Enhancing biodegradation efficiency of PLA/PBAT-ST20 bioplastic using thermophilic bacteria co-culture system: New insight from structural characterization, enzyme activity, and metabolic pathways.

The rising utilization of PLA/PBAT-ST20 presents potential ecological risks stemming from its casual disposal and incomplete degradation. To solve this problem, this study investigated the degradation capabilities of PLA/PBAT-ST20 by a co-culture system comprising two thermophilic bacteria, Pseudomonas G1 and Kocuria G2, selected and identified from the thermophilic phase of compost. Structural characterization results revealed that the strains colonized the PLA/PBAT-ST20's surface, causing holes and cracks, with an increase in the carbonyl index (CI) and polydispersity index (PDI), indicating oxidative degradation. Enzyme activity results demonstrated that the co-culture system significantly enhanced the secretion and activity of proteases and lipases, promoting the breakdown of ester bonds. LC-QTOF-MS results showed that various intermediate products were obtained after degradation, ultimately participating in the TCA cycle (ko00020), further completely mineralized. Additionally, after 15-day compost, the co-culture system achieved a degradation rate of 72.14 ± 2.1 wt% for PBAT/PLA-ST20 films, with a decrease in the abundance of plastic fragments of all sizes, demonstrating efficient degradation of PLA/PBAT-ST20 films. This study highlights the potential of thermophilic bacteria to address plastic pollution through biodegradation and emphasizes that the co-culture system could serve as an ideal solution for the remediation of PLA/PBAT plastics.

In vitro co-culture models for studying organoids-macrophages interaction: the golden technology of cancer immunotherapy.

Macrophages, as the largest immune cell group in tumour tissues, play a crucial role in influencing various malignant behaviours of tumour cells and tumour immune evasion. As the research on macrophages and cancer immunotherapy develops, the importance of appropriate research models becomes increasingly evident. The development of organoids has bridged the gap between traditional two-dimensional (2D) cultures and animal experiments. Recent studies have demonstrated that organoids exhibit similar physiological characteristics to the source tissue and closely resemble the in vivo genome and molecular markers of the source tissue or organ. However, organoids still lack an immune component. Developing a co-culture model of organoids and macrophages is crucial for studying the interaction and mechanisms between tumour cells and macrophages. This paper presents an overview of the establishment of co-culture models, the current research status of organoid macrophage interactions, and the current status of immunotherapy. In addition, the application prospects and shortcomings of the model are explained. Ultimately, it is hoped that the co-culture model will offer a preclinical testing platform for maximising a precise cancer immunotherapy strategy.

Retinal debris triggers cytotoxic damage in cocultivated primary porcine RPE cells.

Introduction: One of the most common causes of vision loss in the elderly population worldwide is age-related macular degeneration (AMD). Subsequently, the number of people affected by AMD is estimated to reach approximately 288 million by the year 2040. The aim of this study was to develop an ex vivo model that simulates various aspects of the complex AMD pathogenesis. Methods: For this purpose, primary porcine retinal pigment epithelial cells (ppRPE) were isolated and cultured. One group was exposed to medium containing sodium iodate (NaIO3) to induce degeneration. The others were exposed to different supplemented media, such as bovine serum albumin (BSA), homogenized porcine retinas (HPR), or rod outer segments (ROOS) for eight days to promote retinal deposits. Then, these ppRPE cells were cocultured with porcine neuroretina explants for another eight days. To assess the viability of ppRPE cells, live/dead assay was performed at the end of the study. The positive RPE65 and ZO1 area was evaluated by immunocytochemistry and the expression of RLBP1, RPE65, and TJP1 was analyzed by RT-qPCR. Additionally, drusen (APOE), inflammation (ITGAM, IL6, IL8, NLRP3, TNF), oxidative stress (NFE2L2, SOD1, SOD2), and hypoxia (HIF1A) markers were investigated. The concentration of the inflammatory cytokines IL-6 and IL-8 was determined in medium supernatants from day 16 and 24 via ELISA. Results: Live/dead assay suggests that especially exposure to NaIO3 and HPR induced damage to ppRPE cells, leading in a significant ppRPE cell loss. All supplemented media resulted in decreased RPE-characteristic markers (RPE65; ZO-1) and gene expression like RLBP1 and RPE65 in the cultured ppRPE cells. Besides, some inflammatory, oxidative as well as hypoxic stress markers were altered in ppRPE cells cultivated with NaIO3. The application of HPR induced an enhanced APOE expression. Pre-exposure of the ppRPE cells led to a diminished number of cones in all supplemented media groups compared to controls. Discussion: Overall, this novel coculture model represents an interesting initial approach to incorporating deposits into coculture to mimic AMD pathogenesis. Nevertheless, the effects of the media used need to be investigated in further studies.

