RESUMEN
BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.
Asunto(s)
Aminoácidos Diaminos , Halomonas , Cloruro de Sodio/farmacología , Cloruro de Sodio/metabolismo , Halomonas/genética , Halomonas/metabolismo , Presión Osmótica , Perfilación de la Expresión Génica , Peroxidasas/metabolismoRESUMEN
Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.
Asunto(s)
Acetatos , Carthamus tinctorius , Ciclopentanos , Oxilipinas , Transferasas , Transferasas/metabolismo , Ácido Clorogénico/metabolismo , Carthamus tinctorius/genética , Simulación del Acoplamiento Molecular , Transcriptoma , Nucleotidiltransferasas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model "difficult-to-express" virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90ß, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other "difficult-to-express" virus capsid proteins in the mammalian cell system.
Asunto(s)
Proteínas de la Cápside , Circovirus , Porcinos , Animales , Humanos , Circovirus/genética , Células HEK293 , Cápside/metabolismo , Proteínas Recombinantes/genética , Anticuerpos Antivirales , MamíferosRESUMEN
Ectoine is generally produced by the fermentation process of Halomonas elongata DSM 2581 T, which is one of the primary industrial ectoine production techniques. To effectively monitor and control the fermentation process, the important parameters require accurate real-time measurement. However, for ectoine fermentation, three critical parameters (cell optical density, glucose, and product concentration) cannot be measured conveniently in real-time due to time variation, strong coupling, and other constraints. As a result, our work effectively created a series of hybrid models to predict the values of these three parameters incorporating both fermentation kinetics and machine learning approaches. Compared with the traditional machine learning models, our models solve the problem of insufficient data which is common in fermentation. In addition, a simple kinetic modeling is only applicable to specific physical conditions, so different physical conditions require refitting the function, which is tedious to operate. However, our models also overcome this limitation. In this work, we compared different hybrid models based on 5 feature engineering methods, 11 machine-learning approaches, and 2 kinetic models. The best models for predicting three key parameters, respectively, are as follows: CORR-Ensemble (R2: 0.983 ± 0.0, RMSE: 0.086 ± 0.0, MAE: 0.07 ± 0.0), SBE-Ensemble (R2: 0.972 ± 0.0, RMSE: 0.127 ± 0.0, MAE: 0.078 ± 0.0), and SBE-Ensemble (R2:0.98 ± 0.0, RMSE: 0.023 ± 0.001, MAE: 0.018 ± 0.001). To verify the universality and stability of constructed models, we have done an experimental verification, and its results showed that our proposed models have excellent performance. KEY POINTS: ⢠Using the kinetic models for producing simulated data ⢠Through different feature engineering methods for dimension reduction ⢠Creating a series of hybrid models to predict the values of three parameters in the fermentation process of Halomonas elongata DSM 2581 T.
Asunto(s)
Aminoácidos Diaminos , Halomonas , Halomonas/genética , Halomonas/metabolismo , FermentaciónRESUMEN
Viral infections cause endoplasmic reticulum (ER) stress and subsequently unfolded protein response (UPR) which restores ER homeostasis. In this study, levels of proteins or transcription of three UPR pathways were examined in suspension-cultured BHK-21 cells to investigate Pseudorabies virus (PRV) infection-induced ER stress, in which glucose-related proteins 78 kD and 94 kD (GRP78 and GRP94) were upregulated. The downstream double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway was activated with upregulation of ATF4, CHOP, and GADD34, and the inositol requiring kinase 1 (IRE1) pathway was triggered by the splicing of X box-binding protein 1 (XBP1) mRNA and the enhanced expression of p58IPK and EDEM. Furthermore, our results showed that the ER stress, induced by 0.005 µM thapsigargin, promoted PRV replication in suspension-cultured BHK-21 cells, and that PRV glycoprotein B (gB) overexpression triggered the PERK and IRE1 pathways.
