From mouth to gut: microfluidic in vitro simulation of human gastro-intestinal digestion and intestinal permeability.

Reproducible in vitro studies of bioaccessibility, intestinal absorption, and bioavailability are key to the successful development of novel food ingredients or drugs intended for oral administration. There is currently a lack of methods that offer the finesse required to study these parameters for valuable molecules typically found in small volumes - as is the case of nanomaterials, which are often used to carry and protect bioactives. Here, we describe a modular microfluidic-based platform for total simulation of the human gastro-intestinal tract. Digestion-chips and cell-based gut-chips were fabricated from PDMS by soft lithography. On-chip digestion was validated using a fluorescently labelled casein derivative, which followed typical Michaelis-Menten kinetics and showed temporal resolution and good agreement with well-established bench-top protocols. Irreversible inhibition of serine proteases using Pefabloc® SC and a 1 : 6 dilution was sufficient to mitigate the cytotoxicity of simulated digestion fluids. Caco-2/HT29-MTX co-cultures were grown on-chip under a continuous flow for 7 days to obtain a differentiated cell monolayer forming a 3D villi-like epithelium with clear tight junction formation, and with an apparent permeability (Papp) of Lucifer Yellow closely approximating values reported ex vivo (3.7 × 10-6 ± 1.4 × 10-6vs. 4.0 × 10-6 ± 2.2 × 10-6). Digesta from the digestion-chips were flowed through the gut-chip, demonstrating the capacity to study sample digestion and intestinal permeability in a single microfluidic platform holding great promise for use in pharmacokinetic studies.

Differences in nevirapine biotransformation as a factor for its sex-dependent dimorphic profile of adverse drug reactions.

OBJECTIVES: Nevirapine is widely used for the treatment of HIV-1 infection; however, its chronic use has been associated with severe liver and skin toxicity. Women are at increased risk for these toxic events, but the reasons for the sex-related differences are unclear. Disparities in the biotransformation of nevirapine and the generation of toxic metabolites between men and women might be the underlying cause. The present work aimed to explore sex differences in nevirapine biotransformation as a potential factor in nevirapine-induced toxicity. METHODS: All included subjects were adults who had been receiving 400 mg of nevirapine once daily for at least 1 month. Blood samples were collected and the levels of nevirapine and its phase I metabolites were quantified by HPLC. Anthropometric and clinical data, and nevirapine metabolite profiles, were assessed for sex-related differences. RESULTS: A total of 52 patients were included (63% were men). Body weight was lower in women (P = 0.028) and female sex was associated with higher alkaline phosphatase (P = 0.036) and lactate dehydrogenase (P = 0.037) levels. The plasma concentrations of nevirapine (P = 0.030) and the metabolite 3-hydroxy-nevirapine (P = 0.035), as well as the proportions of the metabolites 12-hydroxy-nevirapine (P = 0.037) and 3-hydroxy-nevirapine (P = 0.001), were higher in women, when adjusted for body weight. CONCLUSIONS: There was a sex-dependent variation in nevirapine biotransformation, particularly in the generation of the 12-hydroxy-nevirapine and 3-hydroxy-nevirapine metabolites. These data are consistent with the sex-dependent formation of toxic reactive metabolites, which may contribute to the sex-dependent dimorphic profile of nevirapine toxicity.

A miniaturised semi-dynamic in-vitro model of human digestion.