Interspecific cross-talk: The catalyst driving microbial biosynthesis of secondary metabolites.

Microorganisms co-exist and co-evolve in nature, forming intricate ecological communities. The interspecies cross-talk within these communities creates and sustains their great biosynthetic potential, making them an important source of natural medicines and high-value-added chemicals. However, conventional investigations into microbial metabolites are typically carried out in pure cultures, resulting in the absence of specific activating factors and consequently causing a substantial number of biosynthetic gene clusters to remain silent. This, in turn, hampers the in-depth exploration of microbial biosynthetic potential and frequently presents researchers with the challenge of rediscovering compounds. In response to this challenge, the coculture strategy has emerged to explore microbial biosynthetic capabilities and has shed light on the study of cross-talk mechanisms. These elucidated mechanisms will contribute to a better understanding of complex biosynthetic regulations and offer valuable insights to guide the mining of secondary metabolites. This review summarizes the research advances in microbial cross-talk mechanisms, with a particular focus on the mechanisms that activate the biosynthesis of secondary metabolites. Additionally, the instructive value of these mechanisms for developing strategies to activate biosynthetic pathways is discussed. Moreover, challenges and recommendations for conducting in-depth studies on the cross-talk mechanisms are presented.

Accurate Identification of Cancer Cells in Complex Pre-Clinical Models Using a Deep-Learning Neural Network: A Transfection-Free Approach.

3D co-cultures are key tools for in vitro biomedical research as they recapitulate more closely the in vivo environment while allowing a tighter control on the culture's composition and experimental conditions. The limited technologies available for the analysis of these models, however, hamper their widespread application. The separation of the contribution of the different cell types, in particular, is a fundamental challenge. In this work, ORACLE (OvaRiAn Cancer ceLl rEcognition) is presented, a deep neural network trained to distinguish between ovarian cancer and healthy cells based on the shape of their nucleus. The extensive validation that are conducted includes multiple cell lines and patient-derived cultures to characterize the effect of all the major potential confounding factors. High accuracy and reliability are maintained throughout the analysis (F1score> 0.9 and Area under the ROC curve -ROC-AUC- score = 0.99) demonstrating ORACLE's effectiveness with this detection and classification task. ORACLE is freely available (https://github.com/MarilisaCortesi/ORACLE/tree/main) and can be used to recognize both ovarian cancer cell lines and primary patient-derived cells. This feature is unique to ORACLE and thus enables for the first time the analysis of in vitro co-cultures comprised solely of patient-derived cells.

Two cyanobacterial species exhibit stress responses when grown together in visible light or far-red light.

