rBet v 1a-BanLecwt induce upregulation of IL-10 and IFN-γ gene expression in Caco-2/THP-1 co-culture and secretion of IL-10 and IFN-γ/IL-4 levels in PBMCs of birch pollen allergic donors.

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT.

Differentiation of Human-induced Pluripotent Stem Cell-derived Dental Stem Cells through Epithelial-Mesenchymal Interaction.

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal-epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.

Jiawei Taohe Chengqi decoction inhibition of the notch signal pathway affects macrophage reprogramming to inhibit HSCs activation for the treatment of hepatic fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Taohe Chengqi Tang (JTCD) is a modified formulation of Traditional Chinese Medicine (TCM) known as Taohe Chengqi Decoction, which has been described in the ancient TCM literature "Treatise on Febrile Diseases". As a formula that can activate blood circulation and eliminate blood stasis and regulate Yin and Yang in traditional Chinese medicine applications, JTCD has been reported to be effective in the treatment of chronic liver disease and hepatic fibrosis (HF). AIM OF STUDY: The current study aimed to evaluate the effectiveness of JTCD in modulating hepatic macrophages by regulating the Notch signal pathway, and to further investigate the mechanisms underlying macrophage reprogramming that leads to HF. MATERIALS AND METHODS: Molecular assays were performed using in vitro cultures of human mononuclear THP-1 cells and human-derived hepatic stellate cells LX-2. CCl4-induced mice were utilized as an in vivo model to simulate HF. RESULTS: Our results demonstrated that JTCD exhibited dual effects by inhibiting hepatic stellate cell (HSCs) activation and modulating the polarisation of macrophages towards the M2 phenotype while decreasing the M1 phenotype. Network pharmacological analyses and molecular docking studies revealed that the Notch signal pathway was significantly enriched and played a crucial role in the therapeutic response of JTCD against HF. Moreover, through the establishment of a co-culture model, we validated that JTCD inhibited the Notch signal pathway in macrophages, leading to alterations in macrophage reprogramming, subsequent inhibition of HSC activation, and ultimately exerting anti-HF effects. CONCLUSION: In conclusion, our findings provide solid evidence for JTCD in treating HF, as it suppresses the Notch signal pathway in macrophages, regulates macrophage reprogramming, and inhibits HSC activation.

Bioremediation of hydrocarbon by co-culturing of biosurfactant-producing bacteria in microbial fuel cell with Fe2O3-modified anode.

The most common type of environmental contamination is petroleum hydrocarbons. Sustainable and environmentally friendly treatment strategies must be explored in light of the increasing challenges of toxic and critical wastewater contamination. This paper deals with the bacteria-producing biosurfactant and their employment in the bioremediation of hydrocarbon-containing waste through a microbial fuel cell (MFC) with Pseudomonas aeruginosa (exoelectrogen) as co-culture for simultaneous power generation. Staphylococcus aureus is isolated from hydrocarbon-contaminated soil and is effective in hydrocarbon degradation by utilizing hydrocarbon (engine oil) as the only carbon source. The biosurfactant was purified using silica-gel column chromatography and characterised through FTIR and GCMS, which showed its glycolipid nature. The isolated strains are later employed in the MFCs for the degradation of the hydrocarbon and power production simultaneously which has shown a power density of 6.4 W/m3 with a 93% engine oil degradation rate. A biogenic Fe2O3 nanoparticle (NP) was synthesized using Bambusa arundinacea shoot extract for anode modification. It increased the power output by 37% and gave the power density of 10.2 W/m3. Thus, simultaneous hydrocarbon bioremediation from oil-contamination and energy recovery can be achieved effectively in MFC with modified anode.

Antifeedant, Antifungal Cryptic Polyketides with Six Structural Frameworks from Tea Endophyte Daldinia eschscholtzii Propelled by the Antagonistic Coculture with Phytopathogen Colletotrichum pseudomajus and Different Culture Methods.

