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1.
Science ; 371(6533): 1033-1037, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674490

RESUMO

Microbial production of antibiotics is common, but our understanding of their roles in the environment is limited. In this study, we explore long-standing observations that microbes increase the production of redox-active antibiotics under phosphorus limitation. The availability of phosphorus, a nutrient required by all life on Earth and essential for agriculture, can be controlled by adsorption to and release from iron minerals by means of redox cycling. Using phenazine antibiotic production by pseudomonads as a case study, we show that phenazines are regulated by phosphorus, solubilize phosphorus through reductive dissolution of iron oxides in the lab and field, and increase phosphorus-limited microbial growth. Phenazines are just one of many examples of phosphorus-regulated antibiotics. Our work suggests a widespread but previously unappreciated role for redox-active antibiotics in phosphorus acquisition and cycling.


Assuntos
Antibacterianos/biossíntese , Fenazinas/metabolismo , Fósforo/metabolismo , Pseudomonas/metabolismo , Técnicas de Cultura Celular por Lotes , Disponibilidade Biológica , Oxirredução , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento
2.
Food Chem ; 347: 129036, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33508589

RESUMO

3-(Methylthio)-1-propanol, reminiscent of cauliflower and cooked vegetable aroma, is an important sulfur compound in Baijiu. It is important to develop a method to increase 3-(methylthio)-1-propanol content to improve flavor quality of products. In this study, a synthetic microbial community was employed to enhance the content of 3-(methylthio)-1-propanol by multi-module division of labor approach. Firstly, the synthetic pathway of 3-(methylthio)-1-propanol was reconstructed and classified into three modules. Later, the hyper producers in each module were isolated and negative interaction between the members was relieved. Finally, a synthetic microbial community was constructed using three species containing one hyper producer from each module. Furthermore, the transcription characteristics of the species in each module were validated by metatranscriptomic analysis. The constructed synthetic microbial community can be used to biosynthesize 3-(methylthio)-1-propanol for Baijiu. This work provided a novel and workable strategy to design synthetic microbial community to enhance the flavor feature of other fermented foods.


Assuntos
Bacillus/metabolismo , Compostos de Enxofre/metabolismo , Bacillus/genética , Técnicas de Cultura Celular por Lotes , Biomassa , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lactobacillus/genética , Lactobacillus/metabolismo , Metionina/análise , Metionina/metabolismo , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA Fúngico/química , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Compostos de Enxofre/química
3.
Food Chem ; 348: 129116, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33508610

RESUMO

ß-Glucan as a component of grain cell walls is consumed daily. However, little is known about whether ß-glucan is influenced by the gastrointestinal environment. In this study, we aim to investigate the integrated metabolic process of cereal ß-glucan. In vitro simulated digestion and fermentation combined with microbiome and metabolome analysis were used to profile the metabolism of ß-glucan. Intriguingly, we found that ß-glucan was not hydrolyzed by digestive enzymes but partially degraded by gastric acid environment during in vitro digestion. Moreover, ß-glucan was utilized by gut microbiota to produce acetate, propionate and butyrate, concurrently, the relative abundance of Lactobacillus significantly increased and Escherichia-Shigella significantly decreased. The correlation analysis between metabolomics datasets and microorganisms revealed that ß-glucan catabolism was also accompanied by amino acid catabolism and linoleic acid biosynthesis. Our study offered a forceful basis for the further exploration of the role of ß-glucan and gut microbiota in host health.


