Culture Time Research Articles

Land Use Effects on Organic Carbon in Andean Volcanic Ash Soils

ABSTRACTBackgroundBoth organic carbon (OC) stocks and labile OC (LOC) fractions are important indicators of soil health and are sensitive to land use change.AimsTo study the effects of land use change on these indicators in montane volcanic ash soils, a soil transect was surveyed in northern Ecuador.MethodsSamples were collected from 0–30, 30–60, and 60–90 cm soil depth at two agricultural sites with different time of cultivation and at three natural vegetation sites (tropical alpine grassland, páramo). LOC was determined as cold and hot water extractable OC (CWEOC and HWEOC). Molar absorptivity at 254 nm was determined in the extracts as a qualitative measure.ResultsTotal OC stocks were high at the páramo sites (51.3–60.2 kg C m−3) and the younger agricultural site (50.8 kg C m−3; 20 years of cultivation), but significantly lower (30.1 kg C m−3) at the older agricultural site (at least 100 years of cultivation); CWEOC (0.1%–0.7%) and HWEOC (0.6%–4.1%) represented only a small part of the OC. Both LOC pools decreased with increasing cultivation time, with CWEOC reflecting short‐term and HWEOC long‐term effects. In contrast, the molar absorptivity was highest at the oldest site (198–307 L mol−1 cm−1 vs. 36–64 L mol−1 cm−1 at the other sites), indicating that easily degradable labile C was depleted leaving compounds with higher aromaticity.ConclusionsThe conversion of páramo into agricultural land negatively affects OC stocks and soil health, as indicated by reduced OC storage capacities and lower LOC contents.

Thinking Through the Tiger: Korean Cultural Identity, Space, and Time

In the region of Northeast Asia, Korea has been identified as a nation that has a distinctive affinity for tigers. Koreans’ lives are deeply ingrained with emotions and thoughts related to tigers, even though they have completely disappeared from Korea due to historical changes and urbanization. This study first connects the image of tigers in Korean creation myths and folk tales with cultural identity. The analysis uncovers that the satire and humor present in the literature and paintings depicting tigers were employed to surmount the realistic fears associated with these creatures. Subsequently, the study delves into historical perceptions and spatial concepts by examining folk tales and maps of the Korean Peninsula that are symbolized by tigers. During the Japanese colonial period, tigers were regarded as a symbol of Korean national consciousness, and since then, they have played a significant role in disseminating national spirit and identity discourse. Finally, the analysis of Korean proverbs related to tigers reveals the profound influence of these animals on the development of Korean concepts of seasons and time. This study thus demonstrates the significant role of tigers in shaping the cultural identity and conceptual framework of space and time in Korea.

Optimization of Culture Medium Ingredients and Culture Conditions for Bacteriocin Production in Lactococcus lactis NCU036019.

Bacteriocin lactococcin036019 was identified and characterized from Lactococcus lactis NCU036019, which displayed significant antibacterial activity toward foodborne pathogenic bacteria Staphylococcus aureus under various conditions. However, the in situ low-level expression of lactococcin036019 severely limited its wide application in food industry. In this study, we optimized the medium ingredients and culture conditions of L. lactis NCU036019 for maximum production of lactococcin036019. The effects of different carbon sources, nitrogen sources, inorganic salts, growth factors, surfactants, and buffer salts on the production of bacteriocin were studied using antibacterial titer and diameter of inhibitory zone as evaluation indexes. Through single-factor experiments, Plackett-Burman (PB) experiment, steepest ascent experiment and response surface methodology, yeast extract, zinc sulfate, sodium acetate, mannitol, Tween-80, and dipotassium hydrogen phosphate were identified to display significant influence on the production of bacteriocin. By optimizing Man Rogosa and Sharpe (MRS) culture medium ingredients, the antibacterial activity of lactococcin036019 in the cell-free supernatant raised from 46.19 to 300.14Au/mL, namely, 6.5 times increased. Furthermore, the culture conditions, such as inoculation amount, culture time, and culture temperature, were optimized, and this further increased the antibacterial activity to 409Au/mL, namely, 8.8 times increased. This study investigated the effects of culture media and conditions on the production of lactococcin036019, and they were optimized for a maximum harvest of bacteriocin, and the significant increase of bacteriocin production in L. lactis NCU036019 facilitates the application of the antibacterial substance in future work.

