Co-culture Growth Research Articles

The Staphylococcus aureus-antagonizing human nasal commensal Staphylococcus lugdunensis depends on siderophore piracy

BackgroundBacterial pathogens such as Staphylococcus aureus colonize body surfaces of part of the human population, which represents a critical risk factor for skin disorders and invasive infections. However, such pathogens do not belong to the human core microbiomes. Beneficial commensal bacteria can often prevent the invasion and persistence of such pathogens by using molecular strategies that are only superficially understood. We recently reported that the commensal bacterium Staphylococcus lugdunensis produces the novel antibiotic lugdunin, which eradicates S. aureus from the nasal microbiomes of hospitalized patients. However, it has remained unclear if S. lugdunensis may affect S. aureus carriage in the general population and which external factors might promote S. lugdunensis carriage to enhance its S. aureus-eliminating capacity.ResultsWe could cultivate S. lugdunensis from the noses of 6.3% of healthy human volunteers. In addition, S. lugdunensis DNA could be identified in metagenomes of many culture-negative nasal samples indicating that cultivation success depends on a specific bacterial threshold density. Healthy S. lugdunensis carriers had a 5.2-fold lower propensity to be colonized by S. aureus indicating that lugdunin can eliminate S. aureus also in healthy humans. S. lugdunensis-positive microbiomes were dominated by either Staphylococcus epidermidis, Corynebacterium species, or Dolosigranulum pigrum. These and further bacterial commensals, whose abundance was positively associated with S. lugdunensis, promoted S. lugdunensis growth in co-culture. Such mutualistic interactions depended on the production of iron-scavenging siderophores by supportive commensals and on the capacity of S. lugdunensis to import siderophores.EJt4aqYa_PFzdC4ihACHg3Video ConclusionsThese findings underscore the importance of microbiome homeostasis for eliminating pathogen colonization. Elucidating mechanisms that drive microbiome interactions will become crucial for microbiome-precision editing approaches.

Impact of acidic and alkaline conditions on Staphylococcus aureus and Acinetobacter baumannii interactions and their biofilms.

Bacterial biofilms pose significant challenges due to their association with antibiotic resistance, metabolic adaptation, and survival under harsh conditions. Among notable pathogens forming biofilms, Staphylococcus aureus and Acinetobacter baumannii are concerning pathogens in nosocomial settings. However, their behaviour under acidic (pH 4.5) and alkaline (pH10.5) conditions, especially in co-culture setups, remains insufficiently understood. This study investigates these aspects, by examining growth rates, biofilm formation, pH shifts, phenotypic analysis, and gene expression profiles. The results showed A. baumannii exhibited reduced growth and biofilm formation at pH 4.5, while S. aureus showed slow growth and low biofilm formation at pH10.5 in mono-cultures. S. aureus leaned towards an acidic pH (6-6.5), whereas A. baumannii shifted towards an alkaline pH (8-9). In co-culture environments, growth rates and biofilm formation increased across all pH conditions, converging towards a neutral pH over time. Phenotypic motility assays indicated that A. baumannii exhibited greater motility in alkaline conditions, while S. aureus showed increased staphyloxanthin production under acidic conditions. Gene expression analyses revealed that the fibronectin-binding protein A (FnbA) and N-acetylglucosaminyl-transferase (icaA) genes, responsible for initial attachment during biofilm formation, were highly expressed in acidic co-culture condition but poorly expressed in alkaline condition. In A. baumannii, the outer membrane protein A (OmpA) gene associated with adhesion and virulence, was upregulated in co-culture. TheLuxR gene involved in quorum sensing was upregulated in acidic conditions and poorly expressed at pH 10.5. This study elucidates the metabolic adaptability and biofilm formation tendencies of S. aureus towards acidic conditions and A. baumannii towards alkaline conditions, providing insights for better management of biofilm-related infections.

