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RESUMEN Introducción. Las células dendríticas (CD) desempeñan un papel clave en la presentación antigénica y la activación de linfocitos T, pero su función puede ser modulada por el microambiente tumoral, lo que afecta la respuesta inmunitaria antitumoral. Este estudio se centra en la interacción entre las CD y el hepatocarcinoma (HCC), explorando cómo el entorno tumoral influye en la actividad de las CD. Objetivo. Evaluar la variación en la actividad de las CD en respuesta a la expresión de citoquinas proinflamatorias, IL-10 y receptores CXCR4 y CCR7 en un modelo murino de hepatocarcinoma (PM299L). Métodos. Se realizaron ensayos in vitro cocultivando CD murinas y una línea tumoral murina de HCC. Se evaluó la expresión de citoquinas proinflamatorias (IL-12, IL-6, IL-1β, TNF-α), inmunosupresora (IL-10) y receptores asociados a migración y maduración (CXCR4 y CCR7) mediante Qpcr a las 24, 48 y 72 horas. Los ensayos se repitieron tres veces. Resultados. Las CD expuestas al entorno tumoral de HCC mostraron una mayor expresión de citoquinas proinflamatorias y IL-10 en comparación al grupo control. Además, se observó una expresión elevada de receptores CXCR4 y CCR7 en las CD expuestas al HCC. Estos cambios en la expresión de genes ocurrieron en un período de 72 horas de cocultivo. Conclusión. La actividad de las CD se ve influenciada por el entorno tumoral de HCC y el tiempo de interacción, lo que modula su función proinflamatoria y de presentación antigénica. Estos hallazgos destacan la importancia de comprender la dinámica de la respuesta inmunitaria en el hepatocarcinoma.
ABSTRACT Introduction. Dendritic cells (DCs) play a key role in antigen presentation and T cell activation, but their function can be modulated by the tumor microenvironment, affecting the antitumor immune response. This study focuses on the interaction between DCs and hepatocellular carcinoma (HCC), exploring how the tumor environment influences DC activity. Objective. To evaluate the variation in DC activity in response to the expression of proinflammatory cytokines, IL-10 and CXCR4 and CCR7 receptors in a murine model of hepatocellular carcinoma (PM299L). Methods. In vitro assays were performed co-culturing murine DCs and HCC tumor line. The expression of proinflammatory cytokines (IL-12, IL-6, IL-1β, TNF-α), immunosuppressive (IL-10) and receptors associated with migration and maturation (CXCR4 and CCR7) was evaluated by Qpcr at 24, 48 and 72 hours. The tests were repeated three times. Results. DCs exposed to the HCC tumor environment showed increased expression of proinflammatory cytokines and IL-10 compared to the control group. Furthermore, elevated expression of CXCR4 and CCR7 receptors will be observed in DCs exposed to HCC. These changes in gene expression occurred within a 72-h period of coculture. Conclusion. DC activity is influenced by HCC tumor environment and interaction time, which modulates their proinflammatory and antigen presentation function. These findings highlight the importance of understanding the dynamics of the immune response in hepatocarcinoma.
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Marine bacteria, particularly those from the Actinomycetes class, such as Streptomyces spp., represent a promising source of new compounds with various bioactivities. Although the number of reported compounds is increasing, there is still massive potential for producing new compounds if cryptic gene clusters can be activated. Co-culture is one of the techniques used to activate these cryptic gene clusters. However, the results of co-culture may not only be new compounds. Therefore, we collected literature on co-culture of marine bacteria published between 2012 and 2022 from the databases such as Google Scholar, PubMed, and Scopus. The results showed that co-culturing marine bacteria may result in one of the three groups: 1) increased yield of bioactivities and/or bioactive compounds, 2) production of known compounds that were not present in single-strain cultures, and 3) production of previously undescribed compounds. The results suggest that co-culture cannot be universally applied to generate new compounds, as the outcome of the co-culture system is often specific to each individual study.
