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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.

The intestinal epithelium is a highly dynamic and self-renewing tissue that is crucial for maintaining gut homeostasis. It can be cultured in vitro from isolated crypts to form three-dimensional (3D) intestinal organoids. These organoids have the ability to proliferate and differentiate into various epithelial cell lineages, offering a more physiologically relevant model compared to traditional two-dimensional (2D) culture systems. Mesenchymal cells, located near epithelial cells, regulate epithelial behavior through paracrine signaling and provide structural support. Building on recent advances in the biology of epithelial and mesenchymal cells, we have developed a coculture system that integrates intestinal organoids with mesenchymal cells. In this system, intestinal organoids are cultured in direct or indirect contact with mesenchymal cells, allowing for the simulation of signal exchange and interactions within the in vivo-like microenvironment. This coculture system not only preserves the 3D architecture of the organoids but also enhances their physiological relevance by introducing cellular complexity. The system is capable of long-term maintenance and is adaptable to a wide range of experimental manipulations. As such, this coculture model serves as a powerful tool for studying the interactions between the intestinal epithelium and its surrounding stroma, providing new insights into stem cell biology, tissue regeneration, and disease mechanisms. Here, we introduce the methods of mouse crypt isolation, intestinal organoid culture, and its coculture with mesenchymal cell., (© 2024. Springer Science+Business Media, LLC.)

Approximately 50% of patients with surgically resected early-stage lung cancer develop distant metastasis. At present, there is no in vivo metastasis model to investigate the biology of human lung cancer metastases. Using well-characterized lung adenocarcinoma (LUAD) patient-derived organoids (PDOs), we establish an in vivo metastasis model that preserves the biologic features of human metastases. Results of whole-genome and RNA sequencing establish that our in vivo PDO metastasis model can be used to study clonality and tumor evolution and to identify biomarkers related to organotropism. Investigation of the response of KRAS G12C PDOs to sotorasib demonstrates that the model can examine the efficacy of treatments to suppress metastasis and identify mechanisms of drug resistance. Finally, our PDO model cocultured with autologous peripheral blood mononuclear cells can potentially be used to determine the optimal immune-priming strategy for individual patients with LUAD., Competing Interests: Declaration of interests G.R. has financial relationships with Scanlan, AstraZeneca, and Medtronic. S.S. is a member of the AstraZeneca Advisory Board. J.M.I. has stock ownership in LumaCyte and is a consultant/advisory board member for Roche Genentech. M.J.B. is a consultant for AstraZeneca, Iovance Biotherapeutics, and Intuitive Surgical and receives research support from Obsidian Therapeutics. B.T.L. has served as an uncompensated advisor and consultant to Amgen, AstraZeneca, Boehringer Ingelheim, Bolt Biotherapeutics, Daiichi Sankyo, Genentech, and Lilly; has received research grants (institutional) from Amgen, AstraZeneca, Bolt Biotherapeutics, Daiichi Sankyo, Genentech, Hengrui USA, and Lilly; has received academic travel support from Amgen, Jiangsu Hengrui Medicine, and MORE Health; and has intellectual property rights as a book author at Karger Publishers and Shanghai Jiao Tong University Press. M.H.B. receives royalties from Globus Medical and DePuy Synthes. P.S.A. declares research funding from Atara Biotherapeutics; is a scientific advisory board member and consultant for ATARA Biotherapeutics, Bayer, Bio4T2, Carisma Therapeutics, Imugene, ImmPACT Bio, Johnson & Johnson, Orion, and Outpace Bio; has patents, royalties, and intellectual property on mesothelin-targeted chimeric antigen receptor and other T cell therapies, which have been licensed to Atara Biotherapeutics; and has an issued patent method for detection of cancer cells using virus and pending patent applications on PD-1 dominant negative receptor, a wireless pulse-oximetry device, and an ex vivo malignant pleural effusion culture system. MSK has licensed intellectual property related to mesothelin-targeted chimeric antigen receptors and T cell therapies to Atara Biotherapeutics and has associated financial interests. D.R.J. serves on a clinical trial steering committee for AstraZeneca and has research grant support from Merck., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The airway mucus of patients with cystic fibrosis has altered properties, which create a microenvironment primed for chronic infections that are difficult to treat. These complex polymicrobial airway infections and corresponding mammalian-microbe interactions are challenging to model in vitro. Here, we report the development of mucus-like hydrogels with varied compositions and viscoelastic properties reflecting differences between healthy and cystic fibrosis airway mucus. Models of cystic fibrosis and healthy airway microenvironments were created by combining the hydrogels with relevant pathogens, human bronchial epithelial cells, and an antibiotic. Notably, pathogen antibiotic resistance was not solely dependent on the altered properties of the mucus-like hydrogels but was also influenced by culture conditions including microbe species, monomicrobial or polymicrobial culture, and the presence of epithelial cells. Additionally, the cystic fibrosis airway model showed the ability to mimic features characteristic of chronic cystic fibrosis airway infections including sustained polymicrobial growth and increased antibiotic tolerance.

Astrocytes are essential for the formation and maintenance of neural networks. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques, primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing, we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons, consistent with their further maturation. In coculture with human neurons, multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min -1 (hPSC-derived), 2.86 ± 0.64 min -1 (rat); p  = 0.19]. In comparison, we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived), 24.77 ± 4.04 Hz (rat); p  < 0.01], and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived), 1.07 ± 0.14 Hz (rat); p  < 0.001], consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived), 8.39 ± 0.63/100 μm 2 (rat); p  < 0.001]. Taken together, we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation, providing a fully human platform for investigating astrocyte function and neuronal-glial interactions., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Lendemeijer et al.)

In bone tissue engineering, biological scaffolds are designed with structural and functional properties that closely resemble the extracellular environment, aiming to establish a microenvironment conducive to osteogenesis. Macrophages hold significant potential for promoting osteogenesis and modulating the biological behavior of tumor cells. Multiple coculture experiments of macrophages and osteoblasts have demonstrated that macrophage polarization significantly impacts osteogenesis. Therefore, exploring bone biomaterials that can modulate macrophage polarization holds great clinical significance. In this study, heparin was modified with maleimide and was used as a raw material to form a hydrogel with 4-am-PEG-SH. The compound was used to polarize macrophages and promote osteogenesis after combining with interleukin 4 (IL-4) by taking advantage of the electronegativity of heparin. The results revealed overexpressed M2 macrophage-related phenotypic genes and cocultivation with MC3T3-E1 cells demonstrated the osteogenesis-promoting effect of the loaded IL-4 heparin hydrogel. Previous research reported that hydrogel loaded with IL-4 can be used as a biomaterial for osteogenesis promotion. Heparin materials used in this paper are derived from clinically anticoagulant drugs and feature a simple operation. The synthesized hydrogel effectively binds cytokines, regulates macrophages to induce osteogenesis and has many potential clinical applications.