Identification of Yarrowia lipolytica as a platform for designed consortia that incorporate in situ nitrogen fixation to enable ammonia-free bioconversion

Bioconversion processes require nitrogen for growth and production of intracellular enzymes to produce biofuels and bioproducts. Typically, this is supplied as reduced nitrogen in the form of ammonia, which is produced offsite from N2 and H2 via the Haber-Bosch process. While this has revolutionized industries dependent on fixed nitrogen (e.g., modern agriculture), it is highly energy-intensive and its reliance on natural gas combustion results in substantial global CO2 emissions. Here we investigated the feasibility of in situ biological nitrogen fixation from N2 gas as a strategy to reduce greenhouse gas impacts of aerobic bioconversion processes. We developed an efficient and cost-effective method to screen fungal bioconversion hosts for compatibility with the free-living diazotrophic bacterium Azotobacter vinelandii under nitrogen fixing conditions. Our screening revealed that the genus Yarrowia is particularly enriched during co-culture experiments. Follow-up experiments identified four Y. lipolytica strains (NRRL Y-11853, NRRL Y-7208, NRRL Y-7317, and NRRL YB-618) capable of growth in co-culture with A. vinelandii. These strains utilize ammonium secreted during diazotrophic fixation of N2, which is provided as a component of the air input stream during aerobic fermentation. This demonstrates the feasibly of in situ biological nitrogen fixation to support heterotrophic fermentation processes for production of fuels and chemicals.

The Antilisterial Effect of Latilactobacillus sakei CTC494 in Relation to Dry Fermented Sausage Ingredients and Temperature in Meat Simulation Media

Listeria monocytogenes, the causative agent of listeriosis, is a relevant pathogen in dry fermented sausages (DFSs), and the application of antilisterial starter cultures is an effective intervention strategy to control the pathogen during DFS production. The effect of factors in relation to DFS formulation and production, NaCl (0–40 g/L), Mn (0.08–0.32 g/L), glucose (0–40 g/L) and temperature (3–37 °C), on the behaviour of L. monocytogenes when cocultured with Latilactobacillus sakei 23K (non-bacteriocinogenic) and CTC494 (bacteriocinogenic) strains was studied through a central composite design in meat simulation media. L. sakei and L. monocytogenes counts, pH, lactic acid production and bacteriocin activity were determined in mono and coculture. The pH decrease and lactic acid production were highly influenced by glucose, while production of sakacin K by L. sakei CTC494 was observed at moderate (10 and 20 °C), but not at the lowest (3 °C) and highest (37 °C), temperatures. Coculture growth had no effect on the acidification and bacteriocin production but inhibited and inactivated L. monocytogenes when L. sakei 23K entered the early stationary phase and when L. sakei CTC494 produced sakacin K. Optimal conditions for achieving a 5-log units reduction of L. monocytogenes were at 20 °C, 20 g/L of NaCl, 0.20 g/L of Mn and 40 g/L of glucose, those highlighting the importance of considering product formulation and fermentation conditions for bioprotective starter cultures application.

Optogenetic Control of Bacterial Cell-Cell Adhesion Dynamics: Unraveling the Influence on Biofilm Architecture and Functionality.

The transition of bacteria from an individualistic to a biofilm lifestyle profoundly alters their biology. During biofilm development, the bacterial cell-cell adhesions are a major determinant of initial microcolonies, which serve as kernels for the subsequent microscopic and mesoscopic structure of the biofilm, and determine the resulting functionality. In this study, the significance of bacterial cell-cell adhesion dynamics on bacterial aggregation and biofilm maturation is elucidated. Using photoswitchable adhesins between bacteria, modifying the dynamics of bacterial cell-cell adhesions with periodic dark-light cycles is systematic. Dynamic cell-cell adhesions with liquid-like behavior improve bacterial aggregation and produce more compact microcolonies than static adhesions with solid-like behavior in both experiments and individual-based simulations. Consequently, dynamic cell-cell adhesions give rise to earlier quorum sensing activation, better intermixing of different bacterial populations, improved biofilm maturation, changes in the growth of cocultures, and higher yields in fermentation. The here presented approach of tuning bacterial cell-cell adhesion dynamics opens the door for regulating the structure and function of biofilms and cocultures with potential biotechnological applications.