Unbiased identification of cell identity in dense mixed neural cultures.

Induced pluripotent stem cell (iPSC) technology is revolutionizing cell biology. However, the variability between individual iPSC lines and the lack of efficient technology to comprehensively characterize iPSC-derived cell types hinder its adoption in routine preclinical screening settings. To facilitate the validation of iPSC-derived cell culture composition, we have implemented an imaging assay based on cell painting and convolutional neural networks to recognize cell types in dense and mixed cultures with high fidelity. We have benchmarked our approach using pure and mixed cultures of neuroblastoma and astrocytoma cell lines and attained a classification accuracy above 96%. Through iterative data erosion, we found that inputs containing the nuclear region of interest and its close environment, allow achieving equally high classification accuracy as inputs containing the whole cell for semi-confluent cultures and preserved prediction accuracy even in very dense cultures. We then applied this regionally restricted cell profiling approach to evaluate the differentiation status of iPSC-derived neural cultures, by determining the ratio of postmitotic neurons and neural progenitors. We found that the cell-based prediction significantly outperformed an approach in which the population-level time in culture was used as a classification criterion (96% vs 86%, respectively). In mixed iPSC-derived neuronal cultures, microglia could be unequivocally discriminated from neurons, regardless of their reactivity state, and a tiered strategy allowed for further distinguishing activated from non-activated cell states, albeit with lower accuracy. Thus, morphological single-cell profiling provides a means to quantify cell composition in complex mixed neural cultures and holds promise for use in the quality control of iPSC-derived cell culture models.

Assays to Enhance Metabolic Phenotyping in the Kidney.

The kidney is highly metabolically active, and injury induces changes in metabolism that can impact repair and fibrosis progression. Changes in expression of metabolism-related genes and proteins provide valuable data, but functional metabolic assays are critical to confirm changes in metabolic activity. Stable isotope metabolomics are the gold standard, but these involve considerable cost and specialized expertise. Both the Seahorse bioflux assays and substrate oxidation assays in tissues ex vivo are two relatively cost-effective assays for interrogating metabolism. Many institutions have access to Seahorse bioflux analyzers, which can easily and quickly generate data, but guidelines to enhance reproducibility are lacking. We investigate how variables (e.g. primary versus immortalized cells, time in culture) impact the data generated by Seahorse bioflux analyzers. In addition, we show the utility of 3H-palmitate, a new approach for assessing fatty acid oxidation in the kidney, in uninjured and injured kidney cortices. The 3H-palmitate substrate oxidation assays also demonstrate significant sex-dependent and strain-dependent differences in rates of fatty acid oxidation. These data should facilitate metabolic interrogation in the kidney field with enhanced reproducibility.

Event-based time in Polish culture and language

Event-based time in Polish culture and language

Complete genome sequence of a Bangladesh-developed live-attenuated lumpy skin disease (LSD) vaccine strain.

Lumpy skin disease (LSD) in cattle is managed through live-attenuated vaccines. Bangladesh LSD vaccine was developed from LSD virus isolate Bangladesh LSD-29 by passaging 60 times in cell culture. Here, we report the complete genome sequence of Bangladesh LSD vaccine strain.

