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Background: Typical difficulties encountered during in vitro fertilization (IVF) to produce embryos in pigs include poor pronucleus formation and poor-quality fertilized embryos because of high polysperm invasion. In this study, we evaluated the effects of supplementation with apple seed extract (ASE) and coculture systems on porcine in vitro-fertilized embryo culture. Methods: Slaughterhouse-derived ovaries were used to obtain cumulus-oocyte complexes (COCs). COCs were conventionally used to perform IVF. We examined the differences in apoptosis and metabolism during development following addition of ASE to normal culture and coculture systems. Matrix metalloproteinases (MMPs), cell development-related factors, and apoptotic proteins were compared in porcine embryos produced under different conditions. Results: The expression of genes related to insulin-like growth factor (IGF) signaling was increased in the coculture system. In the ASE group, early apoptosis and necrosis were reduced in fertilized embryos and the late survival rate increased. Supplementation of the coculture system with ASE led to increased expression of BCL-2 and decreased expression of Casp-3 in the cytoplasm, thereby lowering the apoptosis rate and inducing MMP expression. In addition, compared with the extract-supplemented group in normal culture, the activity of MMP-2 decreased in the coculture system supplemented with ASE, activity of MMP-9 increased, and the expression of dynactin p62 and BrdU in the cytoplasm was higher than that in the other groups. Conclusions: The coculture system increased the activity of the embryonic cytoplasm compared with the non-coculture system. Supplementation with ASE may induce cell activity and inhibit the expression of apoptotic factors.

IntroductionMicrocystis aeruginosa (M. aeruginosa), one of the most abundant blue-green algae in aquatic environments, produces microcystin by causing harmful algal blooms (HABs). This study investigated the combined effects of nutrients and competition among cyanobacterial subpopulations on the synthesis of microcystin-LR.MethodsUnder varying nitrogen and phosphorus concentrations, cyanobacterial coculture, and the presence of algicidal DCMU, the growth was monitored by optical density analysis or microscopic counting, and the microcystin production was analyzed using high-performance liquid chromatography-UV. Furthermore, growth and toxin production were predicted using a kinetic model.Results and discussionFirst, coculture with the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (S. elongatus) reduced M. aeruginosa biomass and microcystin production at 30°C. Under high nitrogen and low phosphorus conditions, S. elongatus was most effective, limiting M. aeruginosa growth and toxin synthesis by up to 94.7% and 92.4%, respectively. Second, this biological strategy became less effective at 23°C, where S. elongatus grew more slowly. Third, the photosynthesis inhibitor DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) inhibited M. aeruginosa growth (at 0.1 mg/L) and microcystin production (at 0.02 mg/L). DCMU was also effective in controlling microcystin production in S. elongatus–M. aeruginosa cocultures. Based on the experimental results, a multi-substrate, multi-species kinetic model was built to describe coculture growth and population interactions.ConclusionMicrocystin from representative toxin-producing M. aeruginosa can be controlled by coculturing fast-growing benign cyanobacteria, which can be made even more efficient if appropriate algicide is applied. This study improved the understanding of the biological control of microcystin production under complex environmental conditions.

ABSTRACT Many microbial biosynthetic gene clusters (BGCs) are inactive under standard laboratory conditions, making characterization of their products difficult. Silent BGCs are likely activated by specific cues in their natural environment, such as the presence of competitors. Growth conditions such as coculture with other microbes, which more closely mimic natural environments, are practical strategies for inducing silent BGCs. Here, we utilize coculture to activate BGCs in nine actinobacteria strains. We observed increased production of the ferrous siderophores siderochelin A and B during coculture of Amycolatopsis strain WAC04611 and Tsukamurella strain WAC06889b. Furthermore, we identified the siderochelin BGC in WAC04611 and discovered that the GntR-family transcription factor sidR3 represses siderochelin production. Deletion of the predicted aminotransferase sidA abolished production of the carboxamides siderochelin A/B and led to the accumulation of the carboxylate siderochelin D. Finally, we deleted the predicted hydroxylase sidB and established that it is essential for siderochelin production. Our findings show that microbial coculture can successfully activate silent BGCs and lead to the discovery and characterization of unknown BGCs for molecules like siderochelin.IMPORTANCESiderophores are vital iron-acquisition elements required by microbes for survival in a variety of environments. Furthermore, many siderophores are essential for the virulence of various human pathogens, making them a possible target for antibacterials. The significance of our work is in the identification and characterization of the previously unknown BGC for the siderophore siderochelin. Our work adds to the growing knowledge of siderophore biosynthesis, which may aid in the future development of siderophore-targeting pharmaceuticals and inform on the ecological roles of these compounds. Furthermore, our work demonstrates that combining microbial coculture with metabolomics is a valuable strategy for identifying upregulated compounds and their BGCs.

ABSTRACT 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.

Abstract Multi resistant fungi are on the rise, and our arsenal compounds are limited to few choices in the market such as polyenes, pyrimidine analogs, azoles, allylamines, and echinocandins. Although each of these drugs featured a unique mechanism, antifungal resistant strains did emerge and continued to arise against them worldwide. Moreover, the genetic variation between fungi and their host humans is small, which leads to significant challenges in new antifungal drug discovery. Endophytes are still an underexplored source of bioactive secondary metabolites. Many studies were conducted to isolate and screen endophytic pure compounds with efficacy against resistant yeasts and fungi; especially, Candida albicans, C. auris, Cryptococcus neoformans and Aspergillus fumigatus, which encouraged writing this review to critically analyze the chemical nature, potency, and fungal source of the isolated endophytic compounds as well as their novelty features and SAR when possible. Herein, we report a comprehensive list of around 320 assayed antifungal compounds against Candida albicans, C. auris, Cryptococcus neoformans and Aspergillus fumigatus in the period 1980–2024, the majority of which were isolated from fungi of orders Eurotiales and Hypocreales associated with terrestrial plants, probably due to the ease of laboratory cultivation of these strains. 46% of the reviewed compounds were active against C. albicans, 23% against C. neoformans, 29% against A. fumigatus and only 2% against C. auris. Coculturing was proved to be an effective technique to induce cryptic metabolites absent in other axenic cultures or host extract cultures, with Irperide as the most promising compounds MIC value 1 μg/mL. C. auris was susceptible to only persephacin and rubiginosin C. The latter showed potent inhibition against this recalcitrant strain in a non-fungicide way, which unveils the potential of fungal biofilm inhibition. Further development of culturing techniques and activation of silent metabolic pathways would be favorable to inspire the search for novel bioactive antifungals. Graphic abstract

One strain of Hanseniaspora uvarum YT-35 was screened from fermented sediment of wild cherry. Hanseniaspora uvarum YT-35 and commercial Saccharomyces cerevisiae were used as coculture for manufacture of fermented wild cherry beverage. The dynamics of microbial populations, reducing sugars and ethanol were analyzed at different stages of fermentation using single-strain fermentation with 2 strains of bacteria as a control. Meanwhile, the organic acids and volatile aromatic compounds of the fermented beverages were detected by high-performance liquid chromatography (HPLC) and headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS). The results showed that H. uvarum YT-35 dominated in the pre-fermentation stage of co-culture. Compared with single fermentation with S. cerevisiae, the coculture fermentation resulted in lower ethanol content (3.51 g/L). Notably, HPLC results revealed that coculture fermented beverage reduced the yield of citric, malic and quinic acids and increased the yield of glacial acetic acid. HS-SPME/GC-MS results revealed that coculture fermented beverage produced more volatile compounds of esters, such as ethyl caproate, methyl benzoate and isoamyl octanoate and showed enhanced contents of ethyl laurate, ethyl octanoate, phenyl ethyl alcohol, benzyl alcohol, octanoic acid and lauric acid. Meanwhile, clustering analysis revealed that coculture fermentation were correlated with the greatest number of volatile aroma compounds in the fermented wild cherry beverage. This study provides scientific basis and theoretical guidance for the research of coculture strains with different metabolic potential in improving the quality of fruit juice fermented beverage.