Abstract LB307: The role of tumor microenvironment derived growth factors in pediatric brain tumors

Abstract Introduction: Children with H3K27-altered diffuse midline gliomas (DMG-H3K27a), have a 5-year survival rate of only 2% following diagnosis. While in-depth genomic characterization has been performed in DMG-H3K27a, very few druggable targets have been identified. A common feature of DMG-H3K27a is infiltration of microglia, macrophages, other myeloid cells, collectively referred to as GAMs. Cellular crosstalk between non-tumor and tumor cells in the tumor microenvironment (TME) can both promote and or inhibit tumor growth, thus representing an opportunity in the pursuit of novel therapeutics. We have recently determined that DMG-H3K27a tumor cells stimulate microglial secretion of pro-tumor growth factors. Hypothesis: Microglial-derived growth factors activate receptor tyrosine kinases (RTKs) via paracrine signaling in DMG-H3K27a. Methods: We used scRNA-seq and RNA-seq datasets and, in parallel, in vitro studies, such as co-culture studies, ELISA assays, western blotting, and growth assays to test our hypothesis. In addition, we are developing a mouse-human chimeric neutralizing antibodies of our top targets for in vivo studies. Preliminary Results: Microglial cells secrete growth factors into the TME, which promotes DMG-H3K27a cell proliferation and growth. Targeting growth factors with neutralizing antibodies attenuates glioma cell proliferation in co-culture studies. Conclusion: This research uncovers an important signaling network between GAMs and DMG-H3K27a that can be therapeutically targeted. Citation Format: Andrea Cruz, Brenden Johnson, Taylor Gatesman, Mathew Halbert, Suchet Taori, Baoli Hu, Gary Kohanbash, Ian F. Pollack, Sameer Agnihotri. The role of tumor microenvironment derived growth factors in pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB307.

Metformin Treatment of Macrophages Increases Microvessel Growth in Three-Dimensional Hydrogel Coculture.

The global population is aging rapidly, posing unprecedented challenges to health care systems. This study investigates the often-overlooked role of macrophages in microvascular dysfunction associated with aging. We use a three-dimensional in vitro hydrogel model to assess the effects of both age and metformin, an anti-aging therapeutic, on macrophage interactions with microvasculature. Metformin's broad cellular impact is a subject of significant interest, yet its precise mechanisms remain unclear. Our research reveals that metformin treatment enhances genetic pathways associated with macrophage-mediated support of angiogenesis, resulting in increased microvessel density. Of importance, monocyte chemoattractant protein-1 expression is upregulated with metformin treatment and positively correlated with microvascular volume, shedding light on a potential mechanism for metformin's promotion of macrophage support of vasculogenesis. This work not only uncovers metformin's impact on human macrophages but also supports its potential as an antiaging therapeutic, offering new avenues for combating age-related diseases.

In vitro competition between two transmissible cancers and potential implications for their host, the Tasmanian devil.

Since the emergence of a transmissible cancer, devil facial tumour disease (DFT1), in the 1980s, wild Tasmanian devil populations have been in decline. In 2016, a second, independently evolved transmissible cancer (DFT2) was discovered raising concerns for survival of the host species. Here, we applied experimental and modelling frameworks to examine competition dynamics between the two transmissible cancers invitro. Using representative cell lines for DFT1 and DFT2, we have found that in monoculture, DFT2 grows twice as fast as DFT1 but reaches lower maximum cell densities. Using co-cultures, we demonstrate that DFT2 outcompetes DFT1: the number of DFT1 cells decreasing over time, never reaching exponential growth. This phenomenon could not be replicated when cells were grown separated by a semi-permeable membrane, consistent with exertion of mechanical stress on DFT1 cells by DFT2. A logistic model and a Lotka-Volterra competition model were used to interrogate monoculture and co-culture growth curves, respectively, suggesting DFT2 is a better competitor than DFT1, but also showing that competition outcomes might depend on the initial number of cells, at least in the laboratory. We provide theories how the invitro results could be translated to observations in the wild and propose that these results may indicate that although DFT2 is currently in a smaller geographic area than DFT1, it could have the potential to outcompete DFT1. Furthermore, we provide a framework for improving the parameterization of epidemiological models applied to these cancer lineages, which will inform future disease management.