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BACKGROUND:Stem cell transplantation is a new way to prevent and cure intervertebral disc degeneration.However,whether the transplanted stem cells can survive,proliferate,differentiate,and restore the function of nucleus pulposus cells after transplantation,is the key and difficult point to overcome. OBJECTIVE:To explore the effects of Bushenhuoxue decoction on survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells. METHODS:A Transwell chamber was used to construct a co-culture model of human adipose-derived stem cells and human degenerative nucleus pulposus cells.The experiment was divided into control group,model group,drug-containing serum group,and drug-free serum group.Except for the control group,the co-culture system of other groups was treated with 50 μmol/L tert-butyl hydrogen peroxide for 24 hours.The drug-containing serum group and drug-free serum group were treated with DMEM low-glucose complete culture medium containing drug-containing serum of Bushenhuoxue decoction or drug-free serum with 20%volume fraction for 48 hours.The sublayer adipose-derived stem cells were taken.Toluidine blue staining was used to detect proteoglycan synthesis levels.Real-time PCR method was used to detect mRNA expression of type Ⅱ collagen,proteoglycan and SRY-box transcription factor 9.The protein expression of SOX9 was detected by western blot assay.Lactate dehydrogenase assay was used to detect cytotoxicity.Flow cytometry was used to detect reactive oxygen species,and β-galactosidase staining was used to detect cell senescence. RESULTS AND CONCLUSION:(1)Compared with the control group,the proportion of necrotic cells in the model group increased;toluidine blue staining became lighter,and the expression levels of type Ⅱ collagen,proteoglycan,SOX9 mRNA and SOX9 protein decreased(P<0.05).Compared with the model group,the drug-containing serum of Bushenhuoxue decoction could significantly reduce cell injury and promote the expression of type Ⅱ collagen,proteoglycan,SOX9 mRNA,and SOX9 protein(P<0.05),but the improvement in the drug-free serum group was not significant(P>0.05).(2)Compared with the control group,the contents of cytotoxicity,reactive oxygen species,and cell senescence in the model group were significantly increased.Compared with the model group,the microenvironment of the coculture system was significantly improved by drug-containing serum of Bushenhuoxue decoction(P<0.05),while drug-free serum had no significant effect on the microenvironment of the co-culture system(P>0.05).(3)The results show that Bushenhuoxue decoction can promote the survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells.
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BACKGROUND:In the co-culture environment of mesenchymal stem cells and macrophages,mesenchymal stem cells can promote the polarization of macrophages into anti-inflammatory macrophages to reduce inflammation,and macrophages can promote the osteogenic differentiation of mesenchymal stem cells.The co-culture of both plays an important role in regulating the immune system and promoting tissue regeneration. OBJECTIVE:To summarize the methods,influencing factors and possible mechanisms of co-culture between mesenchymal stem cells and macrophages,and to provide theoretical basis and experimental methods for the application of co-culture of mesenchymal stem cells and macrophages in tissue engineering. METHODS:The first author searched the relevant articles published from January 1970 to September 2023 in PubMed and CNK by computer from January to September 2023.The Chinese and English key words were"mesenchymal stem cells,macrophages,co-culture".Finally,63 articles were included and analyzed. RESULTS AND CONCLUSION:(1)In vitro co-culture of mesenchymal stem cells and macrophages can be divided into direct contact co-culture and indirect contact co-culture according to the model,and two-dimensional cell co-culture and three-dimensional cell co-culture according to the dimension.(2)The co-culture of mesenchymal stem cells and macrophages can promote the polarization of macrophages towards M2 type and enhance the osteogenic effect of mesenchymal stem cells.(3)In the co-culture model,the methods of co-culture,the proportion and time of co-culture,the phenotype of macrophages,and the cell source and conditions all affected the immune regulation of macrophages and the osteogenesis of mesenchymal stem cells.(4)Cell interaction in co-culture may regulate the immune function of macrophages,proliferation,migration and osteogenesis of mesenchymal stem cells through cell-secreted soluble factors,extracellular vesicles,cell-cell contact,and metabolic pathways.(5)Mesenchymal stem cells and macrophages can enhance cardiac function after acute myocardial infarction,promote epithelial wound healing,reduce lung inflammation,improve renal function,and accelerate bone repair.(6)There are still some problems in co-culture of mesenchymal stem cells and macrophages,such as the selection of co-culture conditions,the maintenance of good cell state and interaction of co-cultured cells.(7)The co-culture of mesenchymal stem cells and macrophages can improve the local inflammatory microenvironment and promote tissue regeneration and repair,so it will have a broad application prospect in tissue engineering.
