Molecular and cellular neurocardiology in heart disease.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.

Microvascular changes following exposure to iodinated contrast media in vitro. A qualitative comparison to serum creatinine concentrations in post-cardiac catheterization patients.

INTRODUCTION: Contrast-associated acute kidney injury (CA-AKI) is characterized as a loss of renal function following radiological contrast media administration. While all contrast media induce variable changes in microvascular endothelial cells in vitro, only few studies report clinical significance of their findings. A comprehensive assessment of the effect of iodinated contrast media on the renal function in vitro and in vivo is essential. The aim of our study was to morphometrically quantify the effect of two different contrast media (Iobitridol and Iodixanol) on vascular endothelial capillaries in vitro and to analyze their effect on the renal function of patients who underwent cardiac catheterization including the intra-arterial administration of contrast media, by measuring serum creatinine concentration (SCr), a byproduct of muscle metabolism, primarily excreted by the kidneys. Our hypothesis suggests that conducting a qualitative comparison of both outcomes will enable identification of differences and similarities between in vitro and in vivo exposure. MATERIAL AND METHODS: In vitro, co-cultures of human dermal fibroblasts and human dermal microvascular endothelial cells forming capillary beds were exposed to a mixture of phosphate buffered saline and either Iobitridol, Iodixanol, or one of their supplements EDTA or Trometamol for 1.5 or 5 min. Negative control co-cultures were exposed exclusively to phosphate buffered saline. Co-cultures were either directly fixed or underwent a regeneration time of 1, 3 or 7 days. An artificial intelligence software was trained for detection of labeled endothelial capillaries (CD31) on light microscope images and measurements of morphometric parameters. In vivo, we retrospectively analyzed data from patients who underwent intra-arterial administration of contrast media and for whom SCr values were available pre- and post-contrast exposition (1, 3, and 7 days following procedure). Temporal development of SCr and incidence of CA-AKI were assessed. Both exposure types were qualitatively compared. RESULTS: In vitro, Iobitridol, Iodixanol and EDTA induced a strong decrease of two morphometric parameters after 3 days of regeneration. In vivo, a significant increase of SCr and incidence of CA-AKI was observed 3 days following procedure in the post-contrast media patients. No difference was observed between groups. DISCUSSION: Two of the morphometric parameters were inversely proportional to the SCr of the patients. If the endothelial damages observed in vitro occur in vivo, it may result in renal hypoxia, inducing a loss of kidney function clinically translated into an increase of SCr. Further development of our in vitro model could allow closer replication of the internal structure of a kidney and bridge the gap between in vitro studies and their clinical findings.

Methanogenic partner influences cell aggregation and signalling of Syntrophobacterium fumaroxidans.

For several decades, the formation of microbial self-aggregates, known as granules, has been extensively documented in the context of anaerobic digestion. However, current understanding of the underlying microbial-associated mechanisms responsible for this phenomenon remains limited. This study examined morphological and biochemical changes associated with cell aggregation in model co-cultures of the syntrophic propionate oxidizing bacterium Syntrophobacterium fumaroxidans and hydrogenotrophic methanogens, Methanospirillum hungatei or Methanobacterium formicicum. Formerly, we observed that when syntrophs grow for long periods with methanogens, cultures tend to form aggregates visible to the eye. In this study, we maintained syntrophic co-cultures of S. fumaroxidans with either M. hungatei or M. formicicum for a year in a fed-batch growth mode to stimulate aggregation. Millimeter-scale aggregates were observed in both co-cultures within the first 5 months of cultivation. In addition, we detected quorum sensing molecules, specifically N-acyl homoserine lactones, in co-culture supernatants preceding the formation of macro-aggregates (with diameter of more than 20 µm). Comparative transcriptomics revealed higher expression of genes related to signal transduction, polysaccharide secretion and metal transporters in the late-aggregation state co-cultures, compared to the initial ones. This is the first study to report in detail both biochemical and physiological changes associated with the aggregate formation in syntrophic methanogenic co-cultures. KEYPOINTS: • Syntrophic co-cultures formed mm-scale aggregates within 5 months of fed-batch cultivation. • N-acyl homoserine lactones were detected during the formation of aggregates. • Aggregated co-cultures exhibited upregulated expression of adhesins- and polysaccharide-associated genes.

