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\"\"Examines the area known as the Big Quiet, the role of environment, the history of this area in the West, and the power of Western mythology\"--Provided by publisher.

Background Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. Results We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. Conclusions The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Cartilage and bone defects as well as osteoarthritis are prevalent worldwide, affecting individuals across all age groups, from young, active populations to older adults. The standard protocol in cartilage regeneration involves knee replacement surgery through the implantation of an endoprosthesis. Current clinical protocols involving cell-based therapies are associated with limitations, including the lack of functional cartilage-like tissue and dedifferentiation of chondrocyte, particularly during monoculture. Similarly, in bone regeneration, the “gold standard” is the use of bone auto- or allografts, which are associated with immunological rejection, inadequate vascularization, and limited osteogenesis. To overcome these limitations, various co-culture techniques have been introduced as promising strategies for cartilage and bone tissue regeneration. These systems aim to mimic native microenvironments by promoting interactions between chondrocytes and mesenchymal stromal cells (MSCs) in cartilage repair and between osteogenic and angiogenic cells in bone regeneration. This paper introduces different co-culture systems focusing on in vitro crosstalk between MSCs derived from various sources and other somatic cell populations in cartilage and bone regeneration.

Microfluidics is applied in biotechnology research via the creation of microfluidic channels and reaction vessels. Filters are considered to be able to simulate microfluidics. A typical example is the cell culture insert, which comprises two vessels connected by a filter. Cell culture inserts have been used for years to study cell-to-cell communication. These systems generally have a bucket-in-bucket structure and are hereafter referred to as a vertical-type co-culture plate (VTCP). However, VTCPs have several disadvantages, such as the inability to simultaneously observe samples in both containers and the inability of cell-to-cell communication through the filters at high cell densities. In this study, we developed a novel horizontal-type co-culture plate (HTCP) to overcome these disadvantages and confirm its performance. In addition, we clarified the migration characteristics of substances secreted from cells in horizontal co-culture vessels. It is generally assumed that less material is exchanged between the horizontal vessels. However, the extracellular vesicle (EV) transfer was found to be twice as high when using HTCP. Other merits include control of the degree of co-culture via the placement of cells. We believe that this novel HTCP container will facilitate research on cell-to-cell communication in various fields.

Cell culture is a widely used tool for improving our understanding of cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production and the development of tissue engineering. Most research regarding cancer biology is based on experiments using two-dimensional (2D) cell cultures . However, 2D cultures have many limitations, such as the disturbance of interactions between the cellular and extracellular environments, changes in cell morphology, polarity, and method of division. These disadvantages led to the creation of models which are more closely able to mimic conditions . One such method is three-dimensional culture (3D). Optimisation of the culture conditions may allow for a better understanding of cancer biology and facilitate the study of biomarkers and targeting therapies. In this review, we compare 2D and 3D cultures as well as different versions of 3D cultures.

Organoids have certain cellular composition and physiological features in common with real organs, making them promising models of organ formation, function, and diseases. However, Matrigel, the commonly used animal‐derived matrices in which they are developed, has limitations in mechanical adjustability and providing complex physicochemical signals. Here, the incorporation of Ti3C2TxMXene nanomaterial into Matrigel regulates the properties of Matrigel and exhibits satisfactory biocompatibility. The Ti3C2TxMXene Matrigel composites (MXene‐Matrigel) regulate the development of Cochlear Organoids (Cochlea‐Orgs), particularly in promoting the formation and maturation of organoid hair cells. Additionally, regenerated hair cells in MXene‐Matrigel are functional and exhibit better electrophysiological properties compared to hair cells in Matrigel. MXene‐Matrigel potentiates the amycin (mTOR) signaling pathway to promote hair cell differentiation, and mTOR signaling inhibition restrains hair cell differentiation. Moreover, MXene‐Matrigel facilitates innervation establishment between regenerated hair cells and spiral ganglion neurons (SGNs) growing from the Cochlea modiolus in a co‐culture system, as well as promotes synapse formation efficiency. The approach overcomes some limitations of the Matrigel‐dependent culture system and greatly accelerates the application of nanomaterials in organoid development and research on therapies for hearing loss. 3D Ti3C2TxMXene composited Matrigel (MXene‐Matrigel) has biocompatibility to Cochlear Organoids (Cochlea‐Orgs) and potentiates rapamycin activity to promote differentiation and maturation of Cochlea‐Orgs. Regenerated organoid hair cells are functional and comparable to postnatal day 2 native auditory hair cells. Ti3C2TxMXene also benefits the formation of synapse‐like contacts between regenerated hair cells and sensory neurons.

