Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

Mesoscopic Conductance Fluctuations in 2D HgTe Semimetal.

Mesoscopic conductance fluctuations were discovered in a weak localization regime of a strongly disordered two-dimensional HgTe-based semimetal. These fluctuations exist in macroscopic samples with characteristic sizes of 100 µm and exhibit anomalous dependences on the gate voltage, magnetic field, and temperature. They are absent in the regime of electron metal (at positive gate voltages) and strongly depend on the level of disorder in the system. All the experimental facts lead us to the conclusion that the origin of the fluctuations is a special collective state in which the current is conducted through the percolation network of electron resistances. We suppose that the network is formed by fluctuation potential whose amplitude is higher than the Fermi level of electrons due to their very low density.

Effect of Collagen Matrix on Doxorubicin Distribution and Cancer Cells' Response to Treatment in 3D Tumor Model.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells' metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.

FLIM for Evaluation of Difference in Metabolic Status between Native and Differentiated from iPSCs Dermal Papilla Cells.

iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells.

Publisher Correction: Ballistic geometric resistance resonances in a single surface of a topological insulator.

In the original version of this Article, the second and third sentences of the first paragraph of the "Gate voltage and antidot period dependencies" section of the Results originally incorrectly read "The characteristic evolution of the sheet resistance ρ□=ρ□ (B=0) with Vg shown for three antidot samples and an unpatterned reference sample in Fig. 3a. The maxima of ρxx, located between Vg~0.5 and 1 V, reflect the charge neutrality point (CNP), corresponding to an EF position located slightly in the valence band (see band structure in Fig. 3b)." In the corrected version, "[Formula: see text]" is replaced by "[Formula: see text]", and "The maxima of [Formula: see text]" is replaced by "The maxima of [Formula: see text]".

Ballistic geometric resistance resonances in a single surface of a topological insulator.

Transport in topological matter has shown a variety of novel phenomena over the past decade. Although numerous transport studies have been conducted on three-dimensional topological insulators (TIs), study of ballistic motion and thus exploration of potential landscapes on a hundred nanometer scale is for the prevalent TI materials almost impossible due to their low carrier mobility. Therefore, it is unknown whether helical Dirac electrons in TIs, bound to interfaces between topologically distinct materials, can be manipulated on the nanometer scale by local gates or locally etched regions. Here we impose a submicron periodic potential onto a single surface of Dirac electrons in high-mobility strained mercury telluride (HgTe), which is a strong TI. Pronounced geometric resistance resonances constitute the clear-cut observation of a ballistic effect in three-dimensional TIs.

Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein.

A xyloglucanase of the GH74 family was identified in the thermophilic fungus strain Myceliophthora thermophila VKPM F-244, and its gene sequence was optimized for cloning and expression in Pichia pastoris. The recombinant xyloglucanase MtXgh74 exhibited the highest activity toward tamarind seed xyloglucan with a K M value of 0.51 ± 0.06 mg/mL. The activities on barley ß-glucan and carboxymethylcellulose were about 4 and 2%, respectively, compared to xyloglucan. Maximum xyloglucanase activity was observed at 70-75 °C and pH 6.5. After pre-incubation at 50 °C, pH 6.0 for 3 h, the enzyme retained 100% of its activity. The half-life of MtXgh74 at 60 °C, pH 6.0 was 40 min. In P. pastoris, MtXgh74 was produced in glycosylated form. The enzyme production in a 1 L bioreactor resulted in a yield of 118 U/mL or 5.3 g/L after 51 h fermentation. Kinetic studies of the hydrolysis product formation suggest that MtXgh74 has an endo-processive mode of action. The final products were the standard xyloglucan building blocks XXXG, XXLG, XLXG, and XLLG. Additionally, MtXgh74 hydrolyzed various linkages within the xyloglucan building blocks XXXG, XXLG, and XLXG (except XLLG) producing diverse low molecular weight oligosaccharides which may be identified by MALDI-TOF as XG, XX, XXG/GXX/XGX, XXX, LG, LX/XL, XLX/XXL, LLG, GXXXG, GXLLG, XLLGX. The unique combination of different activities within one enzyme along with its high thermostability and specificity toward xyloglucan makes MtXgh74 a promising candidate enzyme for industrial applications.