The repertoire and levels of secondary metabolites in microbial cocultures depend on the inoculation ratio: a case study involving Aspergillus terreus and Streptomyces rimosus.

OBJECTIVE: The aim of this study was to determine the influence of the inoculation volume ratio on the production of secondary metabolites in submerged cocultures of Aspergillus terreus and Streptomyces rimosus. RESULTS: The shake flask cocultures were initiated by using 23 inoculum variants that included different volumes of A. terreus and S. rimosus precultures. In addition, the axenic controls were propagated in parallel with the cocultures. UPLC‒MS analysis revealed the presence of 15 secondary metabolites, 12 of which were found both in the "A. terreus vs. S. rimosus" cocultures and axenic cultures of either A. terreus or S. rimosus. The production of the remaining 3 molecules was recorded solely in the cocultures. The repertoire and quantity of secondary metabolites were evidently dependent on the inoculation ratio. It was also noted that detecting filamentous structures resembling typical morphological forms of a given species was insufficient to predict the presence of a given metabolite. CONCLUSIONS: The modification of the inoculation ratio is an effective strategy for awakening and enhancing the production of secondary metabolites that are not biosynthesized under axenic conditions.

Mesothelioma-Associated Fibroblasts Modulate the Response of Mesothelioma Patient-Derived Organoids to Chemotherapy via Interleukin-6.

Malignant pleural mesothelioma (MPM) remains an incurable disease. This is partly due to the lack of experimental models that fully recapitulate the complexity and heterogeneity of MPM, a major challenge for therapeutic management of the disease. In addition, the contribution of the MPM microenvironment is relevant for the adaptive response to therapy. We established mesothelioma patient-derived organoid (mPDO) cultures from MPM pleural effusions and tested their response to pemetrexed and cisplatin. We aimed to evaluate the contribution of mesothelioma-associated fibroblasts (MAFs) to the response to pemetrexed and cisplatin (P+C). Organoid cultures were obtained from eight MPM patients using specific growth media and conditions to expand pleural effusion-derived cells. Flow cytometry was used to verify the similarity of the organoid cultures to the original samples. MAFs were isolated and co-cultured with mPDOs, and the addition of MAFs reduced the sensitivity of mPDOs to P+C. Organoid formation and expression of cancer stem cell markers such as ABCG2, NANOG, and CD44 were altered by conditioned media from treated MAFs. We identified IL-6 as the major contributor to the attenuated response to chemotherapy. IL-6 secretion by MAFs is correlated with increased resistance of mPDOs to pemetrexed and cisplatin.

Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.

Engineering blood and lymphatic microvascular networks.

The human vasculature plays a crucial role in the blood supply of nearly all organs as well as the drainage of the interstitial fluid. Consequently, if these physiological systems go awry, pathological changes might occur. Hence, the regeneration of existing vessels, as well as approaches to engineer artificial blood and lymphatic structures represent current challenges within the field of vascular research. In this review, we provide an overview of both the vascular blood circulation and the long-time neglected but equally important lymphatic system, with regard to their organotypic vasculature. We summarize the current knowledge within the field of vascular tissue engineering focusing on the design of co-culture systems, thereby mainly discussing suitable cell types, scaffold design and disease models. This review will mainly focus on addressing those subjects concerning atherosclerosis. Moreover, current technological approaches such as vascular organ-on-a-chip models and microfluidic devices will be discussed.

Co-cultures from Plants and Cyanobacteria: A New Way for Production Systems in Agriculture and Bioprocess Engineering.

Due to the global increase in the world population, it is not possible to ensure a sufficient food supply without additional nitrogen input into the soil. About 30-50% of agricultural yields are due to the use of chemical fertilizers in modern times. However, overfertilization threatens biodiversity, such as nitrogen-loving, fast-growing species overgrow others. The production of artificial fertilizers produces nitrogen oxides, which act as greenhouse gases. In addition, overfertilization of fields also releases ammonia, which damages surface waters through acidification and eutrophication. Diazotrophic cyanobacteria, which usually form a natural, stable biofilm, can fix nitrogen from the atmosphere and release it into the environment. Thus, they could provide an alternative to artificial fertilizers. In addition to this, biofilms stabilize soils and thus protect against soil erosion and desiccation. This chapter deals with the potential of cyanobacteria as the use of natural fertilizer is described. Possible partners such as plants and callus cells and the advantages of artificial co-cultivation will be discussed later. In addition, different cultivation systems for studying artificial co-cultures will be presented. Finally, the potential of artificial co-cultures in the agar industry will be discussed.