The antagonistic coculture with tea phytopathogen Colletotrichum pseudomajus induces antifungal cryptic metabolites from isogenesis endophyte Daldinia eschscholtzii against tea phytopathogens. Sixteen new polyketides with six structural frameworks including ten cryptic ones, named coldaldols A-C (1-3), collediol (5), and daldinrins A-L (10-20 and 23), were found from the coculture of C. pseudomajus and D. eschscholtzii by different culture methods. The unique framework of compounds 11 and 12 featured a benzopyran-C7 polyketone hybrid, and compounds 13-16 were characterized by the novel benzopyran dimer. The structures were determined mainly by spectroscopic methods, including extensive one-dimensional (1D), two-dimensional (2D) NMR, high resolution electrospray ionisation mass spectroscopy (HRESIMS), ECD calculation, and single-crystal X-ray diffraction. The configuration of acyclic compounds 5 and 18 were determined by application of the universal NMR database. Most compounds showed significant antifungal activities against the tea pathogens C. pseudomajus and Alternaria sp. with MICs of 1-8 µg/mL. Compound 12 had stronger antifungal activity than that of positive drug nystatin. The ether bond at C-4 of the benzopyran derivative increased the antifungal activity. Compounds 4-9 and 11-23 showed antifeedant activities against silkworms with feeding deterrence indices of 15-100% at the concentration of 50 µg/cm2.

Synergistic effect of the coculture of Leuconostoc pseudomesenteroides and Lactococcus lactis, isolated from honeybees, on the generation of plant-based dairy alternatives based on soy, pea, oat, and potato drinks.

The production of plant-based dairy alternatives has been majorly focused on the improvement of sensorial, technological and nutritional properties, to be able to mimic and replace milk-based fermented products. The presence of off-flavours and antinutrients, the lack of production of dairy-like flavours or the metabolic inaccessibility of plant proteins are some of the challenges to overcome to generate plant-based dairy alternatives. However, in the present study, it is demonstrated how the synergistic effect of two LAB strains, when cocultured, can simultaneously solve those challenges when fermenting in four different plant-based raw materials: soy, pea, oat, and potato drinks (SPOP). The fermentation was performed through the mono- and co-culture of the two LAB strains isolated from Apis mellifera (honeybee): Leuconostoc pseudomesenteroides NFICC 2004 and Lactococcus lactis NFICC 2005. Firstly, the coculture of both strains demonstrated to increase the acidification rate of the four plant matrices. Moreover, L. pseudomesenteroides (LP) demonstrated to in situ produce high concentrations of mannitol when fructose was present as C-source. Furthermore, L. pseudomesenteroides, which encoded for PII-proteinase, demonstrated to break down SPOP proteins, releasing free amino acids that were used by L.lactis (LL) for growth and metabolism. Lastly, the analysis of their co-metabolic volatile performance showed the principal ability of removal of the main off-flavours found in SPOP, such as hexanal, 1-octen-3-ol, 2-pentylfuran, pentanal, octanal, heptanal, and nonanal, mainly led by L. pseudomesenteroides, as well as the production of dairy-like flavours, such as diacetyl and 3-methyl-1-butanol, triggered by L. lactis metabolism. Overall, these findings endorsed the use of honeybee isolated strains as starter cultures, demonstrated the potential of coupling genotypes and phenotypes of multiple strains to improve the organoleptic properties suggesting a potential of combining plant-based matrices for the generation of future high-quality plant-based dairy alternatives.

Hypoglycemic effect and intestinal transport of phenolics-rich extract from digested mulberry leaves in Caco-2/insulin-resistant HepG2 co-culture model.

