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Plants are sessile organisms, often characterized by limited dispersal. Seeds and pollen are the critical stages for gene flow. Here we investigate spatial genetic structure, gene dispersal and the relative contribution of pollen vs seed in the movement of genes in a stable metapopulation of the white campion Silene latifolia within its native range. This short-lived perennial plant is dioecious, has gravity-dispersed seeds and moth-mediated pollination. Direct measures of pollen dispersal suggested that large populations receive more pollen than small isolated populations and that most gene flow occurs within tens of meters. However, these studies were performed in the newly colonized range (North America) where the specialist pollinator is absent. In the native range (Europe), gene dispersal could fall on a different spatial scale. We genotyped 258 individuals from large and small (15) subpopulations along a 60 km, elongated metapopulation in Europe using six highly variable microsatellite markers, two X-linked and four autosomal. We found substantial genetic differentiation among subpopulations (global FST=0.11) and a general pattern of isolation by distance over the whole sampled area. Spatial autocorrelation revealed high relatedness among neighboring individuals over hundreds of meters. Estimates of gene dispersal revealed gene flow at the scale of tens of meters (5-30 m), similar to the newly colonized range. Contrary to expectations, estimates of dispersal based on X and autosomal markers showed very similar ranges, suggesting similar levels of pollen and seed dispersal. This may be explained by stochastic events of extensive seed dispersal in this area and limited pollen dispersal. [ABSTRACT FROM AUTHOR]

Host size is considered a reliable indicator of host quality and an important determinant of parasitoid fitness. Koinobiont parasitoids attack hosts that continue feeding and growing during parasitism. In contrast with hemolymph-feeding koinobionts, tissue-feeding koinobionts face not only a minimum host size for successful development but also a maximum host size, because consumption of the entire host is often necessary for successful egression. Here we study interactions between a generalist tissue-feeding larval endoparasitoid, Hyposoter didymator Thunberg (Hymenoptera: Ichneumonidae) and two of its natural hosts, Spodoptera exigua Hübner and Chrysodeixis chalcites Esper (Lepidoptera: Noctuidae). Larvae of C. chalcites are up to three times larger than corresponding instars of S. exigua and also attain much higher terminal masses before pupation. We hypothesized that the range of host instars suitable for successful parasitism by H. didymator would be much more restricted in the large host C. chalcites than in the smaller S. exigua. To test this hypothesis, we monitored development of H. didymator in all instars of both host species and measured survival, larval development time, and adult body mass of the parasitioid. In contrast with our predictions, C. chalcites was qualitatively superior to S. exigua in terms of the survival of parasitized hosts, the proportion of parasitoids able to complete development, and adult parasitoid size. However, in both hosts, the proportion of mature parasitoid larvae that successfully developed into adults was low at the largest host sizes. Our results suggest that qualitative, as well as quantitative, factors are important in the success of tissue-feeding parasitoids. [ABSTRACT FROM AUTHOR]

The cell-envelope of Gram-negative bacteria contains endotoxic lipopolysaccharides (LPS) that are recognized by the innate immune system via Toll-Like Receptors (TLRs). The intestinal mucosal symbiont Akkermansia muciniphila is known to confer beneficial effects on the host and has a Gram-negative architecture. Here we show that A. muciniphila LPS lacks the O-polysaccharide repeating unit, with the resulting lipooligosaccharide (LOS) having unprecedented structural and signaling properties. The LOS consists of a complex glycan chain bearing two distinct undeca- and hexadecasaccharide units each containing three 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residues. The lipid A moiety appears as a mixture of differently phosphorylated and acylated species and carries either linear or branched acyl moieties. Peritoneal injection of the LOS in mice increased higher gene expression of liver TLR2 than TLR4 (100-fold) and induced high IL-10 gene expression. A. muciniphila LOS was found to signal both through TLR4 and TLR2, whereas lipid A only induced TLR2 in a human cell line. We propose that the unique structure of the A. muciniphila LOS allows interaction with TLR2, thus generating an anti-inflammatory response as to compensate for the canonical inflammatory signaling associated with LOS and TLR4, rationalizing its beneficial host interaction., (© 2024. The Author(s).)

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.

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 (Galβ1-4GlcNAcβ1-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., (© 2024. The Author(s).)

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., (© 2023. The Author(s).)

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.