Assuntos
Avena/metabolismo , beta-Glucanas/metabolismo , Animais , Técnicas de Cultura Celular por Lotes , Digestão , Análise Discriminante , Ácido Gástrico/química , Microbioma Gastrointestinal/efeitos dos fármacos , Hidrólise , Lactobacillus/genética , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Análise dos Mínimos Quadrados , Camundongos , Camundongos Endogâmicos C57BL , Análise de Componente Principal , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , beta-Glucanas/química , beta-Glucanas/farmacologia
4.
Chemosphere ; 270: 129461, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33412355

RESUMO

Benzotriazole (BT) is a corrosion inhibitor widely distributed in aquatic environments. Little is known about the cometabolic capacity of stabilized nitrifying sludge to biotransform BT. The contribution of the nitrification process in the simultaneous oxidation of ammonium and biotransformation of BT (5 mg/L) was evaluated in 49 d batch cultures inoculated with a sludge produced in steady-state nitrification. The nitrifying sludge could consume BT in the obligate presence of ammonium. A higher cometabolic biotransformation capacity was obtained by increasing the initial ammonium concentration (100-300 mg N/L), reaching 2.3- and 5.8-fold increases for efficiency and specific rate of BT removal. At 300 mg NH4+-N/L, the sludge biotransform 40.8% of BT and 77.6% of ammonium which was completely oxidized into nitrate. In assays with allylthiourea added as specific inhibitor of ammonium monooxygenase (AMO), it was shown that the totality of BT cometabolic biotransformation was associated with the AMO activity. The addition of acetate did not favor heterotrophic biotransformation of BT. BT provoked inhibitory effects on nitrification. This is the first study showing the role of ammonium oxidizing bacteria in the cometabolic biotransformation of BT and their potential use for cometabolism application in treatment of wastewater contaminated with ammonium and BT.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Biotransformação , Nitrificação , Esgotos , Triazóis
5.
Methods Mol Biol ; 2183: 83-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959242

RESUMO

The small (S) envelope protein of the Hepatitis B Virus (HBV), HBV-S, has the unique ability to self-assemble into highly immunogenic subviral particles (SVPs), in the absence of other viral factors, in eukaryotic cells, including those of nonhepatic origin. This feature is currently exploited for generation of SVPs exposing heterologous epitopes on their surface that can be used as vaccine candidates to target various diseases. Here, we describe a simple and robust method for production of such chimeric HBV-S protein-based SVPs in transiently transfected HEK293T cells and purification from cell supernatants by ultracentrifugation on sucrose cushion and sucrose step gradients. The SVPs obtained by this methodology have been successfully used in immunogenicity studies in animal models.


Assuntos
Técnicas de Cultura Celular por Lotes , Antígenos de Superfície da Hepatite B/biossíntese , Proteínas Recombinantes de Fusão , Animais , Técnicas de Cultura de Células , Expressão Gênica , Células HEK293 , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vacinas contra Hepatite B/biossíntese , Vacinas contra Hepatite B/imunologia , Humanos , Transfecção
6.
Methods Mol Biol ; 2183: 95-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959243

RESUMO

Several vaccines are already produced using the baculovirus expression vector system (BEVS). This chapter describes methods for generating recombinant baculoviral DNA (also called bacmid) for cultivating Spodoptera frugiperda Sf-9 cells and producing a baculovirus stock from the recombinant bacmid and for producing a protein-based vaccine with the BEVS in a stirred tank reactor.


Assuntos
Antígenos/biossíntese , Antígenos/genética , Baculoviridae/genética , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Vetores Genéticos/genética , Proteínas Recombinantes , Animais , Antígenos/isolamento & purificação , Técnicas de Cultura de Células , Clonagem Molecular , Expressão Gênica , Engenharia Genética , Células Sf9 , Transfecção , Fluxo de Trabalho
7.
Methods Mol Biol ; 2183: 183-203, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959245

RESUMO

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused major outbreaks of disease around the world over the last few years. The infectious ZIKV consists of a structural protein outer shell surrounding a nucleocapsid. Virus-like particles (VLP) consist of the outer structural protein shell, but without the nucleocapsid, and are hence noninfectious. VLP, however, are structurally equivalent to the native virus and thus present a similar antigenic profile. These properties make them good candidates for vaccine development. ZIKV VLP can be generated on a laboratory scale by cloning the relevant structural proteins into a eukaryotic expression vector and transfecting the construct into mammalian cells. The secreted VLP can be harvested from the culture medium and purified by sucrose cushion ultracentrifugation. Validation of the VLP is achieved through western blotting and electron microscopy.