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Objective:To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism.Methods:The LPS induced type Ⅱ alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting.Results:Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2?438.95±64.89, 3?036.41±96.69 vs. 1?736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67?205.60±3?430.40 vs. 56?272.67±7?650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). Conclusion:LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.
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Chronic obstructive pulmonary disease ( COPD ) major chronic disease threatening public health with complex pathological mechanisms. The change of the cell microenvironment of the lung is an important part of the pathophysiology of COPD. Cell culture technology is an important method to investigate the pathological mechanism of COPD and evaluate the pharmacological effect of medicine. Here we introduce the composition of the cell microenvironment of the lung, the change of the cell microenvironment in the pathological process of COPD, and summarize the application of in vitro model mimics cell microenvironment of COPD in the study of mechanism. In addition, we aim to put forward the ideas of the in vitro model establishment of cell microenvironment of COPD.
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Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells.
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BACKGROUND: Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. RESULTS: Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-ß, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. CONCLUSIONS: Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.
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Humans , Interferon Type I , alpha-Synuclein , SARS-CoV-2 , COVID-19 , Virus Replication , Cell Line , Endothelial CellsABSTRACT
Abstract Background: Digestibility of fiber in the rumen is not due to enzymatic activity of individual bacteria, but rather to their interaction, which complements their enzymatic functioning. Thus, efficiency of fiber digestion depends on the diversity and density of cellulolytic bacteria. Objective: To estimate in vitro production of biogas, methane, and fermentative characteristics of cobra grass (Brachiaria hibrido) inoculated with ruminal bacteria (RB) in coculture with isolated cellulolytic bacteria (ICB) from bovine (ICBbov) or water buffalo (ICBbuf). Methods: ICBbov and ICBbuf were isolated from ruminal cellulolytic bacteria consortia using specific culture media for cellulolytic bacteria. Both were morphologically characterized and a Gram stain was performed. In the in vitro gas production test, the substrate was cobra grass and the inocula were ruminal bacteria (RB), ICBbov, ICBbuf, Coculturebov (RB + ICBbov) and Coculturebuf (RB + ICBbuf). Biogas and methane (CH4) production, as well as dry matter degradation (DMD) and neutral detergent fiber degradation (NDFD) were measured. A completely randomized design was used. Results: The ICB obtained were Gram positive cocci. Accumulated biogas production at 72 h from ICBbov and ICBbuf was on average 42.11% of that produced by RB. The Coculturebov produced 14.24% more biogas than RB. The CH4 production was lower in ICBbov and ICBbuf than in RB, Coculturebov and Coculturebuf. The DMD and NDFD were not different among RB, Coculturebov and Coculturebuf. The ICBbov degraded 37.10 and 96.34% more DMD and NDFD than ICBbuf (p<0.05). Conclusion: The use of ICB from bovine or water buffalo in coculture with RB does not improve in vitro production of biogas, DMD or NDFD with respect to RB alone.