Synthetic hydroxyapatite (HA) is a widely studied bioceramic for bone tissue engineering (BTE) due to its similarity to the mineral component of bone. As bone mineral contains various ionic substitutions that play a crucial role in bone metabolism, the bioactivity of HA can be improved by adding small amounts of physiologically relevant ions into its crystal structure, with silicate-substituted HA (Si-HA) showing particularly promising results. Nevertheless, it remains unclear how distinct material characteristics influence the bioactivity due to the intertwined nature of surface properties. A coculture methodology was optimized and applied for in vitro quantification of the biological response. Initially, HA and Si-HA samples were produced and characterized. To compare the bioactivity of the samples, a method was developed to measure interactions in an increasingly complex environment, first including fibronectin (FN) adsorption and subsequently cell adhesion in mono and coculture using primary human osteoblasts (hOBs) and human dermal microvascular endothelial cells (HDMECs), with and without FN precoating. An experimental set-up was designed to assess to what extent different surface features of the samples contribute to the induced biological response. An 8-nm gold sputter coating was applied to eradicate the electrochemical differences and polishing and abrading was used to reduce the differences in surface topographies. Overall, 1.25 wt% Si-HA exhibited most nanoscale variations in surface potential. In terms of bioactivity, 1.25 wt% Si-HA samples induced the highest osteoblast attachment and vessel formation. Additionally, in vitro vessel formation was established on Si-HA surfaces using a hOB:HDMEC cell ratio of 70:30 and a methodology was established that enabled the assessment of the relative effect of topographical and electrochemical features induced by silicon substitution in the HA lattice on their bioactivity. It was found that the difference in the amount of protein attached to HA and 1.25 wt% Si-HA after 2 h was affected by topographical differences. Conversely, electrochemical differences induced different vessel-like structure formation in coculture with a FN precoating. Without an FN precoating, both topographical and electrochemical differences dictated the differences in angiogenic response. Overall, 1.25 wt% Si-HA surface features appear to induce the most favorable protein adsorption and cell adhesion in mono and coculture with and without FN precoating.

The microalgae Isochrysis galbana and Chaetoceros gracilis are part of the basic diet of most bivalve cultures. This study evaluated the supplementation of bacterial strains naturally associated with these microalgae to improve their productivity. Ten bacterial strains collected from I. galbana and C. gracilis commercial cultures used in the larviculture of the Peruvian scallop Argopecten purpuratus were isolated and purified. These strains were individually coinoculated to each microalgal culture to confirm their promoter effect on growth. First, inoculation was carried out in a volume of 500 mL with constant aeration, which allowed the selection of the best bacterial strains. Then, the culture growth performance after transfer from 500 mL to 2 L tanks, with and without bacterial reinoculation during transfer, was monitored. Two bacterial strains, Lysinibacillus fusiformis and Bacillus cereus, significantly improved (α =.05) I. galbana production, whereas three other strains, Lysinibacillus sp., Psychrobacter celer, and Staphylococcus saprophyticus, significantly increased (α =.05) the growth of the C. gracilis culture. The use of microalgae supplemented with bacteria did not significantly affect A. purpuratus larval rearing. Altogether, these results showed the potential of native probiotic bacteria to improve microalgal production within bivalve hatcheries. [ABSTRACT FROM AUTHOR]

The emerging interest in skeletal muscle tissue originates from its unique properties that control body movements. In particular, recent research advances in engineered skeletal muscle tissue have broadened the possibilities of applications in nonclinical models. However, due to the lack of adipose tissue, current engineered skeletal muscle tissue has the limitation of satisfying in vivo‐like position and proportion of intermuscular fat. Adipose tissue within the skeletal muscle affects their functional properties. Here, a fabrication method for cocultured tissue composed of skeletal muscle and adipose tissues is proposed to reproduce the functional and morphological characteristics of muscle. By implementing prematured adipose microfibers in a myoblast‐laden hydrogel sheet, both the accumulation of large lipid droplets and control of the position of adipose tissue within the skeletal muscle tissue becomes feasible. The findings of this study provide helpful information regarding engineered skeletal muscle, which has strong potential in drug screening models. [ABSTRACT FROM AUTHOR]

Epicardial cells (EpiCs) are necessary for myocardium formation, yet little is known about crosstalk between EpiCs and cardiomyocytes (CMs) during development and the potential impact of EpiCs on CM maturation. To investigate the effects of EpiCs on CM commitment and maturation, we differentiated human pluripotent stem cells (hPSCs) to cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs for two weeks. When EpiCs were allowed to form epicardial-derived cells, we observed increased expression of cTnI in developing CMs. In the presence of the TGFβ inhibitor A83-01, EpiCs remained in the epicardial state and induced CM proliferation, increased MLC2v expression, and led to less organized sarcomeres. These effects were not observed if CPCs were treated with EpiC-conditioned medium or if CPCs were indirectly cocultured with EpiCs. Finally, single cell RNA sequencing identified that EpiC-CPC coculture had bi-directional effects on transcriptional programs in EpiCs and CMs, and biased EpiC lineages from a SFRP2 -enriched population to a DLK1 - or C3 -enriched population. This work suggests important crosstalk between EpiCs and CMs during differentiation which can be used to influence cell fate and improve the ability to generate cardiac cells and tissues for in vitro models and development of cardiac cellular therapies. [Display omitted] • Investigate important cardiac crosstalk during development and disease. • Coculture of human pluripotent stem cell cardiomyocytes and epicardial cells. • Crosstalk causes cocultured cardiomyocytes to be more proliferative. • Cocultured epicardial cells undergo lineage bias. • Implications in tissue engineering and development of cardiac therapies. [ABSTRACT FROM AUTHOR]