Lippia graveolens Essential Oil to Enhance the Effect of Imipenem against Axenic and Co-Cultures of Pseudomonas aeruginosa and Acinetobacter baumannii.

This research focuses on assessing the synergistic effects of Mexican oregano (Lippia graveolens) essential oil or carvacrol when combined with the antibiotic imipenem, aiming to reduce the pathogenic viability and virulence of Acinetobacter baumannii and Pseudomonas aeruginosa. The study highlighted the synergistic effect of combining L. graveolens essential oil or carvacrol with imipenem, significantly reducing the required doses for inhibiting bacterial growth. The combination treatments drastically lowered the necessary imipenem doses, highlighting a potent enhancement in efficacy against A. baumannii and P. aeruginosa. For example, the minimum inhibitory concentrations (MIC) for the essential oil/imipenem combinations were notably low, at 0.03/0.000023 mg/mL for A. baumannii and 0.0073/0.000023 mg/mL for P. aeruginosa. Similarly, the combinations significantly inhibited biofilm formation at lower concentrations than when the components were used individually, demonstrating the strategic advantage of this approach in combating antibiotic resistance. For OXA-51, imipenem showed a relatively stable interaction during 30 ns of dynamic simulation of their interaction, indicating changes (<2 nm) in ligand positioning during this period. Carvacrol exhibited similar fluctuations to imipenem, suggesting its potential inhibition efficacy, while thymol showed significant variability, particularly at >10 ns, suggesting potential instability. With IMP-1, imipenem also displayed very stable interactions during 38 ns and demonstrated notable movement and positioning changes within the active site, indicating a more dynamic interaction. In contrast, carvacrol and thymol maintained their position within the active site only ~20 and ~15 ns, respectively. These results highlight the effectiveness of combining L. graveolens essential oil and carvacrol with imipenem in tackling the difficult-to-treat pathogens A. baumannii and P. aeruginosa.

OrganoIDNet: a deep learning tool for identification of therapeutic effects in PDAC organoid-PBMC co-cultures from time-resolved imaging data.

PURPOSE: Pancreatic Ductal Adenocarcinoma (PDAC) remains a challenging disease due to its complex biology and aggressive behavior with an urgent need for efficient therapeutic strategies. To assess therapy response, pre-clinical PDAC organoid-based models in combination with accurate real-time monitoring are required. METHODS: We established stable live-imaging organoid/peripheral blood mononuclear cells (PBMCs) co-cultures and introduced OrganoIDNet, a deep-learning-based algorithm, capable of analyzing bright-field images of murine and human patient-derived PDAC organoids acquired with live-cell imaging. We investigated the response to the chemotherapy gemcitabine in PDAC organoids and the PD-L1 inhibitor Atezolizumab, cultured with or without HLA-matched PBMCs over time. Results obtained with OrganoIDNet were validated with the endpoint proliferation assay CellTiter-Glo. RESULTS: Live cell imaging in combination with OrganoIDNet accurately detected size-specific drug responses of organoids to gemcitabine over time, showing that large organoids were more prone to cytotoxic effects. This approach also allowed distinguishing between healthy and unhealthy status and measuring eccentricity as organoids' reaction to therapy. Furthermore, imaging of a new organoids/PBMCs sandwich-based co-culture enabled longitudinal analysis of organoid responses to Atezolizumab, showing an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added. CONCLUSION: Optimized PDAC organoid imaging analyzed by OrganoIDNet represents a platform capable of accurately detecting organoid responses to standard PDAC chemotherapy over time. Moreover, organoid/immune cell co-cultures allow monitoring of organoid responses to immunotherapy, offering dynamic insights into treatment behavior within a co-culture setting with PBMCs. This setup holds promise for real-time assessment of immunotherapeutic effects in individual patient-derived PDAC organoids.