Reliable in-vitro digestion models that are able to successfully replicate the conditions found in the human gastrointestinal tract are key to assess the fate and efficiency of new formulations aimed for oral consumption. However, current in-vitro models either lack the capability to replicate crucial dynamics of digestion or require large volumes of sample/reagents, which can be scarce when working with nanomaterials under development. Here, we propose a miniaturised digestion system, a digestion-chip, based on incubation chambers integrated on a polymethylmethacrylate device. The digestion-chip incorporates key dynamic features of human digestion, such as gradual acidification and gradual addition of enzymes and simulated fluids in the gastric phase, and controlled gastric emptying, while maintaining low complexity and using small volumes of sample and reagents. In addition, the new approach integrates real-time automated closed-loop control of two key parameters, pH and temperature, during the two main phases of digestion (gastric and intestinal) with an accuracy down to ± 0.1 °C and ± 0.2 pH points. The experimental results demonstrate that the digestion-chip successfully replicates the gold standard static digestion INFOGEST protocol and that the semi-dynamic digestion kinetics can be reliably fitted to a first kinetic order model. These devices can be easily adapted to dynamic features in an automated, sensorised, and inexpensive platform and will enable reliable, low-cost and efficient assessment of the bioaccessibility of new and expensive drugs, bioactive ingredients or nanoengineered materials aimed for oral consumption, thereby avoiding unnecessary animal testing.

Partitioning of Small Hydrophobic Molecules into Polydimethylsiloxane in Microfluidic Analytical Devices.

Polydimethylsiloxane (PDMS) is ubiquitously used in microfluidics. However, PDMS is porous and hydrophobic, potentially leading to small molecule partitioning. Although many studies addressed this issue and suggested surface/bulk modifications to overcome it, most were not quantitative, did not address which variables besides hydrophobicity governed molecule absorption, and no modification has been shown to completely obviate it. We evaluated qualitatively (confocal microscopy) and quantitatively (fluorescence spectroscopy) the effects of solute/solvent pairings, concentration, and residence time on molecule partitioning into PDMS. Additionally, we tested previously reported surface/bulk modifications, aiming to determine whether reduced PDMS hydrophobicity was stable and hindered molecule partitioning. Partitioning was more significant at lower concentrations, with the relative concentration of rhodamine-B at 20 µM remaining around 90% vs. 10% at 1 µM. Solute/solvent pairings were demonstrated to be determinant by the dramatically higher partitioning of Nile-red in a PBS-based solvent as opposed to ethanol. A paraffin coating slightly decreased the partitioning of Nile-red, and a sol-gel modification hindered the rhodamine-B diffusion into the PDMS bulk. However, there was no direct correlation between reduced surface hydrophobicity and molecule partitioning. This work highlighted the need for pre-assessing the absorption of test molecules into the microfluidic substrates and considering alternative materials for fabrication.

A triagem no processamento auditivo central como instrumento na identificaçäo de alteraçöes auditivas em pré-escolares / Selection of the central auditory process as a tool for identification of auditory disorders in preschool students in the city of Marília (SP)

Com o objetivo de utilizar a Triagem do Processamento Auditivo Central como instrumento de identificaçäo de alteraçöes auditivas näo somente centrais, mas também periféricas, realizamos o procedimento em 68 crianças em idade pré-escolar e, em seguida, realizamos uma avaliaçäo para investigar a acuidade auditiva e o processamento auditivo central das crianças que falharam na triagem. A análise dos resultados, verificamos que a triagem apresentou especificidade de 90,9 por cento na identificaçäo de alteraçöes periféricas e/ou centrais

Starvation-induced stress resistance development in a genetically modified pseudomonas fluorescens strain

The response of a genetically modified Pseudomonas flurescens to nutrient starvation and starvation-induced stress cross-protection were investigated. Strain BR12 was starved for carbon, nitrogen, phosphorus and sulphur individually and for all nutrients in defined mineral media and exposed for 6 h to chemical (ethanol 20 percentage), oxidative (H2O220µM), osmotic (NaCl3M), cold shock (0 degree) and heat shock (47 degree C) stresses at different incubationtimes. Response to starvation and stress cross-protection development were evaluated by viable bacteria counts. There was a significant increase in resistance of late phase cultures grown in rich medium to stress, except for ethanol, in all starvation situations. Multiple nutrient starved cultures were more resistant to stress than individual nutrient starved ones. This strain inoculated in oligotrophic stream water microcosms also showed the starvation-induced stress protection mechanism but it presented a higher resistance to ethanol than cultures starved in mineral media. the acquisition of nonspecific resistance to stress can favour the persistance of Genetically Modified Microorganisms (GMMos) in apparently unfavourable.