Assuntos
Técnicas de Cultura Celular por Lotes , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/imunologia , Zika virus/imunologia , Técnicas de Cultura de Células , Clonagem Molecular , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Plasmídeos/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura
8.
Methods Mol Biol ; 2183: 367-390, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959254

RESUMO

The discovery of the genome-editing tool CRISPR-Cas9 is revolutionizing the world of gene therapy and will extend the gene therapy product pipeline. While applying gene therapy products, the main difficulty is an efficient and effective transfer of the nucleic acids carrying the relevant information to their target destination, the nucleus of the cells. Baculoviruses have shown to be very suitable transport vehicles for this task due to, inter alia, their ability to transduce mammalian/human cells without being pathogenic. This property allows the usage of baculovirus-transduced cells as cell therapy products, thus, combining the advantages of gene and cell therapy. To make such pharmaceuticals available for patients, a successful production and purification is necessary. In this chapter, we describe the generation of a pseudotyped baculovirus vector, followed by downstream processing using depth and tangential-flow filtration. This vector is used subsequently to transduce human mesenchymal stem cells. The production of the cells and the subsequent transduction process are illustrated.


Assuntos
Baculoviridae/genética , Técnicas de Transferência de Genes , Engenharia Genética , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Células-Tronco Mesenquimais/metabolismo , Transdução Genética , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Sobrevivência Celular , Células Cultivadas , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/normas , Humanos , Controle de Qualidade , Fluxo de Trabalho
9.
Food Chem ; 340: 128152, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33032150

RESUMO

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.


Assuntos
Lactobacillus helveticus/metabolismo , Proteínas de Soja/metabolismo , Aminoácidos/análise , Técnicas de Cultura Celular por Lotes , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Lactobacillus helveticus/crescimento & desenvolvimento , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Peptídeos/análise , Peptídeos/metabolismo , Proteólise , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/isolamento & purificação , Proteínas de Armazenamento de Sementes/metabolismo , Proteínas de Soja/química , Proteínas de Soja/isolamento & purificação , Espectrometria de Massas em Tandem , Compostos Orgânicos Voláteis/análise
10.
Methods Mol Biol ; 2234: 113-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33165784

RESUMO

This chapter explains how to perform a batch cultivation of Trichoderma reesei in bench top bioreactors, exemplarily using wheat straw as sole carbon source, and a selection of recommended, frequently used analyses to monitor the cultivation (intra- and extracellular as well), which are microscopic analysis, sodium hydroxide soluble protein, Bradford assay, and GC analysis.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Hypocreales/crescimento & desenvolvimento , Bioensaio , Cromatografia Gasosa , Proteínas Fúngicas/metabolismo , Solubilidade
11.
Food Chem ; 336: 127616, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32763733

RESUMO

This study is an example of apple by-products (AP) recycling through a designed fermentation by selected autochthonous Lactobacillus plantarum AFI5 and Lactobacillus fabifermentans ALI6 used singly or as binary cultures with the selected Saccharomyces cerevisiae AYI7. Compared to Raw-, Unstarted- and Chemically Acidified-AP, Fermented-AP promoted the highest levels of total and insoluble dietary fibers, DPPH scavenging capacity, and free phenolics. The binary culture of L. plantarum AFI5 and S. cerevisiae AYI7 had the best effect on the bioavailability phenolic compounds as resulted by the LC-MS/MS validated method. The accumulation of phenolic acids derivatives highlighted the microbial metabolism during AP fermentation. Bio-converted phenolics were likely responsible for the increased DPPH scavenging capacity. The potential health-promoting effects of Fermented-AP were highlighted using Caco-2 cells. With variations among single and binary cultures, fermented-AP counteracted the inflammatory processes and the effects of oxidative stress in Caco-2 cells, and preserved the integrity of tight junctions.


Assuntos
Suplementos Nutricionais/análise , Malus/química , Fenóis/química , Anti-Inflamatórios/análise , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antioxidantes/química , Técnicas de Cultura Celular por Lotes , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Humanos , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Malus/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/análise , Fenóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem
12.
Nat Commun ; 11(1): 5385, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097726

RESUMO

High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).


