Online Measurement System for Dynamic Flow Bioreactors to Study Barrier Integrity of hiPSC-Based Blood–Brain Barrier In Vitro Models

Electrochemical impedance spectroscopy (EIS) is a noninvasive, reliable, and efficient method to analyze the barrier integrity of in vitro tissue models. This well-established tool is used most widely to quantify the transendothelial/epithelial resistance (TEER) of Transwell-based models cultured under static conditions. However, dynamic culture in bioreactors can achieve advanced cell culture conditions that mimic a more tissue-specific environment and stimulation. This requires the development of culture systems that also allow for the assessment of barrier integrity under dynamic conditions. Here, we present a bioreactor system that is capable of the automated, continuous, and non-invasive online monitoring of cellular barrier integrity during dynamic culture. Polydimethylsiloxane (PDMS) casting and 3D printing were used for the fabrication of the bioreactors. Additionally, attachable electrodes based on titanium nitride (TiN)-coated steel tubes were developed to perform EIS measurements. In order to test the monitored bioreactor system, blood–brain barrier (BBB) in vitro models derived from human-induced pluripotent stem cells (hiPSC) were cultured for up to 7 days. We applied equivalent electrical circuit fitting to quantify the electrical parameters of the cell layer and observed that TEER gradually decreased over time from 2513 Ω·cm2 to 285 Ω·cm2, as also specified in the static control culture. Our versatile system offers the possibility to be used for various dynamic tissue cultures that require a non-invasive monitoring system for barrier integrity.

Influence of mechanical and TGF-β3 stimulation on the tenogenic differentiation of tonsil-derived mesenchymal stem cells

Background Organogenesis from tonsil-derived mesenchymal cells (TMSCs) has been reported, wherein tenogenic markers are expressed depending on the chemical stimulation during tenogenesis. However, there are insufficient studies on the mechanical strain stimulation for tenogenic cell differentiation of TMSCs, although these cells possess advantages as a cell source for generating tendinous tissue. The purpose of this study was to investigate the effects of mechanical strain and transforming growth factor-beta 3 (TGF-β3) on the tenogenic differentiation of TMSCs and evaluate the expression of tendon-related genes and extracellular matrix (ECM) components, such as collagen. Results mRNA expression of tenogenic genes was significantly higher when the mechanical strain was applied than under static conditions. Moreover, mRNA expression of tenogenic genes was significantly higher with TGF-β3 treatment than without. mRNA expression of osteogenic and chondrogenic genes was not significantly different among different mechanical strain intensities. In cells without TGF-β3 treatment, double-stranded DNA concentration decreased, while the amount of normalized collagen increased as the intensity of mechanical strain increased. Conclusions Mechanical strain and TGF-β3 have significant effects on TMSC differentiation into tenocytes. Mechanical strain stimulates the differentiation of TMSCs, particularly into tenocytes, and cell differentiation, rather than proliferation. However, a combination of these two did not have a synergistic effect on differentiation. In other words, mechanical loading did not stimulate the differentiation of TMSCs with TGF-β3 supplementation. The effect of mechanical loading with TGF-β3 treatment on TMSC differentiation can be manipulated according to the differentiation stage of TMSCs. Moreover, TMSCs have the potential to be used for cell banking, and compared to other mesenchymal stem cells, they can be procured from patients via less invasive procedures.

Re-Thinking the Roles of Pathogens and Mutualists: Exploring the Continuum of Symbiosis in the Context of Microbial Ecology and Evolution

Systems of classification are important for guiding research activities and providing a common platform for discussion and investigation. One such system is assigning microbial taxa to the roles of mutualists and pathogens. Yet, there are often challenges and even inconsistencies in reports of research findings when microbial taxa display behaviors outside of these two static conditions (e.g. commensal). Over the last two decades, there has been some effort to highlight a continuum of symbiosis, wherein certain microbial taxa may exhibit mutualistic or pathogenic traits depending on environmental contexts, life stages, and plant host associations. However, gaps remain in understanding how to apply the continuum approach to host-microbe pairs across a range of environmental and ecological factors. This commentary presents an alternative framework for evaluating the continuum of symbiosis using dominant archetypes that define symbiotic ranges. We focus particularly on fungi and bacteria, though we recognize that archaea and other microeukaryotes play important roles in host-microbe interactions that may be described by this approach. This framework is centered in eco-evolutionary theory and aims to enhance communication among researchers, as well as prioritize holistic consideration of the factors shaping microbial life strategies. We discuss the influence of plant-mediated factors, habitat constraints, co-evolutionary forces, and the genetic contributions which shape different microbial lifestyles. Looking to the future, using a continuum of symbiosis paradigm will enable greater flexibility in defining the roles of target microbes and facilitate a more holistic view of the complex and dynamic relationship between microbes and plants.

