyBase, Mouse Genome Database, the Zebrafish Information Network, Rat Genome Database, and the Gene Ontology Resource. The Alliance seeks to provide several benefits: better service to the various communities served by these projects; a harmonized view of data for all biomedical researchers, bioinformaticians, clinicians, and students; and a more sustainable infrastructure. The Alliance has harmonized cross-organism data to provide useful comparative views of gene function, gene expression, and human disease relevance. The basis of the comparative views is shared calls of orthology relationships and the use of common ontologies. The key types of data are alleles and variants, gene function based on gene ontology annotations, phenotypes, association to human disease, gene expression, protein-protein and genetic interactions, and participation in pathways. The information is presented on uniform gene pages that allow facile summarization of information about each gene in each of the 7 organisms covered (budding yeast, roundworm Caenorhabditis elegans, fruit fly, house mouse, zebrafish, brown rat, and human). The harmonized knowledge is freely available on the alliancegenome.org portal, as downloadable files, and by APIs. We expect other existing and emerging knowledge bases to join in the effort to provide the union of useful data and features that each knowledge base currently provides.


Assuntos
Bases de Dados Genéticas , Alelos , Animais , Caenorhabditis elegans/genética , Bases de Dados Genéticas/normas , Drosophila/genética , Ontologia Genética , Humanos , Internet , Camundongos/genética , Anotação de Sequência Molecular , Ratos/genética , Saccharomycetales/genética , Peixe-Zebra/genética
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