Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Structural Aspects of P2-Type Na0.67Mn0.6Ni0.2Li0.2O2 (MNL) Stabilization by Lithium Defects as a Cathode Material for Sodium-Ion Batteries

A known strategy for improving the properties of layered oxide electrodes in sodium-ion batteries is the partial substitution of transition metals by Li. Herein, the role of Li as a defect and its impact on sodium storage in P2-Na_0.67Mn_0.6Ni_0.2Li_0.2O₂ is discussed. In tandem with electrochemical studies, the electronic and atomic structure are studied using solid-state NMR, operando XRD, and density functional theory (DFT). For the as-synthesized material, Li is located in comparable amounts within the sodium and the transition metal oxide (TMO) layers. Desodiation leads to a redistribution of Li ions within the crystal lattice. During charging, Li ions from the Na layer first migrate to the TMO layer before reversing their course at low Na contents. There is little change in the lattice parameters during charging/discharging, indicating stabilization of the P2 structure. This leads to a solid-solution type storage mechanism (sloping voltage profile) and hence excellent cycle life with a capacity of 110 mAh g^-1 after 100 cycles. In contrast, the Li-free compositions Na_0.67Mn_0.6Ni_0.4O₂ and Na_0.67Mn_0.8Ni_0.2O₂ show phase transitions and a stair-case voltage profile. The capacity is found to originate from mainly Ni³⁺/Ni⁴⁺ and O^2-/O^2-δ redox processes by DFT, although a small contribution from Mn⁴⁺/Mn⁵⁺ to the capacity cannot be excluded.     

Novel measurement system for respiratory aerosols and droplets in indoor environments

The SARS-CoV-2 pandemic has created a great demand for a better understanding of the spread of viruses in indoor environments. A novel measurement system consisting of one portable aerosol-emitting mannequin (emitter) and a number of portable aerosol-absorbing mannequins (recipients) was developed that can measure the spread of aerosols and droplets that potentially contain infectious viruses. The emission of the virus from a human is simulated by using tracer particles solved in water. The recipients inhale the aerosols and droplets and quantify the level of solved tracer particles in their artificial lungs simultaneously over time. The mobile system can be arranged in a large variety of spreading scenarios in indoor environments and allows for quantification of the infection probability due to airborne virus spreading. This study shows the accuracy of the new measurement system and its ability to compare aerosol reduction measures such as regular ventilation or the use of a room air purifier.     

Sensitization of MCF7 Cells with High Notch1 Activity by Cisplatin and Histone Deacetylase Inhibitors Applied Together

Histone deacetylase inhibitors (HDIs) are promising anti-cancer agents that inhibit proliferation of many types of cancer cells including breast carcinoma (BC) cells. In the present study, we investigated the influence of the Notch1 activity level on the pharmacological interaction between cisplatin (CDDP) and two HDIs, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), in luminal-like BC cells. The type of drug–drug interaction between CDDP and HDIs was determined by isobolographic analysis. MCF7 cells were genetically modified to express differential levels of Notch1 activity. The cytotoxic effect of SAHA or VPA was higher on cells with decreased Notch1 activity and lower for cells with increased Notch1 activity than native BC cells. The isobolographic analysis demonstrated that combinations of CDDP with SAHA or VPA at a fixed ratio of 1:1 exerted additive or additive with tendency toward synergism interactions. Therefore, treatment of CDDP with HDIs could be used to optimize a combined therapy based on CDDP against Notch1-altered luminal BC. In conclusion, the combined therapy of HDIs and CDDP may be a promising therapeutic tool in the treatment of luminal-type BC with altered Notch1 activity.     

Thermoset IM-LSI-based C/C-SiC: Influence of flow direction and weld lines on microstructure and mechanical properties

The production of ceramic matrix composites (CMC) based on C/C-SiC is still very cost-intensive and therefore only economical for a few applications. The fabrication of the preforms involves many costs that need to be reduced. In this work, the shaping of the CFRP-preforms is realized by thermoset injection molding, which enables large-scale production. The polymeric matrix used is a multi-component matrix consisting of novolak resin, curing agent and lubricant. Six millimeter chopped carbon fiber with a proportion of 50 wt.% were used as a reinforcement. These ingredients are processed by an industrial equipment for compounding and injection molding in order to manufacture a CFRP demonstrator representing a brake disc. Test specimens are cut out of the demonstrator in different directions in order to investigate influences of flow direction and weld lines on microstructural and mechanical properties. Afterward, the CFRP samples were converted to C/C-SiC composites by the liquid silicon infiltration process. The article addresses the flow behavior of the compound during the injection molding and the building of the weld lines in the demonstrator. In addition, results of the directional dependence of the microstructural and mechanical properties within the fabricated disc in the different production steps are presented.     