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., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Physician-focused throughput initiatives are intended to mitigate the effects of emergency department (ED) overcrowding. Our tertiary care academic hospital recently piloted an emergency physician leader role intended to improve throughput. Although a separate experimental evaluation of this initiative was undertaken, it was expected that such an evaluation could not alone provide the necessary information to inform 'how' the emergency physician leader intervention worked. The objective of this study was to conduct a realist evaluation of the emergency physician leader. Realist inquiry utilizes Context Mechanism Outcome configurations to determine the impacts of interventions that are often missed by quantitative analysis. Using a realist perspective, this study aimed to evaluate the emergency physician leader initiative's effects on throughput with the goal of building transferable lessons to the implementation of future interventions., Methods: Semi-structured interviews with key stakeholders in the intervention were conducted and analyzed using thematic and template techniques specifically aimed at identifying Context Mechanism Outcomes., Results: 13 interviews were conducted with physicians and nurses who identified contexts and mechanisms which promoted or impeded ED throughput. For example, in situations where there was a clear indication for imaging or management that could not be initiated within the scope of a nursing protocol, the emergency physician leader initiating these orders was felt to promote ED throughput. Conversely, in contexts where there was no nurse available to fulfill early orders, the emergency physician leader's initiation of orders was perceived to impede throughput., Conclusion: This evaluation provides insights into the reasoning and behaviour of individuals involved in the emergency physician leader initiative and provides a systematic approach to unraveling its complex causal pathway. Knowledge of context-mechanism-outcome relationships may help implementers design and measure the impact of future physician-focused throughput interventions., (© 2022. The Author(s), under exclusive licence to Canadian Association of Emergency Physicians (CAEP)/ Association Canadienne de Médecine d'Urgence (ACMU).)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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, β-galactosidases, exo-α-sialidases and β-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.

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.

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.

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.

Objective: A novel approach to trauma team simulation was used to enhance team performance in a cohort of general surgical residents. We implemented data driven debriefing using performance report cards and video footage of the simulations. We wanted to evaluate the technical and nontechnical skills developed by teams using this approach., Design: All surgical residents in an academic program were divided into 5 equal "trauma teams". Throughout the academic year, each team took part in 4 standardized, high fidelity trauma simulations. Rubrics to assess technical efficiency were scored. Each team received individualized feedback in the form of report cards following each simulation. Video recordings of each simulation were analyzed by blinded raters using a validated instrument to assess nontechnical skills/Crisis Resource Management (CRM) skills., Setting: An academic level 1 trauma hospital in Canada., Results: Five teams comprising five residents participated in four simulations each. Learner feedback was universally positive and learning during simulation was rated higher than learning during didactic lecture. The effect of data driven report cards and anonymized ranking was cited by trainees as a motivating factor to improve. CRM scores improved over the course of the academic year for all teams but without reaching statistical significance. A strong positive correlation was measured between technical and CRM skills for all teams., Conclusions: Adding data driven debriefing using performance report cards that assess both technical and CRM skills to a trauma team curriculum is a feasible and acceptable way to influence trainee performance using positive competitive motivation. More data are required to confirm the early patterns of improvement uncovered in CRM scoring. A positive correlation between technical skills and CRM skills raises important questions for future research., (Copyright © 2019. Published by Elsevier Inc.)

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., (Copyright © 2019 American Society for Microbiology.)

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.

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Host size is considered a reliable indicator of host quality and an important determinant of parasitoid fitness. Koinobiont parasitoids attack hosts that continue feeding and growing during parasitism. In contrast with hemolymph-feeding koinobionts, tissue-feeding koinobionts face not only a minimum host size for successful development but also a maximum host size, because consumption of the entire host is often necessary for successful egression. Here we study interactions between a generalist tissue-feeding larval endoparasitoid, Hyposoter didymator Thunberg (Hymenoptera: Ichneumonidae) and two of its natural hosts, Spodoptera exigua Hübner and Chrysodeixis chalcites Esper (Lepidoptera: Noctuidae). Larvae of C. chalcites are up to three times larger than corresponding instars of S. exigua and also attain much higher terminal masses before pupation. We hypothesized that the range of host instars suitable for successful parasitism by H. didymator would be much more restricted in the large host C. chalcites than in the smaller S. exigua. To test this hypothesis, we monitored development of H. didymator in all instars of both host species and measured survival, larval development time, and adult body mass of the parasitioid. In contrast with our predictions, C. chalcites was qualitatively superior to S. exigua in terms of the survival of parasitized hosts, the proportion of parasitoids able to complete development, and adult parasitoid size. However, in both hosts, the proportion of mature parasitoid larvae that successfully developed into adults was low at the largest host sizes. Our results suggest that qualitative, as well as quantitative, factors are important in the success of tissue-feeding parasitoids.