Asunto(s)
Herpesvirus Suido 1 , Animales , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/metabolismo , Retículo Endoplásmico/genética , Respuesta de Proteína Desplegada , Estrés del Retículo Endoplásmico/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The "design-build-test-learn" (DBTL) cycle has been adopted in rational high-throughput screening to obtain high-yield industrial strains. However, the mismatch between build and test slows the DBTL cycle due to the lack of high-throughput analytical technologies. In this study, a highly efficient, accurate, and noninvasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. First, a self-made tool was established to obtain data sets in 24-well plates based on the color of the cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with an R2 value of 0.98430 for the same batch. Finally, a stable genetically high-yield strain (998 U/mL) was successfully screened out from 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new data sets were updated on the model database in order to improve the learning ability of the DBTL cycle.
Asunto(s)
Gentamicinas , Ensayos Analíticos de Alto Rendimiento , Reactores Biológicos , Computadores , Aprendizaje AutomáticoRESUMEN
Orange peel waste (OPW), a discarded part of orange fruit, is a rich source of essential constituents that can be transformed into highly value-added bioproducts. OPW is being generated in million tonnes globally and returns to the environment without complete benefit. Thus, a high volume of annually produced OPW in the industry requires effective valorization. In this regard, limited data is available that summarizes the broader spectrum for the sustainable fate of OPW to produce value-added bioproducts. The main objective of this treatise is to explore the sustainable production of bioproducts from OPW. Therefore, this review covers all the aspects of OPW, from its production to complete valorization. The review encompasses the extraction technologies employed for extracting different valuable bioactive compounds, such as: essential oil (EO), pectin, and carotenoids, from OPW. Furthermore, the suitability of bioconversion technologies (digestion/fermentation) in transforming OPW to other useful bioproducts, such as: biochemicals (lactic acid and succinic acid), biopolysaccharides (xanthan and curdlan gum), and bioenergy (biomethane and bioethanol) is discussed. Also, it includes the concept of OPW-based biorefineries and their development that shall play a definite role in future to cover demands for: food, chemicals, materials, fuels, power, and heat. Lastly, this review focuses on OPW-supplemented functional food products such as: beverages, yogurts, and extruded products. In conclusion, insights provided in this review maximize the potential of OPW for commercial purposes, leading to a safe, and waste-free environment.
Asunto(s)
Citrus sinensis , Aceites Volátiles , Residuos , PectinasRESUMEN
The halophilic bacterium Halomonas elongata DSM 2581T generally adapts well to high level of salinity by biosynthesizing ectoine, which functions as an important compatible solute protecting the cell against external salinity environment. Halophilic bacteria have specific metabolic activities under high-salt conditions and are gradually applied in various industries. The present study focuses on investigating the physiological and metabolic mechanism of H. elongata DSM 2581T driven by the external salinity environment. The physiological metabolic dynamics under salt stress were investigated to evaluate the effect of NaCl stress on the metabolism of H. elongata. The obtained results demonstrated that ectoine biosynthesis transited from a nongrowth-related process to a growth-related process when the NaCl concentration varied from 1% to 13% (w/v). The maximum biomass (Xm = 41.37 g/L), and highest ectoine production (Pm = 12.91 g/L) were achieved under 8% NaCl. Moreover, the maximum biomass (Xm ) and the maximum specific growth rates (µm ) showed a first rising and then declining trend with the increased NaCl stress. Furthermore, the transcriptome analysis of H. elongata under different NaCl concentrations demonstrated that both 8% and 13% NaCl conditions resulted in increased expressions of genes involved in the pentose phosphate pathway, Entner-Doudoroff pathway, flagellar assembly pathway, and ectoine metabolism, but negatively affected the tricarboxylic acid cycle and fatty acid metabolism. At last, the proposed possible adaptation mechanism under the optimum NaCl concentration in H. elongata was described.
