Randomized lasso links microbial taxa with aquatic functional groups inferred from flow cytometry

High-nucleic-acid (HNA) and low-nucleic-acid (LNA) bacteria are two operational groups identified by flow cytometry (FCM) in aquatic systems. A number of reports have shown that HNA cell density correlates strongly with heterotrophic production, while LNA cell density does not. However, which taxa are specifically associated with these groups, and by extension, productivity has remained elusive. Here, we addressed this knowledge gap by using a machine learning-based variable selection approach that integrated FCM and 16S rRNA gene sequencing data collected from 14 freshwater lakes spanning a broad range in physicochemical conditions. There was a strong association between bacterial heterotrophic production and HNA absolute cell abundances (R-2 = 0.65), but not with the more abundant LNA cells. This solidifies findings, mainly from marine systems, that HNA and LNA bacteria could be considered separate functional groups, the former contributing a disproportionately large share of carbon cycling. Taxa selected by the models could predict HNA and LNA absolute cell abundances at all taxonomic levels. Selected operational taxonomic units (OTUs) ranged from low to high relative abundance and were mostly lake system specific (89.5% to 99.2%). A subset of selected OTUs was associated with both LNA and HNA groups (12.5% to 33.3%), suggesting either phenotypic plasticity or within-OTU genetic and physiological heterogeneity. These findings may lead to the identification of system-specific putative ecological indicators for heterotrophic productivity. Generally, our approach allows for the association of OTUs with specific functional groups in diverse ecosystems in order to improve our understanding of (microbial) biodiversity-ecosystem functioning relationships. IMPORTANCE A major goal in microbial ecology is to understand how microbial community structure influences ecosystem functioning. Various methods to directly associate bacterial taxa to functional groups in the environment are being developed. In this study, we applied machine learning methods to relate taxonomic data obtained from marker gene surveys to functional groups identified by flow cytometry. This allowed us to identify the taxa that are associated with heterotrophic productivity in freshwater lakes and indicated that the key contributors were highly system specific, regularly rare members of the community, and that some could possibly switch between being low and high contributors. Our approach provides a promising framework to identify taxa that contribute to ecosystem functioning and can be further developed to explore microbial contributions beyond heterotrophic production

Quantification of the Temporal Evolution of Collagen Orientation in Mechanically Conditioned Engineered Cardiovascular Tissues

Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network

Effect of Strain Magnitude on the Tissue Properties of Engineered Cardiovascular Constructs

Mechanical loading is a powerful regulator of tissue properties in engineered cardiovascular tissues. To ultimately regulate the biochemical processes, it is essential to quantify the effect of mechanical loading on the properties of engineered cardiovascular constructs. In this study the Flexercell FX-4000T (Flexcell Int. Corp., USA) straining system was modified to simultaneously apply various strain magnitudes to individual samples during one experiment. In addition, porous polyglycolic acid (PGA) scaffolds, coated with poly-4-hydroxybutyrate (P4HB), were partially embedded in a silicone layer to allow long-term uniaxial cyclic mechanical straining of cardiovascular engineered constructs. The constructs were subjected to two different strain magnitudes and showed differences in biochemical properties, mechanical properties and organization of the microstructure compared to the unstrained constructs. The results suggest that when the tissues are exposed to prolonged mechanical stimulation, the production of collagen with a higher fraction of crosslinks is induced. However, straining with a large strain magnitude resulted in a negative effect on the mechanical properties of the tissue. In addition, dynamic straining induced a different alignment of cells and collagen in the superficial layers compared to the deeper layers of the construct. The presented model system can be used to systematically optimize culture protocols for engineered cardiovascular tissues

The mechanisms of pharmacokinetic food-drug interactions - A perspective from the UNGAP group

The simultaneous intake of food and drugs can have a strong impact on drug release, absorption, distribution, metabolism and/or elimination and consequently, on the efficacy and safety of pharmacotherapy. As such, food-drug interactions are one of the main challenges in oral drug administration. Whereas pharmacokinetic (PK) food-drug interactions can have a variety of causes, pharmacodynamic (PD) food-drug interactions occur due to specific pharmacological interactions between a drug and particular drinks or food. In recent years, extensive efforts were made to elucidate the mechanisms that drive pharmacokinetic food-drug interactions. Their occurrence depends mainly on the properties of the drug substance, the formulation and a multitude of physiological factors. Every intake of food or drink changes the physiological conditions in the human gastrointestinal tract. Therefore, a precise understanding of how different foods and drinks affect the processes of drug absorption, distribution, metabolism and/or elimination as well as formulation performance is important in order to be able to predict and avoid such interactions. Furthermore, it must be considered that beverages such as milk, grapefruit juice and alcohol can also lead to specific food-drug interactions. In this regard, the growing use of food supplements and functional food requires urgent attention in oral pharmacotherapy. Recently, a new consortium in Understanding Gastrointestinal Absorption-related Processes (UNGAP) was established through COST, a funding organisation of the European Union supporting translational research across Europe. In this review of the UNGAP Working group "Food-Drug Interface", the different mechanisms that can lead to pharmacokinetic food-drug interactions are discussed and summarised from different expert perspectives