Feasibility of rice-crab (Scylla olivacea) co-cultures in the south-west coastal ghers of Bangladesh

To assess the production performance in terms of growth, survival and intactness of mud crab in co-culture with rice plants an experiment was conducted for a period of 120 days in the ghers of two villages under Paikgacha Upazilla, Khulna. The crab juvenile was collected from adjacent Shibsha River and stocked in two experimental ghers of 0.004 ha area each without considering male female ratio. The stocking density was 50 per decimal, 75 per decimal, 100 per decimal in T1, T2 and T3 treatments of two experiments, respectively. Fresh chopped tilapia fish was fed to the crab and the requirement of supplied tilapia fish was adjusted periodically in accordance with the growth performance or body weight of crab. Production and physico-chemical parameters were recorded and analyzed according to standard methods. Among these parameters salinity had significant (p<0.05) role on survival of mud crab. After 120 days of rearing we obtained average final weight of cultured crab (Scylla olivacea) was 154.6±12.19g, 135.46±21.30g and 107.23±14.87g; with the estimated yield of 995.96±193.43 kg/ha, 1179.4±266.68 kg/ha and 1144.22±544.05 kg/ha, survival was 45%, 47% and 42% in T1, T2 and T3 treatments respectively in Bayra village; On the other experimental site in Gopalpur village we obtained average final weight of cultured crab was 169.2±19.20g, 155.22±17.73g and 167.53±18.60g; with the estimated yield of 476.83±160.51 kg/ha, 793.73±361 kg/ha and 1636.19±868.32 kg/ha, survival was 25%, 27% and 22% in T1, T2 and T3 treatments respectively The yield of rice was found 6006±1021 kg/ha, 6026±991 kg/ha, 5995±1005 kg/ha in T1,T2,T3 treatments respectively in the experiment of Bayra village on the other hands in Gopalpur village the yield was 5877±1297 kg/ha, 5743±1350 kg/ ha, 5468±1190 kg/ha in T1,T2,T3 treatments respectively. Results and findings of the present study suggested that yield of rice was not hampered for rice-crab co-culture in ghers. According to the experimental results poor salinity level, temperature, water level fluctuation and winter seasons are the potential constraints in the growth, survival and intactness of rice crab co-culture in the coastal ghers of Bangladesh.

The evolutionary mechanism and function analysis of two subgroups of histamine-producing and non-histamine-producing Tetragenococcus halophilus

Tetragenococcus halophilus is a halophilic bacterium that existed in the fermentation of soy sauce and miso for flavor production and probiotic benefits. However, it is composed of two subgroups, histamine-producing and non-histamine-producing, with the former causing histamine accumulation and offering risks to food safety. Exploring the evolutionary mechanisms and physiological function of histamine-biosynthesis is of significance for understanding the formative mechanism of T. halophilus’s strain-specificity and is helpful for microbial control. Using systematic genomic analysis, we found that plasmid acquisition and loss is the evolutionary form resulting in the two subgroups of T. halophilus. Two plasmids, plasmid α with 30 kb and plasmid β with 4 kb existed in histamine-producing T. halophilus. We investigated the whole genetic information and proposed their genetic function in both two plasmids. The acquisition of histamine-producing plasmid enhanced the acid tolerance of histamine-producing T. halophilus but did not affect salt tolerance. More interestingly, we found that the existence of plasmid will promote the co-culture growth of T. halophilus. This study deepens our understanding of the formative mechanism of microbial species diversity, and provides our knowledge of the physiological function of histamine-producing plasmids.