Osteoarthritis (OA) is a whole-joint disorder involving inflammation in cartilage, synovium, and subchondral bone. An ex vivo inflammatory OA model incorporating various intra-articular tissues could facilitate the development of new OA therapeutics targeting whole joint tissue. In this study, an inflammatory ex vivo coculture system with bovine osteochondral explants (OCEs) and synovial explants (SEs) was induced using 1 ng/mL interleukin one beta and tumor necrosis factor alpha. Furthermore, 5-aminosalicylic acid (5-ASA), a promising OA drug reported previously, was assessed in the coculture system to evaluate the potential of the system to be applied in drug screening for OA treatment. Under inflammatory stimulation, cartilage and synovium tissues in monoculture displayed a strong inflammatory response transcriptionally, biochemically, and histologically. Under inflammatory conditions, coculture elevated the gene expression of IL-6 in synovium tissue compared with monocultured synovium. The synovium tissue in the coculture inflammatory group showed the highest synovitis score. The IL-8 release in the coculture medium was significantly higher than the additive amount in the OCE and SE monoculture groups under inflammatory microenvironment. For drug assessment, 5-ASA-treated group could promote the gene expression of CD163/CD86 and inhibit CD86 expression in synovium compared with inflammatory group. Moreover, the cumulative release of IL-6 into the medium was mitigated upon 5-ASA treatment. This study revealed the interaction between OCEs and SEs in an ex vivo coculture system. This preclinical coculture system could be used to evaluate the effects of novel OA drugs on both cartilage and synovium and explore the crosstalk mechanisms between intra-articular tissues.

Abstract The growing spread of infectious diseases has become a potential global health threat to human beings. According to WHO reports, in this study, we investigated the impact of co-cultivating the isolated endophytic fungus Aspergillus sp. CO2 and Bacillus sp. COBZ21 as a method to stimulate the production of natural bioactive substances. (GC/MS)-based metabolomics profiling of two sponge-associated microbes, Aspergillus sp. CO2 and Bacillus sp. COBZ21, revealed that the co-culture of these two isolates induced the accumulation of metabolites that were not traced in their axenic cultures. By detection of different activities of extracts of Bacillus sp. COBZ21 and Aspergillus sp. CO2 and coculture between Bacillus sp. COBZ21 and Aspergillus sp. CO2. It was noted that the coculture strategy was the reason for a notable increase in some different activities, such as the antimicrobial activity, which showed potent activity against Escherichia coli ATCC 25,922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC 10,231. The antibiofilm activity showed significant biofilm inhibitory activity toward Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10,145, and Staph aureus NRRLB-767, with activity up to 53.66, 71.17, and 47.89%, while it showed low activity against E. coli ATCC 25,922, while the antioxidant activity based on the DPPH assay showed maximum activity (75.25%). GC-MS investigations revealed the presence of variable chemical constituents belonging to different chemical categories, which reflected their chemical diversity. The main components are (+-) cis-Deethylburnamine (2.66%), Bis(3,6,9,12-tetraoxapentaethylene) crowno-N,N,N’,N’-tetra methylpphanediamine (2.48%), and 11-phenyl-2,4,6,8-tetra(2-thienyl)-11-aza-5,13-dithiaeteracyclo[7.3.0.1(2,8)0.0(3,7)] trideca-3,6-diene-10,12,13-trione (3.13%), respectively, for Bacillus sp. axenic culture, Aspergillus sp. CO2, Aspergillus sp. CO2, and Bacillus sp. COBZ21 coculture. By studying the ADME-related physicochemical properties of coculture extract, the compound showed log P o/w values above 5 (8.82). The solubility of the substance was moderate. In order to provide a comprehensive definition of medicinal chemistry and leadlikness, it is important to note that the latter did not meet the criteria outlined in the rule of three (RO3). The toxicity prediction of the coculture extract was performed using the ProTox II web server, which showed that the selected compound has no pronounced toxicity.

Abstract Background Pulp inflammation is complex interactions between different types of cells and cytokines. To mimic the interactions of different types of cells in inflamed dental pulp tissues, dental pulp cells (DPCs) were cocultured with different ratios of macrophages (THP-1) or LPS treatment. Methods DPCs were cocultured with various ratios of THP-1, then photographed cell morphology and determined cell viability by MTT assay at preset times. Total RNA was also extracted to measure the inflammation marker-IL-6 and IL-8 expressions by RT-Q-PCR. The DPCs and THP-1 were treated with 0.01 – 1μg/ml lipopolysaccharide (LPS) and extract RNA at preset times, and detected IL-6 and IL-8 expression. DPCs were cocultured with various ratios of THP-1 with 0.1 μg/mL LPS, and detected IL-6 and IL-8 expression after 24 and 48 h. The data were analyzed by unpaired t-test or Mann-Whitney test. Differences were considered statistically significant when p

Legumes enhance pasture health and soil productivity by fixing atmospheric nitrogen and boosting soil microbiota. We investigated the effects of tropical pasture legumes, including butterfly pea (Clitoria ternatea), seca stylo (Stylosanthes scabra), desmanthus (Desmanthus virgatus), lablab (Lablab purpureus), and Wynn cassia (Chamaecrista rotundifolia), on the soil microbial community and buffel grass (Cenchrus ciliaris) gene expression. Additionally, we explored the impact of a phytogenic bioactive product (PHY) in the coculture system. A pot trial using soil enriched with cow paunch compost included four treatments: monoculture of buffel grass and five legume species with and without PHY supplementation and coculture of buffel grass with each legume species with and without PHY supplementation. Actinobacteriota and Firmicutes were the dominant bacterial phyla. Regardless of PHY application, the coculture of buffel grass with legumes positively influenced microbial composition and diversity. Transcriptomic analysis revealed significant gene expression changes in buffel grass shoots and roots, with each legume uniquely affecting nitrogen metabolism. Lablab and Wynn cassia exhibited similarities in modulating metabolic processes, butterfly pea contributed to mycotoxin detoxification, and desmanthus balanced cell death and growth. Seca stylo enhanced root cell growth and regeneration. These findings offer insights for optimizing legume–grass coculture systems, enhancing soil activity and promoting sustainable agriculture.