Bone drug parathyroid hormone (PTH or teriparatide) has dual functions: it is a widely used anabolic drug resulting in increased bone formation, but it also activates osteoclasts. To further investigate the mechanistic insight of dose, timing, and delivery mode, a suitable cell model, periodontal ligament fibroblast (PDLF), was used to study PTH’s role in osteoclastogenesis and osteogenesis. PDLF were either co-cultured with peripheral blood mononuclear cells (PBMC) to study osteoclastogenesis or cultured under osteogenic conditions to study osteogenesis. A PTH titration (10 –12 to 10 –9  M) for 3 weeks identified the optimal PTH dose. Optimal PTH (10 –9  M) dose was then administered under six regimens: continuous, first week only, second week only, third week only, and intermittent. At day 21, outcomes included osteoclast number and multinuclearity, gene expression (qPCR) for osteoclastogenesis and alkaline phosphatase (ALP) activity, and Alizarin Red staining for osteogenesis. PTH enhanced osteoclast formation in a dose-dependent manner (p = 0.008), with 10 –9  M being most effective. Timing was critical: first- and second-week exposure significantly increased osteoclast number and size, while continuous or late exposure had minimal effect. Larger osteoclasts, with >5 nuclei per osteoclast, were absent in the controls and exclusively present in all PTH-treated conditions. M-CSF and DC-STAMP expression aligned with osteoclastogenesis. Compared to t = 0 (without PBMC), the RANKL/OPG ratio increased in the second-week exposure group and intermittent groups. IL-1β was upregulated, strongest in the second-week exposure group (p < 0.01). Osteogenic effects were modest and donor-dependent; ALP activity was comparable across groups, though a significant decrease was found between the second week and intermittent regimens. Mineralization varied strongly between donors, with responders and non-responders to PTH treatment. PTH exerts time-dependent effects on osteoclastogenesis in human co-cultures, particularly during early phases. Osteogenic responses varied, suggesting a complex interplay of timing, cell context, and donor variability. These findings support further investigation into optimized timing and delivery of PTH in periodontal regeneration.

Mixed microbial communities exhibit emergent biochemical properties not found in clonal monocultures. We report a new type of synthetic genetic interaction, synthetic mutualism in trans (SMIT), in which certain pairs of auxotrophic Escherichia coli mutants complement one another's growth by cross‐feeding essential metabolites. We find significant metabolic synergy in 17% of 1035 such pairs tested, with SMIT partners identified throughout the metabolic network. Cooperative phenotypes show more growth on average by aiding the proliferation of their conjugate partner, thereby expanding the source of their own essential metabolites. We construct a quantitative, predictive, framework for describing SMIT interactions as governed by stoichiometric models of the metabolic networks of the interacting strains.

Gamma-aminobutyric acid (GABA) is widely distributed in nature and considered a potent bioactive compound with numerous and important physiological functions, such as anti-hypertensive and antidepressant activities. There is an ever-growing demand for GABA production in recent years. Lactic acid bacteria (LAB) are one of the most important GABA producers because of their food-grade nature and potential of producing GABA-rich functional foods directly. In this paper, the GABA-producing LAB species, the biosynthesis pathway of GABA by LAB, and the research progress of glutamate decarboxylase (GAD), the key enzyme of GABA biosynthesis, were reviewed. Furthermore, GABA production enhancement strategies are reviewed, from optimization of culture conditions and genetic engineering to physiology-oriented engineering approaches and co-culture methods. The advances in both the molecular mechanisms of GABA biosynthesis and the technologies of synthetic biology and genetic engineering will promote GABA production of LAB to meet people’s demand for GABA. The aim of the review is to provide an insight of microbial engineering for improved production of GABA by LAB in the future.

Here we present a microfluidic model that allows for co-culture of human osteoblasts, chondrocytes, fibroblasts, and macrophages of both quiescent (M0) and pro-inflammatory (M1) phenotypes, maintaining initial viability of each cell type at 24 h of co-culture. We established healthy (M0-based) and diseased (M1-based) joint models within this system. An established disease model based on supplementation of IFN-γ and lipopolysaccharide in cell culture media was used to induce an M1 phenotype in macrophages to recapitulate inflammatory conditions found in Osteoarthritis. Cell viability was assessed using NucBlue™ Live and NucGreen™ Dead fluorescent stains, with mean viability of 83.9% ± 14% and 83.3% ± 12% for healthy and diseased models, respectively, compared with 93.3% ± 4% for cell in standard monoculture conditions. Cytotoxicity was assessed via a lactate dehydrogenase (LDH) assay and showed no measurable increase in lactate dehydrogenase release into the culture medium under co-culture conditions, indicating that neither model promotes a loss of cell membrane integrity due to cytotoxic effects. Cellular metabolic activity was assessed using a PrestoBlue™ assay and indicated increased cellular metabolic activity in co-culture, with levels 5.9 ± 3.2 times mean monolayer cell metabolic activity levels in the healthy joint model and 5.3 ± 3.4 times mean monolayer levels in the diseased model. Overall, these findings indicate that the multi-tissue nature of in vivo human joint conditions can be recapitulated by our microfluidic co-culture system at 24 h and thus this model serves as a promising tool for studying the pathophysiology of rheumatic diseases and testing potential therapeutics.