Microalgae growth-promoting bacteria for cultivation strategies: Recent updates and progress.

Microalgae growth-promoting bacteria (MGPB), both actinobacteria and non-actinobacteria, have received considerable attention recently because of their potential to develop microalgae-bacteria co-culture strategies for improved efficiency and sustainability of the water-energy-environment nexus. Owing to their diverse metabolic pathways and ability to adapt to diverse conditions, microalgal-MGPB co-cultures could be promising biological systems under uncertain environmental and nutrient conditions. This review proposes the recent updates and progress on MGPB for microalgae cultivation through co-culture strategies. Firstly, potential MGPB strains for microalgae cultivation are introduced. Following, microalgal-MGPB interaction mechanisms and applications of their co-cultures for biomass production and wastewater treatment are reviewed. Moreover, state-of-the-art studies on synthetic biology and metabolic network analysis, along with the challenges and prospects of opting these approaches for microalgal-MGPB co-cultures are presented. It is anticipated that these strategies may significantly improve the sustainability of microalgal-MGPB co-cultures for wastewater treatment, biomass valorization, and bioproducts synthesis in a circular bioeconomy paradigm.

Rapid Acidification and Off-Flavor Reduction of Pea Protein by Fermentation with Lactic Acid Bacteria and Yeasts.

Pea protein is widely used as an alternative protein source in plant-based products. In the current study, we fermented pea protein to reduce off-flavor compounds, such as hexanal, and to produce a suitable fermentate for further processing. Laboratory fermentations using 5% (w/v) pea protein suspension were carried out using four selected lactic acid bacteria (LAB) strains, investigating their growth and acidification capabilities in pea protein. Rapid acidification of pea protein was achieved with Lactococcus lactis subsp. lactis strain LTH 7123. Next, this strain was co-inoculated together with either the yeasts Kluyveromyces lactis LTH 7165, Yarrowia lipolytica LTH 6056, or Kluyveromyces marxianus LTH 6039. Fermentation products of the mixed starter cultures and of the single strains were further analyzed by gas chromatography coupled with mass spectrometry to quantify selected volatile flavor compounds. Fermentation with L. lactis LTH 7123 led to an increase in compounds associated with the "beany" off-flavors of peas, including hexanal. However, significant reduction in those compounds was achieved after fermentation with Y. lipolytica LTH 6056 with or without L. lactis LTH 7123. Thus, fermentation using co-cultures of LAB and yeasts strains could prove to be a valuable method for enhancing quality attributes of pea protein-based products.

Breast Tumor Cell Survival and Morphology in a Brain-like Extracellular Matrix Depends on Matrix Composition and Mechanical Properties.

Triple-negative breast cancer (TNBC) is the most invasive type of breast cancer with high risk of brain metastasis. To better understand interactions between breast tumors with the brain extracellular matrix (ECM), a 3D cell culture model is implemented using a thiolated hyaluronic acid (HA-SH) based hydrogel. The latter is used as HA represents a major component of brain ECM. Melt-electrowritten (MEW) scaffolds of box- and triangular-shaped polycaprolactone (PCL) micro-fibers for hydrogel reinforcement are utilized. Two different molecular weight HA-SH materials (230 and 420 kDa) are used with elastic moduli of 148 ± 34 Pa (soft) and 1274 ± 440 Pa (stiff). Both hydrogels demonstrate similar porosities. The different molecular weight of HA-SH, however, significantly changes mechanical properties, e.g., stiffness, nonlinearity, and hysteresis. The breast tumor cell line MDA-MB-231 forms mainly multicellular aggregates in both HA-SH hydrogels but sustains high viability (75%). Supplementation of HA-SH hydrogels with ECM components does not affect gene expression but improves cell viability and impacts cellular distribution and morphology. The presence of other brain cell types further support numerous cell-cell interactions with tumor cells. In summary, the present 3D cell culture model represents a novel tool establishing a disease cell culture model in a systematic way.