Asunto(s)
Halomonas , Cloruro de Sodio/metabolismoRESUMEN
BACKGROUND: Magnetic materials mediated by mechanical forces to combat cancer cells are currently attracting attention. Firstly, the magnetic force penetrates deeper into tissues than the NIR laser alone to destroy tumours. Secondly, the synergistic effect of nano-magnetic-material characteristics results in a viable option for the targeted killing of cancer cells. Therefore, mechanical force (MF) produced by magnetic nanomaterials under low frequency dynamic magnetic field combined with laser technology is the most effective, safe and efficient tool for killing cancer cells and tumour growth. RESULTS: In this study, we synthesized novel urchin-like hollow magnetic microspheres (UHMMs) composed of superparamagnetic Fe3O4. We demonstrated the excellent performance of UHMMs for killing laryngocarcinoma cancer cells through mechanical force and photothermal effects under a vibrating magnetic field and near-infrared laser, respectively. The killing efficiency was further improved after loading the synthesised UHMMs with Chlorin e6 relative to unloaded UHMMs. Additionally, in animal experiments, laryngocarcinoma solid tumour growth was effectively inhibited by UHMMs@Ce6 through magneto-mechanic force, photothermal and photodynamic therapy. CONCLUSIONS: The biocompatibility and high efficiency of multimodal integrated therapy with the UHMMs prepared in this work provide new insights for developing novel nano therapy and drug loading platforms for tumour treatment. In vivo experiments further demonstrated that UHMMs/Ce6 are excellent tools for strongly inhibiting tumour growth through the above-mentioned characteristic effects.
Asunto(s)
Neoplasias , Fotoquimioterapia , Animales , Línea Celular Tumoral , Supervivencia Celular , Fenómenos Magnéticos , Microesferas , Neoplasias/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
The rapid, accurate and noninvasive detection of biomass and plant cell browning can provide timely feedback on cell growth in plant cell culture. In this study, Siraitia grosvenorii suspension cells were taken as an example, a phenotype analysis platform was successfully developed to predict the biomass and the degree of cell browning based on the color changes of cells in computer-aided vision technology. First, a self-made laboratory system was established to obtain images. Then, matrices were prepared from digital images by a self-developed high-throughput image processing tool. Finally, classification models were used to judge different cell types, and then a semi-supervised classification to predict different degrees of cell browning. Meanwhile, regression models were developed to predict the plant cell mass. All models were verified with a good agreement by biological experiments. Therefore, this method can be applied for low-cost biomass estimation and browning degree quantification in plant cell culture.
Asunto(s)
Técnicas de Cultivo de Célula , Cucurbitaceae/citología , Cucurbitaceae/metabolismo , Procesamiento de Imagen Asistido por Computador , Aprendizaje Automático , Células Vegetales/metabolismoRESUMEN
BACKGROUND: Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. RESULTS: In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. CONCLUSIONS: This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.
Asunto(s)
Oxitetraciclina/biosíntesis , Streptomyces rimosus/genética , Familia de Multigenes , Streptomyces rimosus/metabolismoRESUMEN
Lincomycin A is a clinically important antimicrobial agent produced by Streptomyces lincolnensis In this study, a new regulator designated LmbU (GenBank accession no. ABX00623.1) was identified and characterized to regulate lincomycin biosynthesis in S. lincolnensis wild-type strain NRRL 2936. Both inactivation and overexpression of lmbU resulted in significant influences on lincomycin production. Transcriptional analysis and in vivo neomycin resistance (Neor) reporter assays demonstrated that LmbU activates expression of the lmbA, lmbC, lmbJ, and lmbW genes and represses expression of the lmbK and lmbU genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that LmbU can bind to the regions upstream of the lmbA and lmbW genes through the consensus and palindromic sequence 5'-CGCCGGCG-3'. However, LmbU cannot bind to the regions upstream of the lmbC, lmbJ, lmbK, and lmbU genes as they lack this motif. These data indicate a complex transcriptional regulatory mechanism of LmbU. LmbU homologues are present in the biosynthetic gene clusters of secondary metabolites of many other actinomycetes. Furthermore, the LmbU homologue from Saccharopolyspora erythraea (GenBank accession no. WP_009944629.1) also binds to the regions upstream of lmbA and lmbW, which suggests widespread activity for this regulator. LmbU homologues have no significant structural similarities to other known cluster-situated regulators (CSRs), which indicates that they belong to a new family of regulatory proteins. In conclusion, the present report identifies LmbU as a novel transcriptional regulator and provides