Rita Costa

Biosurfactant-producing and oil-degrading Bacillus subtilis strains enhance oil recovery in laboratory sand-pack columns

Microbial Enhanced Oil Recovery (MEOR) technology uses microorganisms and their metabolites to retrieve unrecoverable oil from mature reservoirs. In situ stimulation of biosurfactant-producing and oil-degrading microorganisms reduces the capillary forces retaining the oil inside the reservoir and decreases its viscosity, thus promoting oil flow and consequently production. In this work, a sand-pack column model was designed to simulate oil recovery operations and evaluate mobilization of residual oil by the selected microorganisms. Four different hydrocarbon mixtures and three Bacillus subtilis strains isolated from crude oil samples were used. Additional oil recoveries ranged from 6 to 24% depending on the hydrocarbon mixture and microorganism used. Biosurfactant production was observed with all the microorganisms and hydrocarbon mixtures studied. The oils recovered after incubation with B. subtilis isolates showed a reduction in the percentage of long-chain n-alkanes and lower viscosity when compared with the original oils. The results obtained suggest that stimulation of the selected B. subtilis strains in situ can contribute to mobilize entrapped oil in mature reservoirs.

Three-dimensional co-culture of human hepatocytes and mesenchymal stem cells: improved functionality in long-term bioreactor cultures

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short‐term viability in two‐dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co‐cultured with human bone marrow mesenchymal stem cells (BM‐MSCs) as spheroids in automated, computer‐controlled, stirred‐tank bioreactors with perfusion operation mode. A dual‐step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM‐MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co‐cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated‐dose toxicity testing. Moreover, the excretory activity was maintained in co‐cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM‐MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright

Seroprevalence of Triatoma virus (Dicistroviridae: Cripaviridae) antibodies in Chagas disease patients

BackgroundChagas disease is caused by Trypanosoma cruzi, and humans acquire the parasite by exposure to contaminated feces from hematophagous insect vectors known as triatomines. Triatoma virus (TrV) is the sole viral pathogen of triatomines, and is transmitted among insects through the fecal-oral route and, as it happens with T. cruzi, the infected insects release the virus when defecating during or after blood uptake.MethodsIn this work, we analysed the occurrence of anti-TrV antibodies in human sera from Chagas disease endemic and non-endemic countries, and developed a mathematical model to estimate the transmission probability of TrV from insects to man, which ranged between 0.00053 and 0.0015.ResultsOur results confirm that people with Chagas disease living in Bolivia, Argentina and Mexico have been exposed to TrV, and that TrV is unable to replicate in human hosts.ConclusionsWe presented the first experimental evidence of antibodies against TrV structural proteins in human sera.

Establishing Liver Bioreactors for In Vitro Research.

In vitro systems that can effectively model liver function for long periods of time are fundamental tools for preclinical research. Nevertheless, the adoption of in vitro research tools at the earliest stages of drug development has been hampered by the lack of culture systems that offer the robustness, scalability, and flexibility necessary to meet industrys demands. Bioreactor-based technologies, such as stirred tank bioreactors, constitute a feasible approach to aggregate hepatic cells and maintain long-term three-dimensional cultures. These three-dimensional cultures sustain the polarity, differentiated phenotype, and metabolic performance of human hepatocytes. Culture in computer-controlled stirred tank bioreactors allows the maintenance of physiological conditions, such as pH, dissolved oxygen, and temperature, with minimal fluctuations. Moreover, by operating in perfusion mode, gradients of soluble factors and metabolic by-products can be established, aiming at resembling the in vivo microenvironment. This chapter provides a protocol for the aggregation and culture of hepatocyte spheroids in stirred tank bioreactors by applying perfusion mode for the long-term culture of human hepatocytes. This in vitro culture system is compatible with feeding high-throughput screening platforms for the assessment of drug elimination pathways, being a useful tool for toxicology research and drug development in the preclinical phase.

Prevalence and Level of Antibodies Anti-Plasmodium spp. in Travellers with Clinical History of Imported Malaria

In this study, we show that 40.29% of travellers with a possible history of malaria exposure were positive for anti-Plasmodium spp. antibodies, while these individuals were negative by microscopy. The antibody test described here is useful to elucidate malaria exposure in microscopy-negative travellers from endemic countries.

Evaluation of the impact of matrix stiffness on encapsulated HepaRG spheroids

Background The drug development process is widely hampered by the lack of human models that recapitulate liver functionality and efficiently predict toxicity of new chemical compounds. Moreover, liver failure is a global medical problem, with transplantation being the only effective treatment currently available. The bipotent liver progenitor cell line HepaRG can be differentiated into cholangiocyte and hepatocyte-like cells that express major functions of mature hepatocytes, representing a valuable tool to model hepatic function [1]. Current two-dimensional (2D) protocols for the differentiation into mature hepatocyte-like cells fail to recapitulate the complex cell-cell interactions, which are crucial for maintaining polarity and inherent mature hepatic functionality. Herein, we present a three-dimensional (3D) strategy for the culture of HepaRG cells based on the encapsulation of aggregates. The effect of matrix stiffness on expansion and differentiation was evaluated through encapsulation with different concentrations of alginate (1.1% and 2%). Further characterization of the hepatic features will reveal the extent of the hepatic functionality of the generated spheroids. Materials and methods HepaRG cells were routinely propagated in static conditions as previously described [2]. Briefly, culture medium Williams E was supplemented with 1% (v/v) Glutamax, 1% (v/v) pen/strep, 5 μ g/ml insulin and 50 μ M hydrocortisone hemissuccinate and 10% (v/v) FBS and cultures were maintained at 37 ° C, 5% CO2 .S pinner vessels with ball impeller (Wheaton) were inoculated with inoculums ranging from 5 to 8 × 10 5 cell/mL and an agitation ranging from 35 to 45 rpm to attain the desired aggregation conditions. Aggregate size was determined by measuring Ferret’s diameter using the Image J software (NIH). After 3 days of aggregation, spheroids were encapsulated in 1.1% and 2% (w/v) of Ultra Pure MVG alginate (UP MVG NovaMatrix, Pronova Biomedical) in NaCl 0.9% (w/v) solution. Encapsulation was performed in an electrostatically driven microencapsulation unit VarV1 (Nisco) and cultures were maintained for 14 days in stirred culture conditions. Viability was determined by the double stain viability test - alginate beads were collected from stirred cultures, incubated with fluorescein diacetate (10 μg/ mL) and TO-PRO3 ® (1 μM) and observed on a fluores