Gastrointestinal formulation behavior in real-life dosing conditions

Once marketed, drugs may suffer from suboptimal performance as they are often taken with meals and/or beverages not accounted for during clinical trials. Any ingested formulation will initially reside in the stomach where exposure to a variety of internal (e.g. pH and motility) and external factors (e.g. co-administration of beverages and foods) potentially influences intragastric formulation disintegration and drug dissolution. As this environment dictates how a drug is presented to the intestinal absorption compartment, these real-life dosing conditions can consequently affect systemic drug disposition. Moreover, a long intragastric residence time of a highly permeable drug creates a plausible scenario for gastric drug absorption. This thesis investigated (i) the potential influence of real-life dosing conditions on the gastrointestinal behavior of drugs and (ii) the potential of the gastric mucosa to absorb drugs. In a first set of experiments in this research project, intraluminal ethanol concentrations (stomach and duodenum) were determined in fasted and fed healthy volunteers after the consumption of common alcoholic beverages: beer, wine, and whisky. Upon gastric arrival, a trend was seen where smaller ingested volumes resulted in higher degrees of ethanol dilution, though, interestingly, no clear differences were observed in the degree of gastric dilution between fasted and fed state volunteers. Overall, these studies revealed relatively low and rapidly declining intragastric ethanol concentrations after drinking two standard consumptions of beer, wine or whisky. Compared to the fasted state, postprandial gastric ethanol concentrations remained higher for a longer time following the Cmax. In all cases, lower ethanol concentrations were observed in the duodenum compared to the stomach. Current FDA guidelines for testing the alcohol resistance of formulations state that in vitro tests should investigate the impact of up to 40% ethanol for 2 h. The intragastric ethanol concentrations observed in the present study question the relevance of these strict guidelines. Further studies in this research project investigated the effects of several real-life dosing conditions on the intraluminal behavior of drugs. The first study performed investigated the influence of (i) concomitant proton-pump inhibitor (PPI) intake and (ii) the administration of a liquid meal on the intraluminal dissolution, supersaturation and precipitation behavior of indinavir in healthy volunteers. A fasted state trial arm was included as a reference condition. When indinavir (Crixivan®) was administered to volunteers on a PPI regime, lower intragastric and intraduodenal indinavir concentrations were observed compared to the fasted state. An elevated intragastric pH hampered indinavir release from the capsule and resulted in a low solubilizing capacity for the drug in the stomach. Gastric supersaturation was observed in all volunteers on a PPI regime. The elevated intragastric pH resulted in lower duodenal indinavir concentrations through reduced dissolution and/or increased precipitation of indinavir in the stomach indicating that this real-life dosing condition can have a direct impact on the driving force for intestinal absorption. When indinavir (Crixivan®) was administered to healthy volunteers with a liquid meal (Ensure® Plus) a notable delay in intragastric drug release was observed. The fraction of dissolved indinavir in the fed stomach was similar to the fasted state. Furthermore, reasonably high duodenal indinavir concentrations were observed compared to the fasted state. The bioaccessible fraction of indinavir was determined and appeared lower in a fed state human intestinal sample compared to a fasted state sample indicating micellar entrapment of the drug in the fed state. This difference in bioaccessible fraction of indinavir may cause reduced intestinal absorption irrespective of duodenal concentrations. In conclusion, both real-life dosing conditions tested notably affected intraluminal drug behavior and thus potentially systemic exposure, albeit through different mechanisms. In a next study, the gastrointestinal behavior of the weakly acidic drug diclofenac was studied in healthy volunteers when given a solid meal. Ingestion of the FDA standard meal resulted in a gastrointestinal pH-profile similar to those observed in studies using a liquid meal. A delay in intragastric tablet disintegration was observed suggesting that the formation of a food-dependent precipitation layer on the tablet. Despite similar pH-profiles, the fraction of diclofenac dissolved in the gastrointestinal tract differed compared to observations following intake of the formulation with a liquid meal. Subsequent in vitro tests suggested that the FDA standard meal affects intragastric diclofenac dissolution from a Cataflam® tablet and local diclofenac solubility in a different way compared to the liquid meal. Furthermore, dissolved diclofenac molecules were observed to adsorb to several meal components present in the FDA standard meal. From this study, it becomes clear that meal composition and the consistency of meal (liquid or solid) given can markedly affect intraluminal drug disposition. Literature research in the context of intraluminal ethanol and drug behavior revealed earlier experiments hinting at the ability of the stomach to absorb alcohol and other small molecules. To explore the potential gastric absorption of pharmaceutical compounds, an in situ gastric bolus administration rat model was used. In this model, gastrointestinal transfer was blocked by ligating the pylorus and drugs were injected into the stomach as solution (paracetamol and diclofenac) or suspension (posaconazole). In this set-up, both paracetamol (neutral) and diclofenac (weakly acidic) appeared in the systemic circulation of fasted and fed rats indicating absorption through the gastric mucosa. For paracetamol, the relative contribution of the gastric absorption was higher in the fed state compared to the fasted state. Very low systemic posaconazole concentrations were detected indicating negligible gastric absorption of a weakly basic compounds. Results from this study demonstrated the ability of the stomach to absorb alcohol and pharmaceutical compounds. During drug development, clinical trials do not account for the large variety of beverages and meals taken with drugs, though these real-life dosing conditions can affect systemic drug exposure and thus therapy effectiveness. The potential influence of real-life dosing conditions is clear, yet further research is desired to elaborate this knowledge. Moreover, greater attention for the stomach as an absorptive organ is warranted. The impact of real-life dosing conditions and gastric absorption on gastrointestinal and systemic drug disposition cannot be underrated; further research and discussion regarding potential implications in predictive in vitro and in silico tools are encouraged.status: publishe