Resumen Antecedentes: La digestibilidad ruminal de la fibra no se debe a la actividad enzimática individual de las bacterias sino a su interacción para complementar su funcionamiento enzimático. Así, la eficiencia de digestión de la fibra depende de la diversidad y la densidad de las bacterias celulolíticas. Objetivo: Estimar la producción de biogás, metano, y las características fermentativas in vitro del pasto cobra (Brachiaria hibrido) inoculado con bacterias ruminales (BR) en cocultivo con bacterias celulolíticas aisladas (BCA) de bovino (BCAbov) o búfalo de agua (BCAbuf). Métodos: BCAbov y BCAbuf se aislaron de consorcios bacterianos celulolíticos ruminales usando medios de cultivo específicos para bacterias celulolíticas. Ambas se caracterizaron morfológicamente y realizó tinción de Gram. En la prueba de producción de gas in vitro, el sustrato fue pasto cobra y los inóculos fueron bacterias ruminales (BR), BCAbov, BCAbuf, Cocultivobov (BR + BCAbov) y Cocultivobuf (BR + BCAbuf). Se midió la producción de biogás y metano (CH4), así como la degradación de la materia seca (DMS) y de la fibra detergente neutro (DFDN). El análisis estadístico se basó en un diseño completamente al azar. Resultados: Las BCA resultantes se identificaron como cocos Gram positivos. La producción de biogás acumulada a las 72 h por BCAbov y BCAbuf fue en promedio 42,11% del producido por BR. El cocultivobov produjo 14,24% más biogás que BR. La producción de CH4 fue menor en BCAbov y BCAbuf que en BR, cocultivobov y cocultivobuf. Las DMS y DFDN no mostraron diferencias entre BR, cocultivobov y cocultivobuf. La BCAbov degradó 37,10 y 96,34% más DMS y DFDN que BCAbuf (p<0,05). Conclusión: El uso de BCA de bovino o búfalo de agua en cocultivo con BR no mejora la producción de biogás, DMS o DFDN in vitro respecto a BR.
Resumo Antecedentes: A digestibilidade da fibra no rúmen não se deve à atividade enzimática individual das bactérias, mas sim à sua interação para complementar o seu funcionamento enzimático. Assim, a eficiência da digestão das fibras depende da diversidade e densidade das bactérias celulolíticas. Objetivo: Estimar a produção in vitro de biogás, metano e características fermentativas da gramínea de cobra (Brachiaria hibrido) inoculada com bactéria ruminal (BR) em cocultura com bactérias celulolíticas isoladas (BCI) de bovino (BCIbov) ou búfalo de água (BCIbuf). Métodos: BCIbov e BCIbuf foram isolados a partir de consórcios de bactérias celulolíticas ruminais utilizando meios de cultura específicos para bactérias celulolíticas. Ambos foram caracterizados morfologicamente, e foi realizada uma coloração de Gram. No teste de produção de gás in vitro, o substrato era erva de cobra e os inóculos eram bactérias ruminais (BR), BCIbov, BCIbuf, Cocultivobov (BR + BCIbov) e cocultivobuf (BR + BCIbuf). Foram medidas a produção de biogás e metano (CH4), bem como a degradação da matéria seca (DMS) e a degradação da fibra em detergente neutro (DFDN). Foi utilizado um desenho completamente aleatório. Resultados: BCIs eram cocos Gram positivos. A produção acumulada de biogás a 72 h de BCIbov e BCIbuf foi em média 42,11% da produzida por BR. O cocultivobov produziu 14,24% mais biogás do que o BR. A produção de CH4 foi menor em BCIbov e BCIbuf do que BR, cocultivobov e cocultivobuf. DMS e DFDN não eram diferentes entre BR, cocultivobov e cocultivobuf. O BCIbov degradou 37,10 e 96,34% mais DMS e DFDN do que o BCIbuf (p<0,05). Conclusão: A utilização de BCI de bovino ou búfalo de água em cocultura com BR não melhora a produção in vitro de biogás, DMS ou DFDN no que diz respeito a BR.