Phenolics of mulberry (Morus alba L.) leaves (MLs) have potential anti-diabetic effects, but they may be chemically modified during gastrointestinal digestion so affect their biological activity. In this study, an in vitro digestion model coupled with Caco-2 monolayer and Caco-2/insulin-resistant HepG2 coculture model were used to study the transport and hypoglycemic effects of phenolics in raw MLs (U-MLs) and solid-fermented MLs (F-MLs). The results of LC-MS/MS analysis showed that the Papp (apparent permeability coefficient, 10-6cm/s) of phenolics in digested MLs ranged from 0.002 ± 0.00 (quercetin 3-O-glucoside) to 60.19 ± 0.67 (ferulic acid), indicating higher phenolic acids absorbability and poor flavonoids absorbability. The Papp values of phenolic extracts of F-MLs in Caco-2 monolayer were significantly higher (p > 0.05) than that of U-MLs. Digested phenolic extracts inhibited the activities of sucrase (60.13 ± 2.03 %) and maltase (82.35 ± 0.78 %) and decreased 9.28 ± 0.84 % of glucose uptake in Caco-2 monolayer. Furthermore, a decrease in the mRNA expression of glucose transporters SGLT1 (0.64 ± 0.18), GLUT2 (0.14 ± 0.02) and the sucrase-isomaltase (0.59 ± 0.00) was observed. In Caco-2/insulin-resistant HepG2 co-culture model, phenolic extracts regulated glucose metabolism by up-regulating the mRNA expressions of IRS1 (9.32-fold), Akt (17.07-fold) and GYS2 (1.5-fold), and down-regulating the GSK-3ß (0.22-fold), PEPCK (0.49-fold) and FOXO1 (0.10-fold) mRNA levels. Both U-MLs and F-MLs could improve glucose metabolism, and the partial least squares (PLS) analysis showed that luteoforol and p-coumaric acid were the primary phenolics that strongly correlated with the hypoglycemic ability of MLs. Results suggested that phenolics of MLs can be used as dietary supplements to regulate glucose metabolism.

Common Pollen Modulate Immune Responses against Viral-Like Challenges in Airway Coculture Model.

Recent research indicates that exposure to pollen increases the risk and severity of respiratory infections, while studies also suggest that it may possess a protective function. Our aim was to investigate how exposure to common pollen modifies airway cells' responses to viral- or bacterial-like challenges and vice versa. Cocultured A549 and THP-1 cells were exposed to three doses of four different pollens (Alnus glutinosa, Betula pendula, Phleum pratense, or Ambrosia artemisiifolia) and subsequently to Toll-like receptor (TLR) ligands mimicking bacterial and viral challenges (TLR3, TLR4, TLR7/8). The stimulation experiment was replicated in reverse order. Toxicological and immunological end points were analyzed. When cells were primed with pollen, especially with grass (P. pratense) or weed (A. artemisiifolia), the ability of cells to secrete cytokines in response to bacterial- and viral-like exposure was decreased. In contrast, cells primed with viral ligand TLR7/8 showed greater cytokine responses against pollen than cells exposed to ligands or pollen alone. Our results suggest that pollen exposure potentially weakens immune reactions to bacterial- or viral-like challenges by modulating cytokine production. They also indicate that TLR7/8-mediated viral challenges could elicit exaggerated immune responses against pollen. Both mechanisms could contribute to the acceleration and complication of infections during the pollen season.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