Liver failure represents a critical medical condition with a traditionally grim prognosis, where treatment options have been notably limited. Historically, liver transplantation has stood as the sole definitive cure, yet the stark disparity between the limited availability of liver donations and the high demand for such organs has significantly hampered its feasibility. This discrepancy has necessitated the exploration of hepatocyte transplantation as a temporary, supportive intervention. In light of this, our review delves into the burgeoning field of hepatocyte transplantation, with a focus on the latest advancements in maintaining hepatocyte function, co-microencapsulation techniques, xenogeneic hepatocyte transplantation, and the selection of materials for microencapsulation. Our examination of hepatocyte microencapsulation research highlights that, to date, most studies have been conducted in vitro or using liver failure mouse models, with a notable paucity of experiments on larger mammals. The functionality of microencapsulated hepatocytes is primarily inferred through indirect measures such as urea and albumin production and the rate of ammonia clearance. Furthermore, research on the mechanisms underlying hepatocyte co-microencapsulation remains limited, and the practicality of xenogeneic hepatocyte transplantation requires further validation. The potential of hepatocyte microencapsulation extends beyond the current scope of application, suggesting a promising horizon for liver failure treatment modalities. Innovations in encapsulation materials and techniques aim to enhance cell viability and function, indicating a need for comprehensive studies that bridge the gap between small-scale laboratory success and clinical applicability. Moreover, the integration of bioengineering and regenerative medicine offers novel pathways to refine hepatocyte transplantation, potentially overcoming the challenges of immune rejection and ensuring the long-term functionality of transplanted cells. In conclusion, while hepatocyte microencapsulation and transplantation herald a new era in liver failure therapy, significant strides must be made to translate these experimental approaches into viable clinical solutions. Future research should aim to expand the experimental models to include larger mammals, thereby providing a clearer understanding of the clinical potential of these therapies. Additionally, a deeper exploration into the mechanisms of cell survival and function within microcapsules, alongside the development of innovative encapsulation materials, will be critical in advancing the field and offering new hope to patients with liver failure.

Abstract The lack of human‐derived in vitro models that recapitulate cervical pre‐cancerous lesions has been the bottleneck in researching human papillomavirus (HPV) infection‐associated pre‐cancerous lesions and cancers for a long time. Here, a long‐term 3D organoid culture protocol for high‐grade squamous intraepithelial lesions and cervical squamous cell carcinoma that stably recapitulates the two tissues of origin is described. Originating from human‐derived samples, a small biobank of cervical pre‐tumoroids and tumoroids that faithfully retains genomic and transcriptomic characteristics as well as the causative HPV genome is established. Cervical pre‐tumoroids and tumoroids show differential responses to common chemotherapeutic agents and grow differently as xenografts in mice. By coculture organoid models with peripheral blood immune cells (PBMCs) stimulated by HPV antigenic peptides, it is illustrated that both organoid models respond differently to immunized PBMCs, supporting organoids as reliable and powerful tools for studying virus‐specific T‐cell responses and screening therapeutic HPV vaccines. In this study, a model of cervical pre‐cancerous lesions containing HPV is established for the first time, overcoming the bottleneck of the current model of human cervical pre‐cancerous lesions. This study establishes an experimental platform and biobanks for in vitro mechanistic research, therapeutic vaccine screening, and personalized treatment for HPV‐related cervical diseases.

Abstract Background Acanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions. Grazing bacteria also exhibit diverse effects on the physiological characteristics of amoebae, such as their growth, encystation, and cytotoxicity. Since the composition of amoebae amino acids is closely related to cellular activities, it can indicate the overall responses of A. castellanii to various stimuli. Method A. castellanii was exposed to different culture conditions in low-nutrient medium with heat-killed DH5α to clarify their effects. A targeted metabolomic technique was utilized to evaluate the concentration of cellular amino acids. The amino acid composition and pathways were analyzed by two web-based tools: MetaboAnalyst and Pathview. Then, long-term exposure to A. castellanii was investigated through in silico and in vitro methods to elucidate the homeostasis of amino acids and the growth of A. castellanii. Results Under short-term exposure, all kinds of amino acids were enriched in all exposed groups. In contrast to the presence of heat-killed bacteria, the medium exhibited obvious effects on the amino acid composition of A. castellanii. After long-term exposure, the amino acid composition was more similar to that of the control group. A. castellanii may achieve amino acid homeostasis through pathways related to alanine, aspartate, citrulline, and serine. Discussion Under short-term exposure, compared to the presence of bacteria, the type of medium exerted a more powerful effect on the amino acid composition of the amoeba. Previous studies focused on the interaction of the amoeba and bacteria with effective secretion systems and effectors. This may have caused the effects of low-nutrient environments to be overlooked. Conclusion When A. castellanii was stimulated in the coculture system through various methods, such as the presence of bacteria and a low-nutrient environment, it accumulated intracellular amino acids within a short period. However, different stimulations correspond to different amino acid compositions. After long-term exposure, A. castellanii achieved an amino acid equilibrium by downregulating the biosynthesis of several amino acids.

Fibrosis is common profound in diabetes condition, they characterized by accumulation of extracellular matrix in tissue then cause tissue injury. The study aim to investigate effect of monoculture, conditioned media, and coculture fibroblast of NIH3T3 cells (NIH3T3 cells) in LPS-high glucose media (HG-LPS media) conditions on mRNA TGF-β and collagen secretion. The fibroblast NIH3T3 were cultured with various culture type there is mono culture NIH3T3 cells, conditioned media from macrophage RAW264.7 cells culture media, and coculture with RAW264.7 cells (ratio 1:1 and 1:2) in normal media (media containing 4500 µg/mL of glucose) and HG-LPS media (media containing LPS 0.25 µg/mL and D-glucose 5400 µg/mL). Our findings shows that the TGF-β mRNA expression in RAW 264.7 and NIH 3T3 cell coculture media with a culture ratio of 1:1 (Coculture 1:1) were the highest (p

The strong appeal to reduce animal testing calls for the development and validation of in vitro, in chemico and in silico models that would replace the need for in vivo testing and ex vivo materials. A category that requires such new approach methods is the assessment of immunosuppression that can be induced by chemicals including environmental pollutants. To assess the immunosuppressive action on monocytes and lymphocytes, we mimicked the whole-blood cytokine-release assay by preparing an in vitro coculture of THP-1 and Jurkat cell lines. We optimised its activation and investigated the effects of known immunosuppressive drugs with different mechanisms of action on the release of proinflammatory cytokines. Decreased secretion of IL-8 was achieved by several immunosuppressive mechanisms and was therefore selected as an appropriate marker of immunosuppression. A set of environmentally occurring bisphenols, BPA, BPAP, BPP, BPZ, BPE, TCBPA and BPS-MAE, were then applied to the model and BPP and BPZ were found to act as potent immunosuppressants at micromolar concentrations.

Cell–cell interactions between fibroblasts and immune cells, like macrophages, are influenced by interaction with the surrounding extracellular matrix during wound healing. In vitro hydrogel models that mimic and modulate these interactions, especially of soluble mediators like cytokines, may allow for a more detailed investigation of immunomodulatory processes. In the present study, a biomimetic extracellular matrix model based on fibrillar 3D collagen I networks with a functionalization with heparin or 6-ON-desulfated heparin, as mimics of naturally occurring heparan sulfate, was developed to modulate cytokine binding effects with the hydrogel matrix. The constitution and microstructure of the collagen I network were found to be stable throughout the 7-day culture period. A coculture study of primary human fibroblasts/myofibroblasts and M-CSF-stimulated macrophages was used to show its applicability to simulate processes of progressed wound healing. The quantification of secreted cytokines (IL-8, IL-10, IL-6, FGF-2) in the cell culture supernatant demonstrated the differential impact of glycosaminoglycan functionalization of the collagen I network. Most prominently, IL-6 and FGF-2 were shown to be regulated by the cell culture condition and network constitution, indicating changes in paracrine and autocrine cell–cell communication of the fibroblast–macrophage coculture. From this perspective, we consider our newly established in vitro hydrogel model suitable for mechanistic coculture analyses of primary human cells to unravel the role of extracellular matrix factors in key events of tissue regeneration and beyond.

