The use of efficient and inexpensive substrates (2D matrices) for cultivation and differentiation of nerve cells in vitro is important for the creation of tissue engineering constructs intended for the treatment of nervous system pathologies. Recombinant analogues of the orb-weaver spider dragline-silk proteins spidroins 1 and 2 appear promising in addressing this task. The aim of the study was to evaluate the effect of cell substrates derived from mixtures of recombinant spidroins (RS) rS1/9 and rS2/12 with hybrid proteins (HP) containing rS1/9 monomer fused with biologically active peptides on gene expression levels of key synapse-specific proteins and viability of the human neuroblastoma SH-SY5Y cell line during directed cholinergic differentiation. A two-stage scheme of directed cholinergic differentiation of SH-SY5Y cells using retinoic acid and brain-derived neurotrophic factor (BDNF) was implemented. Cell viability was assessed via MTT assay and crystal violet staining. The mRNA levels of the studied genes were assessed by real-time PCR. Directed differentiation of the SH-SY5Y cells was marked by a significant increase in the gene expression levels of synaptophysin, synapsins I and II, and the postsynaptic protein PSD-95. The highest cell viability and increased PSD-95 expression levels were observed during differentiation on a matrix consisting of RS rS1/9 and rS2/12 mixed with the RGDS peptide (present in extracellular matrix proteins) and heparin-binding peptide (HBP, laminin fragment) containing HPs. The highest efficiency during the differentiation of the SH-SY5Y cells was demonstrated by a matrix consisting of the mixture of RS rS1/9 and rS2/12 and a HP made up by RS rS1/9 monomer fused with RGDS (the ligand of integrins) and HBP (the ligand of growth factors and syndecans). Matrices consisting of RS rS2/12 alone or the mixture of rS2/12 with HP(RGDS) showed lower efficiency, although the use of the GRGGL peptide (which interacts with the neural cell adhesion molecules and is a component of RS rS1/9) led to an increase in efficiency.

The production and transplantation of functionally active human neurons is a promising approach to cell therapy. Biocompatible and biodegradable matrices that effectively promote the growth and directed differentiation of neural precursor cells (NPCs) into the desired neuronal types are very important. The aim of this study was to evaluate the suitability of novel composite coatings (CCs) containing recombinant spidroins (RSs) rS1/9 and rS2/12 in combination with recombinant fused proteins (FP) carrying bioactive motifs (BAP) of the extracellular matrix (ECM) proteins for the growth of NPCs derived from human induced pluripotent stem cells (iPSC) and their differentiation into neurons. NPCs were produced by the directed differentiation of human iPSCs. The growth and differentiation of NPCs cultured on different CC variants were compared with a Matrigel (MG) coating using qPCR analysis, immunocytochemical staining, and ELISA. An investigation revealed that the use of CCs consisting of a mixture of two RSs and FPs with different peptide motifs of ECMs increased the efficiency of obtaining neurons differentiated from iPSCs compared to Matrigel. CC consisting of two RSs and FPs with Arg–Gly–Asp–Ser (RGDS) and heparin binding peptide (HBP) is the most effective for the support of NPCs and their neuronal differentiation.

Recombinant spidroins (RS; the analogues of silk proteins of spider’s web) have multiple properties beneficial for bioengineering, including their suitability for electrospinning and thus, for production of materials with oriented fibers. This makes RS-based matrices potentially effective in stimulating regeneration of peripheral nerves. The restoration of injured nerves also depends on prompt regrowth of blood vessels. Therefore, prospective scaffold materials for neuro-regenerative therapy should positively affect both the nerves and the blood vessels. Currently, the experimental models suitable for culturing and quantitative assessment of the vascular and neuronal cells on the same material are lacking. Here, we assessed the suitability of electrospun RS-based matrices for cultivation of the mouse aorta and dorsal root ganglia (DRG) explants. We also quantified the effects of matrix topography upon both types of tissues. The RS-based materials have effectively supported aortic explants survival and sprouting. The cumulative length of endothelial sprouts on rS1/9-coated inserts was significantly higher as compared to type I collagen coatings, suggesting stimulatory effects on angiogenesis in vitro. In contrast to matrices with random fibers, on matrices with parallel fibers the migration of both smooth muscle and endothelial cells was highly oriented. Furthermore, alignment of RS fibers effectively directs the growth of axons and the migration of Schwann cells from DRGs. Thus, the electrospun RS matrices are highly suitable to culture both, the DRGs and aortic explants and to study the effects of matrix topography on cell migration. This model has a high potential for further endeavor into interactions of nerve and vascular cells and tissues.