STUDY QUESTION: Can in vitro maturation (IVM) and developmental competence of human oocytes be improved by co-culture with ovarian support cells (OSCs) derived from human-induced pluripotent stem cells (hiPSCs)? SUMMARY ANSWER: OSC-IVM significantly improves the rates of metaphase II (MII) formation and euploid Day 5 or 6 blastocyst formation, when compared to a commercially available IVM system. WHAT IS KNOWN ALREADY: IVM has historically shown highly variable performance in maturing oocytes and generating oocytes with strong developmental capacity, while limited studies have shown a positive benefit of primary granulosa cell co-culture for IVM. We recently reported the development of OSCs generated from hiPSCs that recapitulate dynamic ovarian function in vitro. STUDY DESIGN, SIZE, DURATION: The study was designed as a basic science study, using randomized sibling oocyte specimen allocation. Using pilot study data, a prospective sample size of 20 donors or at least 65 oocytes per condition were used for subsequent experiments. A total of 67 oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without hCG triggers and retrieved cumulus-oocyte complexes (COCs) were allocated between the OSC-IVM or control conditions (fetal-like OSC (FOSC)-IVM or media-only IVM) in three independent experimental design formats. The total study duration was 1 April 2022 to 1 July 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte donors between the ages of 19 and 37 years were recruited for retrieval after informed consent, with assessment of anti-Mullerian hormone, antral follicle count, age, BMI and ovarian pathology used for inclusion and exclusion criteria. In experiment 1, 27 oocyte donors were recruited, in experiment 2, 23 oocyte donors were recruited, and in experiment 3, 17 oocyte donors and 3 sperm donors were recruited. The OSC-IVM culture condition was composed of 100 000 OSCs in suspension culture with hCG, recombinant FSH, androstenedione, and doxycycline supplementation. IVM controls lacked OSCs and contained either the same supplementation, FSH and hCG only (a commercial IVM control), or FOSCs with the same supplementation (Media control). Experiment 1 compared OSC-IVM, FOSC-IVM, and a Media control, while experiments 2 and 3 compared OSC-IVM and a commercial IVM control. Primary endpoints in the first two experiments were the MII formation (i.e. maturation) rate and morphological quality assessment. In the third experiment, the fertilization and embryo formation rates were assessed with genetic testing for aneuploidy and epigenetic quality in blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE: We observed a statistically significant improvement (∼1.5×) in maturation outcomes for oocytes that underwent IVM with OSCs compared to control Media-IVM and FOSC-IVM in experiment 1. More specifically, the OSC-IVM group yielded a MII formation rate of 68% ± 6.83% SEM versus 46% ± 8.51% SEM in the Media control (P = 0.02592, unpaired t-test). FOSC-IVM yielded a 51% ± 9.23% SEM MII formation rate which did not significantly differ from the media control (P = 0.77 unpaired t-test). Additionally, OSC-IVM yielded a statistically significant ∼1.6× higher average MII formation rate at 68% ± 6.74% when compared to 43% ± 7.90% in the commercially available IVM control condition (P = 0.0349, paired t-test) in experiment 2. Oocyte morphological quality between OSC-IVM and the controls did not significantly differ. In experiment 3, OSC-IVM oocytes demonstrated a statistically significant improvement in Day 5 or 6 euploid blastocyst formation per COC compared to the commercial IVM control (25% ± 7.47% vs 11% ± 3.82%, P = 0.0349 logistic regression). Also in experiment 3, the OSC-treated oocytes generated blastocysts with similar global and germline differentially methylated region epigenetic profiles compared commercial IVM controls or blastocysts after either conventional ovarian stimulation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: While the findings of this study are compelling, the cohort size remains limited and was powered on preliminary pilot studies, and the basic research nature of the study limits generalizability compared to randomized control trials. Additionally, use of hCG-triggered cycles results in a heterogenous oocyte cohort, and potential differences in the underlying maturation state of oocytes pre-IVM may limit or bias findings. Further research is needed to clarify and characterize the precise mechanism of action of the OSC-IVM system. Further research is also needed to establish whether these embryos are capable of implantation and further development, a key indication of their clinical utility. WIDER IMPLICATIONS OF THE FINDINGS: Together, these findings demonstrate a novel approach to IVM with broad applicability to modern ART practice. The controls used in this study are in line with and have produced similar to findings to those in the literature, and the outcome of this study supports findings from previous co-culture studies that found benefits of primary granulosa cells on IVM outcomes. The OSC-IVM system shows promise as a highly flexible IVM approach that can complement a broad range of stimulation styles and patient populations. Particularly for patients who cannot or prefer not to undergo conventional gonadotropin stimulation, OSC-IVM may present a viable path for obtaining developmentally competent, mature oocytes. STUDY FUNDING/COMPETING INTEREST(S): A.D.N., A.B.F., A.G., B.P., C.A., C.C.K., F.B., G.R., K.S.P., K.W., M.M., P.C., S.P., and M.-J.F.-G. are shareholders in the for-profit biotechnology company Gameto Inc. P.R.J.F. declares paid consultancy for Gameto Inc. P.C. also declares paid consultancy for the Scientific Advisory Board for Gameto Inc. D.H.M. has received consulting services from Granata Bio, Sanford Fertility and Reproductive Medicine, Gameto, and Buffalo IVF, and travel support from the Upper Egypt Assisted Reproduction Society. C.C.K., S.P., M.M., A.G., B.P., K.S.P., G.R., and A.D.N. are listed on a patent covering the use of OSCs for IVM: U.S. Provisional Patent Application No. 63/492,210. Additionally, C.C.K. and K.W. are listed on three patents covering the use of OSCs for IVM: U.S. Patent Application No. 17/846,725, U.S Patent Application No. 17/846,845, and International Patent Application No.: PCT/US2023/026012. C.C.K., M.P.S., and P.C. additionally are listed on three patents for the transcription factor-directed production of granulosa-like cells from stem cells: International Patent Application No.: PCT/US2023/065140, U.S. Provisional Application No. 63/326,640, and U.S. Provisional Application No. 63/444,108. The remaining authors have no conflicts of interest to declare.