new insights into regulation of lincomycin biosynthesis in S. lincolnensisIMPORTANCE Although lincomycin biosynthesis has been extensively studied, its regulatory mechanism remains elusive. Here, a novel regulator, LmbU, which regulates transcription of its target genes in the lincomycin biosynthetic gene cluster (lmb gene cluster) and therefore promotes lincomycin biosynthesis, was identified in S. lincolnensis strain NRRL 2936. Importantly, we show that this new regulatory element is relatively widespread across diverse actinomycetes species. In addition, our findings provide a new strategy for improvement of yield of lincomycin through manipulation of LmbU, and this approach could also be evaluated in other secondary metabolite gene clusters containing this regulatory protein.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Lincomicina/biosíntesis , Streptomyces/genética , Streptomyces/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Saccharopolyspora/genética , Metabolismo Secundario , Factores de Transcripción/genéticaRESUMEN
Global regulator BldA, the only tRNA for a rare leucine codon UUA, is best known for its ability to affect morphological differentiation and secondary metabolism in the genus Streptomyces. In this study, we confirmed the regulatory function of the bldA gene (Genbank accession no. EU124663.1) in Streptomyces lincolnensis. Disruption of bldA hinders the sporulation and lincomycin production, that can recur when complemented with a functional bldA gene. Western blotting assays demonstrate that translation of the lmbB2 gene which encodes a L-tyrosine hydroxylase is absolutely dependent on BldA; however, mistranslation of the lmbU gene which encodes a cluster-situated regulator (CSR) is observed in a bldA mutant. Intriguingly, when the preferential cognate codon CTG was used, the expression level of LmbU was not the highest compared to the usage of rare codon TTA or CTA, indicating the rare codon in this position is significant for the regulation of lmbU expression. Moreover, replacement of TTA codons in both genes with another leucin codon in the bldA mutant did not restore lincomycin production. Thus, we believe that the bldA gene regulates lincomycin production via controlling the translation of not only lmbB2 and lmbU, but also the other TTA-containing genes. In conclusion, the present study demonstrated the importance of the bldA gene in morphological differentiation and lincomycin production in S. lincolnensis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Lincomicina/biosíntesis , ARN de Transferencia de Leucina/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , ARN Bacteriano/metabolismo , Streptomyces/citologíaRESUMEN
BACKGROUND: Riboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work. RESULTS: In this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating 13C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden-Meyerhof-Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD+ ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively. CONCLUSIONS: This study shows that integration of transcriptomics, 13C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin's regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD-ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Oxígeno/metabolismo , Riboflavina/biosíntesis , Acetolactato Sintasa/genética , Acetolactato Sintasa/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fermentación , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Glucólisis , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas , Metabolómica , Oxígeno/farmacología , Vía de Pentosa Fosfato , Riboflavina/metabolismoRESUMEN
Bone marrow mesenchymal stem cells (BM-MSCs) are considered as a promising option in the field of regenerative medicine and tissue engineering. However, little is known about how TM4 mouse Sertoli cells, which are known to enhance stem cells proliferation in co-culture, may influence the proliferation of BM-MSCs and which signaling pathways are involved in. To address these questions, an in vitro transwell system was used. We found that TM4 cells could produce soluble factors which enhanced the growth of BM-MSCs without inhibiting the multipotency. Furthermore, cell cycle analysis showed that co-culture with the TM4 cells accelerated the progress of BM-MSCs from the G1 to the S phase. The expression of the phospho-akt, mdm2, as well as pho-CDC2, and cyclin D1 were markedly upregulated in co-cultured BM-MSCs. The observed promoting effect was significantly inhibited by the administration of the PI3K/AKT inhibitor, LY294002. Among the various growth factors produced by TM4 cells, the epithelial growth factor (EGF) stimulated the proliferation of the BM-MSCs more significantly compared with the other growth factors examined in this study. Neutralization of EGF via a blocking antibody significantly limited the promoting growth effect in BM-MSCs. These results suggest that TM4 cells provide a favorable in vitro environment for BM-MSCs growth and imply the involvement of the EGF/PI3K/AKT pathway.