Although most cyanobacteria grow in visible light (VL; λ = 400-700 nm), some cyanobacteria can also use far-red light (FRL; λ = 700-800 nm) for oxygenic photosynthesis by performing far-red light photoacclimation. These two types of cyanobacteria can be found in the same environment. However, how they respond to each other remains unknown. Here, we reveal that coculture stresses FRL-using Chlorogloeopsis fritschii PCC 9212 and VL-using Synechocystis sp. PCC 6803. No significant growth difference was found in Synechocystis sp. PCC 6803 between the coculture and the monoculture. Conversely, the growth of Chlorogloeopsis fritschii PCC 9212 was suppressed in VL under coculture. According to transcriptomic analysis, Chlorogloeopsis fritschii PCC 9212 in coculture shows low transcript levels of metabolic activities and high transcript levels of ion transporters, with the differences being more noticeable in VL than in FRL. The transcript levels of stress responses in coculture were likewise higher than in monoculture in Synechocystis sp. PCC 6803 under FRL. The low transcript level of metabolic activities in coculture or the inhibition of cyanobacterial growth indicates a possible negative interaction between these two cyanobacterial strains.IMPORTANCEThe interaction between two cyanobacterial species is the primary focus of this study. One species harvests visible light, while the other can harvest far-red and visible light. Prior research on cyanobacteria interaction concentrated on its interactions with algal, coral, and fungal species. Interactions between cyanobacterial species were, nevertheless, rarely discussed. Thus, we characterized the interaction between two cyanobacterial species, one capable of photosynthesis using far-red light and the other not. Through experimental and bioinformatic approaches, we demonstrate that when one cyanobacterium thrives under optimal light conditions, it stresses the remaining cyanobacterial species. We contribute to an ecological understanding of these two kinds of cyanobacteria distribution patterns. Cyanobacteria that utilize far-red light probably disperse in environments with limited visible light to avoid competition with other cyanobacteria. From a biotechnological standpoint, this study suggests that the simultaneous cultivation of two cyanobacterial species in large-scale cultivation facilities may reduce the overall biomass yield.

Carbon Nanotube Immunotoxicity in Alveolar Epithelial Type II Cells Is Mediated by Physical Contact-Independent Cell-Cell Interaction with Macrophages as Demonstrated in an Optimized Air-Liquid Interface (ALI) Coculture Model.

There is a need for the assessment of respiratory hazard potential and mode of action of carbon nanotubes (CNTs) before their approval for technological or medical applications. In CNT-exposed lungs, both alveolar macrophages (MФs), which phagocytose CNTs, and alveolar epithelial type II cells (AECII cells), which show tissue injury, are impacted but cell-cell interactions between them and the impacted mechanisms are unclear. To investigate this, we first optimized an air-liquid interface (ALI) transwell coculture of human AECII cell line A549 (upper chamber) and human monocyte cell line THP-1 derived macrophages (lower chamber) in a 12-well culture by exposing macrophages to CNTs at varying doses (5-60 ng/well) for 12-48 h and measuring the epithelial response markers for cell differentiation/maturation (proSP-C), proliferation (Ki-67), and inflammation (IL-1ß). In optimal ALI epithelial-macrophage coculture (3:1 ratio), expression of Ki-67 in AECII cells showed dose dependence, peaking at 15 ng/well CNT dose; the Ki-67 and IL-1ß responses were detectable within 12 h, peaking at 24-36 h in a time-course. Using the optimized ALI transwell coculture set up with and without macrophages, we demonstrated that direct interaction between CNTs and MФs, but not a physical cell-cell contact between MФ and AECII cells, was essential for inducing immunotoxicity (proliferative and inflammatory responses) in the AECII cells.

Chondrosarcoma Co-Culture 3D Model─An Insight to Evaluate Drugs Acting on TAMs.

Chondrosarcoma (CHS), also known as malignant cartilage tumors, is the second most common bone cancer after osteosarcoma. This tumor is particularly chemo- and radioresistant, and the only therapeutic alternative is surgery with wide margins. The tumor immune microenvironment reveals an infiltration of tumor-associated macrophages (TAMs) sometimes approaching 50% of the tumor mass, mainly differentiated into M2-like phenotype and correlated with poor prognosis and metastasis. Thus, macrophage-targeting therapies could have an interest in the management of CHS. To evaluate these strategies, we propose here the development of a three-dimensional (3D) tumoroid co-culture model between two human CHS cell lines (JJ012 and CH2879) and a human leukemia monocytic cell line (THP-1) in a methylcellulose matrix. These two models were compared to the in vivo xenograft models in terms of macrophage phenotypes, proteoglycans, MMP-9, and COX-2 expression. Finally, mifamurtide, an immunomodulator acting on TAMs, was evaluated on the most in vitro relevant model: 3D co-culture CH2879 model. Our results showed that it is now possible to develop 3D models that very accurately mimic what is found in vivo with the possibility of evaluating treatments specific to a tumor cell component.