Microbiome and transcriptome analyses reveal the influence of calcined dolomite application on Eriocheir sinensis in a rice-crab co-culture system.

Co-culture systems of rice and aquatic animals can contribute to the ecological intensification of agriculture by reducing nutrient loss and the need for N fertilizer application and by enhancing nutrient-use efficiency. However, the input of high-protein diets into paddy fields, to facilitate the growth of aquatic animals, has been found to increase N pollution and acidification of the soil. Although soil amendments have been widely used to ameliorate acidic soils, reduce N2O emissions, and improve agronomic production, the relationship between soil amendments and aquatic animal remains unclear. Therefore, this study investigated the effects of calcined dolomite (hereafter referred to as dolomite) as an acidic soil amendment and Ca-Mg supplement in rice-crab co-culture using Eriocheir sinensis crabs (Chinese mitten crabs). High-throughput sequencing was used to examine crab bacterial community composition and crab hepatopancreas biology. Although the water pH was significantly increased in the dolomite group, the number, composition, and diversity of bacteria identified in crab gut microbiome did not vary significantly between the dolomite and control groups. In the dolomite group, the probiotic agents Candidatus Hepatoplasma and Lactobacillus were highly abundant in the crab gut, and immune- and retinol metabolism-related genes were significantly upregulated in the crab hepatopancreas. Overall, dolomite application increased crab health and water pH. Dolomite is a low-cost amendment, with better stability, compared to other soil amendments, thus making it ideal for sustainable and clean rice-aquatic animal co-culture.

Simultaneous bioreduction of tellurite and selenite by Yarrowia lipolytica, Trichosporon cutaneum, and their co-culture along with characterization of biosynthesized Te-Se nanoparticles.

BACKGROUND: Natural and anthropogenic activities, such as weathering of rocks and industrial processes, result in the release of toxic oxyanions such as selenium (Se) and tellurium (Te) into the environment. Due to the high toxicity of these compounds, their removal from the environment is vital. RESULTS: In this study, two yeast strains, Yarrowia lipolytica and Trichosporon cutaneum, were selected as the superior strains for the bioremediation of tellurium and selenium. The reduction analyses showed that exposure to selenite induced more detrimental effects on the strains compared to tellurite. In addition, co-reduction of pollutants displayed almost the same results in selenite reduction and more than ~ 20% higher tellurite reduction in 50 h, which shows that selenite triggered higher tellurite reduction in both strains. The selenite and tellurite kinetics of removal were consistent with the first-order model because of their inhibitory behavior. The result of several characterization experiments, such as FE-SEM (Field emission scanning electron microscopy), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), and dispersive X-ray (EDX) on Te-Se nanoparticles (NPs) revealed that the separated Te-Se NPs were needle-like, spherical, and amorphous, consisted of Te-Se NPs ranging from 25 to 171 nm in size, and their surface was covered with different biomolecules. CONCLUSIONS: Remarkably, this work shows, for the first time, the simultaneous bioreduction of tellurite and selenite and the production of Te-Se NPs using yeast strains, indicating their potential in this area, which may be applied to the nanotechnology industry and environmental remediation.

Garlic extract enhances bioceramic bone scaffolds through upregulating ALP & BGLAP expression in hMSC-monocyte co-culture.