The interaction of neural progenitor cells (NPCs) with the extracellular matrix (ECM) plays an important role in neural tissue regeneration. Understanding which motifs of the ECM proteins are crucial for normal NPC adhesion, proliferation, and differentiation is important in order to create more adequate tissue engineered models of neural tissue and to efficiently study the central nervous system regeneration mechanisms. We have shown earlier that anisotropic matrices prepared from a mixture of recombinant dragline silk proteins, such as spidroin 1 and spidroin 2, by electrospinning are biocompatible with NPCs and provide good proliferation and oriented growth of neurites. This study objective was to find the effects of spidroin-based electrospun materials, modified with peptide motifs of the extracellular matrix proteins (RGD, IKVAV, and VAEIDGIEL) on adhesion, proliferation, and differentiation of directly reprogrammed neural precursor cells (drNPCs). The structural and biomechanical studies have shown that spidroin-based electrospun mats (SBEM), modified with ECM peptides, are characterized by a uniaxial orientation and elastic moduli in the swollen state, comparable to those of the dura mater. It has been found for the first time that drNPCs on SBEM mostly preserve their stemness in the growth medium and even in the differentiation medium with brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, while addition of the mentioned ECM-peptide motifs may shift the balance toward neuroglial differentiation. We have demonstrated that the RGD motif promotes formation of a lower number of neurons with longer neurites, while the IKVAV motif is characterized by formation of a greater number of NF200-positive neurons with shorter neurites. At the same time, all the studied matrices preserve up to 30% of neuroglial progenitor cells, phenotypically similar to radial glia derived from the subventricular zone. We believe that, by using this approach and modifying spidroin by various ECM-motifs or other substances, one may create an in vitro model for the neuroglial stem cell niche with the potential control of their differentiation.

Simple, rapid, and efficient techniques of DNA isolation from a wide range of organisms are currently in demand in biotechnology and bioinformatics. Cell-wall destruction, as well as the subsequent DNA extraction from the disintegrated cells, is key (and often limiting) step. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: the treatment of cells or tissue samples with ammonium acetate, followed by cell lysis in low-salt buffer with added SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for the isolation of large amounts of high-molecular native DNA from bacteria, ascomycetes, yeasts, and mammalian blood; it is also useful for the express analysis of environmental microbial isolates and for plasmid extraction in two-hybrid library screening.

It has been shown that recombinant spidroins rS1/9 and rS2/12 that we previously developed exhibit adhesive properties with respect to both inorganic and organic substrates. It is well known that the adhesive properties of mussel foot proteins are associated with the level of DOPA, which is formed as a result of the post-translational modification of tyrosine residues by the tyrosinase enzyme. Therefore, we used recombinant tyrosinase for in vitro modification of tyrosine residues in the recombinant rS1/9 and rS2/12 spidroins to increase their adhesion capacity. As expected, the conversion of tyrosine residues into DOPA led to an increase in the adhesion properties of these proteins, which was demonstrated in experiments on gluing plates of polyvinyl chloride, aluminum, polylactic acid, and tubular pork bone. The molecules of recombinant spidroins retained their inherent properties to form supramolecular structures, hydrogels (microgels), transparent films, and 3D matrices. Interestingly, tyrosinase-modified proteins exhibited increased cohesion in experiments on bonding different materials in the presence of water.

A dataset of four draft genome sequences of Bifidobacterium strains is presented. All four genome assemblies are high-quality drafts characterized by high completeness and low contamination levels. GC content of the genomes varied in the range between 59.27% and 62.77%. Genome sequences were annotated for further functional and taxonomical analyses of the respective Bifidobacterium strains. Genetic determinants of probiotic capabilities, including the genes, related to utilization of human milk oligosaccharides and mucin, as well as the genes, encoding bile salt hydrolase were identified. The genome of B. bifidum VKPM=Ac-1784 has been shown to possess two bacteriocin gene clusters. The dataset expands knowledge on genomic diversity of probiotic strains of Bifidobacterium genus. The dataset is available under PRJNA656137 accession number in NCBI database and under zyv26t6x5r accession number in Mendeley Data repository.

We have designed a novel two-component matrix (SPRPix) for the encapsulation of directly reprogrammed human neural precursor cells (drNPC). The matrix is comprised of 1) a solid anisotropic complex scaffold prepared by electrospinning a mixture of recombinant analogues of the spider dragline silk proteins – spidroin 1 (rS1/9) and spidroin 2 (rS2/12) - and polycaprolactone (PCL) (rSS-PCL), and 2) a “liquid matrix” based on platelet-rich plasma (PRP). The combination of PRP and spidroin promoted drNPC proliferation with the formation of neural tissue organoids and dramatically activated neurogenesis. Differentiation of drNPCs generated large numbers of βIII-tubulin and MAP2 positive neurons as well as some GFAP-positive astrocytes, which likely had a neuronal supporting function. Interestingly the SPRPix microfibrils appeared to provide strong guidance cues as the differentiating neurons oriented their processes parallel to them. Implantation of the SPRPix matrix containing human drNPC into the brain and spinal cord of two healthy Rhesus macaque monkeys showed good biocompatibility: no astroglial and microglial reaction was present around the implanted construct. Importantly, the human drNPCs survived for the 3 month study period and differentiated into MAP2 positive neurons. Tissue engineered constructs based on SPRPix exhibits important attributes that warrant further examination in spinal cord injury treatment.