Hierarchical Coherent Phonons in a Superatomic Semiconductor

The coupling of phonons to electrons and other phonons plays a defining role in material properties, such as charge and energy transport, light emission, and superconductivity. In atomic solids, phonons are delocalized over the 3D lattice, in contrast to molecular solids where localized vibrations dominate. Here, a hierarchical semiconductor that expands the phonon space by combining localized 0D modes with delocalized 2D and 3D modes is described. This material consists of superatomic building blocks (Re₆Se₈) covalently linked into 2D sheets that are stacked into a layered van der Waals lattice. Using transient reflectance spectroscopy, three types of coherent phonons are identified: localized 0D breathing modes of isolated superatom, 2D synchronized twisting of superatoms in layers, and 3D acoustic interlayer deformation. These phonons are coupled to the electronic degrees of freedom to varying extents. The presence of local phonon modes in an extended crystal opens the door to controlling material properties from hierarchical phonon engineering.     

Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.     

Trans-differentiation of human adipose-derived mesenchymal stem cells into cardiomyocyte-like cells on decellularized bovine myocardial extracellular matrix-based films

In this study, we aimed at fabricating decellularized bovine myocardial extracellular matrix-based films (dMEbF) for cardiac tissue engineering (CTE). The decellularization process was carried out utilizing four consecutive stages including hypotonic treatment, detergent treatment, enzymatic digestion and decontamination, respectively. In order to fabricate the dMEbF, dBM were digested with pepsin and gelation process was conducted. dMEbF were then crosslinked with N-hydroxysuccinimide/1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (NHS/EDC) to increase their durability. Nuclear contents of native BM and decellularized BM (dBM) tissues were determined with DNA content analysis and agarose-gel electrophoresis. Cell viability on dMEbF for 3rd, 7th, and 14th days was assessed by MTT assay. Cell attachment on dMEbF was also studied by scanning electron microscopy. Trans-differentiation capacity of human adipose-derived mesenchymal stem cells (hAMSCs) into cardiomyocyte-like cells on dMEbF were also evaluated by histochemical and immunohistochemical analyses. DNA contents for native and dBM were, respectively, found as 886.11?±?164.85 and 47.66?±?0.09?ng/mg dry weight, indicating a successful decellularization process. The results of glycosaminoglycan and hydroxyproline assay, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), performed in order to characterize the extracellular matrix (ECM) composition of native and dBM tissue, showed that the BM matrix was not damaged during the proposed method. Lastly, regarding the histological study, dMEbF not only mimics native ECM, but also induces the stem cells into cardiomyocyte-like cells phenotype which brings it the potential of use in CTE.     

Thermodynamic performance analysis of three solid oxide fuel cell and gas microturbine hybrid systems for application in auxiliary power units

In this study, three different configurations of a solid oxide fuel cell and gas microturbine hybrid system are evaluated for application in auxiliary power units. The first configuration is a common hybrid system in auxiliary power units, utilizing a fuel cell stack in the structure of the gas turbine cycle. The other configurations use two series and parallel fuel cell stacks in the structure of the gas turbine cycle. The main purpose of this research is thermodynamic analysis, evaluation of the performance of the proposed hybrid systems in similar conditions, and selection of an appropriate system in terms of efficiency, power generation, and entropy generation rate. In this study, the utilized fuel cells were subjected to electrochemical, thermodynamic, and thermal analyses and their working temperatures were calculated under various working conditions. Results indicate that the hybrid system with two series stacks had maximum power generation and efficiency compared with the other two cases. Moreover, the simple hybrid system and the system with two parallel stacks had relatively equal pure power generation and efficiency. According to the investigations, hybrid system with two series fuel cell stacks, which had 3424 and 1712 cells, respectively, can achieve the electrical efficiency of over 48%. A hybrid system with two parallel fuel cell stacks, in which each stack had 2568 cells, had the electrical efficiency of 46.3%. Findings suggested that maximum electrical efficiency occurred between the pressure ratios of 5–6 in the proposed hybrid systems.