Converting plant biomass into biofuels and biochemicals via microbial fermentation has received considerable attention in the quest for finding renewable energies and materials. Most approaches have so far relied on cultivating a single microbial strain, tailored for a specific purpose. However, this contrasts to how nature works, where microbial communities rather than single species perform all tasks. In artificial coculture systems, metabolic synergies are rationally designed by carefully selecting and simultaneously growing different microbes, taking advantage of the broader metabolic space offered by the use of multiple organisms. 1-propanol and 2-propanol, as biofuels and precursors for propylene, are interesting target molecules to valorize plant biomass. Some solventogenic Clostridia can naturally produce 2-propanol in the so-called Isopropanol-Butanol-Ethanol (IBE) fermentation, by coupling 2-propanol synthesis to acetate and butyrate reduction into ethanol and 1-butanol. In this work, we hypothesized propanoate would be converted into 1-propanol by the IBE metabolism, while driving at the same time 2-propanol synthesis. We first verified this hypothesis and chose two propionic acid bacteria (PAB) strains as propanoate producers. While consecutive PAB and IBE fermentations only resulted in low propanol titers, coculturing Propionibacterium freudenreichii and Clostridium beijerinckii at various inoculation ratios yieldedmuch higher solvent concentrations, with asmuch as 21 g/l of solvents (58% increase compared to C. beijerinckii monoculture) and 12 g/l of propanol (98% increase). Taken together, our results underline how artificial cocultures can be used to foster metabolic synergies, increasing fermentative performances and orienting the carbon flow towards a desired product. [ABSTRACT FROM AUTHOR]

Pancreatic cancer remains largely unresponsive to immune modulatory therapy attributable in part to an immunosuppressive, desmoplastic tumor microenvironment. Here, we analyze mechanisms of cancer cellautonomous resistance to T cells. We used a 3D co-culture model of cancer cell spheroids from the KPC (LSL-KrasG12D/+/LSL-Trp53R172H/+/p48-Cre) pancreatic ductal adenocarcinoma (PDAC) model, to examine interactions with tumor-educated T cells isolated from draining lymph nodes of PDAC-bearing mice. Subpopulations of cancer cells resistant to these tumor-educated T cells were isolated from the in vitro coculture and their properties compared with sensitive cancer cells. In co-culture with resistant cancer cell subpopulations, tumor-educated T cells showed reduced effector T cell functionality, reduced infiltration into tumor cell spheroids and decreased induction of apoptosis. A combination of comparative transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to dissect the role of differential gene expression and signaling pathways between sensitive and resistant cells. A decreased expression of the chemokine CXCL12 (SDF-1) was revealed as a common feature in the resistant cell subpopulations. Adding back CXCL12 reversed the resistant phenotype and was inhibited by the CXCR4 inhibitor AMD3100 (plerixafor). We conclude that reduced CXCL12 signaling contributes to PDAC subpopulation resistance to T cell-mediated attack. [ABSTRACT FROM AUTHOR]

All-time preservation of an intact mucosal barrier is crucial to ensuring intestinal homeostasis and, hence, the organism’s overall health maintenance. This complex process relies on an equilibrated signaling system between the intestinal epithelium and numerous cell populations inhabiting the gut mucosa. Any perturbations of this delicate cross talk, particularly regarding the immune cell compartment and microbiota, may sustainably debilitate the intestinal barrier function. As a final joint event, a critical rise in epithelial permeability facilitates the exposure of submucosal immunity to microbial antigens, resulting in uncontrolled inflammation, collateral tissue destruction, and dysbiosis. Organoid-derived intestinal coculture models have established themselves as convenient tools to reenact such pathophysiological events, explore interactions between selected cell populations, and assess their roles with a central focus on intestinal barrier recovery and stabilization. [ABSTRACT FROM AUTHOR]

Introduction: Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is extremely malignant and lethal. GBM tumors are highly heterogenous, being comprised of cellular and matrix components, which contribute to tumor cell invasion, cancer stem cell maintenance, and drug resistance. Here, we developed a heterotypic 3D spheroid model integrating GBM cells with astrocytes and endothelial cells (ECs) to better simulate the cellular components of the tumor microenvironment and investigate their impact on the stemness marker expression of GBM cells, which has not been previously investigated. Methods: We used U87 GBM cells, C8-D1A mouse astrocytes, and human umbilical vein ECs to construct co- and tri-culture spheroid models in low-attachment U-well plates. We characterized the expression of known stemness markers NESTIN, SOX2, CD133, NANOG, and OCT4 in these models and compared it to respective mixed monoculture spheroids (control) using qRT-PCR and immunostaining. Results: We incorporated GBM cells and astrocytes/ECs in 1:1, 1:2, 1:4, and 1:9 ratio and observed spontaneous self-assembled spheroids in all coculture conditions. We observed changing spheroid size dynamics over 7 days and an increased expression in stemness markers in GBM-astrocyte and GBM-EC coculture spheroids in 1:4 and 1:9 coculture conditions, respectively. In a triculture model employing GBM cells, astrocytes, and ECs in a 1:4:9 ratio, we found an increased expression of all the stemness markers. Conclusions: We elucidated the impact of astrocytes and ECs on GBM stemness marker expression. This multicellular spheroid model may provide an important tool for investigating the crosstalk between cell types in GBM. [ABSTRACT FROM AUTHOR]

Hepatocytes, the main epithelial cell type in the liver, perform most of the biochemical functions of the liver. Thus, maintenance of a primary hepatocyte phenotype is crucial for investigations of in vitro drug metabolism, toxicity, and development of bioartificial liver constructs. Here, we report the impact of topographic cues alone and in combination with soluble signals provided by encapsulated feeder cells on maintenance of the primary hepatocyte phenotype. Topographic features were 300 nm deep with pitches of either 400, 1400, or 4000 nm. Hepatocyte cell attachment, morphology and function were markedly better on 400 nm pitch patterns compared with larger scale topographies or planar substrates. Interestingly, topographic features having biomimetic size scale dramatically increased cell adhesion whether or not substrates had been precoated with collagen I. Albumin production in primary hepatocytes cultured on 400 nm pitch substrates without collagen I was maintained over 10 days and was considerably higher compared to albumin synthesis on collagen-coated flat substrates. In order to investigate the potential interaction of soluble cytoactive factors supplied by feeder cells with topographic cues in determining cell phenotype, bioactive heparin-containing hydrogel microstructures were molded (100 μm spacing, 100 μm width) over the surface of the topographically patterned substrates. These hydrogel microstructures either carried encapsulated fibroblasts or were free of cells. Hepatocytes cultured on nanopatterned substrates next to fibroblast carrying hydrogel microstructures were significantly more functional than hepatocytes cultured on nanopatterned surfaces without hydrogels or stromal cells significantly elevated albumin expression and cell junction formation compared to cells provided with topographic cues only. The simultaneous presentation of topographic biomechanical cues along with soluble signaling molecules provided by encapsulated fibroblasts cells resulted in optimal functionality of cultured hepatocytes. The provision of both topographic and soluble signaling cues could enhance our ability to create liver surrogates and inform the development of engineered liver constructs.