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-1β). 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-1β 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.

The brackish water-induced lands are abandoned by their owners because the land is unproductive. These lands are adjacent to the pond. This study aims to evaluate the brackish water-induced land through the rice-tiger shrimp coculture system. The land was reconstructed using excavators into 70 % rice fields and 30 % caren for shrimp cultivation. On this land, cultivation was carried out with Irrigated Inbred Rice (Inpari 34) and tiger shrimp (P) and Inpari 35 and tiger shrimp (T). Each treatment is two replicates. Salt-tolerant rice seeds used are varieties of Inpari 34 and Inpari 35 planted on rice fields after seedbeds for 25 days. Post larvae (PL) of tiger shrimp were adapted to a salinity of 7 ppt for 30 days, to be stocked at a density of 4 ind/m2. The growth of rice and tiger shrimp growth and water quality were monitored at a 14-day interval. Production of rice and tiger shrimp was counted at the end of the study. The results showed that salt-tolerant rice varieties Inpari 34 grew from 40 to 108 cm in height and Inpari 35 grew from 43 to 126 cm with production reach 5.060 ± 64.28 and 4.324 ± 19.09 t/ha, respectively, during 105 days of culture period. The survival rate and body weight of tiger shrimp are 20.19 ± 0.43 % and 11.56 ± 2.36 g for Inpari 34 and 20.83 ± 31.26 % and 12.40 ± 2.42 g for Inpari 35 for 65 days culture period, with production estimated 0.176 ± 0.054 (P) and 0.134 ± 0.010 t/ha (T). Inpari 34 and Inpari 35 rice varieties can be cocultured with tiger shrimp on the brackish water-induced land. Rice-tiger shrimp coculture system can be an alternative technology to restore land function that is intrusive by brackish water-induced land and support food security.

In this study, we established a coculture model comprising human neuroblastoma SH-SY5Y cells and induced pluripotent stem cell-derived astrocytes, faithfully replicating the human brain environment for in vitro neurotoxicity assessment. We optimized the cell differentiation duration and cell ratios to obtain images conducive to neurite outgrowth evaluation. Subsequently, the neurotoxic effects in the coculture and monoculture of SH-SY5Y cells were confirmed using neurotoxic agents such as acrylamide (ACR) and hydrogen peroxide (H2O2). Disparities in the neurotoxic impacts of ACR and H2O2 within the coculture were mirrored in the expression of genes associated with early neuronal injury. Notably, the reduction in neurite outgrowth induced by neurotoxic agents revealed the coculture's lower sensitivity compared to monocultures. Furthermore, the coculture system exhibited distinct effects of test agents on nerve damage and manifested protective influences on nerve cells. The proposed methodology holds promise for large-scale chemical neurotoxicity screening through neurite change measurements. This in vitro coculture model, accounting for cell interactions, emerges as a valuable tool in toxicity testing, offering insights into the potential effects of chemicals within the human body.

ABSTRACTMethanogens, reductive acetogens and sulfate-reducing bacteria play an important role in disposing of hydrogen in gut ecosystems. However, how they interact with each other remains largely unknown. This in vitro study cocultured Blautia hydrogenotrophica (reductive acetogen), Desulfovibrio piger (sulfate reducer) and Methanobrevibacter smithii (methanogen). Results revealed that these three species coexisted and did not compete for hydrogen in the early phase of incubations. Sulfate reduction was not affected by B. hydrogenotrophica and M. smithii. D. piger inhibited the growth of B. hydrogenotrophica and M. smithii after 10 h incubations, and the inhibition on M. smithii was associated with increased sulfide concentration. Remarkably, M. smithii growth lag phase was shortened by coculturing with B. hydrogenotrophica and D. piger. Formate was rapidly used by M. smithii under high acetate concentration. Overall, these findings indicated that the interactions of the hydrogenotrophic microbes are condition-dependent, suggesting their interactions may vary in gut ecosystems.

The accumulation of cadmium (Cd) in plants is strongly impacted by soil microbes, but its mechanism remains poorly understood. Here, we report the mechanism of reduced Cd accumulation in rice by coculture of Enterobacter and Comamonas species. In pot experiments, inoculation with the coculture decreased Cd content in rice grain and increased the amount of nonbioavailable Cd in Cd-spiked soils. Fluorescence in situ hybridization and scanning electron microscopy detection showed that the coculture colonized in the rhizosphere and rice root vascular tissue and intercellular space. Soil metagenomics data showed that the coculture increased the abundance of sulfate reduction and biofilm formation genes and related bacterial species. Moreover, the coculture increased the content of organic matter, available nitrogen, and potassium and increased the activities of arylsulfatase, β-galactosidase, phenoloxidase, arylamidase, urease, dehydrogenase, and peroxidase in soils. In subsequent rice transcriptomics assays, we found that the inoculation with coculture activated a hypersensitive response, defense-related induction, and mitogen-activated protein kinase signaling pathway in rice. Heterologous protein expression in yeast confirmed the function of four Cd-binding proteins (HIP28-1, HIP28-4, BCP2, and CID8), a Cd efflux protein (BCP1), and three Cd uptake proteins (COPT4, NRAM5, and HKT6) in rice. Succinic acid and phenylalanine were subsequently proved to inhibit rice divalent Cd [Cd(II)] uptake and activate Cd(II) efflux in rice roots. Thus, we propose a model that the coculture protects rice against Cd stress via Cd immobilization in soils and reducing Cd uptake in rice. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Background Energy is the basis and assurance for a world's stable development; however, as traditional non-renewable energy sources deplete, the development and study of renewable clean energy have emerged. Using microalgae as a carbon source for anaerobic bacteria to generate biohydrogen is a clean energy generation system that both local and global peers see as promising. Results Klebsiella pneumonia , Enterobacter cloacae, and their coculture were used to synthesize biohydrogen using Oscillatoria acuminata biomass via dark fermentation. The total carbohydrate content in O. acuminata was 237.39 mg/L. To enhance the content of fermentable reducing sugars, thermochemical, biological, and biological with magnesium zinc ferrite nanoparticles (Mg-Zn Fe2O4-NPs) pretreatments were applied. Crude hydrolytic enzymes extracted from Trichoderma harzianum of biological pretreatment were enhanced by Mg-Zn Fe2O4-NPs and significantly increased reducing sugars (230.48 mg/g) four times than thermochemical pretreatment (45.34 mg/g). K. pneumonia demonstrated a greater accumulated hydrogen level (1022 mLH2/L) than E. cloacae (813 mLH2/L), while their coculture showed superior results (1520 mLH2/L) and shortened the production time to 48 h instead of 72 h in single culture pretreatments. Biological pretreatment + Mg-Zn Fe2O4 NPs using coculture significantly stimulated hydrogen yield (3254 mLH2/L), hydrogen efficiency)216.9 mL H2/g reducing sugar( and hydrogen production rate (67.7 mL/L/h) to the maximum among all pretreatments. Conclusion These results confirm the effectiveness of biological treatments + Mg-Zn Fe2O4-NPs and coculture dark fermentation in upregulating biohydrogen production.

