Strategic guidelines for the development of enterprises of the construction sector

The current trend of globalization of the world economy necessitates the use of high-tech developments and innovations that allow achieving strategic goals at the national, regional, and sectoral levels. The prerequisites of the study are determined by the urgency of finding solutions to problematic issues of formation and implementation of priority strategic guidelines for the development of enterprises of the construction sector, designed to ensure an adequate contribution to the strategic vector of advanced industrial, technological and socio-economic development of the construction industry and the national economy. This determines the need to find a solution to the problem of forming and implementing priority strategic guidelines for the development of enterprises mainly by increasing technological and innovative potentials that form the economic potential of the development of enterprises by the type of activity "Construction". The purpose of the study is to identify strategic guidelines for the development of enterprises of the construction sector that meet the targets of the fourth scientific and technological revolution and the achievement of strategic goals for the development of national economies. The findings of the paper outline the key signs of development, inherent in the nature of the development of material objects and economic entities of the economy are revealed. This allowed us to propose a systematization of the formation of priority strategic guidelines for the economic development of construction enterprises, reflecting the relationship with the targets for achieving national goals and strategic objectives for the development of economies of various countries and meeting the targets of the fourth scientific and technological revolution Industry 4.0. The practical implications refer to enterprises of the construction sector

Cadmium SAD phasing at CuKα wavelength [version 1; peer review: 2 approved]

Single-wavelength anomalous diffraction (SAD) is the most common method for de novo elucidation of macromolecular structures by X-ray crystallography. It requires an anomalous scatterer in a crystal to calculate phases. A recent study by Panneerselvam et al. emphasized the utility of cadmium ions for SAD phasing at the standard synchrotron wavelength of 1 Å. Here we show that cadmium is also useful for phasing of crystals collected in-house with CuKα radiation. Using a crystal of single-domain antibody as an experimental model, we demonstrate how cadmium SAD can be conveniently employed to solve a CuKα dataset. We then discuss the factors which make this method generally applicable

Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm2. Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.</p

Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm2. Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.</p

Insights into the mechanisms of light‐oxygen‐voltage domain color tuning from a set of high‐resolution X‐ray structures

Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins

Targeting ErbB3 Receptor in Cancer with Inhibitory Antibodies from Llama

The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli, as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3+ cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC50 in the range of 0.1–25 μg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies

Rational Design of a Split Flavin-Based Fluorescent Reporter

Protein-fragment complementation assays are used ubiquitously for probing protein–protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from Chloroflexus aggregans, can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aβ-Bβ, Eα-Fα, and Hβ-Iβ, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent in vivo in E. coli and human cells and have low background fluorescence. Our results enable probing protein–protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools

Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans

Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2◦ C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Dataset from "Revealing the Oligomerization of Channelrhodopsin-2 in the Cell Membrane using Photo-Activated Localization Microscopy" (wtChR2-mEos3.2)

This dataset contains raw microscopy data from the article "Revealing the Oligomerization of Channelrhodopsin-2 in the Cell Membrane using Photo-Activated Localization Microscopy" (doi.org/10.1101/2023.05.24.542088) Zip file contains PALM measurements of HEK293 cells expressing ChR2WT fused with mEos3.2. Each folder in .zip file contains one PALM measurement (TIFF format) and recording settings (.xml) automatically created by acquiring software. For technical reasons, single measurements are divided into TIFF files of =<4088 frames. Detailed protocols for sample preparation and data acquisition are described in the article. Some details are listed below. SAMPLE PREPARATION cell line: Flp-In™ T-REx™ 293 transfection method: modified calcium-phosphate transient transfection [1] fixation method: 4% paraformaldehyde solution in PBS for 30 min at room temperature imaging buffer: PBS PALM ACQUISITION A custom-built setup for single-molecule localization microscopy is described in [2]. mEos3.2 was simultaneously photoconverted, imaged and photobleached by gradually increasing UV illumination (405 nm; up to 1 mW at the sample) and continuous excitation at 561 nm (approximately 50 mW at the sample). PALM movies of cell plasma membrane were recorded in total internal reflection fluorescence (TIRF) mode using an EMCCD camera: exposure time = 100 ms frame size = 512x512 pixels pixel size = 80 nm [1] Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Molecular and cellular biology. 1987 Aug 1;7(8):2745-52. [2] Tang Y, Dai L, Zhang X, Li J, Hendriks J, Fan X, Gruteser N, Meisenberg A, Baumann A, Katranidis A, Gensch T. SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy. Scientific reports. 2015 Jun 22;5(1):1107