The Impact of Culture Time on Genistin and Genistein Accumulation in Flemingia macrophylla (Willd.) Prain Seedlings and the Differentially Expressed Genes

Background Genistein and genistin are the main active constituents of Flemingia macrophylla (Willd.) Prain and the amount of active constituents in the roots varies depending on the cultivation time. The harvesting period has a great influence on its quality, and there are fewer studies on the optimal harvesting time of F. macrophylla, which is urgently needed to explore the optimal harvesting period to improve its use value. Purpose F. macrophylla (Willd.) Prain is an important medicinal plant distributed in China. This study aimed to enhance the accumulation of active ingredients genistin and genistein in F. macrophylla, and explore the mechanism of biological synthesis of these active compounds at molecular levels. Methods Transcriptome sequencing and high-performance liquid chromatography (HPLC) technology were applied to F. macrophylla tissue culture seedlings for 30 days after culturing (DAC), 45 DAC, 60 DAC, and 75 DAC. Then, gene expression, representative active substances, and important phenotypic indexes were analyzed. Results The highest fresh weight and root thickness were attained at 45 DAC, the highest plant height at 60 DAC, and the maximum rooting rate at 75 DAC. The genistin content reached the highest level at 45 DAC, and the content of genistein reached the highest level at 30 DAC. The total content of genistin and genistein achieved the maximum levels at 45 DAC. A total of 7,735 differently expressed genes (DEGs) in the three comparable groups, including T45d vs T30d, T60d vs T30d, and T60d vs T45d, were identified. Through the Kyoto Encyclopedia Genes and Genomes (KEGG) enrichment analysis of DEGs, isoflavonoid biosynthesis and phenylpropanoid biosynthesis pathways were significantly enriched in two groups, including T45d vs T30d and T60d vs T30d. A total of 41 DEGs involved in the production of isoflavones were discovered in the three groups. Conclusion The optimism culture time for the accumulation of genistein and genistin in F. macrophylla seedlings ranged from 30 to 45 days. Forty-one DEGs in the genistein and genistin synthesis pathways were found through transcriptomics analysis. This study sheds light on the biosynthesis of genistin and genistein in F. macrophylla.

Vechur cow milk yoghurt: response surface methodology-based process optimization and storage studies.

In this research article, response surface methodology (RSM) based optimization of three production parameters namely temperature, time and amount of starter culture of Vechur cow milk yoghurt (VCMY) on the basis of sensory evaluation responses comparing cross-bred cow milk yoghurt (CCMY) as the control is reported. The optimized values of production parameters were 2.15 per cent rate of inoculation, 42°C incubation temperature and 4 h incubation period. The optimized product exhibited significantly lower syneresis, a*, b* values and higher L* values than CCMY. Physico-chemical, microbiological, textural and sensory properties of both VCMY and CCMY during room temperature and refrigerated storage were assessed daily until the onset of spoilage (room temperature) or at five day intervals over a period of 15 d (refrigerated). Both room temperature stored products were graded undesirable by the sensory panel upon one day of storage. Significant reduction was observed in the fat, SNF, total solids, protein and pH content and all the tested colour parameters of the optimized product during refrigerated storage. Total viable counts as well as yeast and mould counts and lactic acid bacteria counts of both VCMY and CCMY progressively increased over the 15 d of storage. Significant reductions were observed in the flavour (P < 0.01), body and texture, colour and appearance and overall acceptability (P < 0.05) scores of both the samples over a period of 15 d. During storage, hardness and adhesiveness values showed an increasing trend whereas the cohesiveness showed a decreasing trend. Storage studies revealed significant differences in the acidity, pH, syneresis, tyrosine value, colour parameters and sensorial attributes of both the yoghurt samples. During the 15 d refrigerated storage period, the VCMY exhibited superior technological attributes to CCMY in terms of lower syneresis %, acidity, microbial population, firmer and less cohesive texture, better flavour, colour and appearance scores. Being the first comprehensive study on the utilization of Vechur cow milk for the preparation of yoghurt, the data generated in the current study would provide a solid base for the exploration of fermentation as a means of value addition of milk of this very rare indigenous cattle breed.

Extrusion-Based Printing of Myoblast-Loaded Fibrin Microthreads to Induce Myogenesis.