Astrocytes and microglia play an orchestrated role following spinal cord injury; however, the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood. Herein, microglia were pharmacologically depleted and the effects on the astrocytic response were examined. We further explored the potential mechanisms involving the signal transducers and activators of transcription 3 (STAT3) pathway. For in vivo experiments, we constructed a contusion spinal cord injury model in C57BL/6 mice. To deplete microglia, all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397, starting 2 weeks prior to surgery until they were sacrificed. Cell proliferation was examined by 5-ethynyl-2-deoxyuridine (EdU) and three pivotal inflammatory cytokines were detected by a specific Bio-Plex ProTM Reagent Kit. Locomotor function, neuroinflammation, astrocyte activation and phosphorylated STAT3 (pSTAT3, a maker of activation of STAT3 signaling) levels were determined. For in vitro experiments, a microglia and astrocyte coculture system was established, and the small molecule STA21, which blocks STAT3 activation, was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia. PLX3397 administration disrupted glial scar formation, increased inflammatory spillover, induced diffuse tissue damage and impaired functional recovery after spinal cord injury. Microglial depletion markedly reduced EdU+ proliferating cells, especially proliferating astrocytes at 7 days after spinal cord injury. RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397. Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes. Importantly, in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration. These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation, and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.

Abstract The manufacturing of viable and functional β‐cell spheroids is required for diabetes cell therapy and drug testing. Mesenchymal stromal/stem cells (MSCs) are known to improve β‐cell viability and functionality. We therefore investigated the aggregation behavior of three different β‐cell lines (rat insulinoma‐1 cell line [INS‐1], mouse insulinoma‐6 cell line [MIN6], and a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells [1.1B4]), two MSC types, and mixtures of β‐cells and MSCs under different conditions. We screened several static systems to produce uniform β‐cell and MSC spheroids, finding cell‐repellent plates the most suitable. The three different β‐cell lines differed in their aggregation behavior, spheroid size, and growth in the same static environment. We found no major differences in spheroid formation between primary MSCs and an immortalized MSC line, although both differed with regard to the aggregation behavior of the β‐cell lines. All spheroids showed a reduced viability due to mass transfer limitations under static conditions. We therefore investigated three dynamic systems (shaking multi‐well plates, spinner flasks, and shaking flasks). In shaking flasks, there were no β‐cell‐line‐dependent differences in aggregation behavior, resulting in uniform and highly viable spheroids. We found that the aggregation behavior of the β‐cell lines changed in a static coculture with MSCs. The β‐cell/MSC coculture conditions must be refined to avoid a rapid segregation into distinct populations under dynamic conditions.

Resveratrol, a valuable plant-derived polyphenolic compound with various bioactivities, has been widely used in nutraceutical industries. Microbial production of resveratrol suffers from metabolic burden and low malonyl-CoA availability, which is a big challenge for synthetic biology. Herein, we took advantage of coculture engineering and divided the biosynthetic pathway of resveratrol into the upstream and downstream strains. By enhancing the supply of malonyl-CoA via CRISPRi system and fine-tuning the expression intensity of the synthetic pathway genes, we significantly improved the resveratrol productivity of the downstream strain. Furthermore, we developed a resveratrol addiction circuit that coupled the growth of the upstream strain and the resveratrol production of the downstream strain. The bidirectional interaction stabilized the coculture system and increased the production of resveratrol by 74%. Moreover, co-utilization of glucose and arabinose by the coculture system maintained the growth advantage of the downstream strain for production of resveratrol throughout the fermentation process. Under optimized conditions, the engineered E. coli coculture system produced 204.80 mg/L of resveratrol, 12.8-fold improvement over monoculture system. This study demonstrates the promising potential of coculture engineering for efficient production of natural products from biomass.

Lactiplantibacillus plantarum OV50 is a novel strain that was isolated from Algerian olives. Prior to its use as a natural biopreservative, OV50 underwent characterization for various functions. OV50 shows no proteolytic, lipolytic, or hemolytic activity. In addition, it is non-cytotoxic to eukaryotic cells and does not exhibit acquired antibiotic resistance. OV50 was tested with Pseudomonas aeruginosa ATCC 27835, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Vibrio cholerae ATCC 14035 in a sardine based-medium at 37 °C and 7 °C. At 37 °C, OV50 completely inhibited the growth of these foodborne pathogens for a maximum of 6 h. At 7 °C, it suppressed their growth for a maximum of 8 days, except for S. aureus ATCC 6538, whose growth was reduced from 4 to 2 log CFU/mL. Microbiological counts, total volatile basic nitrogen (TVB-N), and peroxide values (PV) concentrations were determined in fresh sardines inoculated with OV50 and kept at 7 °C for 12 days. The inoculated sardines showed a significant reduction in TVB-N levels at D8 (34.9 mg/100 g) compared to the control (59.73 mg/100 g) and in PV concentrations at D4 (6.67 meq/kg) compared to the control (11.44 meq/kg), as well as a significant reduction in the numbers of Enterobacterales, Coliforms, Pseudomonas spp., Vibrio spp., and S. aureus At D8 and D12 compared to the control. Taken together, these results indicate that OV50 can improve the microbiological safety, freshness, and quality of sardines.

Abstract Background Zinc oxide nanoparticles (ZnO NP) offer beneficial properties for many applications, especially in the food sector. Consequently, as part of the human food chain, they are taken up orally. The toxicological evaluation of orally ingested ZnO NP is still controversial. In addition, their physicochemical properties can change during digestion, which leads to an altered biological behaviour. Therefore, the aim of our study was to investigate the fate of two different sized ZnO NP (

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

ABSTRACT The dynamic structures of microbial communities emerge from the complex network of interactions between their constituent microorganisms. Quantitative measurements of these interactions are important for understanding and engineering ecosystem structure. Here, we present the development and application of the BioMe plate, a redesigned microplate device in which pairs of wells are separated by porous membranes. BioMe facilitates the measurement of dynamic microbial interactions and integrates easily with standard laboratory equipment. We first applied BioMe to recapitulate recently characterized, natural symbiotic interactions between bacteria isolated from the Drosophila melanogaster gut microbiome. Specifically, the BioMe plate allowed us to observe the benefit provided by two Lactobacillus strains to an Acetobacter strain. We next explored the use of BioMe to gain quantitative insight into the engineered obligate syntrophic interaction between a pair of Escherichia coli amino acid auxotrophs. We integrated experimental observations with a mechanistic computational model to quantify key parameters associated with this syntrophic interaction, including metabolite secretion and diffusion rates. This model also allowed us to explain the slow growth observed for auxotrophs growing in adjacent wells by demonstrating that, under the relevant range of parameters, local exchange between auxotrophs is essential for efficient growth. The BioMe plate provides a scalable and flexible approach for the study of dynamic microbial interactions. IMPORTANCE Microbial communities participate in many essential processes from biogeochemical cycles to the maintenance of human health. The structure and functions of these communities are dynamic properties that depend on poorly understood interactions among different species. Unraveling these interactions is therefore a crucial step toward understanding natural microbiota and engineering artificial ones. Microbial interactions have been difficult to measure directly, largely due to limitations of existing methods to disentangle the contribution of different organisms in mixed cocultures. To overcome these limitations, we developed the BioMe plate, a custom microplate-based device that enables direct measurement of microbial interactions, by detecting the abundance of segregated populations of microbes that can exchange small molecules through a membrane. We demonstrated the possible application of the BioMe plate for studying both natural and artificial consortia. BioMe is a scalable and accessible platform that can be used to broadly characterize microbial interactions mediated by diffusible molecules.