Real-Time Flow Cytometry to assess qualitative and quantitative responses of oral pathobionts during exposure to antiseptics

AbstractAntiseptics are widely used in oral healthcare to prevent or treat oral diseases, such as gingivitis and periodontitis. However, the incidence of bacteria being tolerant to standard antiseptics has sharply increased over the last few years. This stresses the urgency for surveillance against tolerant organisms, as well as the discovery of novel antimicrobials. Traditionally, susceptibility to antimicrobials is assessed by broth micro-dilution or disc diffusion assays, both of which are time-consuming, labor-intensive and provide limited information on the mode of action of the antimicrobials. The above-mentioned limitations highlight the need for the development of new methods to monitor and further understand antimicrobial susceptibility. Here, we used real-time flow cytometry, combined with membrane permeability staining, as a quick and sensitive technology to study the quantitative and qualitative response of two oral pathobionts to different concentrations of chlorhexidine, cetylpyridinium chloride or triclosan. Apart from the real-time monitoring of cell damage, we further applied a phenotypic fingerprint method to differentiate between the bacterial subpopulations that arose due to treatment. We quantified the pathobiont damage rate of different antiseptics at different concentrations within 15 minutes of exposure and identified the conditions under which the bacteria were most susceptible. Moreover, we detected species-specific and treatment-specific phenotypic subpopulations. This proves that real-time flow cytometry can provide information on the susceptibility of different microorganisms in a short time frame while differentiating between antiseptics and thus could be a valuable tool in the discovery of novel antimicrobial compounds while at the same time deciphering their mode of action.</jats:p

Randomized Lasso Links Microbial Taxa with Aquatic Functional Groups Inferred from Flow Cytometry

A major goal in microbial ecology is to understand how microbial community structure influences ecosystem functioning. Various methods to directly associate bacterial taxa to functional groups in the environment are being developed. In this study, we applied machine learning methods to relate taxonomic data obtained from marker gene surveys to functional groups identified by flow cytometry. This allowed us to identify the taxa that are associated with heterotrophic productivity in freshwater lakes and indicated that the key contributors were highly system specific, regularly rare members of the community, and that some could possibly switch between being low and high contributors. Our approach provides a promising framework to identify taxa that contribute to ecosystem functioning and can be further developed to explore microbial contributions beyond heterotrophic production.</jats:p

Quantification of MgO surface excess on the SnO2 nanoparticles and relationship with nanostability and growth

AbstractIn this work, we experimentally showed that the spontaneous segregation of MgO as surface excess in MgO doped SnO2 nanoparticles plays an important role in the system's energetics and stability. Using X-ray fluorescence in specially treated samples, we quantitatively determined the fraction of MgO forming surface excess when doping SnO2 with several different concentrations and established a relationship between this amount and the surface energy of the nanoparticles using the Gibbs approach. We concluded that the amount of Mg ions on the surface was directly related to the nanoparticles total free energy, in a sense that the dopant will always spontaneously distribute itself to minimize it if enough diffusion is provided. Because we were dealing with nanosized particles, the effect of MgO on the surface was particularly important and has a direct effect on the equilibrium particle size (nanoparticle stability), such that the lower the surface energy is, the smaller the particle sizes are, evidencing and quantifying the thermodynamic basis of using additives to control SnO2 nanoparticles stability