A portable six-wheeled mobile robot with reconfigurable body and self-adaptable obstacle-climbing mechanisms

Mobile robots can replace rescuers in rescue and detection missions in complex and unstructured environments and draw the interest of many researchers. This paper presents a novel six-wheeled mobile robot with a reconfigurable body and self-adaptable obstacle-climbing mechanisms, which can reconfigure itself to three locomotion states to realize the advantages of terrain adaptability, obstacle crossing ability and portability. Design criteria and mechanical design of the proposed mobile robot are firstly presented, based on which the geometry of the robot is modelled and the geometric constraint, static conditions and motion stability condition for obstacle crossing of the robot are derived and formulated. Numerical simulations are then conducted to verify the geometric passing capability, static passing capability and motion stability and find feasible structure parameters of the robot in obstacle crossing. Further, a physical prototype of the proposed mobile robot is developed and integrated with mechatronic systems and remote control. Using the prototype, field experiments are carried out to verify the feasibility of the proposed design and theoretical derivations. The results show that the proposed mobile robot satisfies all the criteria set and is feasible for applications in disastrous rescuing scenarios.

Synthesis, Hydrophilicity and Micellization of Coil–Brush Polystyrene-b-(polyglycidol-g-polyglycidol) Copolymer—Comparison with Linear Polystyrene-b-Polyglycidol

In this paper, an original method of synthesis of coil–brush amphiphilic polystyrene-b-(polyglycidol-g-polyglycidol) (PS-b-(PGL-g-PGL)) block copolymers was developed. The hypothesis that their hydrophilicity and micellization can be controlled by polyglycidol blocks architecture was verified. The research enabled comparison of behavior in water of PS-b-PGL copolymers and block–brush copolymers PS-b-(PGL-g-PGL) with similar composition. The coil–brush copolymers were composed of PS-b-PGL linear core with average DPn of polystyrene 29 and 13 of polyglycidol blocks. The DPn of polyglycidol side blocks of coil–b–brush copolymers were 2, 7, and 11, respectively. The copolymers were characterized by 1H and 13C NMR, GPC, and FTIR methods. The hydrophilicity of films from the linear and coil–brush copolymers was determined by water contact angle measurements in static conditions. The behavior of coil–brush copolymers in water and their critical micellization concentration (CMC) were determined by UV-VIS using 1,6-diphenylhexa-1,3,5-trien (DPH) as marker and by DLS. The CMC values for brush copolymers were much higher than for linear species with similar PGL content. The results of the copolymer film wettability and the copolymer self-assembly studies were related to fraction of hydrophilic polyglycidol. The CMC for both types of polymers increased exponentially with increasing content of polyglycidol.

Impact of Electrospun Piezoelectric Core–Shell PVDFhfp/PDMS Mesh on Tenogenic and Inflammatory Gene Expression in Human Adipose-Derived Stem Cells: Comparison of Static Cultivation with Uniaxial Cyclic Tensile Stretching

Specific microenvironments can trigger stem cell tenogenic differentiation, such as specific substrates or dynamic cell cultivation. Electrospun meshes composed by core–shell fibers (random or aligned; PDMS core; piezoelectric PVDFhfp shell) were fabricated by coaxial electrospinning. Elastic modulus and residual strain were assessed. Human ASCs were seeded on such scaffolds either under static conditions for 1 week or with subsequent 10% dynamic stretching for 10,800 cycles (1 Hz, 3 h), assessing load elongation curves in a Bose® bioreactor system. Gene expression for tenogenic expression, extracellular matrix, remodeling, pro-fibrotic and inflammatory marker genes were assessed (PCR). For cell-seeded meshes, the E modulus increased from 14 ± 3.8 MPa to 31 ± 17 MPa within 3 h, which was not observed for cell-free meshes. Random fibers resulted in higher tenogenic commitment than aligned fibers. Dynamic cultivation significantly enhanced pro-inflammatory markers. Compared to ASCs in culture flasks, ASCs on random meshes under static cultivation showed a significant upregulation of Mohawk, Tenascin-C and Tenomodulin. The tenogenic commitment expressed by human ASCs in contact with random PVDFhfp/PDMS paves the way for using this novel highly elastic material as an implant to be wrapped around a lacerated tendon, envisioned as a functional anti-adhesion membrane.

