Systematic comparison of transcriptomes of Caco-2 cells cultured under different cellular and physiological conditions.

There is a need for standardized in vitro models emulating the functionalities of the human intestinal tract to study human intestinal health without the use of laboratory animals. The Caco-2 cell line is a well-accepted and highly characterized intestinal barrier model, which has been intensively used to study intestinal (drug) transport, host-microbe interactions and chemical or drug toxicity. This cell line has been cultured in different in vitro models, ranging from simple static to complex dynamic microfluidic models. We aimed to investigate the effect of these different in vitro experimental variables on gene expression. To this end, we systematically collected and extracted data from studies in which transcriptome analyses were performed on Caco-2 cells grown on permeable membranes. A collection of 13 studies comprising 100 samples revealed a weak association of experimental variables with overall as well as individual gene expression. This can be explained by the large heterogeneity in cell culture practice, or the lack of adequate reporting thereof, as suggested by our systematic analysis of experimental parameters not included in the main analysis. Given the rapidly increasing use of in vitro cell culture models, including more advanced (micro) fluidic models, our analysis reinforces the need for improved, standardized reporting protocols. Additionally, our systematic analysis serves as a template for future comparative studies on in vitro transcriptome and other experimental data.

Peptidoglycan from Akkermansia muciniphila MucT: chemical structure and immunostimulatory properties of muropeptides.

Akkermansia muciniphila is an intestinal symbiont known to improve the gut barrier function in mice and humans. Various cell envelope components have been identified to play a critical role in the immune signaling of A. muciniphila, but the chemical composition and role of peptidoglycan (PG) remained elusive. Here, we isolated PG fragments from A. muciniphila MucT (ATCC BAA-835), analyzed their composition and evaluated their immune signaling capacity. Structurally, the PG of A. muciniphila was found to be noteworthy due of the presence of some nonacetylated glucosamine residues, which presumably stems from deacetylation of N-acetylglucosamine. Some of the N-acetylmuramic acid (MurNAc) subunits were O-acetylated. The immunological assays revealed that muropeptides released from the A. muciniphila PG could both activate the intracellular NOD1 and NOD2 receptors to a comparable extent as muropeptides from Escherichia coli BW25113. These data challenge the hypothesis that non-N-acetylattion of PG can be used as a NOD-1 evasion mechanism. Our results provide new insights into the diversity of cell envelope structures of key gut microbiota members and their role in steering host-microbiome interactions.

A Systematic Search and Mapping Review of Studies on Intracerebral Microdialysis of Amino Acids, and Systematized Review of Studies on Circadian Rhythms.

BACKGROUND: Microdialysis can be used to measure amino acids in the extracellular space in vivo, based on the principle of diffusion. Variations in experimental set-up result in variations in baseline levels of the compounds measured. Variations may also be due to circadian rhythms. METHOD: We systematically searched and mapped the literature on all studies reporting baseline microdialysis measurements of histamine and the amino acids asparagine, aspartate, GABA, glutamate, glutamine, glycine, proline and taurine. We fully reviewed the studies describing circadian rhythms for histamine and the selected amino acids. RESULTS: We retrieved 2331 papers describing baseline measurements of one or more of the compounds of interest. We provide a numerical summary and lists of the publications by compound. We retrieved 11 references describing studies on the circadian rhythms of the compounds of interest. Aspartate, glutamate and histamine are generally higher during the dark than during the light phase in nocturnal rodents. For glutamine, no rhythmicity was observed. For GABA, the results were too inconsistent to generalise. For asparagine, glycine, proline and taurine, insufficient data are available. CONCLUSION: The literature on intracerebral microdialysis measurements of the amino acids is vast, but certain primary studies are still warranted. Future systematic reviews on the individual compounds can shed light on the effects of experimental variations on baseline concentrations.

Binding of Akkermansia muciniphila to mucin is O-glycan specific.

The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O-glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O-glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galß1-4GlcNAcß1-R) disaccharide selectively on core2 and core3 O-glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium.

Revealing Glycosylation Patterns in In Vitro-Produced Mucus Exposed to Pasteurized Mucus-Associated Intestinal Microbes by MALDI-TOF-MS and PGC-LC-MS/MS.