AIM: To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor A (VEGF-A) in human retinal vascular endothelial cells (HRECs). METHODS: Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis. HRECs were randomly divided into a normal control group (group A), a high glucose model group (group B), a high glucose group with 25 μg/mL (group C), 50 μg/mL (group D), and 100 μg/mL exosomes (group E). Twenty-four hours after coculture, the cell proliferation rate was detected using flow cytometry, and the VEGF-A level was detected using immunofluorescence. After coculture 8, 16, and 24h, the expression levels of VEGF-A in each group were detected using PCR and Western blots. RESULTS: The characteristic morphology (membrane structured vesicles) and size (diameter between 50 and 200 nm) were observed under transmission electron microscopy. The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis (NTA). The exosomal markers CD9, CD63, and HSP70 were strongly detected. The proliferation rate of the cells in group B increased after 24h of coculture. Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes (F=39.03, P

Oncolytic virotherapy is one of the emerging biological therapeutics that needs a more efficient in vitro tumor model to overcome the two-dimensional (2D) monolayer tumor cell culture model’s inability to maintain tissue-specific structure. This is to offer significant prognostic preclinical assessment findings. One of the best models that can mimic the in vivo model in vitro are the three-dimensional (3D) tumor–normal cell coculture systems, which can be employed in preclinical oncolytic virus therapeutics. Thus, we developed our 3D coculture system in vitro using two types of breast cancer cell lines showing different receptor statuses cocultured with adipose tissue–derived mesenchymal stem cells. The cells were cultured in a floater tissue culture plate to allow spheroids formation, and then the spheroids were collected and transferred to a scaffold spheroids dish. These 3D culture systems were used to evaluate oncolytic Newcastle disease virus AMHA1 strain infectivity and antitumor activity using a tracking system of the Newcastle disease virus (NDV) labeled with fluorescent PKH67 linker to follow the virus entry into target cells. This provides evidence that the NDV AMHA1 strain is an efficient oncolytic agent. The fluorescently detected virus particles showed high intensity in both coculture spheres. Strategies for chemically introducing fluorescent dyes into NDV particles extract quantitative information from the infected cancer models. In conclusion, the results indicate that the NDV AMHA1 strain efficiently replicates and induces an antitumor effect in cancer–normal 3D coculture systems, indicating efficient clinical outcomes.

Interactions between fibroblasts and immune cells play an important role in tissue inflammation. Previous studies have found that eosinophils activated with interleukin-3 (IL-3) degranulate on aggregated immunoglobulin G (IgG) and release mediators that activate fibroblasts in the lung. However, these studies were done with eosinophil-conditioned media that have the capacity to investigate only one-way signaling from eosinophils to fibroblasts. Here, we demonstrate a coculture model of primary normal human lung fibroblasts (HLFs) and human blood eosinophils from patients with allergy and asthma using an open microfluidic coculture device. In our device, the two types of cells can communicate via two-way soluble factor signaling in the shared media while being physically separated by a half wall. Initially, we assessed the level of eosinophil degranulation by their release of eosinophil-derived neurotoxin (EDN). Next, we analyzed the inflammation-associated genes and soluble factors using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiplex immunoassays, respectively. Our results suggest an induction of a proinflammatory fibroblast phenotype of HLFs following the coculture with degranulating eosinophils, validating our previous findings. Additionally, we present a new result that indicate potential impacts of activated HLFs back on eosinophils. This open microfluidic coculture platform provides unique opportunities to investigate the intercellular signaling between the two cell types and their roles in airway inflammation and remodeling.

Bioreactor cocultures involving Penicillium rubens and Streptomyces rimosus were investigated with regard to secondary metabolite production, morphological development, dissolved oxygen levels, and carbon substrate utilization. The production profiles of 22 secondary metabolites were analyzed, including penicillin G and oxytetracycline. Three inoculation approaches were tested, i.e., the simultaneous inoculation of P. rubens with S. rimosus and the inoculation of S. rimosus delayed by 24 or 48 h relative to P. rubens. The delayed inoculation of S. rimosus into the P. rubens culture did not prevent the actinomycete from proliferating and displaying its biosynthetic repertoire. Although a period of prolonged adaptation was needed, S. rimosus exhibited growth and the production of secondary metabolites regardless of the chosen delay period (24 or 48 h). This promising method of coculture initiation resulted in increased levels of metabolites tentatively identified as rimocidin B, 2-methylthio-cis-zeatin, chrysogine, benzylpenicilloic acid, and preaustinoid D relative to the values recorded for the monocultures. This study demonstrates the usefulness of the delayed inoculation approach in uncovering the metabolic landscape of filamentous microorganisms and altering the levels of secondary metabolites.

The blood–brain barrier (BBB) is the bottleneck in the development of new drugs to reach the brain. Due to the BBB, toxic substances cannot enter the brain, but promising drug candidates also pass the BBB poorly. Suitable in vitro BBB models are therefore of particular importance during the preclinical development process, as they can not only reduce animal testing but also enable new drugs to be developed more quickly. The aim of this study was to isolate cerebral endothelial cells, pericytes, and astrocytes from the porcine brain to produce a primary model of the BBB. Additionally, as primary cells are well suited by their properties but the isolation is complex and better reproducibility with immortalized cells must be ensured, there is a high demand for immortalized cells with suitable properties for use as a BBB model. Thus, isolated primary cells can also serve as the basis for a suitable immortalization technique to generate new cell lines. In this work, cerebral endothelial cells, pericytes, and astrocytes were successfully isolated and expanded using a mechanical/enzymatic method. Furthermore, in a triple coculture model, the cells showed a significant increase in barrier integrity compared with endothelial cell monoculture, as determined by transendothelial electrical resistance measurement and permeation studies using sodium fluorescein. The results demonstrate the opportunity to obtain all three cell types significantly involved in BBB formation from one species, thus providing a suitable tool for testing the permeation properties of new drug candidates. In addition, the protocols are a promising starting point to generate new cell lines of BBB-forming cells as a novel approach for BBB in vitro models.

Topical antiseptics are essential in wound treatment, and adipose-derived stem cells (ADSCs) have recently been proven to facilitate healing. However, the impact of antiseptics on ADSCs has not been fully elucidated, especially in relation to other relevant cell types present in the wound microenvironment, e.g., fibroblasts. This study evaluated the effects of chlorhexidine and povidone–iodine on four cellular constructs in 2D and 3D in vitro culture systems. Cell constructs were treated with two concentrations of each antiseptic, after which cell migration activity, α-SMA, and Ki67 marker expressions were assessed and compared. Both tested concentrations of povidone–iodine impaired migration and sprouting compared to chlorhexidine, which had minimal effects when used in low concentrations. The gap in the wound healing assay did not close after 24 h of povidone–iodine treatment, although, at the lower concentration, cells started to migrate in a single-cell movement pattern. Similarly, in 3D culture systems, sprouting with reduced spike formation was observed at high povidone–iodine concentrations. Both antiseptics modulated α-SMA and Ki67 marker expressions at 5 days following treatment. Although both antiseptics had cytotoxic effects dependent on drug concentration and cell type, povidone–iodine contributed more substantially to the healing process than chlorhexidine, acting especially on fibroblasts.