Large skeletal muscle injuries such as volumetric muscle loss (VML) disrupt native tissue structures, including biophysical and biochemical signaling cues that promote the regeneration of functional skeletal muscle. Various biofabrication strategies have been developed to create engineered skeletal muscle constructs that mimic native matrix and cellular microenvironments to enhance muscle regeneration; however, there remains a need to create scalable engineered tissues that provide mechanical stability as well as structural and spatiotemporal signaling cues to promote cell-mediated regeneration of contractile skeletal muscle. We describe a novel strategy for bioprinting multifunctional myoblast-loaded fibrin microthreads (myothreads) that recapitulate the cellular microniches to drive myogenesis and aligned myotube formation. We characterized myoblast alignment, myotube formation, and tensile properties of myothreads as a function of cell-loading density and culture time. We showed that increasing myoblast loading densities enhances myotube formation. Additionally, alignment analyses indicate that the bioprinting process confers myoblast alignment in the constructs. Finally, tensile characterizations suggest that myothreads possess the structural stability to serve as a potential platform for developing scalable muscle scaffolds. We anticipate that our myothread biofabrication approach will enable us to strategically investigate biophysical and biochemical signaling cues and cellular mechanisms that enhance functional skeletal muscle regeneration for the treatment of VML.

Formation of a Novel Antagonistic Bacterial Combination to Enhance Biocontrol for Cucumber Fusarium Wilt.

Paenibacillus polymyxa strain PJH16, isolated and tested by our team, suppresses cucumber Fusarium wilt as an efficient biocontrol agent. For further investigation, the strain has been combined with two other Bacillus strains (Bacillus velezensis VJH504 and Bacillus subtilis JNF2) to enhance biocontrol ability, which formed high-efficiency microbial agents in the current study. The methodological target taken is based on achieving the optimal growth conditions of the combined microbial agents; hence, the medium composition and culture conditions were optimized through a single-factor test, orthogonal test and response surface methodology. Following this, the effectiveness of the microbial combination was assessed through pot experiments, which provided a theoretical foundation for the synthesis of microbial flora to significantly control cucumber Fusarium wilt. The results showed excellent compatibility, proving suitable for the proliferation and growth of Paenibacillus polymyxa PJH16, Bacillus velezensis VJH504, and Bacillus subtilis JNF2 strains together, specifically, when the inoculation amounts were adjusted to 4% of each. Using the single-factor test and orthogonal test analysis, the optimum composition of culture medium for the composite strain was identified as 3% glucose as the optimal carbon source, 2% yeast extract powder as the preferred nitrogen source, and 1% dipotassium hydrogen phosphate as the most suitable inorganic salt. Furthermore, the optical density (OD600) of the composite strain solution reached its highest level at 3.16 under the following culture conditions: inoculation volume of 200 µL, 171 rpm culture speed, 21.6 h culture time, 30 °C cultural temperature, and an initial pH of 7.0. The pot experiment demonstrated that the mixed bacterial solution achieved a relative control efficacy of 93.4% against cucumber Fusarium wilt, which was significantly superior to that of single- strain or pesticide treatment, and also promoted cucumber growth. In summary, the microbial flora synthesized by the three Bacillus strains displayed a high bacterial concentration, following the optimization of culture conditions, and exerted remarkable control and growth-promoting effects on cucumber Fusarium wilt. This finding holds great significance for future developments of composite microbial agents.

Effects of Different Types of Microplastics on Cold Seep Microbial Diversity and Function.

The massive production and widespread use of plastics have resulted in a growing marine plastic pollution problem. Cold seep ecosystems are maintained by microorganisms related to nitrogen and carbon cycling that occur in deep-sea areas, where cold hydrocarbon-rich water seeps from the ocean floor. Little is known about plastic pollution in this ecosystem. To fill this knowledge gap, we collected sediment and seawater samples from the Haima cold seep and conducted laboratory cultivation experiments, simulating in situ environmental conditions. Environmental factors and microbial genetics were analyzed at different stages over a 2-month cultivation period. Our main conclusions are as follows: (1) When microplastics (MPs) were added to sediment and seawater environments, the microbial communities most closely resembled those of the original habitat. The changes in the plastisphere communities were mainly associated with the culture time. (2) The co-occurrence network of the plastisphere was more fragile than that of environments. (3) Multiple environmental factors determined the community composition, whereas a small number of environmental variables drove the community function. MPs affected nitrogen cycling and methane metabolism and might aggregate pathogenic species. This work provides a better perspective of the effect of MPs on the community structure and function in cold seeps.