A rich-nutritious cultured meat via bovine myocytes and adipocytes co-culture: Novel Prospect for cultured meat production techniques.

Cultured meat, an emerging meat production technology, has reduced environmental burden as well as provide healthier and more sustainable method of meat culture. Fat in cultured meat is essential for enhancing texture, taste, and tenderness. However, current cultured meat production method is limited to single-cell type. To meet the consumer demands for cultured meat products, it is crucial to develop new methods for producing cultured meat products that contain both muscle and fat. In this study, cell viability and differentiation were promoted by controlling the ratio and cultivation conditions of myocytes and adipocytes. The total digestibility of cultured meat exceeded 37%, higher than that of beef (34.7%). Additionally, the texture, appearance, and taste of the co-cultured meat were improved. Collectively, this research has great promise for preparing rich-nutritious and digestion cultured meat.

Understanding the sources, function, and irreplaceable role of cobamides in organohalide-respiring bacteria.

Halogenated organic compounds are persistent pollutants that pose a serious threat to human health and the safety of ecosystems. Cobamides are essential cofactors for reductive dehalogenases (RDase) in organohalide-respiring bacteria (OHRB), which catalyze the dehalogenation process. This review systematically summarizes the impact of cobamides on organohalide respiration. The catalytic processes of cobamide in dehalogenation processes are also discussed. Additionally, we examine OHRB, which cannot synthesize cobamide and must obtain it from the environment through a salvage pathway; the co-culture with cobamide producer is more beneficial and possible. This review aims to help readers better understand the importance and function of cobamides in reductive dehalogenation. The presented information can aid in the development of bioremediation strategies.

Search for natural products from actinomycetes of the genus Nocardia.

The genus Nocardia are gram-positive bacteria, many of which possess pathogenicity and infect human lungs, skin, brain, and other organs. Since research on the genus Nocardia has not progressed as rapidly as that on the genus Streptomyces, the genus Nocardia is considered a useful undeveloped resource for exploring natural products. On the other hand, when the genus Nocardia infects the human body, the strains are attacked by immune cells such as macrophages. Therefore, we suggested a new method for screening natural products by culturing the genus Nocardia in the presence of animal cells. In this review, we describe our recent results in searching for natural products from the genus Nocardia.

The systemic cellular immune response against allogeneic mesenchymal stem cells is influenced by inflammation, differentiation and MHC compatibility: in vivo study in the horse.

The effectiveness and safety of allogeneic mesenchymal stem/stromal cells (MSCs) can be affected by patient's immune recognition. Thus, MSC immunogenicity and their immunomodulatory properties are crucial aspects for therapy. Immune responses after allogeneic MSC administration have been reported in different species, including equine. Interactions of allogenic MSCs with the recipient's immune system can be influenced by factors like matching or mismatching for the major histocompatibility complex (MHC) between donor-recipient, and by the levels of MHC expression in MSCs. The latter can vary upon MSC inflammatory exposure or differentiation, such as chondrogenic induction, making both priming and differentiation interesting therapeutic strategies. This study investigated the systemic in vivo immune cellular response against allogeneic equine MSCs in these situations. Either MSCs in basal conditions (MSC-naïve), pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were repeatedly administered subcutaneously into autologous, MHC-matched or MHC-mismatched allogeneic equine recipients. At different time-points after each administration, lymphocytes were obtained from recipient horses and exposed in vitro to the same type of MSCs to assess the proliferative response of different T cell subsets (cytotoxic, helper, regulatory), B cells, and interferon gamma (IFNγ) secretion. Higher proliferative response of helper and cytotoxic T lymphocytes and IFNγ secretion was observed in response to all types of MHC-mismatched MSCs over MHC-matched ones. MSC-primed produced the highest immune response, followed by MSC-naïve, and MSC-chondro. However, MSC-primed activated Treg and had a mild effect on B cells, and the response after their second administration was similar to the first one. On the other hand, both MSC-chondro and MSC-naïve barely induced Treg response but promoted B lymphocyte activation, and proportionally induced a higher cell response after the second administration. In conclusion, both the type of MSC conditioning and the MHC compatibility influenced systemic immune recognition of equine MSCs after single and repeated administrations, but the response was different. Selecting MHC-matched donors would be particularly recommended for MSC-primed and repeated MSC-naïve administrations. While MHC-mismatching in MSC-chondro would be less critical, B cell response should not be ignored. Comprehensively investigating the in vivo immune response against equine allogeneic MSCs is crucial for advancing veterinary cell therapies.