Abstract Background Escherichia coli (E. coli) is the principal pathogen that causes biofilm formation. Biofilms are associated with infectious diseases and antibiotic resistance. This study employed proteomic analysis to identify differentially expressed proteins after coculture of E. coli with Lactobacillus rhamnosus GG (LGG) microcapsules. Methods To explore the relevant protein abundance changes after E. coli and LGG coculture, label-free quantitative proteomic analysis and qRT-PCR were applied to E. coli and LGG microcapsule groups before and after coculture, respectively. Results The proteomic analysis characterised a total of 1655 proteins in E. coli K12MG1655 and 1431 proteins in the LGG. After coculture treatment, there were 262 differentially expressed proteins in E. coli and 291 in LGG. Gene ontology analysis showed that the differentially expressed proteins were mainly related to cellular metabolism, the stress response, transcription and the cell membrane. A protein interaction network and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis indicated that the differentiated proteins were mainly involved in the protein ubiquitination pathway and mitochondrial dysfunction. Conclusions These findings indicated that LGG microcapsules may inhibit E. coli biofilm formation by disrupting metabolic processes, particularly in relation to energy metabolism and stimulus responses, both of which are critical for the growth of LGG. Together, these findings increase our understanding of the interactions between bacteria under coculture conditions.

Abstract Background The porcine respiratory tract harbours multiple microorganisms, and the interactions between these organisms could be associated with animal health status. Pasteurella multocida is a culturable facultative anaerobic bacterium isolated from healthy and diseased porcine respiratory tracts. The interaction between P. multocida and other aerobic commensal bacteria in the porcine respiratory tract is not well understood. This study aimed to determine the interactions between porcine P. multocida capsular serotype A and D strains and other culturable aerobic bacteria isolated from porcine respiratory tracts using a coculture assay in conditioned media followed by calculation of the growth rates and interaction parameters. Results One hundred and sixteen bacterial samples were isolated from five porcine respiratory tracts, and 93 isolates were identified and phylogenetically classified into fourteen genera based on 16S rRNA sequences. Thirteen isolates from Gram-negative bacterial genera and two isolates from the Gram-positive bacterial genus were selected for coculture with P. multocida. From 17 × 17 (289) interaction pairs, the majority of 220 pairs had negative interactions indicating competition for nutrients and space, while 17 pairs were identified as mild cooperative or positive interactions indicating their coexistence. All conditioned media, except those of Acinetobacter, could inhibit P. multocida growth. Conversely, the conditioned media of P. multocida also inhibited the growth of nine isolates plus themselves. Conclusion Negative interaction was the major interactions among the coculture of these 15 representative isolates and the coculture with P. multocida. The conditioned media in this study might be further analysed to identify critical molecules and examined by the in vivo experiments. The study proposed the possibility of using these molecules in conditioned media to control P. multocida growth.

Direct contact-based coculture of human dermal fibroblasts and epidermal keratinocytes has been a long-standing and challenging issue owing to different serum and growth factor requirements of the two cell types. Existing protocols employ high serum concentrations (up to 10% fetal bovine serum), complex feeder systems and a range of supplemental factors. These approaches are technically demanding and labor intensive, and pose scientific and ethical limitations associated with the high concentrations of animal serum. On the other hand, serum-free conditions often fail to support the proliferation of one or both cell types when they are cultured together. We have developed two reduced serum approaches (1–2% serum) that support the contact-based coculture of human dermal fibroblasts and immortalized keratinocytes and enable the study of cell migration and wound closure.

Liver transplantation is currently the only option for patients with end-stage liver disease. Thus, other alternate therapeutic strategies are needed. Bone marrow mesenchymal stem cells (BM-MSCs) are nonhematopoietic cells present in the bone marrow stroma that serve as precursors cells for various other cells. In this study, we evaluated the differentiation of porcine BM-MSCs into hepatocyte-like cells using three types of culture systems: hepatic induction medium (HIM), HIM/primary hepatocyte culture supernatant (HCS; 1:1 ratio), and a hepatocyte coculture system (HCCS; primary hepatocytes in the upper chamber, and BM-MSCs in the lower chamber). Primary hepatocytes were isolated from anesthetized healthy 1-month-old pigs by enzymatic digestion. Hepatic-specific marker expression (albumin [ALB], transferrin [TF], α-fetoprotein [AFP]), glycogen storage, low-density lipoprotein, and indocyanine green uptake were evaluated. Upregulation of hepatic-specific markers (ALB, TF, and AFP) was observed by real-time polymerase chain reaction in the HCCS group. Periodic acid-Schiff staining revealed enhanced glycogen storage in hepatocyte-like cells from the HCCS group compared with that from the HIM/HCS group. Furthermore, hepatocyte like-cells in the HCCS group showed improved LDL and ICG uptake than those in the other groups. Overall, our current study revealed that indirect coculture of primary hepatocytes and BM-MSCs enhanced the differentiation efficacy of BM-MSCs into hepatocyte-like cells by unknown useful soluble factors, including paracrine factors.

Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smoke (WCS). Coculture of ALI-PBEC with lipopolysaccharide (LPS)-activated M(GM-CSF) macrophages increased the expression of DEFB4A, CXCL8, and IL6 at 24 h in the ALI-PBEC, whereas LPS-activated M(M-CSF) macrophages only increased epithelial IL6 expression. Furthermore, wound repair was accelerated by coculture with both activated M(GM-CSF) and M(M-CSF) macrophages, also following WCS exposure. Coculture of ALI-PBEC and M(GM-CSF) macrophages resulted in increased CAMP expression in M(GM-CSF) macrophages, which was absent in M(M-CSF) macrophages. CAMP encodes LL-37, an antimicrobial peptide with immune-modulating and repair-enhancing activities. In conclusion, dynamic crosstalk between ALI-PBEC and macrophages enhances epithelial innate immunity and wound repair, even upon concomitant cigarette smoke exposure.

Production of lignocellulolytic enzymes by filamentous fungi have a great potential at industrial level due to their widespread applications. Mixed fungal cultures and particularly mixed fungal biofilms constitute a promising fermentation system for an enhanced enzyme production. However, it has not been addressed how much of this enhancement depends on the mixed biomass proportion. In this sense, the aim of this study was to develop a method to specifically and accurately quantify mixed fungal biomass. For this purpose, mixed biofilm cultures composed of Aspergillus niger and Trichoderma reesei, two filamentous fungi used industrially for cellulase production, were collected from 48 to 120 h of growth; mycelia were pulverized, and DNA was extracted for qPCR assays with specific primers for each fungus. Primers were designed from non-conserved regions of sequences of actin and β-tubulin genes of both A. niger and T. reesei. Specificity of these primers was tested in silico and experimentally. A statistically significant correlation was obtained between qPCR-calculated biomass and dry weight biomass data. By this method, it was possible to detect changes on mycelia proportions in biofilms over time, suggesting a competitive interaction between these two fungi. In conclusion, this method allows a specific and accurate quantification of mixed fungal biomass and could be also applied to different mixed culture systems for studying microbial interactions.