Bone homeostasis is predicated by osteoblast and osteoclast cell cycles where gene expressions are responsible for their differentiation from human mesenchymal stem cells (hMSC) and monocytes, respectively. The pro-osteogenic potential of an hMSC-monocyte co-culture can be measured through complementary DNA (mRNA synthesis) within the nucleus, known as quantitative polymerase chain reaction (qPCR). Through this technique, the effects of garlic extract (allicin) release from calcium phosphate bone scaffolds on gene expression of bone forming and bone remodeling cells was explored. Results show this complex biomaterial system enhances hMSC differentiation through the upregulation of bone-forming proteins. Osteoblastic gene markers alkaline phosphatase (ALP) and osteocalcin (BGLAP), are respectively upregulated by 3-fold and 1.6-fold by day 14. These mature osteoblasts then upregulate the receptor activator of nuclear factor-kB ligand (RANKL) which recruits osteoclast cells, as captured by a nearly 2-fold higher osteoclast expression of tartrate-resistance acid-phosphatase (ACP5). This also activates antagonist osteoprotegerin (OPG) expression in osteoblasts, decreasing osteoclast resorption potential and ACP5 expression by day 21. The pro-osteogenic environment with garlic extract release is further quantified by a 4× increase in phosphatase activity and visibly captured in immunofluorescent tagged confocal images. Also corroborated by enhanced collagen formation in a preliminary in vivo rat distal femur model, this work collectively reveals how garlic extract can enhance bioceramic scaffolds for bone tissue regenerative applications.

Micropatterned primary hepatocyte co-culture (HEPATOPAC) for fatty liver disease modeling and drug screening.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, progressive disorder and growing public health concern. To address this issue considerable research has been undertaken in pursuit of new NAFLD therapeutics. Development of effective, high-throughput in vitro models is an important aspect of drug discovery. Here, a micropatterned hepatocyte co-culture (MPCC) was used to model liver steatosis. The MPCC model (HEPATOPACTM) is comprised of hepatocytes and 3T3-J2 mouse stromal cells plated onto a patterned standard 96-well or 24-well plate, allowing the cultures to be handled and imaged in a standardized multi-well format. These studies employed high content imaging (HCI) analysis to assess lipid content in cultures. HCI analysis of lipid accumulation allows large numbers of samples to be imaged and analyzed in a relatively short period of time compared to manual acquisition and analysis methods. Treatment of MPCC with free fatty acids (FFA), high glucose and fructose (HGF), or a combination of both induces hepatic steatosis. MPCC treatment with ACC1/ACC2 inhibitors, as either a preventative or reversal agent, showed efficacy against FFA induced hepatic steatosis. Drug induced steatosis was also evaluated. Treatment with valproic acid showed steatosis induction in a lean background, which was significantly potentiated in a fatty liver background. Additionally, these media treatments changed expression of fatty liver related genes. Treatment of MPCC with FFA, HGF, or a combination reversibly altered expression of genes involved in fatty acid metabolism, insulin signaling, and lipid transport. Together, these data demonstrate that MPCC is an easy to use, long-term functional in vitro model of NAFLD having utility for compound screening, drug toxicity evaluation, and assessment of gene regulation.

Co-culture of Acinetobacter sp. and Scedosporium sp. immobilized beads for optimized biosurfactant production and degradation of crude oil.

The widespread exploration and exploitation of crude oil has increased the prevalence of petroleum hydrocarbon pollution in the marine and coastal environment. Bioremediation of petroleum hydrocarbons using cell immobilization techniques is gaining increasing attention. In this study, the crude oil degradation performance of bacterial and fungal co-culture was optimized by entrapping both cells in sodium-alginate and polyvinyl alcohol composite beads. Results indicate that fungal cells remained active after entrapment and throughout the experiment, while bacterial cells were non-viable at the end of the experimental period in treatments with the bacterial-fungal ratio of 1:2. A remarkable decrease in surface tension from 72 mN/m to 36.51 mN/m was achieved in treatments with the bacterial-fungal ratio of 3:1. This resulted in a significant (P < 0.05) total petroleum hydrocarbon (TPH) removal rate of 89.4%, and the highest degradation of n-alkanes fractions (from 2129.01 mg/L to 118.53 mg/L), compared to the other treatments. Whereas PAHs removal was highest in treatments with the most fungal abundance (from 980.96 µg/L to 177.3 µg/L). Furthermore, enzymes analysis test revealed that catalase had the most effect on microbial degradation of the target substrate, while protease had no significant impact on the degradation process. High expression of almA and PAH-RHDa genes was achieved in the co-culture treatments, which correlated significantly (P < 0.05) with n-alkanes and PAHs removal, respectively. These results indicate that the application of immobilized bacterial and fungal cells in defined co-culture systems is an effective strategy for enhanced biodegradation of petroleum hydrocarbons in aqueous systems.