Assuntos
Piperidonas/metabolismo , Pseudomonas putida/metabolismo , Biologia Sintética/métodos , Técnicas de Cultura Celular por Lotes , Biomassa , Reatores Biológicos/microbiologia , Carbono/metabolismo , Meios de Cultura , Fermentação , Técnicas de Inativação de Genes , Engenharia Genética , Genoma Bacteriano , Glucose/metabolismo , Microbiologia Industrial , Pseudomonas putida/genética
13.
PLoS One ; 15(10): e0234372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33091058

RESUMO

There arose one of the most important ecological transitions in Earth's history approximately 750 million years ago during the middle Neoproterozoic Era (1000 to 541 million years ago, Ma). Biomarker evidence suggests that around this time there was a rapid shift from a predominantly bacterial-dominated world to more complex ecosystems governed by eukaryotic primary productivity. The resulting 'Rise of the algae' led to dramatically altered food webs that were much more efficient in terms of nutrient and energy transfer. Yet, what triggered this ecological shift? In this study we examined the theory that it was the alleviation of phosphorus (P) deficiency that gave eukaryotic alga the prime opportunity to flourish. We performed laboratory experiments on the cyanobacterium Synechocystis salina and the eukaryotic algae Tetraselmis suecica and examined their ability to compete for phosphorus. Both these organisms co-occur in modern European coastal waters and are not known to have any allelopathic capabilities. The strains were cultured in mono and mixed cultures in chemostats across a range of dissolved inorganic phosphorus (DIP) concentrations to reflect modern and ancient oceanic conditions of 2 µM P and 0.2 µM P, respectively. Our results show that the cyanobacteria outcompete the algae at the low input (0.2 µM P) treatment, yet the eukaryotic algae were not completely excluded and remained a constant background component in the mixed-culture experiments. Also, despite their relatively large cell size, the algae T. suecica had a high affinity for DIP. With DIP input concentrations resembling modern-day levels (2 µM), the eukaryotic algae could effectively compete against the cyanobacteria in terms of total biomass production. These results suggest that the availability of phosphorus could have influenced the global expansion of eukaryotic algae. However, P limitation does not seem to explain the complete absence of eukaryotic algae in the biomarker record before ca. 750 Ma.


Assuntos
Clorófitas/crescimento & desenvolvimento , Fósforo/metabolismo , Synechocystis/crescimento & desenvolvimento , Algoritmos , Fosfatase Alcalina/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Clorofila A/metabolismo , Clorófitas/metabolismo , Meios de Cultura/química , Synechocystis/metabolismo
14.
Int J Food Microbiol ; 335: 108886, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-32950916

RESUMO

Tarhana is produced at batch systems in which the microbiota has changed accordingly to the microbial load from ingredients. In order to stabilize the microbiota, the effects of backslopping carried out under different temperature regimes (25 and 30 °C), pH (3.70 and 4.00) and inoculation rates (5, 10 and 15%) on lactic acid bacteria (LAB) diversity were determined in tarhana dough. LAB and Total Aerobic Mesophilic Bacteria (TAMB) numbers increased in all tarhana dough samples subjected to backslopping. Temperature and pH significantly affected the microbiological diversity of tarhana whereas the different inoculation rates did not. Tarhana dough showed complex tarhana microbiota following backslopping at pH 4.00 independently on the temperature applied. When backslopping was carried out at pH 3.70 and 25 °C, tarhana microbiota stabilized and became steady after several cycles. The LAB species found in all dough samples after the final backslopping were Lactobacillus plantarum, Lactobacillus alimentarius and Lactobacillus brevis which were able to carry out the fermentation in all conditions tested. In order to obtain a stable presence of LAB populations at industrial level for tarhana production, this work showed that backslopping is recommended at pH 3.70 and 25 °C with any inoculation ratios.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Biodiversidade , Alimentos e Bebidas Fermentados/microbiologia , Lactobacillales/isolamento & purificação , Pão/microbiologia , Fermentação , Concentração de Íons de Hidrogênio , Lactobacillales/classificação , Lactobacillales/genética , Lactobacillales/metabolismo , Microbiota , Temperatura
15.
PLoS One ; 15(9): e0239392, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970721