Miniaturization and characterization of patient derived hepatocellular carcinoma tumor organoid cultures for cancer drug discovery applications.

Patient derived tumor organoid (PDTO) models retain the structural, morphological, genetic, and clonal heterogeneity of the original tumors. The ability to efficiently generate, expand, and biobank PDTOs has the potential to make the clinical diversity of cancer accessible for personalized medicine assay guided therapeutic drug selection and drug discovery. We describe the miniaturization and growth in 96- and 384-well formats of a single non-tumor liver and two Hepatocellular carcinoma (HCC) organoids derived from cryopreserved PDTO cells and the application of high content imaging (HCI) to characterize the models and enhance drug sensitivity testing. Non-invasive sequentially acquired transmitted light images showed that seeding cryopreserved cells from non-tumoral and HCC PDTOs into 96- or 384-well plates in reduced growth factor Matrigel (rgf-MG) that were fed with growth medium every 3 days supported organoid growth up to 15 days. The number and sizes of organoids increased with longer times in culture. HCC PDTO's had more heterogeneous morphologies than non-tumor organoids with respect to size, shape, and optical density. Organoids cultured in rgf-MG could be stained in situ with HCI reagents without mechanical, chemical or enzymatic disruption of the hydrogel matrices and quantitative data extracted by image analysis. Hoechst and live/dead reagents provided organoid numbers and viability comparisons. HCC PDTO's stained with phalloidin or immuno-stained with α-tubulin antibodies revealed F-actin and microtubule cytoskeleton organization. HCC PDTO's stained with antibodies to signaling pathway proteins and their phosphorylation status allowed comparisons of relative expression levels and inference of pathway activation. Images of HCC PDTO's exposed to ellipticine showed that drugs penetrate Matrigel hydrogels and accumulate in organoid cells. 9-day 384-well HCC organoid cultures exhibited robust and reproducible growth signals suitable for cancer drug testing. Complimenting cell viability readouts with multiple HCI parameters including morphological features and dead cell staining improved the analysis of drug impact and enhanced the value that could be extracted from these more physiologically relevant three-dimensional HCC organoid cultures.

Growth and bioactivity of Phellinus linteus in the mushroom farm, at different cultivation times

Growth and bioactivity of <i>Phellinus linteus</i> in the mushroom farm, at different cultivation times

Accelerating CAR T cell manufacturing with an automated next-day process.

The traditional method of CAR T cell production involves lengthy ex-vivo culture times which can result in the reduction of crucial naïve T cell subsets. Moreover, traditional CAR T cell therapy manufacturing processes can prolong time-to-patient, potentially delaying patient treatment, and contribute to disease progression. In this study, we describe an innovative and semi-automated 24-hour CAR T manufacturing process that yields a higher percentage of naïve/stem-cell like T cells which showed high cytotoxic activity and cytokine release in vitro. The data supports the feasibility of implementing this streamlined manufacturing process in clinics. This approach also has the potential to enhance CAR T therapy efficacy and improve patient access to therapy.

Formation of epithelial polarity on the fluorinated-oil microdropletsurfacebyregulating cell adhesion.

Polarized epithelial cells are compartmentalized into apical and basement membranes with asymmetrically distributed proteins. This study aimed to establish a method for culturing epithelial cells at the fluorinated oil (Novec-7500) microdroplet surface for the formation of epithelial polarity, which is desirable for regenerative medicine and drug discovery research. Microdroplet surfaces treated with fibronectin, which regulates a variety of cell behaviors through direct interactions with cell surface integrin receptors, were prepared for culturing epithelial cells. The cells adhered rapidly to the fibronectin-coated fluorinated oil-medium interface and reached confluence. However, as the culture time progressed, the cells began to detach from the microdroplet surface. To promote adhesion to the microdroplet interface, the cells were exposed to the Rho-associated protein kinase inhibitor Y27632, which increased the frequency of microdroplets with cells adhering to the liquid-liquid interface by 1.63-fold. However, continuous exposure to Y27632 caused the cells to detach from the microdroplet surface. When the drug was switched from Y27632 to forskolin, which enhances cell-cell and cell-substrate adhesion, the cells remained as a monolayer on the microdroplet interface. Na+/K+-ATPase and zonula occludens-2 were localized to the apical and lateral membranes of the cells, respectively, while paxillin co-localized with fibronectin at the microdroplet interface. This suggests that the cells exhibited epithelial polarity. These findings indicate that the regulation of cell-cell and cell-substrate adhesion is crucial for establishing epithelial polarity in cells cultured on the microdroplet interface.