Evaluation of co-culture of cellulolytic fungi for enhanced cellulase and xylanase activity and saccharification of untreated lignocellulosic material.

Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.

Addressing the Mechanical Interaction Between Cancer-Associated Fibroblasts and Cancer Cells by Laser Ablation.

Cells within a tumor interact by generating, transmitting, and sensing mechanical forces. Among all the cells of the tumor microenvironment, cancer-associated fibroblasts (CAFs) are a paradigmatic example of mechanical communication. In different steps of tumor progression, CAFs pull and push on cancer cells, regulating cancer cell migration, invasion, compartmentalization, and signaling. There is thus an increasing need to experimentally address mechanical interactions within a tumor. A common technique to measure these interactions is laser ablation. Cutting a tissue region with a high-power laser triggers a sudden tissue displacement whose direction and magnitude reveal the local mechanical stresses. In this chapter, we provide a detailed protocol to perform laser ablations in vitro and ex vivo. First, we describe how to prepare cocultures of primary CAFs and cancer cells and tumor explants. Then, we explain how to perform laser ablations in these two systems and how to analyze the induced tissue displacements using particle image velocimetry (PIV). Overall, we provide a workflow to perform, analyze, and interpret laser ablations to explore tumor mechanical interactions.

Investigating the paracrine and juxtacrine abilities of adipose-derived stromal cells in angiogenesis triple cell co-cultures.

The pro-angiogenic abilities of adipose-derived stromal cells (ASCs) make them attractive candidates for cellular therapy, especially for ischemic disease indications. However, details regarding the underlying mechanisms remain elusive. Therefore, this study aimed to investigate paracrine and juxtacrine abilities of ASCs in angiogenesis triple cell co-cultures by detailed image analysis of the vascular-like structures. Fibroblast-endothelial cell co-cultures were established, and ASCs were added directly or indirectly through inserts. The cultures were treated with antibodies or subjected to analyses using ELISA and RT2 PCR Arrays. The model consistently generated vascular-like structures. ASCs increased the total branch lengths equally well in paracrine and juxtacrine conditions, by increasing the number of branches and average branch lengths (ABL). In contrast, addition of VEGF to the model increased the number of branches, but not the ABL. Still, ASCs increased the VEGF levels in supernatants of paracrine and juxtacrine co-cultures, and anti-VEGF treatment decreased the sprouting. ASCs themselves up-regulated collagen type V in response to paracrine signals from the co-cultures. The results suggest that ASCs initiate sprouting through secretion of several paracrine factors, among which VEGF is identified, but VEGF alone does not recapitulate the paracrine actions of ASCs. By employing neutralizing antibodies and dismantling common model outputs using image analysis, the triple cell co-culture is an attractive tool for discovery of the paracrine factors in ASCs' secretome which act in concert with VEGF to improve angiogenesis.

TempEasy 3D Hydrogel Coculture System Provides Mechanistic Insights into Prostate Cancer Bone Metastasis.

Patients diagnosed with advanced prostate cancer (PCa) often experience incurable bone metastases; however, a lack of relevant experimental models has hampered the study of disease mechanisms and the development of therapeutic strategies. In this study, we employed the recently established Temperature-based Easy-separable (TempEasy) 3D cell coculture system to investigate PCa bone metastasis. Through coculturing PCa and bone cells for 7 days, our results showed a reduction in PCa cell proliferation, an increase in neovascularization, and an enhanced metastasis potential when cocultured with bone cells. Additionally, we observed increased cell proliferation, higher stemness, and decreased bone matrix protein expression in bone cells when cocultured with PCa cells. Furthermore, we demonstrated that the stiffness of the extracellular matrix had a negligible impact on molecular responses in both primary (PCa cells) and distant malignant (bone cells) sites. The TempEasy 3D hydrogel coculture system is an easy-to-use and versatile coculture system that provides valuable insights into the mechanisms of cell-cell communication and interaction in cancer metastasis.

Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria.