Co-Culture Device for in vitro High Throughput Analysis of Cancer-Associated Fibroblast and Cancer Cell Interactions

Introduction: Cancers in general, and specifically lung cancer, continue to have low patient survival rates when the patient is at an advanced stage when diagnosed. It appears that the local environment, especially fibroblasts and their signaling molecules, tends to induce metastasis, increase cancer cell resistance to treatment, and aid in tumor growth rates. Since 3-D models quickly become too complex and/or expensive and therefore rarely leave the lab they are developed in, it is interesting to develop a 2-D model that more closely mimics clustered tumor formation and bulk interaction with a surrounding fibroblast environment. Methods: In the present study, we utilize an off-the-shelf stereolithography 3-D printer, standard use well plates, magnets, and metallic tubes to create a customizable 2-D co-culture system capable of being analyzed quantitatively with staining and qualitatively with standard fluorescent/brightfield microscopy to determine cancer-fibroblast interactions while also being able to test chemotherapeutic drugs in a high-throughput manner with standard 96-well plates. Results: Comparisons from monoculture and co-culture growth rates show that the presence of fibroblasts allows for significantly increased growth rates for H460 cancer. Additionally, the viability of cancer cells can be quantified with simple cell staining methods, and morphology and cell-cell interactions can be observed and studied. Discussion: The high throughput model demonstrates that boundary condition changes can be observed between cancer cells and fibroblasts based upon the different chemotherapeutics that have been administered.

Mr. Toad's wild fungi: Fungal isolate diversity on Colorado boreal toads and their capacity for pathogen inhibition

The amphibian skin pathogen Batrachochytrium dendrobatidis (Bd) has caused an ongoing biodiversity crisis, including in the locally endangered Colorado boreal toad (Anaxyrus boreas). Although researchers have investigated the bacteria living on amphibian skin and how they interact with Bd, there is less information about fungal community members. This study describes (1) the diversity of culturable fungi from boreal toad skin, (2) which subset of these isolates is Bd-inhibitory, and (3) how Bd affects these isolates' growth and morphology. Most isolates were from the orders Capnodiales, Helotiales, and Pleosporales. Of 16 isolates tested for Bd-inhibition, two from the genus Neobulgaria and three from Pseudeurotium inhibited Bd. Fungal growth in co-culture with Bd varied with weak statistical support for Neobulgaria sp. (isolate BTF_36) and cf Psychrophila (isolate BTF_60) (p-values = 0.076 and 0.092, respectively). Fungal morphology remained unchanged in co-culture with Bd, however, these results could be attributed to low replication per isolate. Nonetheless, two fungal isolates’ growth may have been affected by Bd, implying that fungal growth changes in Bd co-culture could be a variable worth measuring in the future (with higher replication). These findings add to the sparse but growing literature on amphibian-associated fungi and suggest further study may uncover the relevance of fungi to amphibian health and Bd infection.

2-Fucosyllactose Metabolism by Bifidobacteria Promotes Lactobacilli Growth in Co-Culture.

Breastfeeding is recognized as the gold standard in infant nutrition, not only because of breastmilk's intrinsic nutritional benefits but also due to the high content of different bioactive components such as 2-fucosyllactose (2'FL) in the mother's milk. It promotes the growth of its two major consumers, Bifidobacterium longum ssp. infantis and Bifidobacterium bifidum, but the effect on other intestinal microorganisms of infant microbiota remains incompletely understood. pH-uncontrolled fecal cultures from infants donors identified as "fast 2'FL -degrader" microbiota phenotype were used for the isolation of 2'FL-associated microorganisms. The use of specific selective agents allowed the successful isolation of B. bifidum IPLA20048 and of Lactobacillus gasseri IPLA20136. The characterization of 2'FL consumption and its moieties has revealed more pronounced growth, pH drop, and lactic acid production after 2'FL consumption when both microorganisms were grown together. The results point to an association between B. bifidum IPLA20048 and L. gasseri IPLA20136 in which L. gasseri is able to use the galactose from the lactose moiety after the hydrolysis of 2'FL by B. bifidum. The additional screening of two groups of bifidobacteria (n = 38), fast and slow degraders of 2'FL, in co-culture with lactobacilli confirmed a potential cross-feeding mechanism based on degradation products released from bifidobacterial 2'FL break-down. Our work suggests that this phenomenon may be widespread among lactobacilli and bifidobacteria in the infant gut. More investigation is needed to decipher how the ability to degrade 2'FL and other human milk oligosaccharides could influence the microbiota establishment in neonates and the evolution of the microbiota in adult life.