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.

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.

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 FROM AUTHOR]

Studies have shown that human umbilical cord mesenchymal stem cells (hUCMSCs) could ameliorate liver fibrosis (LF) through inhibiting the activation of hepatic stellate cells (HSCs). However, the specific mechanisms have not been studied clearly. The purpose of this study was to explore the possible mechanism of hepatocyte growth factor (HGF)-transfected hUCMSCs in inhibiting the proliferation and activation of HSCs-T6. The upper and lower double-cell coculture system was established among HGF-hUCMSCs, LV5-NC-hUCMSCs, hUCMSCs, and HSCs-T6 in experimental groups; HSCs-T6 were cultured alone as control group. After coculturing for 1, 2, and 3 days, results showed that HGF-transfected hUCMSCs could decrease cell viability of HSCs-T6 and promote apoptosis; inhibit their activation and reduce the expression of Collagen I, Collagen III, TGF-β1, Smad2 and Smad3, which may be related to inhibiting the activation of TGF-β1/Smads signaling pathway. These findings suggested that HGF-transfected hUCMSCs may be used as an alternative and novel therapeutic approach for the treatment of LF. [ABSTRACT FROM AUTHOR]

Prevascularization and mechanical stimulation have been reported as effective methods for the construction of functional bone tissue. However, their combined effects on osteogenic differentiation and its mechanism remain to be explored. Here, the effects of fluid shear stress (FSS) on osteogenic differentiation of rat bone-marrow-derived mesenchymal stem cells (BMSCs) when cocultured with human umbilical vein endothelial cells (HUVECs) were investigated, and underlying signaling mechanisms were further explored. FSS stimulation for 1-4 h/day increased alkaline phosphatase (ALP) activity and calcium deposition in coculture systems and promoted the proliferation of cocultured cells. FSS stimulation for 2 h/day was selected as the optimized protocol according to osteogenesis in the coculture. In this situation, the mRNA levels of ALP, runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and protein levels of OCN and osteopontin (OPN) in BMSCs were upregulated. Furthermore, FSS and coculture with HUVECs synergistically increased integrin ß1 expression in BMSCs and further activated focal adhesion kinases (FAKs) and downstream extracellular signalrelated kinase (ERK), leading to the enhancement of Runx2 expression. Blocking the phosphorylation of FAK abrogated FSS-induced ERK phosphorylation and inhibited osteogenesis of cocultured BMSCs. These results revealed that FSS and coculture with HUVECs synergistically promotes the osteogenesis of BMSCs, which was mediated by the integrin ß1-FAK-ERK signaling pathway. [ABSTRACT FROM AUTHOR]

Methane-oxidizing bacteria (methanotrophs) often coexist with methylotrophs that utilize methanol excreted by methanotrophs. Recently, we found that a facultative methylotroph, Methyloceanibacter caenitepidi Gela4T, possibly utilizes acetate rather than methanol in the coculture with a methanotroph, Methylocaldum marinum S8. Here, we examined the effects of oxygen concentrations on growth of and acetate excretion by M. marinum S8 in pure culture and the coculture with M. caenitepidi Gela4T. M. marinum S8 excreted acetate during the exponential growth phase not only under microaerobic (O2 concentrations of 3.5%-6%) but also under aerobic (O2 concentrations of 20%-31%) conditions. RNA-Seq analyses of M. marinum S8 cells grown under aerobic conditions suggested that phosphoketolase and acetate kinase were candidate genes involved in acetate production. Nonmethylotrophic bacteria, Cupriavidus necator NBRC 102504, could grow when cocultured with M. marinum S8, also supporting the existence of methanol-independent cross-feeding from M. marinum S8 under aerobic conditions. Acetate excretion by Methylocaldum marinum S8 and methanol-independent cross-feeding between strain S8 and Methyloceanibacter caenitepidi Gela4T occurred under both aerobic and microaerobic conditions. [ABSTRACT FROM AUTHOR]

[Display omitted] • Total acid content decreased significantly while esters increased with batches. • The ketone and terpene contents of first batch was enhanced by coculturing. • Volatiles were closely related to functional microbes in various fermentations modes. • Key volatiles were major lied on the microbial community and their metabolism. This study aimed to investigate the effect of repeated batch fermentation of cocultured Saccharomyces bayanus Y4 and Torulaspora delbrueckii Y7 on the quality of greengage wine. The acidity, as well as citric acid and L-malic acid content, decreased significantly with the batches. The citric acid content of the single culture in each batch was 58.59, 26.27, and 8.45 g/L, respectively. Additionally, the ester content increased with the batches. The respective values were 1627.93, 2249.46, and 2491.17 μg/L for the prophase of coculture. However, volatile content in the fresh wine of Y4 yeast in each batch was high. Ketone and terpene contents in the first batch of coculture significantly increased from 31.64 to 95.60 μg/L and 58.58–95.13 μg/L, respectively. Furthermore, a significant difference was observed between the microbial communities fermented using single culture and coculture. The change in fungal community diversity for coculture pattern was higher than that in the mash-inoculated single strain, which decreased the abundance of dominant fungi. Pichia , Stropharia , Suillus , Malassezia , Trichoderma , Faecalibacterium , and Butyricicoccus were associated with esters, terpenes, phenylethyl alcohol, and β-ionone. Our study provides a new avenue to improve the flavour and quality of wine brewed using a characteristic fruit. [ABSTRACT FROM AUTHOR]

Controlled wound healing requires a temporal and spatial coordination of cellular activities within the surrounding extracellular matrix (ECM). Disruption of cell–cell and cell–matrix communication results in defective repair, like chronic or fibrotic wounds. Activities of macrophages and fibroblasts crucially contribute to the fate of closing wounds. To investigate the influence of the ECM as an active part controlling cellular behavior, coculture models based on fibrillar 3D biopolymers such as collagen have already been successfully used. With well-defined biochemical and biophysical properties such 3D scaffolds enable in vitro studies on cellular processes including infiltration and differentiation in an in vivo like microenvironment. Further, paracrine and autocrine signaling as well as modulation of soluble mediator transport inside the ECM can be modeled using fibrillar 3D scaffolds. Herein, we review the usage of these scaffolds in in vitro coculture models allowing in-depth studies on the crosstalk between macrophages and fibroblasts during different stages of cutaneous wound healing. A more accurate mimicry of the various processes of cellular crosstalk at the different stages of wound healing will contribute to a better understanding of the impact of biochemical and biophysical environmental parameters and help to develop further strategies against diseases such as fibrosis. [ABSTRACT FROM AUTHOR]