RESUMO

The purpose of the present study was to evaluate yellow mombin (Spondias mombin L.) juice as a vehicle for the Lactobacillus acidophilus NRRL B-4495 probiotic. The initial pH and fermentation temperature conditions were optimized by central composite rotational design. The beverage was evaluated for its chemical composition, bioactive properties, microbiological stability, survival in simulated gastrointestinal conditions and sensory analysis. The ideal conditions for probiotic juice production were an initial pH of 6.4 and 16 h of fermentation, with maximum viability of 12.9 ± 0.4 Log CFU/mL. The fermented juice showed a total phenolic concentration of 94.90 ± 7.12 GAE/mL and antioxidant activity, as measured by DPPH (0.31 ± 0.00 µmol TE/mL) and ABTS sequestration (2.59 ± 0.30 µmol TE/mL). Antibacterial activity could also be observed against S. aureus, E. coli and K. pneumoniae. The obtained formulation showed good microbiological stability when stored at 4ºC for 28 days. In addition, there was no significant change in viability after exposure to simulated gastrointestinal conditions. The sensory analysis showed that the probiotic beverage was not well accepted. However, the Just-About-Right (JAR) ideal scale test enabled identifying the specific attributes which need to be improved from the tasters' point of view so that it is possible to improve product acceptance.


Assuntos
Anacardiaceae/química , Sucos de Frutas e Vegetais/análise , Lactobacillus acidophilus/crescimento & desenvolvimento , Anacardiaceae/metabolismo , Antioxidantes/química , Técnicas de Cultura Celular por Lotes , Escherichia coli/efeitos dos fármacos , Armazenamento de Alimentos , Concentração de Íons de Hidrogênio , Lactobacillus acidophilus/fisiologia , Testes de Sensibilidade Microbiana , Fenóis/química , Fenóis/metabolismo , Fenóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Temperatura
16.
J Biosci Bioeng ; 130(5): 525-532, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32800439

RESUMO

Chinese hamster ovary (CHO) cells are used as host cells for biopharmaceutical production, including monoclonal antibodies (mAbs). Arresting the cell cycle with chemical compounds is an effective approach to improve biopharmaceutical productivity. In a previous study, potential new cell cycle-arresting compounds were screened from marine-derived microorganism culture extracts, and it was suggested that staurosporine might improve mAb productivity in CHO cells via cell cycle arrest. The purpose of this study was to demonstrate the effectiveness of staurosporine as a cell-cycle arresting compound to improve mAb productivity. The optimal staurosporine concentration range was initially investigated using batch cultures. Thereafter, the effects on the culture profile and mAb productivity were evaluated using fed-batch cultures. Staurosporine at concentrations ≥10 nM induced cell death, but at concentrations ≤5 nM did not. In the range of 2-4 nM, cell growth was inhibited, whereas the specific production rate (Qp) and cell longevity were improved in a dose-dependent manner. The Qp and maximum mAb concentration with 4 nM staurosporine improved by 36.3 and 5.2%, respectively, compared to those with control conditions. Cell viability post-culture without staurosporine was 40.0 ± 0.3%, whereas with 4 nM staurosporine, it was 90.1 ± 1.0%. Flow cytometric analysis indicated cell-cycle arrest at the G1/G0 phase with 4 nM staurosporine addition. The present study highlighted the efficacy of staurosporine in improving mAb production by causing cell-cycle arrest. Further research into staurosporine analogs and how to use them will lead to development of more effective industrial production technologies of biopharmaceuticals.


Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas Recombinantes/biossíntese , Estaurosporina/farmacologia , Animais , Técnicas de Cultura Celular por Lotes , Células CHO , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Proteínas Recombinantes/genética
17.
Sci Adv ; 6(30): eaba6884, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32832666

RESUMO

More than 1050 clinical trials are registered at FDA.gov that explore multipotent mesenchymal stromal cells (MSCs) for nearly every clinical application imaginable, including neurodegenerative and cardiac disorders, perianal fistulas, graft-versus-host disease, COVID-19, and cancer. Several companies have or are in the process of commercializing MSC-based therapies. However, most of the clinical-stage MSC therapies have been unable to meet primary efficacy end points. The innate therapeutic functions of MSCs administered to humans are not as robust as demonstrated in preclinical studies, and in general, the translation of cell-based therapy is impaired by a myriad of steps that introduce heterogeneity. In this review, we discuss the major clinical challenges with MSC therapies, the details of these challenges, and the potential bioengineering approaches that leverage the unique biology of MSCs to overcome the challenges and achieve more potent and versatile therapies.


Assuntos
Betacoronavirus , Infecções por Coronavirus/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Pneumonia Viral/terapia , Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Infecções por Coronavirus/virologia , Doença Enxerto-Hospedeiro/terapia , Humanos , Engenharia Metabólica/métodos , Pandemias , Pneumonia Viral/virologia , Transplantados
18.
J Biosci Bioeng ; 130(4): 402-408, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32669208

RESUMO

Aerobic fed-batch cultures were studied as a means of suppressing the production of lactate, which inhibits the growth of lactic acid bacteria (LAB). LAB produce lactate via lactate dehydrogenase (LDH), regenerating nicotinamide adenine dinucleotide (NAD+) consumed during glycolysis. Therefore, we focused on NADH oxidase (NOX), employing oxygen as an electron acceptor, as an alternative pathway to LDH for NAD+ regeneration. To avoid glucose repression of NOX and NAD+ consumption by glycolysis exceeding NAD+ regeneration by NOX, glucose was fed gradually. When Lactococcus lactis MG 1363 was aerobically fed at a specific growth rate of 0.2 h-1, the amount of lactate produced per amount of grown cell was reduced to 12% of that in anaerobic batch cultures. Metabolic flux analysis revealed that in addition to NAD+ regeneration by NOX, ATP acquisition by production of acetate and NAD+ regeneration by production of acetoin and 2,3-butanediol contributed to suppression of lactate production.


Assuntos
Técnicas de Cultura Celular por Lotes , Ácido Láctico/biossíntese , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/metabolismo , Aerobiose , Glucose/metabolismo , Glicólise , L-Lactato Desidrogenase/metabolismo , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo
19.
PLoS One ; 15(6): e0234077, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559199

RESUMO

Geobacter spp. enrichment biofilms were cultivated in batch using one-chamber and two-chamber bioelectrochemical reactors. Time-resolved substrate quantification was performed to derive physiological parameters as well as incremental coulombic efficiency (i.e., coulombic efficiency during one batch cycle, here every 6h) during early stage biofilm development. The results of one-chamber reactors revealed an intermediate acetate increase putatively due to the presence of acetogens. Total coulombic efficiencies of two-chamber reactors were considerable lower (19.6±8.3% and 49.3±13.2% for 1st and 2nd batch cycle, respectively) compared to usually reported values of mature Geobacter spp. enrichment biofilms presumably reflecting energetic requirements for biomass production (i.e., cells and extracellular polymeric substances) during early stages of biofilm development. The incremental coulombic efficiency exhibits considerable changes during batch cycles indicating shifts between phases of maximizing metabolic rates and maximizing biomass yield. Analysis based on Michaelis-Menten kinetics yielded maximum substrate uptake rates (vmax,Ac, vmax,I) and half-saturation concentration coefficients (KM,Ac,KM,I) based on acetate uptake or current production, respectively. The latter is usually reported in literature but neglects energy demands for biofilm growth and maintenance as well as acetate and electron storage. From 1st to 2nd batch cycle, vmax,Ac and KM,Ac, decreased from 0.0042-0.0051 mmol Ac- h-1 cm-2 to 0.0031-0.0037 mmol Ac- h-1 cm-2 and 1.02-2.61 mM Ac- to 0.28-0.42 mM Ac-, respectively. Furthermore, differences between KM,Ac/KM,I and vmax,Ac/vmax,I were observed providing insights into the physiology of Geobacter spp. enrichment biofilms. Notably, KM,I considerably scattered while vmax,Ac/vmax,I and KM,Ac remained rather stable indicating that acetate transport within biofilm only marginally affects reaction rates. The observed data variation mandates the requirement of a more detailed analysis with an improved experimental system, e.g., using flow conditions and a comparison with Geobacter spp. pure cultures.