Promoting Magnusiomyces spicifer AW2 Cell-Bound Lipase Production by Co-culturing with Staphylococcus hominis AUP19 and Its Application in Solvent-Free Biodiesel Synthesis.

Yeast-bacterium interaction has recently been investigated to benefit the production of cell-bound lipases (CBLs). Staphylococcus hominis AUP19 supported the growth of Magnusiomyces spicifer AW2 in a palm oil mill effluent (POME) medium to produce CBLs through a bioremediation approach, including oil and grease (O&G) and chemical oxygen demand (COD) removals. This research used the yeast-bacterium co-culture to optimize CBLs and cell biomass (CBM) productions through bioremediation using the statistical Plackett-Burman design and response surface methodology-central composite design. The CBLs were finally applied in biodiesel synthesis. The CBM of 13.8 g/L with CBLs activity at 3391 U/L was achieved after incubation at room temperature (RT, 30 ± 2 °C) for 140 h in 50% POME medium, pH 7.0, containing 1.23% (w/v) ammonium sulfate. Bacterium promoted yeast growth to achieve bioremediation with 87.9% O&G removal and 84.5% COD removal. Time course study showed that the CBLs activity was highest at 24 h cultivation (4103 U/L) and retained 80% and 60% of activities at 4 °C and RT after 5 weeks of storage. The CBLs application successfully yielded 77.3% biodiesel from oleic acid (esterification) and 86.4% biodiesel from palm oil (transesterification) within 72 h in solvent-free systems. This study highlights that yeast-bacterium co-culture and POME should receive more attention for potential low-cost CBLs production through bioremediation, i.e., O&G and COD removals, while the CBLs as biocatalysts are promising for significant contribution to an effective strategy for economic green biodiesel production.

Culture Media Supplemented With 10% Equine Serum Provided Chondroprotection in an In Vitro Co-Culture of Cartilage and Synovial Membrane.

No studies have evaluated the effect of culture in serum-free media (SF) vs. media supplemented with equine serum (ES) on co-culture of synovial membrane and cartilage tissue explants. The study objective was to evaluate the effects of equine serum supplementation on induced production of inflammatory and catabolic mediators from articular cartilage and synovial explants while in co-culture. Articular cartilage and synovial membrane explants were harvested from femoropatellar joints of five adult horses. Cartilage and synovial explants were harvested from the stifle of five horses, placed in co-culture, stimulated with IL-1ß (10 ng/ml) and maintained in culture for 3, 6 and 9 days in 10% ES or SF. At each time point, media was harvested for analysis of cellular viability (Lactate dehydrogenase) and elution of glycosaminoglycans (Dimethylene Blue Binding Assay). Tissue explants were harvested for histopathologic and gene expression analyses. No differences in cell viability were observed between SF and ES groups. SF culture produced an upregulation of TNF-α in synovial membrane and ADAMTS-4 and five in articular cartilage at 9 days of culture. ES produced an upregulation of aggrecan expression in cartilage at 9 days of culture. No differences in tissue viability were found between culture media, but SF media produced a higher glycosaminoglycan concentration in media at 3 days of culture. The addition of 10% ES produced a slight chondroprotective effect in an inflamed co-culture system. This effect should be considered when designing studies evaluating treatment of serum or plasma-based orthobiologic studies in vitro.

Performance of co-culture strategy on nutrient removal and biogas upgrading by strigolactone induction.