Investigating Yeast–Lactobacilli Interactions through Co-Culture Growth and Metabolite Analysis

The various forms of interactions that microorganisms engage in within fermented foods result in distinct metabolic product patterns. Fermentation products often feature both yeasts and bacteria, each possessing characteristics that can enhance the overall quality of the food, thus benefiting consumers. Kefir, a fermented milk originating from grains containing a unique and intricate blend of bacteria and yeasts living in a symbiotic relationship, is a valuable model for studying the evolution of the interactions between yeasts and bacteria. Targeted metagenomics was applied to investigate the microbiome of a batch of traditional Romanian kefir and further examine the growth and metabolic properties of the dominant yeast and bacterial strains isolated from this batch. In contrast to yeast, which is either unaffected or harmed by the presence of bacteria, our study revealed that a specific strain of Lactobacillus (L. rhamnosus) derived from the kefir batch benefited from the presence of Saccharomyces cerevisiae. The analysis of short-chain fatty acids (SCFAs) produced by in vitro cultures of these two chosen strains indicated significant changes in SCFA levels compared to single cultures. The dynamic interactions described in this and other studies on kefir emphasize the importance of a more profound comprehension of the ecological mechanisms governing interactions between yeast, bacterial, and mammalian cells.

Endophytic fungus-suspension cell co-culture: A new strategy for more rapid and sustainable screening of plant beneficial microorganisms

Microbial fertilizers are increasingly used as alternatives to conventional fertilizers, while the screening process for beneficial microorganisms is inefficient and time-consuming, often taking months or years. This study aimed to evaluate whether the endophytic fungus-suspension cell co-culture strategy could be used for rapid screening of plant beneficial microorganisms. Therefore, we investigated the effects and molecular mechanisms of Fusarium oxysporum GU-7 and GU-60 on the growth and accumulation of secondary metabolites in Glycyrrhiza uralensis suspension cells and potted seedlings. GU-7 significantly promoted the growth of suspension cells and potted seedlings, which may be related to its strong phosphorus-solubilizing and indole-3-acetic acid production capacity. In contrast to GU-60, GU-7 promoted the accumulation of flavonoids and glycyrrhizin at different co-culture times and growth times. Both strains accelerated the accumulation of liquiritigenin, liquiritin, and glycyrrhizin in suspension cells after 24 h of co-culture. Meanwhile, they differentially altered the expression of critical genes. The defense response of suspension cells was enhanced at 72 h, the antioxidant enzyme activity and flavonoid aglycone content were increased and the flavonoid glycoside content was decreased. RNA-seq revealed that the two strains had five times as many glycoside hydrolases as glycosyltransferases and differed in biological processes and biosynthetic pathways involved. Conclusively, the effects of these two strains on suspension cells were consistent with those on potted seedlings, while different strains produced different effects. These results suggest that co-culture with suspension cells is a promising strategy for rapid screening of beneficial microorganisms, even for conspecific fungi. GU-7 has the potential to be further developed as a microbial fertilizer to improve the growth and secondary metabolite accumulation of cultivated licorice.

Regulated bacterial interaction networks: A mathematical framework to describe competitive growth under inclusion of metabolite cross-feeding.