Assuntos
Biofilmes/crescimento & desenvolvimento , Geobacter/fisiologia , Acetatos/análise , Acetatos/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Cromatografia Líquida de Alta Pressão , Transporte de Elétrons , Geobacter/metabolismo , Cinética
20.
Appl Environ Microbiol ; 86(16)2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32561578

RESUMO

Phloem-limited bacterial "Candidatus Liberibacter" species are associated with incurable plant diseases worldwide. Antimicrobial treatments for these pathogens are challenging due to the difficulty of reaching the vascular tissue they occupy at bactericidal concentrations. Here, in vitro antimicrobial mechanisms of Zinkicide TMN110 (ZnK), a nonphytotoxic zinc oxide (ZnO)-based nanoformulation, were compared to those of bulk ZnO (b-ZnO) using as a model the only culturable species of the genus, Liberibacter crescens Minimum bactericidal concentration (MBC) determination and time-kill assays showed that ZnK has a bactericidal effect against L. crescens, whereas b-ZnO is bacteriostatic. When ZnK was used at the MBC (150 ppm), its antimicrobial mechanisms included an increase in Zn solubility, generation of intracellular reactive oxygen species, lipid peroxidation, and cell membrane disruption; all of these were of greater intensity than those of b-ZnO. Inhibition of biofilms, which are important during insect vector colonization, was stronger by ZnK than by b-ZnO at concentrations between 2.5 and 10 ppm in batch cultures; however, neither ZnK nor b-ZnO removed L. crescens preformed biofilms when applied between 100 and 400 ppm. In microfluidic chambers simulating source-to-sink phloem movement, ZnK significantly outperformed b-ZnO in Zn mobilization and bactericidal activity against L. crescens planktonic cells in sink reservoirs. In microfluidic chamber assays assessing antibiofilm activity, ZnK displayed a significantly enhanced bactericidal activity against L. crescens individual attached cells as well as preformed biofilms compared to that of b-ZnO. The superior mobility and antimicrobial activity of ZnK in microenvironments make this formulation a promising product to control plant diseases caused by "Candidatus Liberibacter" species and other plant vascular pathogens.IMPORTANCE "Candidatus Liberibacter" species are associated with incurable plant diseases that have caused billions of dollars of losses for United States and world agriculture. Chemical control of these pathogens is complicated, because their life cycle combines intracellular vascular stages in plant hosts with transmission by highly mobile insect vectors. To date, "Candidatus Liberibacter" species are mostly unculturable, except for Liberibacter crescens, a member of the genus that has been used as a model for in vitro assays. Here, we evaluated the potential of Zinkicide (ZnK) as an antimicrobial against "Candidatus Liberibacter" species in batch cultures and under flow conditions, using L. crescens as a biological model. ZnK displayed bactericidal activity against L. crescens in batch cultures and showed increased mobility and bactericidal activity in microfluidic devices resembling "Candidatus Liberibacter" species natural habitats. ZnK performance observed here against L. crescens makes this compound a promising candidate to control plant diseases caused by vascular pathogens.


Assuntos
Antibacterianos/farmacologia , Citrus/microbiologia , Nanopartículas Metálicas , Floema/microbiologia , Doenças das Plantas/prevenção & controle , Rhizobiaceae/efeitos dos fármacos , Óxido de Zinco/farmacologia , Técnicas de Cultura Celular por Lotes , Microfluídica , Doenças das Plantas/microbiologia
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