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Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis (RA), but the relationship between the two phenomena remains unclear. We explored whether and how cholinergic dysfunction accelerates protein citrullination and consequently drives the development of RA. Cholinergic function and protein citrullination levels in patients with RA and collagen-induced arthritis (CIA) mice were collected. In both neuron-macrophage coculture system and CIA mice, the effect of cholinergic dysfunction on protein citrullination and expression of peptidylarginine deiminases (PADs) was assessed by immunofluorescence. The key transcription factors for PAD4 expression were predicted and validated. Cholinergic dysfunction in the patients with RA and CIA mice negatively correlated with the degree of protein citrullination in synovial tissues. The cholinergic or alpha7 nicotinic acetylcholine receptor (α7nAChR) deactivation and activation resulted in the promotion and reduction of protein citrullination in vitro and in vivo, respectively. Especially, the activation deficiency of α7nAChR induced the earlier onset and aggravation of CIA. Furthermore, deactivation of α7nAChR increased the expression of PAD4 and specificity protein-3 (SP3) in vitro and in vivo. Our results suggest that cholinergic dysfunction-induced deficient α7nAChR activation, which induces the expression of SP3 and its downstream molecule PAD4, accelerating protein citrullination and the development of RA.
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Bibenzyls, a kind of important plant polyphenols, have attracted growing attention for their broad and remarkable pharmacological activities. However, due to the low abundance in nature, uncontrollable and environmentally unfriendly chemical synthesis processes, these compounds are not readily accessible. Herein, one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes. Three types of efficiently post-modifying modular strains were engineered by employing methyltransferases, prenyltransferase, and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules. Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes. Especially, a prenylated bibenzyl derivative ( 12) was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke. RNA-seq, quantitative RT-PCR, and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor, mitochondria associated 3 (Aifm3), suggesting that Aifm3 might be a new target in ischemic stroke therapy. This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.
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Organoids are three-dimensional structures formed by self-organizing growth of cells in vitro, which own many structures and functions similar with those of corresponding in vivo organs. Although the organoid culture technologies are rapidly developed and the original cells are abundant, the organoid cultured by current technologies are rather different with the real organs, which limits their application. The major challenges of organoid cultures are the immature tissue structure and restricted growth, both of which are caused by poor functional vasculature. Therefore, how to develop the vascularization of organoids has become an urgent problem. We presently reviewed the progresses on the original cells of organoids and the current methods to develop organoids vascularization, which provide clues to solve the above-mentioned problems.
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Humans , Organoids , Neovascularization, Pathologic , TechnologyABSTRACT
Diesel exhaust (DE) is an important pollution source widely existing in the living and production environment, which is closely related to the health of the public and occupational groups. The International Agency for Research on Cancer has classified DE as a Group 1 carcinogen. Considering the negative health impacts on the respiratory system due to DE exposure in vitro, it is crucial to apply reliable test systems allowing accurate assessment of the biological effects of DE. The exposure technology of respiratory system in vitro is considered as one of the feasible measures to implement the 3R (reduce, refine, and replace) principle in animal experiments. Compared with the traditional submerged culture in vitro models, the air-liquid interface (ALI) exposure technology has the advantages including fewer influencing factors, easier exposure condition control, and shorter exposure cycle. ALI has become an important tool to study molecular events associated with physiology and pathology of respiratory system, and action modes and interactions of different cell types. Also, ALI has been increasingly widely used because it can simulate the actual processes of human respiratory system cells and/or tissues to DE exposure. This review was intended to introduce the development and advantages of ALI exposure technology, and further summarized the application progress of ALI exposure technology in studying the respiratory toxicity induced by DE exposure in vitro, so as to provide new ideas and pathways for the use of ALI exposure technology in the study of biomarkers and mechanisms of respiratory toxicity associated with DE exposure, and provide basic data to screen and promote biomarkers for exposed populations.