When bacterial species with the same resource preferences share the same growth environment, it is commonly believed that direct competition will arise. A large variety of competition and more general 'interaction' models have been formulated, but what is currently lacking are models that link monoculture growth kinetics and community growth under inclusion of emerging biological interactions, such as metabolite cross-feeding. In order to understand and mathematically describe the nature of potential cross-feeding interactions, we design experiments where two bacterial species Pseudomonas putida and Pseudomonas veronii grow in liquid medium either in mono- or as co-culture in a resource-limited environment. We measure population growth under single substrate competition or with double species-specific substrates (substrate 'indifference'), and starting from varying cell ratios of either species. Using experimental data as input, we first consider a mean-field model of resource-based competition, which captures well the empirically observed growth rates for monocultures, but fails to correctly predict growth rates in co-culture mixtures, in particular for skewed starting species ratios. Based on this, we extend the model by cross-feeding interactions where the consumption of substrate by one consumer produces metabolites that in turn are resources for the other consumer, thus leading to positive feedback in the species system. Two different cross-feeding options were considered, which either lead to constant metabolite cross-feeding, or to a regulated form, where metabolite utilization is activated with rates according to either a threshold or a Hill function, dependent on metabolite concentration. Both mathematical proof and experimental data indicate regulated cross-feeding to be the preferred model to constant metabolite utilization, with best co-culture growth predictions in case of high Hill coefficients, close to binary (on/off) activation states. This suggests that species use the appearing metabolite concentrations only when they are becoming high enough; possibly as a consequence of their lower energetic content than the primary substrate. Metabolite sharing was particularly relevant at unbalanced starting cell ratios, causing the minority partner to proliferate more than expected from the competitive substrate because of metabolite release from the majority partner. This effect thus likely quells immediate substrate competition and may be important in natural communities with typical very skewed relative taxa abundances and slower-growing taxa. In conclusion, the regulated bacterial interaction network correctly describes species substrate growth reactions in mixtures with few kinetic parameters that can be obtained from monoculture growth experiments.

Iron and nitrogen regulate carbon transformation in a methanotroph-microalgae system

Nutrient supply is important for maintaining a methanotroph and microalgae (MOB-MG) system for biogas valorization. However, there is a lack of understanding regarding how key elements regulate the growth of a MOB-MG coculture. In this study, a MOB-MG coculture with high protein content (0.47 g/g biomass) was established from waste activated sludge using synthetic biogas. An increase in iron availability substantially stimulated the specific growth rate (from 0.18 to 0.62 day−1) and biogas conversion rate (from 26.81 to 106.57 mg-C L−1 day−1) of the coculture. Moreover, the protein content remained high (0.51 g/g biomass), and the total lipid content increased (from 0.09 to 0.14 g/g biomass). Nitrogen limitation apparently constrained the specific growth rate (from 0.64 to 0.28 day−1) and largely reduced the protein content (from 0.51 to 0.31 g/g biomass) of the coculture. Intriguingly, the lipid content remained unchanged after nitrogen was depleted. The eukaryotic community was consistently dominated by MG belonging to Chlorella, while the populations of MOB shifted from Methylococcus/Methylosinus to Methylocystis due to iron and nitrogen amendment. In addition, diverse non-methanotrophic heterotrophs were present in the community. Their presence neither compromised the performance of the coculture system nor affected the protein content of the biomass. However, these heterotrophs may contribute to high carbon conversion efficiency by utilizing the dissolved organic carbon released by MOB and MG. Overall, the findings highlight the vital roles of iron and nitrogen in achieving efficient conversion of biogas, fast growth of cells, and optimal biomass composition in a MOB-MG coculture system.

Targeting hyaluronic acid synthase-3 (HAS3) for the treatment of advanced renal cell carcinoma