Detection of aflatoxin B1 level and revelation of its dynamic accumulation process using visible/near-infrared hyperspectral and microscopic imaging.

Understanding and controlling the dynamic process of aflatoxin B1 (AFB1) accumulation by Aspergillus flavus (A. flavus) remains challenging. In this study, the A. flavus development and AFB1 accumulation were investigated using visible/near-infrared (Vis/NIR) hyperspectral imaging (HSI) on culture media, Potato Dextrose Agar (PDA), PDA+l-glutamine (Gln), and PDA+rapamycin (RAPA). In addition, the levels of AFB1 in various heterogeneous regions of colonies were measured and their microscopic morphology was characterized. In the temporal and spatial domains, fungal colonies exhibited a concentric circular response pattern. A continuous increase in AFB1 content was observed in the PDA and PDA+Gln groups as culture time increased. The growth of A. flavus and aflatoxin accumulation were promoted by adding Gln to PDA. However, adding RAPA inhibited the development of fungi and the production of AFB1. The distribution of AFB1 across the fungal colony was uneven, and this heterogeneity was associated with the aging and autolysis of the hyphae. Principal component analysis showed that spectral bands of 480, 623, 674, 726nm were related to the color changes of hyphae and spores during colony growth; however, wavelengths of 840, 882, 867, 972nm were key bands related to changes in nutritional composition. Multiple preprocessing techniques and modeling methods employed to construct regression models for predicting AFB1 contents showed that the first-derivative and partial least squares regression (PLSR) provided the best results. A visualization map of AFB1 levels established using the optimal model showed a spatial pattern similar to the measurement results. This study highlights the application potential of Vis/NIR HSI for monitoring A. flavus growth and AFB1 content.

Wasting Time as a Cultural Practice in Contemporary Serbia

The expression "wasting time" is a colloquial label for periods of time spent waiting to receive services in various social situations in which people find themselves in state or private institutions, such as the Ministry of Internal Affairs or health institutions, banks and technical inspections of vehicles, etc. In this way, the superiority of social or cultural time over individual time is manifested. Cultural use of time represents the tendency to manage linear time by repeating events anchored in certain time segments, such as rituals, music festivals, sports events, or national holidays. Individuals perceive time based on cultural learning, understanding sociocultural time as a framework for organizing their personal time. This means that when they are not engaged in activities arising from collective time, individuals tend to organize their time according to their personal needs and intentions - as much as possible.Wasting time is therefore perceived as time not spent on activities related to work or free time that are the consequence of personal choice or existential necessity. The term most often refers to the waiting periods, which are apparently not regarded as integral to completing a task, such as a medical examination or obtaining personal documents. The concept of wasting time emerges as an uncreative, imposed, wasted time spent on completing tasks that could be organized differently. It is an empty time, devoid of important events and which is contrasted with periods of activity. The idea of ​​wasting time corresponds to the personal experience of time. The given expression is a consequence of evaluating whether or not a certain period of time was spent usefully, viewed from a personal perspective. Everyone's experience suggests that time seems to pass more slowly when we can do nothing but wait. The networking of such experiences, arising from a multitude of social situations that impose prolonged waiting as a procedural part to obtain what is considered useful or necessary for individuals, can be characterised as a cultural practice. The contributors to this practice include all social institutions that provide services to users through direct contact and hold a societal monopoly of performing their activities.