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A BBB co-culture cell model consisting of rat brain microvascular endothelial cells (BMEC) and astrocytes (AS) was established to study the effect of Angelica dahurica coumarins on the transport behavior of puerarin across blood-brain barrier (BBB) in vitro and in vivo. The barrier function of this model was evaluated by measuring the transendothelial resistance, phenol red permeability and BBB related protein expression. The permeability assay and western blot methods were performed to study the effects of Angelica dahurica coumarins on the BBB permeability and the expression of BBB related protein. The animal experiment protocols in this study were approved by the Animal Ethics Committee of Xi'an Jiaotong University (Animal Ethics No.: 2021-1329). The results showed that the established BMEC/AS co-culture model could be used to evaluate drug transport across BBB in vitro. After combined with Angelica dahurica coumarins, the transport capacity of puerarin was significantly increased in vitro and in vivo. Additionally, Angelica dahurica coumarins enhanced BBB permeability and inhibited the protein expression of P-glycoprotein (P-gp), zonula occludens-1 (ZO-1) and occludin. Angelica dahurica coumarins might increase BBB permeability by inhibiting the expression of P-gp and tight junction protein, thereby increasing the content of puerarin in brain tissue.
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A new siderophore chelate (1) and 8 known compounds were identified from the liquid co-cultures of the marine-derived Streptomyces sp. IMB18-531 and Cladosporium sp. IMB19-099 by a combination of chromatography methods, including C18 reversed-phase medium pressure chromatography, gel column chromatography and HPLC. Their structures were determined by spectroscopic analysis and chemical methods as aluminioxamine E (1), desferrioxamine E (2), ferrioxamine E (3), terragine E (4), capsimicin (5), cyclo(L-prolinyl-L-tyrosine) (6), anthranilic acid (7), (Z)-14-methylpentadec-9-enoic acid (8), and (Z)-hexadec-8-enoic acid (9). Compound 2 showed inhibitory activities against the expression of liver fibrosis related genes COL1A1, MMP2, and TIMP2. Compounds 5, 8, and 9 displayed antibacterial activities against methicillin-resistant Staphylococcus aureus, S. epidermidis and Bacillus subtilis, with MICs of 16-64 μg·mL-1. Compound 5 showed cytotoxicities against human pancreatic cancer MIA Paca-2 and human colon cancer HT-29 cell lines with IC50 of 2.9 and 6.3 μmol·L-1, respectively.
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Objective@#To study the effect of stem cell factor (SCF) on the angiogenic ability of cocultured dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs).@*Methods @#This study has been reviewed and approved by the Ethics Committee. The experiment was split into the HUVECs, SCF+HUVECs, DPSCs+HUVECs, and SCF+DPSCs+HUVECs groups. A mixture of SCF and culture medium was used to prepare a mixed culture medium with an SCF concentration of 100 ng/mL. In vitro coculture of DPSCs and HUVECs was performed at a 1∶5 ratio. CCK-8 proliferation assay was used to observe the proliferative capacity of cells in each group on days 1, 3, 5, and 7. Wound healing and Transwell migration assays were used to detect the effect of SCF on cell migration under either direct or indirect coculture conditions, respectively. In vitro angiogenesis experiments were performed to detect the angiogenic capacity of the cells in each group. The vascular endothelial growth factor A (VEGFA) concentration in the cell culture supernatant was detected using ELISAs, and the protein expression levels of CD31, CD34, and VEGFA were detected using Western blot analysis. @*Results @# Wound healing and Transwell migration experiments showed that SCF significantly promoted the migration of cocultured DPSCs and HUVECs (P<0.05). The in vitro angiogenesis experiment showed that the number of branches and the total length of branches of tubular structures in the SCF+DPSCs+HUVECs group were significantly greater than those of the other groups (P<0.05), and the expression levels of the vascular-related proteins CD31, CD34, and VEGFA in this group were greater (P<0.01). @*Conclusion @# SCF can enhance the migration and in vitro angiogenesis of cocultured DPSCs and HUVECs.
