Characterization of Oxygen Levels in an Uninfected and Infected Human Blood-Cerebrospinal-Fluid-Barrier Model

The host–pathogen interaction during meningitis can be investigated with blood-cerebrospinal-fluid-barrier (BCSFB) cell culture models. They are commonly handled under atmospheric oxygen conditions (19–21% O2), although the physiological oxygen conditions are significantly lower in cerebrospinal fluid (CSF) (7–8% O2). We aimed to characterize oxygen levels in a Streptococcus (S.) suis-infected BCSFB model with transmigrating neutrophils. A BCSFB model with human choroid plexus epithelial cells growing on transwell-filters was used. The upper “blood”-compartment was infected and blood-derived neutrophils were added. S. suis and neutrophils transmigrated through the BCSFB into the “CSF”-compartment. Here, oxygen and pH values were determined with the non-invasive SensorDish® reader. Slight orbital shaking improved the luminescence-based measurement technique for detecting free oxygen. In the non-infected BCSFB model, an oxygen value of 7% O2 was determined. However, with S. suis and transmigrating neutrophils, the oxygen value significantly decreased to 2% O2. The pH level decreased slightly in all groups. In conclusion, we characterized oxygen levels in the BCSFB model and demonstrated the oxygen consumption by cells and bacteria. Oxygen values in the non-infected BCSFB model are comparable to in vivo values determined in pigs in the CSF. Infection and transmigrating neutrophils decrease the oxygen value to lower values.

Mechanical Stress Induces Ca2+-Dependent Signal Transduction in Erythroblasts and Modulates Erythropoiesis

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca2+-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca2+-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.

Ciprofloxacin Release from Polymeric Films. Modeling and Pharmaceutical Parameters Determination

Ciprofloxacin (Cipro) is a broad-spectrum antibiotic used against both Gram (+) and Gram (−) bacteria. Its biological half-life is very short (4–5 h) and its conventional administration forms present a limited absorption efficiency. For this reason, the aim of this work was to study other administration strategies based on topical films. Sodium alginate (SA), a naturally occurring polymer, and a recombinant elastin-like polymer (rELP) produced by advanced genetic engineering techniques were evaluated as potential carrier systems. The films were obtained by the casting technique, adding the Cipro by direct dispersion in the polymer solution using 16.6% w/w rELP or 1.5% w/w SA. The in vitro release assays were performed at 37 °C in physiological solution and with orbital shaking at 90 rpm. Cipro concentration was determined by ultraviolet (UV) spectrophotometry at 276 nm. The release profiles were analyzed and adjusted using the Lumped model developed and validated by our research group. Pharmaceutical interest parameters were calculated and compared for both polymer-Cipro systems: the time required to reach 80% of the drug dissolved (t80%), the dissolution efficiency (DE) and the mean dissolution time (MDT). The SA-Cipro platform released the 80% of the drug in 35 min, while this parameter was 209 min for the rELP-Cipro system. The MDT80% was 8.9 and 53 min for the SA-Cipro and rELP-Cipro, respectively, while the DE, evaluated at 200 min, was 66.6 and 58.8 for each platform, respectively. These parameter values demonstrate that the rELP films were able to modulate the drug release rate and for the SA ones, release can be considered immediate. Therefore, both systems are promising strategies for the topical application of Cipro.

Acidithiobacillus thiooxidans DSM 26636: An Alternative for the Bioleaching of Metallic Burrs

Metallic wastes from the metal-mechanic industry represent a serious environmental problem. The possible strategies to reduce the metal content of these industrial wastes is their biotreatment by means of sulfur-oxidizing bacteria, such as Acidithioobacillus thiooxidans DSM 26636, which has been reported as an excellent metal-leaching microorganism by its capability to oxide sublimed sulfur and produce sulfuric acid in the presence of metallic burrs, and leach metals. The metallic composition of burrs was determined by ICP-OES before and after its exposure to biological treatment. The bioleaching process was kept for 21 days at 30 °C at an orbital shaking of 150 rev/min by using Erlenmeyer flasks of 125 mL containing 30 mL of Starkey-modified media added with 0.33 g (1% w/v) of sublimed sulfur and 0.33 g (1% w/v) of metal burrs, and 3 mL of inoculum at logarithmic phase. Results showed that A. thiooxidans was able to grow at these conditions with a maximum sulfate production of 11,028 mg/L, sulfuric acid corresponded to 0.16 M, but no statistical difference was observed for days 14 and 21. A reduction in pH was observed from 2.5 to 1.3 units. Metal bioleaching in mg/kg corresponded Fe (4658.5 ± 291), Cr (237 ± 46), Al (185 ± 12), Si (71 ± 10.3), Mo (63 ± 3.6), Mn (46 ± 3.3), V (18 ± 0.94), Mg (22.2 ± 3.7), Ni (15.8 ± 1.5), and Cu (5.7 ± 1.9). Results showed that A. thiooxidans DSM 26636 was able to grow in the presence of metal-containing wastes, and although metal removal was feasible, more studies are needed to enhance metal removal.