Carbon loss in the form of CO 2 is an intrinsic and persistent challenge faced during conventional and advanced biofuel production from biomass feedstocks. Current mechanisms for increasing carbon conservation typically require the provision of reduced co-substrates as additional reducing equivalents. This need can be circumvented, however, by exploiting the natural heterogeneity of lignocellulosic sugars mixtures and strategically using specific fractions to drive complementary CO 2 emitting vs. CO 2 fixing pathways. As a demonstration of concept, a coculture-coproduction system was developed by pairing two catabolically orthogonal Escherichia coli strains; one converting glucose to ethanol (G2E) and the other xylose to succinate (X2S). 13C-labeling studies reveled that G2E + X2S cocultures were capable of recycling 24% of all evolved CO 2 and achieved a carbon conservation efficiency of 77%; significantly higher than the 64% achieved when all sugars are instead converted to just ethanol. In addition to CO 2 exchange, the latent exchange of pyruvate between strains was discovered, along with significant carbon rearrangement within X2S. [Display omitted] • Heterotrophic coculture-coproduction system developed for direct CO 2 -recycling. • Maximum carbon conversion efficiency of 77% achieved. • Reassimilated 24% of CO 2 generated during ethanol production into succinate. • Feedstock distribution for coproduction offers potential to decrease CO 2 emissions. [ABSTRACT FROM AUTHOR]

[Display omitted] • Host Paris polyphylla medium, and coculture led to seventeen novel metabolites. • Nine cryptic compounds were produced by the inductions of host medium and coculture. • The antiphytopathogenic activities of all compounds were investigated. • Antifungal metabolites showed the chemical interaction of the endophytes, and host. The synergism of host Paris polyphylla medium, the monoculture, and the coculture led to seventeen new metabolites, including eight sesquiterpenes, 1 – 7 having uncommon structural motifs compared to similar caryophyllene derivatives, 8 with an unprecedented bicyclic framework, and three xyloketals (13 – 15) with unprecedented frameworks from Nigrospora lacticolonia ; one polyketide, 17 with novel bicyclo [2.2.2] undecane skeleton, and five polyketide-terpenoid hybrids, 20 (one novel sulfated), 21 – 24 from Penicillium rubens. The structures were determined mainly by the NMR, HRESIMS, ECD calculation, and single-crystal X-ray diffraction. Nine cryptic compounds (2 – 4 , 5 , 12 – 15, 17) were produced by the inductions of host medium and the coculture. The compounds 13 from N. lacticolonia , 24 – 26 , 28 , 29 , and 31 from P. rubens indicated significant antiphytopathogenic activities against N. lacticolonia with MICs at 2–4 μg/mL. Moreover, compounds 22 – 26 , 28 , 29 , and 31 from P. rubens showed antifungal activities against P. rubens with MICs at 2–4 μg/mL. The synergistic effects of host medium and the coculture can induce the structural diversity of metabolites. [ABSTRACT FROM AUTHOR]

Crop cocultures (the growing of one crop with another plant or animal species as partner) are known to increase yield and other ecosystem services. However, whether and how partner species (i.e., the species grown together with the focal crop) affect coculture effects is poorly understood. Here, we test the effects of partner species using a meta-analysis. Two types of cocultures (i.e., crop-crop and rice-fish cocultures) with 19 cereals as focal crops were considered, and 136 plant or animal species were involved. We found that cocultures increased focal crop yield, the land equivalent ratio and soil organic matter (SOM), and decreased insect pest infestation. Farming practices (i.e., fertilization, pesticide use, and irrigation) did not alter these coculture effects on yield, but fertilization decreased the coculture effect on SOM, and pesticide use reduced the coculture effect on pest control. Without irrigation, cocultures increased yield under drought conditions compared to normal moisture conditions. Beneficial coculture effects were obtained with both plant and animal partner species and under both patterns of partner species grown with focal crops (species addition and species replacement). Functional types of partner plants (i.e., C 3 , C 4 , legume and non-legume plants) and taxonomic groups of partner animals (i.e., crabs, fish, shrimps, and turtles) affected coculture effects on yield, SOM, and insect pests. Both multiple functional trait distance and single functional trait distance between the focal crops and plant partners correlated with coculture effect on yield. The results suggest that biological types of partner species are important in obtaining beneficial effects of cocultures. • Global crop coculture effects and the role of partner species were evaluated. • Cocultures increase yield and soil organic matter, and decrease pest infestation. • Both plant and animal partner species increased yield of focal crop in cocultures. • Plant functional types and animal taxonomic groups affect outcome of coculture. • Functional trait dissimilarity between the cocultured crops benefit yield increase. [ABSTRACT FROM AUTHOR]

Pediococcus pentosaceus has a promising potential to improve the quality and flavor of Chinese steamed bread (CSB), but its synergistic effect with Saccharomyces cerevisiae is still unclear. In this study, P. pentosaceus was cocultured with three S. cerevisiae isolates separately, which had different leavening ability. The times required for the fermentation of dough by 2 times the volume of S. cerevisiae isolates ACX 0089, ACX 0490 and ACX 0078 were 46, 88 and 120 min, respectively. The results showed that the leavening ability of S. cerevisiae had a significant impact on the quality of CSBs. Compared other S. cerevisiae isolates, the ACX 0490 cocultured with P. pentosaceus increased the contents of bound water and free water in CSB, decreased the content of semibound water, and produced larger and regular porosity, thus endowed a greater specific volume and softer texture. A total of 36 volatile organic compounds (VOCs) were found in CSBs, with alcohols accounting for the main component. The coculture of P. pentosaceus with the intermediate leavening ability of S. cerevisiae ACX 0490 produced the highest proportion of esters among all CSBs. Moreover, S. cerevisiae ACX 0490 endowed the CSB unique VOCs, including cyclodecanol, ethylacetate, and 3,5-dimethylbenzaldehyde. The results of sensory evaluation also showed that S. cerevisiae ACX0490 cocultured CSB obtained the highest scores among all the test samples. The results provided a reference for selecting S. cerevisiae isolates with different leavening ability and the combination of P. pentosaceus to achieve the best quality and flavor of CSB. [Display omitted] • The quality and flavor of CSB cocultured by P. pentosaceus and S. cerevisiae with different leavening ability were compared. • The hardness of CSB cocultured by S. cerevisiae ACX0490 was the lowest. • The contents of volatile esters in CSBs cocultured by S. cerevisiae ACX 0490 were the highest. • CSB cocultured by S. cerevisiae ACX 0490 and P. pentosaceus had the best sensory quality. [ABSTRACT FROM AUTHOR]