The human intestinal mucus layer protects against pathogenic microorganisms and harmful substances, whereas it also provides an important colonization niche for mutualistic microbes. The main functional components of mucus are heavily glycosylated proteins, called mucins. Mucins can be cleaved and utilized by intestinal microbes. The mechanisms between intestinal microbes and the regulation of mucin glycosylation are still poorly understood. In this study, in vitro mucus was produced by HT29-MTX-E12 cells under Semi-Wet interface with Mechanical Stimulation. Cells were exposed to pasteurized nonpathogenic bacteria Akkermansia muciniphila, Ruminococcus gnavus, and Bacteroides fragilis to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in in vitro produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. These results highlight the influence of pasteurized bacteria on glycosylation patterns, stress the intrinsic relationship between glycosylation and microbiota, and show the potential of using in vitro produced mucus to study glycosylation behavior.

Comparative Genomics and Physiology of Akkermansia muciniphila Isolates from Human Intestine Reveal Specialized Mucosal Adaptation.

Akkermansia muciniphila is a champion of mucin degradation in the human gastrointestinal tract. Here, we report the isolation of six novel strains from healthy human donors and their genomic, proteomic and physiological characterization in comparison to the type-strains A. muciniphila MucT and A. glycaniphila PytT. Complete genome sequencing revealed that, despite their large genomic similarity (>97.6%), the novel isolates clustered into two distinct subspecies of A. muciniphila: Amuc1, which includes the type-strain MucT, and AmucU, a cluster of unassigned strains that have not yet been well characterized. CRISPR analysis showed all strains to be unique and confirmed that single healthy subjects can carry more than one A. muciniphila strain. Mucin degradation pathways were strongly conserved amongst all isolates, illustrating the exemplary niche adaptation of A. muciniphila to the mucin interface. This was confirmed by analysis of the predicted glycoside hydrolase profiles and supported by comparing the proteomes of A. muciniphila strain H2, belonging to the AmucU cluster, to MucT and A. glycaniphila PytT (including 610 and 727 proteins, respectively). While some intrinsic resistance was observed among the A. muciniphila straind, none of these seem to pose strain-specific risks in terms of their antibiotic resistance patterns nor a significant risk for the horizontal transfer of antibiotic resistance determinants, opening the way to apply the type-strain MucT or these new A. muciniphila strains as next generation beneficial microbes.

Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation.

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

Animal models for cystic fibrosis: a systematic search and mapping review of the literature. Part 2: nongenetic models.

Various animal models are available to study cystic fibrosis (CF). These models may help to enhance our understanding of the pathology and contribute to the development of new treatments. We systematically searched all publications on CF animal models. Because of the large number of models retrieved, we split this mapping review into two parts. Previously, we presented the genetic CF animal models. In this paper we present the nongenetic CF animal models. While genetic animal models may, in theory, be preferable for genetic diseases, the phenotype of a genetic model does not automatically resemble human disease. Depending on the research question, other animal models may thus be more informative.We searched Pubmed and Embase and identified 12,303 unique publications (after duplicate removal). All references were screened for inclusion by two independent reviewers. The genetic animal models for CF (from 636 publications) were previously described. The non-genetic CF models (from 189 publications) are described in this paper, grouped by model type: infection-based, pharmacological, administration of human materials, xenografts and other. As before for the genetic models, an overview of basic model characteristics and outcome measures is provided. This CF animal model overview can be the basis for an objective, evidence-based model choice for specific research questions. Besides, it can help to retrieve relevant background literature on outcome measures of interest.

Intercellular communication between artificial cells by allosteric amplification of a molecular signal.

Multicellular organisms rely on intercellular communication to coordinate the behaviour of individual cells, which enables their differentiation and hierarchical organization. Various cell mimics have been developed to establish fundamental engineering principles for the construction of artificial cells displaying cell-like organization, behaviour and complexity. However, collective phenomena, although of great importance for a better understanding of life-like behaviour, are underexplored. Here, we construct collectives of giant vesicles that can communicate with each other through diffusing chemical signals that are recognized and processed by synthetic enzymatic cascades. Similar to biological cells, the Receiver vesicles can transduce a weak signal originating from Sender vesicles into a strong response by virtue of a signal amplification step, which facilitates the propagation of signals over long distances within the artificial cell consortia. This design advances the development of interconnected artificial cells that can exchange metabolic and positional information to coordinate their higher-order organization.

Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro.