In Vitro Hypoxia Responsiveness of [18F]FDG and [18F]FAZA Retention: Influence of Shaking Versus Stagnant Conditions, Glass Versus Polystyrene Substrata and Cell Number Down-Scaling

Background. In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including 18F-fluoro-2-deoxyglucose ([18F]FDG) and hypoxia-selective 2-nitroimidazoles (e.g., 18F-fluoroazomycin-arabinoside, [18F]FAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention. Methods. Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2h to 21%, 0.5% or 0% O2 and [18F]FDG or [18F]FAZA was added, followed by cell harvest and analysis of radioactivity 1h ([18F]FDG) or 3h ([18F]FAZA) after. Experiments were conducted with/without orbital shaking. Results. The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven [18F]FAZA, and to some extent [18F]FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy. Conclusions. Tracer retention was similar under stagnant and forced convection conditions suggesting that the former approach may be appropriate even when accurate control of oxygen and tracer availability is required. In contrast, conventional plasticware should be used with caution when studying tracers and drugs that are metabolized and retained or activated at low O2 levels. Downscaling of cell number, by reducing the effective growth area, was feasible, without compromising accuracy.

In Vitro Hypoxia Responsiveness Of Fdg And Faza Retention: Influence Of Shaking Versus Stagnant Conditions, Glass Versus Polystyrene Substrata And Cell Number Down-Scaling

Background. In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including 18F-fluoro-2-deoxyglucose (FDG) and hypoxia-selective 2-nitroimidazoles (e.g., 18F-fluoroazomycin-arabinoside, FAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention. Methods. Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2h to 21%, 0.5% or 0% O2 and FDG or FAZA was added, followed by cell harvest and analysis of radioactivity 1h (FDG) or 3h (FAZA) after. Experiments were conducted with/without orbital shaking. Results. The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven FAZA, and to some extent FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy. Conclusions. Most experiments can be performed appropriately in the absence of shaking and with downscaling of cell number but the use of conventional plasticware is highly problematic for studies on tracers and drugs that are metabolized and retained or activated at low O2 levels.

In Vitro Hypoxia Responsiveness Of Fdg And Faza Retention: Influence Of Shaking Versus Stagnant Conditions, Glass Versus Polystyrene Substrata And Cell Number Down-Scaling

Background. In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including 18F-fluoro-2-deoxyglucose (FDG) and hypoxia-selective 2-nitroimidazoles (e.g., 18F-fluoroazomycin-arabinoside, FAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention. Methods. Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2h to 21%, 0.5% or 0% O2 and FDG or FAZA was added, followed by cell harvest and analysis of radioactivity 1h (FDG) or 3h (FAZA) after. Experiments were conducted with/without orbital shaking. Results. The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven FAZA, and to some extent FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy. Conclusions. Most experiments can be performed appropriately in the absence of shaking and with downscaling of cell number but the use of conventional plasticware is highly problematic for studies on tracers and drugs that are metabolized and retained or activated at low O2 levels.

Effects of Different Softening Processes on the Hardness and Quality of Thawed Neritic Squid (Uroteuthis edulis) Muscle

While attempting to develop a soft, seafood-based product as a potential food item for senior citizens, we evaluated the effects of different softening methods on the hardness and quality of thawed muscular mantle tissue of the neritic squid (Uroteuthis edulis) versus controls. Comparisons were made among injection with proteolytic enzymes (papain, bromelain); soaking in these enzymes or in alkali solutions (NaHCO3, NaOH); various soaking regimes combined with either orbital shaking under vacuum, ultrasonic processing, or ultrasonic cleaning; or hot-air drying and rehydration. Elderly panelists’ sensory impressions of thawed and heat-sterilized squid mantle subjected to these treatments were recorded, together with the total volatile basic nitrogen (TVBN), pH, color, protein breakdown profile (SDS-PAGE), and histological characteristics of thawed squid mantle subjected to the same treatments but not heat-sterilized. TVBN measurements showed that squid mantle remained in a close-to-fresh state under all treatments except for hot-air drying. The pH and hardness decreased and the muscles turned white when soaked in either enzymes or alkali. Orbital shaking under vacuum while soaking in 0.3% papain for 12 h produced the softest product, the next softest being obtained by injection with 0.3% papain. After orbital shaking under vacuum in 1.0% papain, protein degradation was confirmed by SDS-PAGE, and broken muscle fibers were evident in histological sections. Sensory evaluation panelists with unimpeded chewing ability rated mushy, papain-treated squid mantle poorly. Soaking in 2% NaHCO3 in an ultrasonic processor, following by washing out of the alkali, proved to be a better tenderizing method than either enzyme treatment or hot-air drying for neritic squid mantle intended for consumption by senior citizens.