Whey is a by-product of the dairy industry with considerable nutritional value but tends to be discarded with industrial effluents. One of the alternatives for its use is the production of whey cheeses, although they need to add extra ingredients to raise the yields, which elevates production costs. A less expensive alternative is using exopolysaccharides (EPS), carbohydrate polymers microorganisms produce. This research aimed to formulate and evaluate the stability of a Requeson-type cheese employing whey that has EPS produced biogenically by in situ fermentation with lactic acid bacteria. Two EPS-producing strains, Lactobacillus delbrueckii subsp bulgaricus NCFB 2772 and Streptococcus thermophilus SY-102, were used separately and in coculture (1:1 ratio). We found that coculture had the highest EPS production (1.49 mg/mL of whey) compared to the monocultures. This influenced the yield of the processed Requeson-type cheese (5.56%), which was higher than the produced with unfermented whey (3.77%). Similarly, the physicochemical and sensory parameters of the developed product showed differences. Furthermore, none of the Requeson-type cheeses made with fermented whey showed microbial growth after 21 days of storage (4 °C). Therefore, EPS is a potential strategy for producing Requeson-type whey cheeses with a high yield and stability. [ABSTRACT FROM AUTHOR]

Critical-sized bone defects can lead to significant morbidity, and interventions are limited by the availability and donor-site morbidity of bone grafts. Polymer scaffolds seeded with cells have been explored to replace bone grafts. Adipose-derived stem cells have shown great promise for vascularization and osteogenesis of these constructs, and cocultures of differentiated stem cells are being explored to augment vessel and bone formation. Adipose-derived stem cells were differentiated into endothelial cells and osteoblasts, and in vitro studies showed increased proliferation of cocultured cells compared with undifferentiated adipose-derived stem cells and monocultures of endothelial cells and osteoblasts. The cells were seeded into polylactic acid gas-plasma-treated scaffolds as cocultures and monocultures and then implanted into critical-sized rat calvarial defects. The cocultures were in a 1:1 osteoblast to endothelial cell ratio. The increase in proliferation seen by the cocultured cells in vitro did not translate to increased vascularization and osteogenesis in vivo. In vivo, there were trends of increased vascularization in the endothelial cell group and increased osteogenesis in the osteoblast and endothelial monoculture groups, but no increase was seen in the coculture group compared with the undifferentiated adipose-derived stem cells. Endothelial cells enhance vascularization and osteoblast and endothelial cell monocultures enhance bone formation in the polymer scaffold. Predifferentiation of adipose-derived stem cells is promising for improving vascularization and osteogenesis in polymer scaffolds but requires future evaluation of coculture ratios to fully characterize this response.

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.

Abstract Background Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. Results A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. Conclusions The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

Chemical investigation of a coculture of the marine-derived fungi Beauveria felina KMM 4639 and Aspergillus carneus KMM 4638 led to the identification of three new drimane-type sesquiterpenes, asperflavinoids B, D and E (2, 4, 5), and nine previously reported related compounds. The structures of these compounds were established using spectroscopic methods and by comparison with known analogues. We also investigated the cytotoxic activity of the isolated compounds against several cancer and normal cell lines. Asperflavinoid C (3) and ustusolate E (9) exerted a significant effect on human breast cancer MCF-7 cell viability, with IC50 values of 10 µM, and induced in caspase-dependent apoptosis and arrest of the MCF-7 cell cycle in the G2/M phase in these cells.

Leishmania parasites present astonishing adaptative abilities that represent a matter of life or death within disparate environments during the heteroxenous parasite life cycle. From an evolutionary perspective, organisms develop methods of overcoming such challenges. Strategies that extend beyond the genetic diversity have been discussed and include variability between parasite cells during the infections of their hosts. The occurrence of Leishmania subpopulation fluctuations with variable structural genomic contents demonstrates that a single strain might shelter the variability required to overcome inconsistent environments. Such intrastrain variability provides parasites with an extraordinary ability to adapt and thus survive and propagate. However, different perspectives on this evolution have been proposed. Strains or species living in the same environment can cooperate but also compete. These interactions might increase the replication rate of some parasites but cause the loss of more aggressive competitors for others. Adaptive responses to intra- and interspecific competition can evolve as a fixed strategy (replication is adapted to the average genetic complexity of infections) or an optional strategy (replication varies according to the genetic complexity of the current infection). This review highlights the complexity of interspecies and intrastrain interactions among Leishmania parasites as well as the different factors that influence this interplay.

Background: Baicalin (BCL), a candidate drug for ischemic stroke, has been indicated to protect neurons by promoting brain-derived neurotrophic factor (BDNF). However, the cellular source of BDNF release promoted by baicalin and its detailed protective mechanism after ischemia/reperfusion remains to be studied. The aim of this study was to investigate the neuroprotective mechanisms of baicalin against oxygen–glucose deprivation/reoxygenation (OGD/R) in a neuron–astrocyte coculture system and to explore whether the BDNF-TrkB pathway is involved.Methods and Results: A neuron–astrocyte coculture system was established to elucidate the role of astrocytes in neurons under OGD/R conditions. The results demonstrated that astrocytes became reactive astrocytes and released more BDNF in the coculture system to attenuate neuronal apoptosis and injury after OGD/R. BCL maintained the characteristics of reactive astrocytes and obviously increased the expression of cyclic AMP response element-binding protein (CREB) and the levels of BDNF in the coculture system after OGD/R. To further verify whether BDNF binding to its receptor tyrosine kinase receptor B (TrkB) was required for the neuroprotective effect of baicalin, we examined the effect of ANA-12, an antagonist of TrkB, on NA system injury, including oxidative stress, inflammation, and apoptosis induced by OGD/R. The results showed that treatment of NA systems with ANA-12 significantly attenuated the neuroprotection of BCL. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways are two important downstream cascades of signaling pathways activated by BDNF binding to TrkB. We investigated the expressions of TrkB, PI3K, Akt, MAPK, and ERK. The results demonstrated that baicalin significantly increased the expressions of TrkB, PI3K/AKT, and MAPK/ERK.Conclusion: The neuroprotective effects of baicalin against oxidative stress, inflammation, and apoptosis were improved by astrocytes, mainly mediated by increasing the release of BDNF and its associated receptor TrkB and downstream signaling regulators PI3K/Akt and MAPK/ERK1/2.