Effects of Saprolegnia parasitica on pathological damage and metabolism of Epithelioma papulosum cyprini cell.

Saprolegniasis is a common fungal disease in aquaculture. It will form white flocculent hyphae on the skin of fish, and the hyphae may grow inward and penetrate into muscle tissue, which will reduce the immunity of the body and eventually lead to death. However, there are still some gaps in the mechanism of the fish body surface against the invasion of Saprolegnia. This study explored the defense mechanism of Epithelioma papulosum cyprini cell (EPC) in the process of Saprolegnia parasitica infection from the perspective of pathogenic bacteria and host cells, so as to provide a theoretical basis for further exploring the mechanism of host resistance to S. parasitica invasion. The EPC cell was used as the research object. The EPC cells were treated with 1×106 CFU/mL of S. parasitica for 0, 6, 12, 24, 48 and 72 h. Cell viability and cell membrane damage were detected, and the non-specific immune enzyme activity in the cells was detected. Based on the above research, the apoptosis genes and antioxidant genes in the cells were detected to analyze the effect of S. parasitica on the metabolism of the EPC cells. The results showed that with the prolongation of the co-culture time of S. parasitica and cells, the cell viability gradually decreased and the cell membrane integrity was destroyed, but at the same time, the activity of non-specific immune enzymes increased to resist the infection of S. parasitica. In addition, the detection of EPC apoptosis gene casp3a and CTSD showed that the relative content of casp3a gene increased significantly at 24 h and reached the maximum value of the culture time (P<0.05). The content of CTSD gene increased significantly at 12 h and reached the maximum value (P<0.05). The results of antioxidant immune genes serpinh1a and gpx1a were opposite to the structure of apoptotic genes. The content of serpinh1a and gpx1a genes decreased significantly at 12 h (P<0.05), but with the prolongation of culture time, the content increased significantly at 24 h and 48 h (P<0.05). After stimulation of EPC cells by S. parasitica, the differential metabolites were mainly concentrated in Lipids, Compounds with biological roles and Phytochemical compounds. The KEGG pathway mainly focused on ABC transporters, Glycerophospholipid metabolism, Cysteine and methionine metabolism, Glycine, serine and threonine metabolism, Purine metabolism. In general, S. parasitica can affect cell activity, destroy the cell membrane of EPC cells, and cause apoptosis. However, EPC cells can also resist the invasion of S. parasitica by regulating their own non-specific immunity and their own metabolites, thereby protecting the body from the infection of S. parasitica.

A new human autologous hepatocyte/macrophage co-culture system that mimics drug-induced liver injury-like inflammation.

The development of in vitro hepatocyte cell culture systems is crucial for investigating drug-induced liver injury (DILI). One prerequisite for monitoring DILI related immunologic reactions is the extension of primary human hepatocyte (PHH) cultures towards the inclusion of macrophages. Therefore, we developed and characterized an autologous co-culture system of PHH and primary human hepatic macrophages (hepM) (CoC1). We compared CoC1 with a co-culture of the same PHH batch + M0 macrophages derived from THP1 cells (CoC2) in order to represent a donor independent macrophage reaction. Then, we treated the mono- and co-cultures with drugs that cause DILI-menadione (MEN, 1 or 10µM, 3h), diclofenac (DIC, 0.5 or 5mM, 6h), or acetaminophen (APAP, 0.5 or 5mM, 6h)-and assessed culture stability, cell activity, macrophage differentiation, cytokine production and cell viability. Without drug treatment, CoC1 was the most stable over a culture time of up to 60h. Cytokine array analysis revealed a proinflammatory profile of PHH mono-cultures due to isolation stress but showed different influences of hepM and M0 on the cytokine profile in the co-cultures. MEN, DIC and APAP treatment led to donor-dependent signs of cell stress and toxicity. HepM can either promote or reduce the DILI effects donor dependently in CoC1. CoC2 are slightly less sensitive than CoC1 in representing DILI. In summary, we present a new autologous co-culture system that can mimic DILI in a donor-dependent manner. This cellular system could be useful for new drug testing strategies and reducing animal testing.