Pyoverdine binding aptamers and label-free electrochemical detection of pseudomonads.

Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil.

Transport of Maternally Administered Pharmaceutical Agents Across the Placental Barrier In Vitro.

To understand the transport of pharmaceutical agents and their effects on developing fetus, we have created a placental microsystem that mimics structural phenotypes and physiological characteristic of a placental barrier. We have shown the formation of a continuous network of epithelial adherens junctions and endothelial cell-cell junctions confirming the integrity of the placental barrier. More importantly, the formation of elongated microvilli under dynamic flow condition is demonstrated. Fluid shear stress acts as a mechanical cue triggering the microvilli formation. Pharmaceutical agents were administered to the maternal channel, and the concentration of pharmaceutical agents in fetal channel for coculture and control models were evaluated. In fetal channel, the coculture model exhibited about 2.5 and 2.2% of the maternal initial concentration for naltrexone and 6ß-naltrexol, respectively. In acellular model, fetal channel showed about 10.5 and 10.3% of the maternal initial concentration for naltrexone and 6ß-naltrexol, respectively. Gene expressions of epithelial cells after direct administration of naltrexone and 6ß-naltrexol to the maternal channel and endothelial cells after exposure due to transport through placental barrier are also reported.

Microfluidic Seeding of Cells on the Inner Surface of Alginate Hollow Microfibers.

Mimicking microvascular tissue microenvironment in vitro calls for a cytocompatible technique of manufacturing biocompatible hollow microfibers suitable for cell-encapsulation/seeding in and around them. The techniques reported to date either have a limit on the microfiber dimensions or undergo a complex manufacturing process. Here, a microfluidic-based method for cell seeding inside alginate hollow microfibers is designed whereby mouse astrocytes (C8-D1A) are passively seeded on the inner surface of these hollow microfibers. Collagen I and poly-d-lysine, as cell attachment additives, are tested to assess cell adhesion and viability; the results are compared with nonadditive-based hollow microfibers (BARE). The BARE furnishes better cell attachment and higher cell viability immediately after manufacturing, and an increasing trend in the cell viability is observed between Day 0 and Day 2. Swelling analysis using percentage initial weight and width is performed on BARE microfibers furnishing a maximum of 124.1% and 106.1%, respectively. Degradation analysis using weight observed a 62% loss after 3 days, with 46% occurring in the first 12 h. In the frequency sweep test performed, the storage modulus (G') remains comparatively higher than the loss modulus (G″) in the frequency range 0-20 Hz, indicating high elastic behavior of the hollow microfibers.

Behavior of Neural Cells Post Manufacturing and After Prolonged Encapsulation within Conductive Graphene-Laden Alginate Microfibers.

Engineering conductive 3D cell scaffoldings offer advantages toward the creation of physiologically relevant platforms with integrated real-time sensing capabilities. Dopaminergic neural cells are encapsulated into graphene-laden alginate microfibers using a microfluidic approach, which is unmatched for creating highly-tunable microfibers. Incorporating graphene increases the conductivity of the alginate microfibers by 148%, creating a similar conductivity to native brain tissue. The cell encapsulation procedure has an efficiency of 50%, and of those cells, ≈30% remain for the entire 6-day observation period. To understand how the microfluidic encapsulation affects cell genetics, tyrosine hydroxylase, tubulin beta 3 class 3, interleukin 1 beta, and tumor necrosis factor alfa are analyzed primarily with real-time reverse transcription-quantitative polymerase chain reaction and secondarily with enzyme-linked immunosorbent assay, immediately after manufacturing, after encapsulation in polymer matrix for 6 days, and after encapsulation in the graphene-polymer composite for 6 days. Preliminary data shows that the manufacturing process and combination with alginate matrix affect the expression of the studied genes immediately after manufacturing. In addition, the introduction of graphene further changes gene expressions. Long-term encapsulation of neural cells in alginate and 6-day exposure to graphene also leads to changes in gene expressions.

Contribution of Physical Methods in Decellularization of Animal Tissues.

Biologic scaffolds composed of extracellular matrix (ECM) are frequently used for clinical purposes of tissue regeneration. Different methods have been developed for this purpose. All methods of decellularization including chemical and physical approaches leave some damage on the ECM; however, the effects of these methods are different which make some of these procedures more proper to maintain ECM structure than other methods. This review is aimed to introduce and compare new physical methods for the decellularization of different tissues and organs in tissue engineering. All recent reports and research that have used at least one physical method in the procedure of decellularization, were included and evaluated in this paper. The advantages and drawbacks of each method were examined and compared considering the effectiveness. This review tried to highlight the prospective potentials and benefits of applying physical methods for decellularization protocols in tissue engineering instead of the current chemical methods. These chemical methods are harsh in nature and were shown to be destructive and harmful to essential substances of ECM and scaffold structure. Therefore, using physical methods as a partial or even a whole protocol could save time, costs, and quality of the final acellular tissue in complicated decellularization procedures. Moreover, regarding the control factor that could be achieved easily with physical methods, optimization of different decellularization protocols would be quite satisfactory. Combined methods take advantage of both chemical and physical approaches.

Advancement of Sensor Integrated Organ-on-Chip Devices.

Organ-on-chip devices have provided the pharmaceutical and tissue engineering worlds much hope since they arrived and began to grow in sophistication. However, limitations for their applicability were soon realized as they lacked real-time monitoring and sensing capabilities. The users of these devices relied solely on endpoint analysis for the results of their tests, which created a chasm in the understanding of life between the lab the natural world. However, this gap is being bridged with sensors that are integrated into organ-on-chip devices. This review goes in-depth on different sensing methods, giving examples for various research on mechanical, electrical resistance, and bead-based sensors, and the prospects of each. Furthermore, the review covers works conducted that use specific sensors for oxygen, and various metabolites to characterize cellular behavior and response in real-time. Together, the outline of these works gives a thorough analysis of the design methodology and sophistication of the current sensor integrated organ-on-chips.