Despite studies reporting functional differentiation of liver cells, a three‐dimensional, vascularized liver organ has yet to be developed from mesenchymal stem cells. We investigated whether treatment with photobiomodulation (PBM) before three‐dimensional liver spheroid transplantation improved the recovery of liver function via stimulation of angiogenesis and hepatocyte differentiation. Liver spheroids composed of hepatic, endothelial, and mesenchymal cells were subjected to PBM therapy. To evaluate the in vivo therapeutic effect of the liver spheroids treated with PBM, phosphate‐buffered saline, liver spheroid, and PBM‐treated liver spheroid were transplanted into a damaged host liver using conventional chimeric mouse models. To further characterize the maturation of transplanted PBM‐liver spheroid compared with the newly generated non‐PBM‐liver spheroid or human liver tissues, the expression profiles of mature liver signature genes were analyzed. Liver spheroids expressed hepatocyte growth factors, including vascular endothelial growth factor and angiogenic factors. The cells in liver spheroid compensated for the low viability and improved the function of hepatocytes. Here, we demonstrate the formation of vascularized and functional human liver spheroid from human adipose‐derived stem cells by transplantation of liver tissue created in vitro. Albumin secretion by PBM‐treated liver spheroid was higher on Day 28 compared with liver spheroid‐seeded transplant group. PBM‐liver spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside the implanted tissue constructs. The vasculature in the liver spheroid transplants became functional by connecting to the host vessels within 48 h. These PBM‐liver spheroids may be useful in designing artificial three‐dimensional hepatic tissue constructs and in cell therapy with limited numbers of human hepatocytes. [ABSTRACT FROM AUTHOR]

In this study, the microbial interactions among cocultures of Streptococcus thermophilus (St) with potential probiotics of Bifidobacterium animalis ssp. lactis (Ba) and Lactiplantibacillus plantarum (Lp) in fermented milk were investigated during a storage period of 21 d at 4°C, in terms of acidifying activity (pH and titratable acidity), viable counts, and metabolites. A nontargeted metabolomics approach based on ultra-high-performance liquid chromatography coupled with mass spectrometry was employed for mapping the global metabolite profiles of fermented milk. Probiotic strains cocultured with St accelerated milk acidification, and improved the microbial viability compared with the single culture of St. The St–Ba/Lp treatment manifested a higher bacteria viability and acidification ability in comparison with the St–Ba or the St–Lp treatment. Relative quantitation of 179 significant metabolites was identified, including nucleosides, AA, short peptides, organic acids, lipid derivatives, carbohydrates, carbonyl compounds, and compounds related to energy metabolism. The principal component analysis indicated that St treatment and coculture treatments displayed a complete distinction in metabolite profiles, and Lp had a larger effect than Ba on metabolic profiles of fermented milk produced by cofermentation with St during storage. The heat map in combination with hierarchical cluster analysis showed that the abundance of metabolites significantly varied with the starter cultures over the storage, and high abundance of metabolites was observed in either St or coculture samples. The St-Ba/Lp treatment showed relatively high abundance for the vast majority of metabolites. These findings suggest that the profile of the metabolites characterizing fermented milk samples may depend on the starter cultures, and incorporation of probiotics may considerably influence the metabolomic activities of fermented milks. [ABSTRACT FROM AUTHOR]

Synthetic microbial communities have the potential to enable new platforms for bioproduction of biofuels and biopharmaceuticals. However, using engineered communities is often assumed to be difficult because of anticipated challenges in establishing and controlling community composition. Cross‐feeding between microbial auxotrophs has the potential to facilitate coculture growth and stability through a mutualistic ecological interaction. We assessed cross‐feeding between 13 Escherichia coli amino acid auxotrophs paired with a leucine auxotroph of Bacillus megaterium. We developed a minimal medium capable of supporting the growth of both bacteria and used the media to study coculture growth of the 13 interspecies pairs of auxotrophs in batch and continuous culture, as well as on semi‐solid media. In batch culture, 8 of 13 pairs of auxotrophs were observed to grow in coculture. We developed a new metric to quantify the impact of cross‐feeding on coculture growth. Six pairs also showed long‐term stability in continuous culture, where coculture growth at different dilution rates highlighted differences in cross‐feeding amongst the pairs. Finally, we found that cross‐feeding‐dependent growth on semi‐solid media is highly stringent and enables identification of the most efficient pairs. These results demonstrate that cross‐feeding is a viable approach for controlling community composition within diverse synthetic communities. [ABSTRACT FROM AUTHOR]

This study investigated the chondrogenic activity of encapsulated mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) and its impact on the mechanical properties of injectable poly(N‐isopropylacrylamide)‐based dual‐network hydrogels loaded with poly(l‐lysine) (PLL). To this effect, an ex vivo study model was employed to assess the behavior of the injected hydrogels—specifically, their surface stiffness and integration strength with the surrounding cartilage. The highest chondrogenic activity was observed from AC‐encapsulated hydrogels, while the effect of PLL on MSC chondrogenesis was not apparent from biochemical analyses. Mechanical testing showed that there were no significant differences in either surface stiffness or integration strength among the different study groups. Altogether, the results suggest that the ex vivo model can allow further understanding of the relationship between biochemical changes within the hydrogel and their impact on the hydrogel's mechanical properties. [ABSTRACT FROM AUTHOR]

In order to explore the abilities of an integrated three‐dimensional micro‐nano topography in immunomodulation and promoting bone formation, present study focuses on the titanium sheets used in the micro‐nano topography by treating them with the sandblasted, large‐grit and acid‐etched (SLA)and alkaline thermal reaction. Further, we characterized and obtained the surface morphology, roughness, and hydrophilicity of the titanium sheets. Moreover, we detected their in vitro cytocompatibility and cell proliferation as well. In addition, investigation was carried out for the immunomodulatory ability of the titanium sheets in a micro‐nano topography by observing the expression of M1 (classical activated macrophage) and M2 (alternatively activated macrophage) type marker factors, inflammatory factors, and morphological changes of RAW264.7 cells cultured on the titanium sheets in different topographies. Through cell migration experiments and coculture, we observed the effects of different titanium sheet immune environments on osteoblast migration, extracellular matrix mineralization, and osteoblast gene expression. These results showed that the micro‐nano topography constructed through SLA and alkaline thermal treatment improved the hydrophilicity and promoted the cell proliferation. Moreover, it promoted RAW264.7 cells to polarize as M2 phenotype, thereby leading to the anti‐inflammatory effect of local microenvironments. This facilitated osteoblasts to secrete bone morphogenetic protein‐2 (BMP2) and vascular endothelial growth factor. Nonetheless, these findings provided a theoretical basis for the molecular biological mechanism related to implants in a micro‐nano topography which promoted the osteointegration while offering a meaningful theoretical basis for the clinical treatment of such implants. [ABSTRACT FROM AUTHOR]