Generating Neuroimmune Assembloids Using Human Induced Pluripotent Stem Cell (iPSC)-Derived Cortical Organoids and Microglia.

The emergence of brain organoids has revolutionized our understanding of neurodevelopment and neurological diseases by providing an in vitro model system that recapitulates key aspects of human brain development. However, conventional organoid protocols often overlook the role of microglia, the resident immune cells of the central nervous system. Microglia dysfunction is implicated in various neurological disorders, highlighting the need for their inclusion in organoid models. Here, we present a novel method for generating neuroimmune assembloids using human-induced pluripotent stem cell (iPSC)-derived cortical organoids and microglia. Building upon our previous work generating myelinating cortical organoids, we extend our methodology to include the integration of microglia, ensuring their long-term survival and maturation within the organoids. We describe two integration methods: one involving direct addition of microglia progenitors to the organoids and an alternative approach where microglia and dissociated neuronal progenitors are aggregated together in a defined ratio. To facilitate downstream analysis, we also describe a dissociation protocol for single-cell RNA sequencing (scRNA-seq) and provide guidance on fixation, cryosectioning, and immunostaining of assembloid structures. Overall, our protocol provides a comprehensive framework for generating neuroimmune assembloids, offering researchers a valuable tool for studying the interactions between neural cell types and immune cells in the context of neurological diseases.

Comprehensive transcriptomic and metabolomic insights into simultaneous CO2 sequestration and nitrate removal by the Chlorella vulgaris and Pseudomonas sp. consortium.

Simultaneous CO2 sequestration and nitrate removal can be achieved by co-cultivation of Chlorella vulgaris with Pseudomonas sp. However, a comprehensive understanding of the synergistic mechanism between C. vulgaris and Pseudomonas sp. remains unknown. In this study, transcriptomics and metabolomics analysis were employed to elucidate the synergistic mechanism of C. vulgaris and Pseudomonas sp. Transcriptomic and metabolomic analyses identified 3664 differentially expressed genes and 314 metabolites. Transcriptome analysis revealed that co-culture with Pseudomonas sp. promoted the photosynthesis of C. vulgaris by promoting the synthesis of photosynthetic pigments and photosynthesis-antenna proteins. Furthermore, it stimulated pathways associated with energy metabolism from carbon sources, such as the Calvin cycle, glycolytic pathway, and TCA cycle. Additionally, Pseudomonas sp. reduced nitrate levels in the co-culture system by denitrification, and microalgae regulated nitrate uptake by down-regulating the transcript levels of nitrate transporter genes. Metabolomic analysis indicated that nutrient exchange was conducted between algae and bacteria, and amino acids, phytohormones, and organic heterocyclic compounds secreted by the bacteria promoted the growth metabolism of microalgae. After supplementation with differential metabolites, the carbon fixation rate and nitrate removal rate of the co-culture system reached 0.549 g L-1 d-1 and 135.4 mg L-1 d-1, which were increased by 20% and 8%, respectively. This study provides a theoretical insight into microalgae-bacteria interaction and its practical application, as well as a novel perspective on flue gas treatment management.

Astrocytes facilitate gabazine-evoked electrophysiological hyperactivity and distinct biochemical responses in mature neuronal cultures.