In this study, we investigated the performance and elucidated the synergistic effects of microalgae-fungi symbionts co-cultured with 10-7 and 10-9  mol L-1 of GR24 and supplemented with endophytic bacteria, multi-walled carbon nanotubes (MWCNTs) or vitamin B12 (VB12), on nutrient removal and biogas upgrading. The results showed that the microalgae-fungi-bacteria symbiotic system co-cultured with 10-9  mol L-1 GR24 presented the optimal growth performance of 0.368 ± 0.04 d-1 , chlorophyll a of 249.36 ± 22.31 µg L-1 , and extracellular carbonic anhydrase activity of 42.55 ± 3.755 enzyme units. In this co-culture system, the organic matter, nutrients, and CO2 purification obtained the highest removal efficiency, with 81.35 ± 7.96% for chemical oxygen demand, 83.56 ± 7.91% total nitrogen, 84.17 ± 7.95% total phosphorus, and 63.72 ± 6.06% CO2 . The symbiont system also greatly increased the methane content in the biogas by 30.67%. The remarkable performance of the microalgae-fungi-bacteria symbiotic system shows its ability to be broadly applied in simultaneous biogas upgrading and wastewater treatment. PRACTITIONER POINTS: The optimal GR24 concentration for microalgae-fungi consortia was 10-9  M. Endophytic bacteria were superior to MWCNTs and VB12. Fungi-algae-bacteria consortia presented excellent growth and removal performance. Removal efficiencies of COD, TN, and TP were about 81% under optimum treatment.

Medulloblastoma and high-grade glioma organoids for drug screening, lineage tracing, co-culture and in vivo assay.

Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60-65 d for cancer organoids generation and from 1-4 weeks for downstream applications. The protocol requires at least 3-6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.

An endophytic fungus Schizophyllum commune isolated from Panax ginseng enhances hairy roots growth and ginsenoside biosynthesis.

Using endophytic fungal elicitors to increase the accumulation of valuable secondary metabolites in plant tissue culture is an effective biotechnology strategy. In this study, a collection of 56 strains of endophytic fungi were isolated from different organs of cultivated Panax ginseng, of which seven strains can be symbiotically co-cultured with the hairy roots of P. ginseng. Further experiments observed that strain 3R-2, identified as endophytic fungus Schizophyllum commune, can not only infect hairy roots but also promote the accumulation of specific ginsenosides. This was further verified because S. commune colonization significantly affected the overall metabolic profile of ginseng hairy roots. By comparing the effects of S. commune mycelia and its mycelia extract (EM) on ginsenoside production in P. ginseng hairy roots, the EM was confirmed to be a relatively better stimulus elicitor. Additionally, the introduction of EM elicitor can significantly enhance the expressions of key enzyme genes of pgHMGR, pgSS, pgSE, and pgSD involved in the biosynthetic pathway of ginsenosides, which was deemed the most relevant factor for promoting ginsenosides production during the elicitation period. In conclusion, this study is the first to show that the EM of endophytic fungus S. commune can be considered as an effective endophytic fungal elicitor for increasing the biosynthesis of ginsenosides in hairy root cultures of P. ginseng.

Protist-Lactic Acid Bacteria Co-Culture as a Strategy to Bioaccumulate Polyunsaturated Fatty Acids in the Protist Aurantiochytrium sp. T66.

Thraustochytrids are aquatic unicellular protists organisms that represent an important reservoir of a wide range of bioactive compounds, such as essential polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), which are involved in the regulation of the immune system. In this study, we explore the use of co-cultures of Aurantiochytrium sp. and bacteria as a biotechnological tool capable of stimulating PUFA bioaccumulation. In particular, the co-culture of lactic acid bacteria and the protist Aurantiochytrium sp. T66 induce PUFA bioaccumulation, and the lipid profile was evaluated in cultures at different inoculation times, with two different strains of lactic acid bacteria capable of producing the tryptophan dependent auxins, and one strain of Azospirillum sp., as a reference for auxin production. Our results showed that the Lentilactobacillus kefiri K6.10 strain inoculated at 72 h gives the best PUFA content (30.89 mg g-1 biomass) measured at 144 h of culture, three times higher than the control (8.87 mg g-1 biomass). Co-culture can lead to the generation of complex biomasses with higher added value for developing aquafeed supplements.