Background: Baicalin (BCL), a candidate drug for ischemic stroke, has been indicated to protect neurons by promoting brain-derived neurotrophic factor (BDNF). However, the cellular source of BDNF release promoted by baicalin and its detailed protective mechanism after ischemia/reperfusion remains to be studied. The aim of this study was to investigate the neuroprotective mechanisms of baicalin against oxygen–glucose deprivation/reoxygenation (OGD/R) in a neuron–astrocyte coculture system and to explore whether the BDNF-TrkB pathway is involved. Methods and Results: A neuron–astrocyte coculture system was established to elucidate the role of astrocytes in neurons under OGD/R conditions. The results demonstrated that astrocytes became reactive astrocytes and released more BDNF in the coculture system to attenuate neuronal apoptosis and injury after OGD/R. BCL maintained the characteristics of reactive astrocytes and obviously increased the expression of cyclic AMP response element-binding protein (CREB) and the levels of BDNF in the coculture system after OGD/R. To further verify whether BDNF binding to its receptor tyrosine kinase receptor B (TrkB) was required for the neuroprotective effect of baicalin, we examined the effect of ANA-12, an antagonist of TrkB, on NA system injury, including oxidative stress, inflammation, and apoptosis induced by OGD/R. The results showed that treatment of NA systems with ANA-12 significantly attenuated the neuroprotection of BCL. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways are two important downstream cascades of signaling pathways activated by BDNF binding to TrkB. We investigated the expressions of TrkB, PI3K, Akt, MAPK, and ERK. The results demonstrated that baicalin significantly increased the expressions of TrkB, PI3K/AKT, and MAPK/ERK. Conclusion: The neuroprotective effects of baicalin against oxidative stress, inflammation, and apoptosis were improved by astrocytes, mainly mediated by increasing the release of BDNF and its associated receptor TrkB and downstream signaling regulators PI3K/Akt and MAPK/ERK1/2. [ABSTRACT FROM AUTHOR]

Among 39 pentose-utilizing lactic acid bacteria (LAB) selected from acid-forming bacteria from the midgut of Eri silkworm, the isolate WX1 was selected with the highest capability to produce optically pure L-lactic acid (L-LA) from glucose, xylose and arabinose with furfural-tolerant properties. The isolate WX1 was identified as Enterococcus mundtii based on 16S rDNA sequence analysis. The conversion yields of L-LA from glucose and xylose by E. mundtii WX1 were 0.97 and 0.68 g/g substrate, respectively. Furthermore, L-LA production by E. mundtii WX1 in various glucose-xylose mixtures indicated glucose repression effect on xylose consumption. The coculture of E. mundtii WX1 and Lactobacillus rhamnosus SCJ9, a homofermentative LAB capable of producing L-LA from glucose clearly showed an improvement of L-LA production from 30 g/L total glucose-xylose (6:4). The results from Plackett–Burman design (PBD) indicated that Tween 80, MnSO4 and yeast extract (YE) were three medium components that significantly influenced (p < 0.05) L-LA production using the coculture strategy in the presence of 2 g/L furfural. Optimal concentrations of these variables revealed by central composite design (CCD) and response surface methodology (RSM) were 20.61 g/L YE, 1.44 g/L Tween 80 and 1.27 g/L MnSO4 . Based on the optimized medium with 30 g/L total glucose-xylose (6:4), the maximum experimental L-LA value of 23.59 g/L reflecting 0.76 g/g substrate were achieved from 48 h fermentation at 37 ◦C. L-LA produced by coculture cultivated under standard MRS medium and new optimized conditions were 1.28 and 1.53 times higher than that obtained from single culture by E. mundtii WX1, respectively. This study provides the foundations for practical applications of coculture in bioconversion of lignocellulose particularly glucose-xylose-rich corn stover to L-LA. [ABSTRACT FROM AUTHOR]

Macrofungi, which are also known as mushrooms, can produce various bioactive constituents and have become promising resources as lead drugs and foods rich in nutritional value. However, the production of these bioactive constituents under standard laboratory conditions is inefficiency due to the silent expression of their relevant genes. Coculture, as an important activation strategy that simulates the natural living conditions of macrofungi, can activate silent genes or clusters through interspecific interactions. Coculturing not only can trigger the biosynthesis of diverse secondary metabolites and enzymes of macrofungi, but is also useful for uncovering the mechanisms of fungal interspecific interactions and novel gene functions. In this paper, coculturing among macrofungi or between macrofungi and other microorganisms, the triggering and upregulation of secondary metabolites and enzymes, the potential medicinal applications, and the fungal–fungal interaction mechanisms are reviewed. Finally, future challenges and perspectives in further advancing coculture systems are discussed.

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.

ObjectiveTo determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns.DesignIn vitro study.SettingUniversity research laboratory.Patient(s)Endometrial biopsies were obtained from premenopausal women.Intervention(s)Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls.Main outcome measure(s)Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures.Result(s)Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion.Conclusion(s)This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro.

7.87 to 10.5 eV vacuum ultraviolet (VUV) photon energies were used in laser desorptionpostionization mass spectrometry (LDPI-MS) to analyze biofilms comprised of binary cultures of interacting microorganisms. The effect of photon energy was examined using both tunable synchrotron and laser sources of VUV radiation. Principal components analysis (PCA) was applied to the MS data to differentiate species in Escherichia coli-Saccharomyces cerevisiae coculture biofilms. PCA of LDPI-MS also differentiated individual E. coli strains in a biofilm comprised of two interacting gene deletion strains, even though these strains differed from the wild type K-12 strain by no more than four gene deletions each out of approximately 2000 genes. PCA treatment of 7.87 eV LDPI-MS data separated the E. coli strains into three distinct groups two ?pure? groups and a mixed region. Furthermore, the ?pure? regions of the E. coli cocultures showed greater variance by PCA when analyzed by 7.87 eV photon energies than by 10.5 eV radiation. Comparison of the 7.87 and 10.5 eV data is consistent with the expectation that the lower photon energy selects a subset of low ionization energy analytes while 10.5 eV is more inclusive, detecting a wider range of analytes. These two VUV photon energies therefore give different spreads via PCA and their respective use in LDPI-MS constitute an additional experimental parameter to differentiate strains and species.