BackgroundHyaluronic acid (HA) promotes cancer metastasis; however, the currently approved treatments do not target HA. Metastatic renal carcinoma (mRCC) is an incurable disease. Sorafenib (SF) is a modestly effective antiangiogenic drug for mRCC. Although only endothelial cells express known SF targets, SF is cytotoxic to RCC cells at concentrations higher than the pharmacological-dose (5-µM). Using patient cohorts, mRCC models, and SF combination with 4-methylumbelliferone (MU), we discovered an SF target in RCC cells and targeted it for treatment.MethodsWe analyzed HA-synthase (HAS1, HAS2, HAS3) expression in RCC cells and clinical (n = 129), TCGA-KIRC (n = 542), and TCGA-KIRP (n = 291) cohorts. We evaluated the efficacy of SF and SF plus MU combination in RCC cells, HAS3-transfectants, endothelial-RCC co-cultures, and xenografts.ResultsRCC cells showed increased HAS3 expression. In the clinical and TCGA-KIRC/TCGA-KIRP cohorts, higher HAS3 levels predicted metastasis and shorter survival. At > 10-µM dose, SF inhibited HAS3/HA-synthesis and RCC cell growth. However, at ≤ 5-µM dose SF in combination with MU inhibited HAS3/HA synthesis, growth of RCC cells and endothelial-RCC co-cultures, and induced apoptosis. The combination inhibited motility/invasion and an HA-signaling-related invasive-signature. We previously showed that MU inhibits SF inactivation in RCC cells. While HAS3-knockdown transfectants were sensitive to SF, ectopic-HAS3-expression induced resistance to the combination. In RCC models, the combination inhibited tumor growth and metastasis with little toxicity; however, ectopic-HAS3-expressing tumors were resistant.ConclusionHAS3 is the first known target of SF in RCC cells. In combination with MU (human equivalent-dose, 0.6–1.1-g/day), SF targets HAS3 and effectively abrogates mRCC.

Impact of rare yeasts in Saccharomyces cerevisiae wine fermentation performance: Population prevalence and growth phenotype of Cyberlindnera fabianii, Kazachstania unispora, and Naganishia globosa

Saccharomyces cerevisiae is a highly fermentative species able to complete the wine fermentation. However, the interaction with other non-Saccharomyces yeasts can determine the fermentation performance of S. cerevisiae. We have characterised three rare non-Saccharomyces yeasts (Cyberlindnera fabianii, Kazachstania unispora and Naganishia globosa), studying their impact on S. cerevisiae fitness and wine fermentation performance. Using a wide meta-taxonomic dataset of wine samples, analysed through ITS amplicon sequencing, we show that about a 65.07% of wine samples contains Naganishia spp., a 27.21% contains Kazachstania spp., and only a 4.41% contains Cyberlindnera spp; in all cases with average relative abundances lower than 1% of total fungal populations. Although the studied N. globosa strain showed a limited growth capacity in wine, both K. unispora and C. fabianii showed a similar growth phenotype to that of S. cerevisiae in different fermentation conditions, highlighting the outstanding growth rate values of K. unispora. In mixed fermentations with S. cerevisiae, the three yeast species affected co-culture growth parameters and wine chemical profile (volatile compounds, polysaccharides and proteins). K. unispora DN201 strain presents an outstanding capacity to compete with S. cerevisiae strains during the first stage of wine fermentation, causing stuck fermentations in both synthetic and natural grape musts.

Towards the human nasal microbiome: Simulating D. pigrum and S. aureus.

The human nose harbors various microbes that decisively influence the wellbeing and health of their host. Among the most threatening pathogens in this habitat is Staphylococcus aureus. Multiple epidemiological studies identify Dolosigranulum pigrum as a likely beneficial bacterium based on its positive association with health, including negative associations with S. aureus. Carefully curated GEMs are available for both bacterial species that reliably simulate their growth behavior in isolation. To unravel the mutual effects among bacteria, building community models for simulating co-culture growth is necessary. However, modeling microbial communities remains challenging. This article illustrates how applying the NCMW fosters our understanding of two microbes' joint growth conditions in the nasal habitat and their intricate interplay from a metabolic modeling perspective. The resulting community model combines the latest available curated GEMs of D. pigrum and S. aureus. This uses case illustrates how to incorporate genuine GEM of participating microorganisms and creates a basic community model mimicking the human nasal environment. Our analysis supports the role of negative microbe-microbe interactions involving D. pigrum examined experimentally in the lab. By this, we identify and characterize metabolic exchange factors involved in a specific interaction between D. pigrum and S. aureus as an in silico candidate factor for a deep insight into the associated species. This method may serve as a blueprint for developing more complex microbial interaction models. Its direct application suggests new ways to prevent disease-causing infections by inhibiting the growth of pathogens such as S. aureus through microbe-microbe interactions.