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain that binds to GABA receptors and hyperpolarizes the postsynaptic neuron. Gabazine acts as a competitive antagonist to type A GABA receptors (GABAAR), thereby causing diminished neuronal hyperpolarization and GABAAR-mediated inhibition. However, the biochemical effects and the potential regulatory role of astrocytes in this process remain poorly understood. To address this, we investigated the neuronal responses of gabazine in rat cortical cultures containing varying ratios of neurons and astrocytes. Electrophysiological characterization was performed utilizing microelectrode arrays (MEAs) with topologically controlled microcircuit cultures that enabled control of neuronal network growth. Biochemical analysis of the cultures was performed using traditional dissociated cultures on coverslips. Our study indicates that, upon gabazine stimulation, astrocyte-rich neuronal cultures exhibit elevated electrophysiological activity and tyrosine phosphorylation of tropomyosin receptor kinase B (TrkB; receptor for brain-derived neurotrophic factor), along with distinct cytokine secretion profiles. Notably, neurons lacking proper astrocytic support were found to experience synapse loss and decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, astrocytes contributed to neuronal viability, morphology, vascular endothelial growth factor (VEGF) secretion, and overall neuronal network functionality, highlighting the multifunctional role of astrocytes.

Isolation, Culture, and Phenotypic Analysis of Murine Lung Organoids.

Three-dimensional (3D) organoid cultures retain self-renewing stem cells that differentiate into multiple cell types that display spatial organization and functional key features, providing a highly physiological relevant system. Here we describe a strategy for the generation of 3D murine lung organoids derived from freshly isolated primary tracheal and distal lung epithelial stem cells. Isolated tracheas are subjected to enzymatic digestion to release the epithelial layer that is then dissociated into a single cell suspension for organoid culture. Lung epithelial cells are obtained from dissected lobes, which are applied to mechanical and enzymatic dissociation. After flow sorting, organoids are established from tracheal basal, secretory club, and alveolar type 2 cells in the defined conditioned medium that is required to sustain organoid growth and generate the differentiated cells. Multi-cell-type organoid co-culture replicates niches for distal epithelial stem cells to differentiate into bronchiolar and alveolar cell types. Established organoids can be fixed for wholemount staining and paraffin embedding, or passaged for further culture. Taken together, this protocol provides an efficient and validated approach to generate murine lung organoids, as well as a platform for further analysis.

Potential impact of NOTCH1 activation on venetoclax sensitivity in chronic lymphocytic Leukaemia: In vitro insights and clinical implications.

Despite significant progress in treating chronic lymphocytic leukaemia (CLL), resistance to therapy remains challenging. NOTCH1 activation, common in CLL, confers adverse prognosis. This study explores the impact of NOTCH1 signalling on venetoclax sensitivity in vitro. Although NOTCH1 activation minimally impaired the susceptibility of CLL cells to venetoclax, ex vivo cell competition studies reveal that cells with constitutive NOTCH1 activation outgrew their wild-type counterparts in the presence of ongoing venetoclax exposure. Our findings suggest that while NOTCH1 activation is insufficient to confer venetoclax refractoriness, there is enhanced potential for cells with NOTCH1 activation to escape and thus become fully resistant to venetoclax.

The Effects of Endometrial Mesenchymal Stem Cells on The In Vitro Maturation of Germinal Vesicle Oocytes in Hanging Drop and Sodium Alginate Hydrogel Co-Culture Systems.