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@#Introduction: Breast cancer is the most common cancer in women and the world’s second leading cause of death in women, after lung cancer. Calreticulin (CRT), an endoplasmic reticulum (ER) multipurpose protein, has been proposed as a potential biomarker for breast cancer. However, reports on the correlation between CRT expression and cell invasiveness in breast cancer micro-tissues are scarce. Thus, in the current study, we analyzed the potential correlation between CRT and invasiveness of breast cancer in a biological scaffold-based 3D co-culture system. Methods: MCF7, MDA-MB-231 and MCF-10A breast cell lines were co-cultured in a 3-dimensional (3D) system with MRC-5 lung fibroblast cell line in the cell density ratio of 3:1. Thereafter, calreticulin gene and protein expression levels were determined based on quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively. Moreover, via RT-qPCR analysis, the gene expression levels of calreticulin-related candidate metastasis genes in breast cancer micro-tissues were carried out. Results: The results showed occasional foci of lumen-like morphology in the non-cancerous breast micro-tissues and the formation of solid clusters for breast cancer micro-tissues. Moreover, immunohistochemistry results revealed protein expression of calreticulin in non-cancerous and cancerous breast micro-tissues with cytoplasmic and nucleic acid localizations. It was found that PCMT1 and ER-α genes were significantly downregulated (p < 0.01) in invasive breast cancer micro-tissues. Conclusion: This study suggests that CRT and CRT-related candidate metastasis genes may potentially serve as prognostic biomarkers in invasive breast carcinoma.
ABSTRACT
@#Antimicrobial resistance (AMR) is a global health crisis. Despite the drug discovery efforts, AMR is increasing, and discoveries are nearly nil. It is thus critical to design new strategies. Probiotics are tapped as alternatives to antibiotics for the treatment of gut-associated diseases. Lactobacillus species, common in food products, can inhibit the growth of gut pathogens. Here, we demonstrate the antimicrobial activities of Lactobacillus species – Lactobacillus paracasei, Lactobacillus casei, and Lactobacillus delbrueckii subsp. bulgaricus are enhanced when cocultured with Salmonella enterica subsp. enterica serovar Typhimurium. Cell-free culture supernatants (CFCS) from cocultures of Lactobacillus spp. and Salmonella enterica serovar Typhimurium more potently inhibit pathogen growth than their monoculture counterparts. Interestingly, we discovered that Salmonella enterica serovar Typhimurium could enhance the production of antimicrobials from Lactobacillus spp., most evident in L. delbrueckii subsp. bulgaricus. Also, L. delbrueckii subsp. bulgaricus CFCS upregulates key Salmonella virulence genes, hilA and sipA. Whether this increases Salmonella’s pathogenicity in vivo or reduces pathogen fitness and growth inhibition in vitro warrants further investigation. We propose that these probiotic isolates may be utilized for innovative natural food processing and preservation strategies to control Salmonella food contaminations. Importantly, our findings that Salmonella elicits an enhanced antimicrobial activity from Lactobacillus spp. provide evidence of a pathogen-mediated elicitation of antimicrobial production. Therefore, extending this phenomenon to other microbial interactions may help augment the strategies for drug discovery.
ABSTRACT
Macrophages are important cells of the immune system. Tumor-associated macrophages are enriched macrophages near tumor cells or tissues. Their role is mainly to promote the construction of tumor inflammatory microenvironment and inhibit tumor immune response. Cell co-culture system is a symbiotic culture system formed by mimicking the internal environment of the body in vitro. The co-culture condition is relatively consistent with the environment in vivo, enabling better information exchange and material exchange between cells, which is a supplement to the monolayer cell culture and animal experiments. Tumor-associated macrophages and tumor cells co-exist in the tumor microenvironment. Thus, constructing a co-culture system for tumor-associated macrophages and tumor cells would be conducive to studying the antitumor effect of tumor-associated macrophages and developing new immunotherapy drugs. The co-culture system would provide a new direction for treating malignant tumors. This article mainly reviewed the co-culture patterns of macrophages and the antitumor effects of different phenotypes of macrophages, and highlighted the importance of using immunotherapy to treat malignant tumors in the tumor microenvironment.
ABSTRACT
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/ LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKA and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.