In eutrophic water, attached bacteria of Microcystis play an important role in the formation, development, and degradation of Microcystis blooms. A novel actinobacterium, designated as JXJ CY 35T, was isolated from the culture mass of Microcystis aeruginosa FACHB-905 (Maf) collected from Lake Dianchi, Yunnan Province, China. Strain JXJ CY 35T was gram-positive, acid-fast staining, aerobic, with short rod-shaped cells, positive for catalase, and negative for oxidase. The isolate was able to grow at 10.0–36.0°C, pH 4.0–10.0, and tolerate up to 5.0% (w/v) NaCl, with optimal growth at 28°C, pH 7.0–8.0, and 0% (w/v) NaCl. Cell-wall peptidoglycan contains aspartic acid, glutamic acid, glycine, and alanine, with mannose, ribose, galactose, and arabinose as whole-cell sugars. Polar lipids consist of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), glycolipid (GL1-3), phosphoglycolipid (PGL), phosphatidylinositol (PI), and unidentified lipid (L1). The predominant menaquinone was MK-9. Major fatty acids (>10%) were C17:1ω7c (37.0%) and C18:1ω9c (18.9%). The complete genome sequence of strain JXJ CY 35T was 6,138,096 bp in size with a DNA G + C content of 68.3%. Based on 16S rRNA gene sequences, it has 98.2% similarity to Mycolicibacterium arabiense JCM 18538T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain JXJ CY 35T and the closest five type strains M. arabiense JCM 18538T, M. goodii ATCC 700504T, M. mageritense DSM 44476T, M. austroafricanum DSM 44191T, and Mycobacterium neglectum CECT 8778T were 52.1, 20.3, 20.3, 20.6, and 19.8%, and 92.7, 75.5, 75.6, 76.0, and 75.2%, respectively. On the basis of the above taxonomic data and differences in physiological characteristics from the closely related type strain, strain JXJ CY 35T was determined to represent a novel species of genus Mycolicibacterium, for which the name Mycolicibacterium lacusdiani sp. nov., is proposed. The type strain is JXJ CY 35T (=KCTC 49379T = CGMCC 1.17501T). Different inoculation dosages of the type strain JXJ CY 35T could exhibit different effects on the growth of Maf and its toxin synthesis and release. Strain JXJ CY 35T could promote the growth of Maf by providing it with available phosphorus, nitrogen, probably vitamins, and plant growth hormones.

We described a novel HIV autologous isolation method based in coculturing macrophages and CD4+T-cell-enriched fractions from peripheral blood collected from antiretroviral-treated (ART) HIV patients. This method allows the isolation of high viral titers of autologous viruses, over 1010HIV RNA copies/ml, and reduces the time required to produce necessary amounts for virus for use as antigens presented by monocyte-derived myeloid cells in HIV therapeutic vaccine approaches. By applying these high titer and autologous virus produced in the patient-derived cells, we intended to elicit a boost of the immunological system response in HIV therapeutic vaccines in clinical trials.

ABSTRACT The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, the candidate phyla radiation (CPR) group has been recognized as representing a large proportion of the microbial kingdom (>26%). CPR are characterized by their obligate symbiotic or exoparasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the coculture of CPR with a suitable bacterial host. Based on various sputum samples, we tried to enrich CPR (Saccharibacteria members) and to cocultivate them with pure hosts (Schaalia odontolytica). This protocol was monitored by TaqMan real-time quantitative PCR (qPCR) using a system specific for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in coculturing and sequencing the complete genomes of two new Saccharibacteria species, “Candidatus Minimicrobia naudis” and “Candidatus Minimicrobia vallesae.” In addition, we noticed a decrease in the CT values of Saccharibacteria and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members, and in the future, their currently unknown characteristics may be revealed. IMPORTANCE In this study, the first TaqMan real-time quantitative PCR (qPCR) system, targeting Saccharibacteria phylum, has been developed. This technique can specifically quantify Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific, and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol facilitates subsequent studies of the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.

Background: Stem cells have been proposed to be one of the potent sources for treatment applications. Among diverse types of stem cells, stem cells derived from human exfoliated deciduous teeth (SHEDs) are known as the immature stem cell population, which are easily isolated, fast, and without ethical implications. SHEDs could induce pluripotent stem cells and show differentiation in chondrocytes, adipocytes, osteoblasts, neural cells, hepatocytes, myocytes, odontoblasts, and skin cells. Materials and Methods: In the current study, we investigated the effects of SHED on osteosarcoma cells (Saos-II) following 3 and 5 days indirect coculture. Results: Our results showed that indirect coculture of SHED with Saos-II cells could promote or inhibit Saos-II cells' growth in a concentration (the number of SHED vs. Saos-II cells) and time (days of indirect co-culture) dependent manner. Conclusion: Our findings suggested that, indirectly, SHED co-culture with the Soas-II cells might functions as a tumor suppressor where a higher number of SHEDs are used in the culture in comparison with the one cultured in the absence of/or fewer SHED incubation.

The exosome precipitation method affects the purity of the exosome and the quality of the downstream application. Polymer-based precipitation is a cost-effective method widely used in different research fields. The percentage of the polymer should be modified in different cell types or liquid biopsy before precipitation. This study aimed to optimize the protocol of the poly(ethylene glycol) (PEG)-based approach for extracting oral squamous cell carcinoma (OSCC)-derived exosomes, and its downstream applications. We used 8%, 10%, and 12% PEG to isolate the exosomes from the culture medium and compared the purity with that of the ultracentrifugation method. In addition, we extracted exosomal protein, DNA, and RNA, and tested the cell transfection efficiency for downstream application. The results reveal that 8% PEG and the medium mixture incubated at 4 °C overnight effectively precipitated exosomes of higher purity and more proper size and particle numbers compared with the ultracentrifuge method. PEG-precipitated exosomes cocultured with fibroblasts showed better transfection efficiency compared to exosomes alone. Therefore, 8% PEG is ideal for OSCC-derived exosome isolation and downstream applications. We recommend that the cost-effective PEG precipitation method be used for precipitating exosomes from OSCC cell experiments.

We established a syntrophic coculture of Syntrophobacter fumaroxidans MPOBT (SF) and Geobacter sulfurreducens PCAT (GS) growing on propionate and Fe(III). Neither of the bacteria was capable of growth on propionate and Fe(III) in pure culture. Propionate degradation by SF provides acetate, hydrogen, and/or formate that can be used as electron donors by GS with Fe(III) citrate as electron acceptor. Proteomic analyses of the SF-GS coculture revealed propionate conversion via the methylmalonyl-CoA (MMC) pathway by SF. The possibility of interspecies electron transfer (IET) via direct (DIET) and/or hydrogen/formate transfer (HFIT) was investigated by comparing the differential abundance of associated proteins in SF-GS coculture against (i) SF coculture with Methanospirillum hungatei (SF-MH), which relies on HFIT, (ii) GS pure culture growing on acetate, formate, hydrogen as propionate products, and Fe(III). We noted some evidence for DIET in the SF-GS coculture, i.e., GS in the coculture showed significantly lower abundance of uptake hydrogenase (43-fold) and formate dehydrogenase (45-fold) and significantly higher abundance of proteins related to acetate metabolism (i.e., GltA; 62-fold) compared to GS pure culture. Moreover, SF in the SF-GS coculture showed significantly lower abundance of IET-related formate dehydrogenases, Fdh3 (51-fold) and Fdh5 (29-fold), and the rate of propionate conversion in SF-GS was 8-fold lower than in the SF-MH coculture. In contrast, compared to GS pure culture, we found lower abundance of pilus-associated cytochrome OmcS (2-fold) and piliA (5-fold) in the SF-GS coculture that is suggested to be necessary for DIET. Furthermore, neither visible aggregates formed in the SF-GS coculture, nor the pili-E of SF (suggested as e-pili) were detected. These findings suggest that the IET mechanism is complex in the SF-GS coculture and can be mediated by several mechanisms rather than one discrete pathway. Our study can be further useful in understanding syntrophic propionate degradation in bioelectrochemical and anaerobic digestion systems.

Abstract Background Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. Results We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. Conclusions We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

Legionellosis was diagnosed in an immunocompromised 3-year-old girl in Canada. We traced the source of the bacterium through co-culture with an ameba collected from a hot tub in her home. We identified Legionella pneumophila serogroup 6, sequence type 185, and used whole-genome sequencing to confirm the environmental and clinical isolates were of common origin.