Asunto(s)
Proliferación Celular/genética , Factor de Crecimiento Epidérmico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células de Sertoli/citología , Animales , Células de la Médula Ósea/citología , Diferenciación Celular/genética , Técnicas de Cocultivo , Humanos , Masculino , Células Madre Mesenquimatosas , Ratones , Transducción de Señal/genética , Ingeniería de TejidosRESUMEN
Greater exposure to Pb(â ¡) increases the likelihood of harmful effects in the environment. In this study, the aquatic unicellular alga Chlorella protothecoides (C. protothecoides) and Chlorella vulgaris (C. vulgaris) were chosen to assess the acute and chronic toxicity of Pb(â ¡) exposure. Results of the observations show dose-response relationships could be clearly observed between Pb(â ¡) concentration and percentage inhibition (PI). Exposure to Pb(â ¡) increased malondialdehyde (MDA) content by up to 4.22 times compared with the control, suggesting that there was some oxidative damage. ANOVA analysis shows that Pb(â ¡) decreased chlorophyll (chl) content, indicating marked concentration-dependent relationships, and the lowest levels of chl a, chl b, and total-chl were 14.53, 18.80, and 17.95% of the controls, respectively. A real-time PCR assay suggests the changes in transcript abundances of three photosynthetic-related genes. After 120 h exposure Pb(â ¡) reduced the transcript abundance of rbcL, psaB, and psbC, and the relative abundances of the three genes of C. protothecoides and C. vulgaris in response to Pb(â ¡) were 54.66-98.59, 51.68-95.59, 37.89-95.48, 36.04-94.94, 41.19-91.20, and 58.75-96.80% of those of the controls, respectively. As for 28 d treatments, the three genes displayed similar inhibitory trend. This research provides a basic understanding of Pb(â ¡) toxicity to aquatic organisms.
Asunto(s)
Chlorella/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Plomo/toxicidad , Malondialdehído/metabolismo , Fotosíntesis/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Chlorella/fisiología , Chlorella vulgaris/efectos de los fármacos , Chlorella vulgaris/fisiología , Clorofila/análogos & derivados , Clorofila/metabolismo , Clorofila A , Oxidación-Reducción , Fotosíntesis/genética , Especificidad de la EspecieRESUMEN
The acute and chronic toxic effects of Bisphenol A (BPA) on Chlorella pyrenoidosa (C. pyrenoidosa) and Scenedesmus obliquus (S. obliquus) were not well understood. The indoor experiments were carried out to observe and analyze the BPA-induced changes. Results of the observations showed that in acute tests BPA could significantly inhibit the growth of both algae, whereas chronic exposure hardly displayed similar trend. Superoxide dismutase (SOD) and Catalase (CAT) activities of both algae were promoted in all the treatments. Chlorophyll a synthesis of the two algae exhibited similar inhibitory trend in short-term treatments, and in chronic tests C. pyrenoidosa hardly resulted in visible influence, whereas in contrast, dose-dependent inhibitory effects of S. obliquus could be clearly observed. The experimental results indicated that the growth and Chlorophyll a syntheses of S.obliquus were more sensitive in response to BPA than that of C. pyrenoidosa, whereas for SOD andCAT activities, C. pyrenoidosa was more susceptible. This research provides a basic understanding of BPA toxicity to aquatic organisms.
Asunto(s)
Compuestos de Bencidrilo/toxicidad , Chlorella/efectos de los fármacos , Fenoles/toxicidad , Scenedesmus/efectos de los fármacos , Catalasa/metabolismo , Chlorella/crecimiento & desarrollo , Clorofila/análisis , Clorofila A , Relación Dosis-Respuesta a Droga , Scenedesmus/crecimiento & desarrollo , Superóxido Dismutasa/metabolismo , Pruebas de Toxicidad Aguda , Pruebas de Toxicidad CrónicaRESUMEN
Artificial Intelligence (AI) technology is spearheading a new industrial revolution, which provides ample opportunities for the transformational development of traditional fermentation processes. During plasmid fermentation, traditional subjective process control leads to highly unstable plasmid yields. In this study, a multi-parameter correlation analysis was first performed to discover a dynamic metabolic balance among the oxygen uptake rate, temperature, and plasmid yield, whilst revealing the heating rate and timing as the most important optimization factor for balanced cell growth and plasmid production. Then, based on the acquired on-line parameters as well as outputs of kinetic models constructed for describing process dynamics of biomass concentration, plasmid yield, and substrate concentration, a machine learning (ML) model with Random Forest (RF) as the best machine learning algorithm was established to predict the optimal heating strategy. Finally, the highest plasmid yield and specific productivity of 1167.74 mg L-1 and 8.87 mg L-1/OD600 were achieved with the optimal heating strategy predicted by the RF model in the 50 L bioreactor, respectively, which was 71% and 21% higher than those obtained in the control cultures where a traditional one-step temperature upshift strategy was applied. In addition, this study transformed empirical fermentation process optimization into a more efficient and rational self-optimization method. The methodology employed in this study is equally applicable to predict the regulation of process dynamics for other products, thereby facilitating the potential for furthering the intelligent automation of fermentation processes.