Akkermansia muciniphila is a well-studied anaerobic bacterium specialized in mucus degradation and associated with human health. Because of the structural resemblance of mucus glycans and free human milk oligosaccharides (HMOs), we studied the ability of A. muciniphila to utilize human milk oligosaccharides. We found that A. muciniphila was able to grow on human milk and degrade HMOs. Analyses of the proteome of A. muciniphila indicated that key-glycan degrading enzymes were expressed when the bacterium was grown on human milk. Our results display the functionality of the key-glycan degrading enzymes (α-L-fucosidases, ß-galactosidases, exo-α-sialidases and ß-acetylhexosaminidases) to degrade the HMO-structures 2'-FL, LNT, lactose, and LNT2. The hydrolysation of the host-derived glycan structures allows A. muciniphila to promote syntrophy with other beneficial bacteria, contributing in that way to a microbial ecological network in the gut. Thus, the capacity of A. muciniphila to utilize human milk will enable its survival in the early life intestine and colonization of the mucosal layer in early life, warranting later life mucosal and metabolic health.

A Systematic Review Comparing Experimental Design of Animal and Human Methotrexate Efficacy Studies for Rheumatoid Arthritis: Lessons for the Translational Value of Animal Studies.

Increased awareness and understanding of current practices in translational research is required for informed decision making in drug development. This paper describes a systematic review of methotrexate for rheumatoid arthritis, comparing trial design between 147 animal and 512 human studies. Animal studies generally included fewer subjects than human studies, and less frequently reported randomisation and blinding. In relation to life span, study duration was comparable for animals and humans, but included animals were younger than included humans. Animal studies often comprised males only (61%), human studies always included females (98% included both sexes). Power calculations were poorly reported in both samples. Analyses of human studies more frequently comprised Chi-square tests, those of animal studies more frequently reported analyses of variance. Administration route was more variable, and more frequently reported in animal than human studies. Erythrocyte sedimentation rate and c-reactive protein were analysed more frequently in human than in animal studies. To conclude, experimental designs for animal and human studies are not optimally aligned. However, methotrexate is effective in treating rheumatoid arthritis in animal models and humans. Further evaluation of the available evidence in other research fields is needed to increase the understanding of translational success before we can optimise translational strategies.

Animal models for cystic fibrosis: A systematic search and mapping review of the literature - Part 1: genetic models.

Animal models for cystic fibrosis (CF) have enhanced our understanding of the pathology and contributed to the development of new treatments. In the field of CF, many animal models have been developed and described. To our knowledge, thus far, none of the reviews of CF animal models has used a systematic methodology. A systematic approach to creating model overviews can lead to an objective, evidence-based choice of an animal model for new research questions. We searched Pubmed and Embase for the currently available animal models for CF. Two independent reviewers screened the results. We included all primary studies describing an animal model for CF. After duplicate removal, 12,304 publications were left. Because of the large number of models, in the current paper, only the genetic models are presented. A total of 636 publications were identified describing genetic animal models for CF in mice, pigs, ferrets, rats and zebrafish. Most of these models have an altered Cftr gene. An overview of basic model characteristics and outcome measures for these genetic models is provided, together with advice on using these data. As far as the authors are aware, this is one of the largest systematic mapping reviews on genetic animal models for CF. It can aid in selecting a suitable model and outcome measures. In general, the reporting quality of the included publications was poor. Further systematic reviews are warranted to determine the quality and translational value of these models further.

The Use of Defined Microbial Communities To Model Host-Microbe Interactions in the Human Gut.

The human intestinal ecosystem is characterized by a complex interplay between different microorganisms and the host. The high variation within the human population further complicates the quest toward an adequate understanding of this complex system that is so relevant to human health and well-being. To study host-microbe interactions, defined synthetic bacterial communities have been introduced in gnotobiotic animals or in sophisticated in vitro cell models. This review reinforces that our limited understanding has often hampered the appropriate design of defined communities that represent the human gut microbiota. On top of this, some communities have been applied to in vivo models that differ appreciably from the human host. In this review, the advantages and disadvantages of using defined microbial communities are outlined, and suggestions for future improvement of host-microbe interaction models are provided. With respect to the host, technological advances, such as the development of a gut-on-a-chip system and intestinal organoids, may contribute to more-accurate in vitro models of the human host. With respect to the microbiota, due to the increasing availability of representative cultured isolates and their genomic sequences, our understanding and controllability of the human gut "core microbiota" are likely to increase. Taken together, these advancements could further unravel the molecular mechanisms underlying the human gut microbiota superorganism. Such a gain of insight would provide a solid basis for the improvement of pre-, pro-, and synbiotics as well as the development of new therapeutic microbes.

miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells.

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.