BACKGROUND: Lignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock for producing chemicals by microbial cell factories. Because of the heterogeneous nature of the lignin feedstocks, the biological upgrading of lignin relying on the metabolic routes of aerobic bacteria is currently considered as the most promising approach. However, the limited substrate range and the inefficient catabolism of the production hosts hinder the upgrading of lignin-related aromatics. Particularly, the aerobic O-demethylation of the methoxyl groups in aromatic substrates is energy-limited, inhibits growth, and results in carbon loss in the form of CO2. RESULTS: In this study, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic O-demethylation; in the process, the carbon from the methoxyl groups is fixed together with CO2 to form acetate, while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. By utilizing this cocultivation approach, we demonstrated an upgrading of guaiacol, an abundant but inaccessible substrate to most microbes, into a plastic precursor muconate, with a nearly equimolar yields (0.9 mol/mol in a small-scale cultivation and 1.0 mol/mol in a one-pot bioreactor cultivation). The process required only a minor genetic engineering, namely a single gene knock-out. Noticeably, by employing a metabolic integration of the two bacteria, it was possible to produce biomass and muconate by utilizing only CO2 and guaiacol as carbon sources. CONCLUSIONS: By the novel approach, we were able to overcome the issues related to aerobic O-demethylation of methoxylated aromatic substrates and demonstrated carbon-wise conversion of lignin-related aromatics to products with yields unattainable by aerobic processes. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

3D Cell Migration Chip (3DCM-Chip): A New Tool toward the Modeling of 3D Cellular Complex Systems.

3D hydrogel-based cell cultures provide models for studying cell behavior and can efficiently replicate the physiologic environment. Hydrogels can be tailored to mimic mechanical and biochemical properties of specific tissues and allow to produce gel-in-gel models. In this system, microspheres encapsulating cells are embedded in an outer hydrogel matrix, where cells are able to migrate. To enhance the efficiency of such studies, a lab-on-a-chip named 3D cell migration-chip (3DCM-chip) is designed, which offers substantial advantages over traditional methods. 3DCM-chip facilitates the analysis of biochemical and physical stimuli effects on cell migration/invasion in different cell types, including stem, normal, and tumor cells. 3DCM-chip provides a smart platform for developing more complex cell co-cultures systems. Herein the impact of human fibroblasts on MDA-MB 231 breast cancer cells' invasiveness is investigated. Moreover, how the presence of different cellular lines, including mesenchymal stem cells, normal human dermal fibroblasts, and human umbilical vein endothelial cells, affects the invasive behavior of cancer cells is investigated using 3DCM-chip. Therefore, predictive tumoroid models with a more complex network of interactions between cells and microenvironment are here produced. 3DCM-chip moves closer to the creation of in vitro systems that can potentially replicate key aspects of the physiological tumor microenvironment.

Metabolite cross-feeding enables concomitant catabolism of chlorinated methanes and chlorinated ethenes in synthetic microbial assemblies.

Isolate studies have been a cornerstone for unraveling metabolic pathways and phenotypical (functional) features. Biogeochemical processes in natural and engineered ecosystems are generally performed by more than a single microbe and often rely on mutualistic interactions. We demonstrate the rational bottom-up design of synthetic, interdependent co-cultures to achieve concomitant utilization of chlorinated methanes as electron donors and organohalogens as electron acceptors. Specialized anaerobes conserve energy from the catabolic conversion of chloromethane or dichloromethane to formate, H2, and acetate, compounds that the organohalide-respiring bacterium Dehalogenimonas etheniformans strain GP requires to utilize cis-1,2-dichloroethenene and vinyl chloride as electron acceptors. Organism-specific qPCR enumeration matched the growth of individual dechlorinators to the respective functional (i.e. dechlorination) traits. The metabolite cross-feeding in the synthetic (co-)cultures enables concomitant utilization of chlorinated methanes (i.e. chloromethane and dichloromethane) and chlorinated ethenes (i.e. cis-1,2-dichloroethenene and vinyl chloride) without the addition of an external electron donor (i.e. formate and H2). The findings illustrate that naturally occurring chlorinated C1 compounds can sustain anaerobic food webs, an observation with implications for the development of interdependent, mutualistic communities, the sustenance of microbial life in oligotrophic and energy-deprived environments, and the fate of chloromethane/dichloromethane and chlorinated electron acceptors (e.g. chlorinated ethenes) in pristine environments and commingled contaminant plumes.