BACKGROUND: The aim of this study is to investigate the co-culture effects of human endometrial mesenchymal stem cells (EnMSCs) with mouse oocytes to enhance their maturation and development by using the hanging drop and sodium alginate hydrogel methods. MATERIALS AND METHODS: In this experimental study, we prepared human EnMSCs (2.5×105 cells/mL) and co-cultured them with partially denuded mouse oocytes by the hanging drop (n=120) and sodium alginate hydrogel (n=120) methods. Control oocytes (n=230, total) were cultured in both systems in the absence of human EnMSCs for 18 hours. Both survival and maturation rates of the oocytes were analysed morphologically. After insemination with capacitated sperm, the fertilization and development of the embryos up to the blastocyst stage were assessed and compared statistically for all of the study groups via one-way ANOVA and the t tests. RESULTS: Oocytes cultured in the hanging drop method had a significantly higher survival rate than their control group (92.60 ± 4.36% vs. 84.20 ± 3.12%, P=0.018). There were no significant differences between the two experimental groups in terms of survival. The mean percent of oocytes that reached the metaphase II (MII) stage was 64.35 ± 3.19% and fertilised was 62.25 ± 4.43% in the hanging drop method; these rates were 63.43 ± 1.92% and 58.14 ± 4.14 in sodium alginate hydrogel method, respectively. These rates were higher than their controls (P<0.050), but there were no statistical differences between the two experimental groups (P>0.050). Among the studied groups, the highest significant blastocyst rate (32.55 ± 2.18%) was observed in the hanging drop experimental group (P=0.0017). CONCLUSION: The results of this study show that human EnMSCs improve the survival, maturation, and development rates of oocytes and they could have future clinical applications.

Transcriptome analysis revealing the effect of Bupleurum scorzonerifolium Willd association with endophytic fungi CHS3 on the production of saikosaponin D.

Saikosaponin D (SSd) is a naturally active product with strong pharmacological activity found in Bupleurum scorzonerifolium Willd. Studies have shown that endophytic fungi have great potential as sources of natural medicines. Fusarium acuminatum (CHS3), an SSd-producing endophytic fungus, was isolated from B. scorzonerifolium. To elucidate the effect of host plants on the production of SSd in CHS3, CHS3 was co-cultured with suspension cells of B. scorzonerifolium and SSd was detected using high-performance liquid chromatography (HPLC). Transcriptome sequencing (RNA-Seq) of CHS3 before and after co-culture was performed using an Illumina HiSeq 2500 platform. The results indicated that the content of SSd synthesised by CHS3 increased after co-culture with suspension cells of B. scorzonerifolium. Transcriptome analysis of CHS3 with differentially expressed genes (DEGs) showed that 1202 and 1049 genes were upregulated and downregulated, respectively, after co-culture. Thirty genes associated with SSd synthesis and 11 genes related to terpene backbone biosynthesis were annotated to the Kyoto Encyclopaedia of Genes and Genomes (KEGG). Combined with transcriptome data, it was speculated that the mevalonate (MVA) pathway is a possible pathway for SSd synthesis in CHS3, and the expression of key enzyme genes (HMGR, HMGCS, GGPS1, MVK, FDFT1, FNTB) was validated by qRT-PCR. In conclusion, the endophytic fungus CHS3 can form an interactive relationship with its host, thereby promoting SSd biosynthesis and accumulation by upregulating the expression of key enzyme genes in the biosynthesis pathway.

Development of Imiquimod-induced HaCaT-THP-1 co-culture for modeling of psoriasis.

Psoriasis is one of the most prevalent and chronic inflammatory disease of the skin, associated with disrupted barrier function. Currently, a widely accepted, generally usable cell culture model has not been developed yet. In the present work, we aimed to establish a co-culture model with human keratinocyte (HaCaT) and human monocyte cells (THP-1) induced by Imiquimod (IMQ), which acts on the TLR7 receptor. The role of TLR7 expressed on THP-1 cells was confirmed by immunofluorescence staining of NF-κB activation. Chloroquine (CH) was used as a receptor inhibitor, in the presence or absence of which the NF-κB pathway was activated. We determined the most effective proliferation-stimulating IMQ concentration by RTCA method and the hyperproliferative effect was investigated by wound-healing test. The effect of IMQ was compared with the effects of the anthocyanin (AC) components from the anti-inflammatory sour cherry extract that we have already studied. We found that IMQ significantly increased the migration rate however, the combined treatment resulted in a decreased migration rate compared to the IMQ treatment alone. Inflammatory cytokines were measured from the supernatant of co-culture by ELISA. During the development of the co-culture intended to model psoriasis, we confirmed the induction effect of IMQ and in the case of AC treatment, we supported the stabilizing effect of the barrier.