Asunto(s)
Reactores Biológicos , Escherichia coli , Fermentación , Aprendizaje Automático , Plásmidos , Plásmidos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Reactores Biológicos/microbiología , Técnicas de Cultivo Celular por Lotes/métodos , BiomasaRESUMEN
This study aimed to evaluate the hydrolysates from orange peel waste (OPW) as the low-cost carbon source for lycopene production. Initially, the dilute acid pretreatment combined with enzymatic hydrolysis of OPW resulted in a total sugar concentration of 62.18 g/L. Meanwhile, a four-month adaptive laboratory evolution (ALE) experiment using a d-galacturonic acid minimal medium resulted in an improvement in the growth rate of our previously engineered Escherichia coli strain for lycopene production. After evolutionary adaptation, response surface methodology (RSM) was adapted to optimize the medium composition in fermentation. The results obtained from RSM analysis revealed that the 5.53 % carbon source of orange peel hydrolysate (OPH), 6.57 g/L nitrogen source, and 30 °C temperature boosted lycopene production in the final strain. Subsequently, the optimized treatment for lycopene fermentation was then conducted in a 5 L batch fermenter under the surveillance of a kinetic model that uses the Logistic equation for strain growth (µm = 0.441 h-1), and Luedeking-Piret equations for lycopene production (Pm = 1043 mgL-1) with growth rate constant (α = 0.1491). At last, lycopene biosynthesized from OPH was extracted and analyzed for qualitative validation. Likewise, its data on phytic acid (between 1.01 % and 0.86 %) and DPPH radical scavenging (between 38.06 % and 29.08 %) highlighted the better antioxidant capacity of lycopene. In conclusion, the OPH can be used as a fermentation feedstock which opens new possibilities of exploiting fruit crop residues for food and pharmaceutical applications.
RESUMEN
BACKGROUND: Adipose-derived stem cells (ADSCs) have been extensively used in preclinical and clinical trials for treating various diseases. However, the differences between ADSCs from lean individuals (L-ADSCs) and those from obese individuals (O-ADSCs) have not been thoroughly investigated, particularly regarding their mitochondrial and lysosomal functions. Therefore, this study aims to evaluate the differences between L-ADSCs and O-ADSCs in terms of cell biological activity, mitochondria, and lysosomes. METHODS: We first isolated and cultured L-ADSCs and O-ADSCs. We then compared the differences between the two groups in terms of biological activity, including cell proliferation, differentiation potential, and their effect on the polarization of macrophages. Additionally, we observed the mitochondrial and lysosomal morphology of ADSCs using an electronic microscope, MitoTracker Red, and lysotracker Red dyes. We assessed mitochondrial function by examining mitochondrial membrane potential and membrane fluidity, antioxidative ability, and cell energy metabolism. Lysosomal function was evaluated by measuring autophagy and phagocytosis. Finally, we performed transcriptome analysis of the ADSCs using RNA sequencing. RESULTS: The biological activities of O-ADSCs were decreased, including cell immunophenotypic profiles, cell proliferation, and differentiation potential. Furthermore, compared to L-ADSCs, O-ADSCs promoted M1-type macrophage polarization and inhibited M2-type macrophage polarization. Additionally, the mitochondrial morphology of O-ADSCs was altered, with the size of the cells becoming smaller and mitochondrial fragments increasing. O-ADSCs also exhibited decreased mitochondrial membrane potential and membrane fluidity, antioxidative ability, and energy metabolism. With respect to lysosomes, O-ADSCs contained ungraded materials in their lysosomes, enhanced lysosomal permeability, and reduced autophagy and phagocytosis ability. RNA sequence analysis indicated that the signalling pathways related to cell senescence, cancer, and inflammation were upregulated, whereas the signalling pathways associated with stemness, cell differentiation, metabolism, and response to stress and stimuli were downregulated. CONCLUSIONS: This study indicates that ADSCs from individuals (BMI > 30 kg/m2) exhibit impaired mitochondrial and lysosomal function with decreased biological activity.