Brucella abortus Encodes an Active Rhomboid Protease: Proteome Response after Rhomboid Gene Deletion

Rhomboids are intramembrane serine proteases highly conserved in the three domains of life. Their key roles in eukaryotes are well understood but their contribution to bacterial physiology is still poorly characterized. Here we demonstrate that Brucella abortus, the etiological agent of the zoonosis called brucellosis, encodes an active rhomboid protease capable of cleaving model heterologous substrates like Drosophila melanogaster Gurken and Providencia stuartii TatA. To address the impact of rhomboid deletion on B. abortus physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. About 50% of the B. abortus predicted proteome was identified by quantitative proteomics under two experimental conditions and 108 differentially represented proteins were detected. Membrane associated proteins that showed variations in concentration in the mutant were considered as potential rhomboid targets. This class included nitric oxide reductase subunit C NorC (Q2YJT6) and periplasmic protein LptC involved in LPS transport to the outer membrane (Q2YP16). Differences in secretory proteins were also addressed. Differentially represented proteins included a putative lytic murein transglycosylase (Q2YIT4), nitrous-oxide reductase NosZ (Q2YJW2) and high oxygen affinity Cbb3-type cytochrome c oxidase subunit (Q2YM85). Deletion of rhomboid had no obvious effect in B. abortus virulence. However, rhomboid overexpression had a negative impact on growth under static conditions, suggesting an effect on denitrification enzymes and/or high oxygen affinity cytochrome c oxidase required for growth in low oxygen tension conditions.

Using natural aluminosilicate (vermiculite) sorbent for purifying waste water from antibiotics

The problem of wastewater treatment from residual antibiotics is of particular relevance, since these drugs are used in many agricultural sectors. Antibiotics get into water, animal and human bodies, where they can accumulate negatively affecting health. The aim of this article is to study the possibility of using natural aluminosilicate vermiculite sorbent from the Koksharovskoye field (Primorsky Region) for purifying fish processing and fish farming enterprises’ waste water from antibiotics (chloramphenicol, tetracycline, cefazolin, cefuroxime, ceftriaxone, cefepime and and ciprofloxacin) under static and dynamic conditions. The study was carried out on a model wastewater system with injected antibiotics. The purification ability of the model system using the method of spectrophotometric antibiotics detection is analyzed. Under static conditions, the total content of antibiotics varied from 0.25 mg to 1.00 mg per 1 g of sorbent. Under dynamic conditions, the antibiotic content was 0.025 mg per 1 g of sorbent. High values of absorption for all studied antibiotics, except for chloramphenicol, were achieved both in static and dynamic modes. For chloramphenicol, when examined under static conditions, the maximum absorption rate was 45% with the minimum total concentration of antibiotics. With an increase in the load on the sorbent, the degree of absorption decreased to 3%. Thus, vermiculite modified with 7% hydrochloric acid is a promising sorbent for cleaning water bodies from residual antibiotics.

Advanced understanding of prokaryotic biofilm formation using a cost-effective and versatile multi-panel adhesion (mPAD) mount

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δ phz ) form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δ phz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that H. volcanii mutants that lack archaella are impaired in early stages of biofilm formation under shaking conditions. Importance: Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

Вариационный подход к определению динамической прочности материала

The problem of the determination of material strength properties through the Kolsky experimental technique is considered. Small size specimens of M1 copper alloy are tested on a split Hopkinson pressure bars equipment. The experimental data of tensile tests observed under both dynamic and quasi-static conditions are analysed within the framework of the incubation time criterion and the Sign-Perturbed Sums method. It is shown that the influence of a test performance error is considered in the data treatment procedure based on the developed method.