We studied the effect of non-thermal argon plasma on proliferative activity of bone marrow multipotent stromal cells in vitro . Treatment of stromal cell suspension with pure argon did not affect their proliferation. The cells treated with non-thermal argon plasma and explanted in the treatment medium demonstrated growth inhibition by 30-40% in comparison with the control. Multipotent stromal cells treated with plasma and after centrifugation explanted in normal medium within 12 min demonstrated accelerated growth. The total cell growth from the pellet and supernatant significantly exceeded the control values. We also analyzed adhesion and proliferative activity of multipotent stromal cells treated with non-thermal plasma on bioresorbable carriers. The cells adhered and proliferated on all types of studied samples. Adhesion properties of scaffolds differed. Caprolactone was found to be the most suitable material for adhesion and proliferation of multipotent stromal cells.

Employing optical spectroscopy we have performed a comparative study of the dielectric response of extracellular matrix and filaments of electrogenic bacteria Shewanella oneidensis MR-1, cytochrome c, and bovine serum albumin. Combining infrared transmission measurements on thin layers with data of the terahertz spectra, we obtain the dielectric permittivity and AC conductivity spectra of the materials in a broad frequency band from a few cm−1 up to 7000 cm−1 in the temperature range from 5 to 300 K. Strong absorption bands are observed in the three materials that cover the range from 10 to 300 cm−1 and mainly determine the terahertz absorption. When cooled down to liquid helium temperatures, the bands in Shewanella oneidensis MR-1 and cytochrome c reveal a distinct fine structure. In all three materials, we identify the presence of liquid bound water in the form of librational and translational absorption bands at ≈ 200 and ≈ 600 cm−1, respectively. The sharp excitations seen above 1000 cm−1 are assigned to intramolecular vibrations.

For decades respiratory chain and photosystems were the main firing field of the studies devoted to mechanisms of electron transfer in proteins. The concept of conjugated lateral electron and transverse proton transport during cellular respiration and photosynthesis, which was formulated in the beginning of 1960-s, has been confirmed by thousands of experiments. However, charge transfer in recently discovered bacterial nanofilaments produced by various electrogenic bacteria is regarded currently outside of electron and proton conjugation concept. Here we report the new study of charge transfer within nanofilaments produced by Shewanella oneidensis MR-1 conducted in atmosphere of different relative humidity (RH). We utilize impedance spectroscopy and DC (direct current) transport measurements to find out the peculiarities of conductivity and Raman spectroscopy to analyze the nanofilaments' composition. Data analysis demonstrates that apparent conductivity of nanofilaments has crucial sensitivity to humidity and contains several components including one with unusual behavior which we assign to electron transport. We demonstrate that in the case of Shewanella oneidensis MR-1 charge transfer within these objects is strongly mediated by water. Basing on current data analysis of conductivity we conclude that the studied filaments of Shewanella oneidensis MR-1 are capable of hybrid (conjugated) electron and ion conductivity.

The electrodynamics of metals is well understood within the Drude conductivity model; properties of insulators and semiconductors are governed by a gap in the electronic states. But there is a great variety of disordered materials that do not fall in these categories and still respond to external field in an amazingly uniform manner. At radiofrequencies delocalized charges yield a frequency-independent conductivity σ 1(ν) whose magnitude exponentially decreases while cooling. With increasing frequency, dispersionless conductivity starts to reveal a power-law dependence σ 1(ν)∝ν s with s < 1 caused by hopping charge carriers. At low temperatures, such Universal Dielectric Response can cross over to another universal regime with nearly constant loss ε″∝σ1/ν = const. The powerful research potential based on such universalities is widely used in condensed matter physics. Here we study the broad-band (1–1012 Hz) dielectric response of Shewanella oneidensis MR-1 extracellular matrix, cytochrome C and serum albumin. Applying concepts of condensed matter physics, we identify transport mechanisms and a number of energy, time, frequency, spatial and temperature scales in these biological objects, which can provide us with deeper insight into the protein dynamics.

Ac conductivity and dielectric permittivity of bovine serum albumin, cytochrome c and extracellular matrix synthesized by electrogenic bacteria Shewanella oneidensis MR-1 are measured at frequencies 1 - 10(12) Hz and temperatures 10-300 K. Two types of conductivity mechanisms are observed as well as nearly constant loss (NCL) phenomenon, universal dielectric response (UDR) and signatures of boson peak depending on the temperature and water/ion contents in the samples.

The fermentation of a Saccharomyces cerevisiae strain producing recombinant spidroin IF9 was optimized. A simplified two-stage scheme of the process was developed; the effect of sucrose, glucose, fructose, and galactose on the efficiency of the process was investigated. The optimal concentration of sucrose in the medium and replenishment was determined. The influence of peptone, tryptone, and casein hydrolysate of various brands on the effectiveness of the fermentation was analyzed. The optimal concentrations of peptone and yeast extract in the medium and replenishment were determined. As a result, owing to the optimization, the process of fermentation was simplified, a new composition of a complex replenishment was designed, and sucrose-specific consumption was reduced by two times, whereas the expenses of peptone and yeast extract were decreased by about 2.5 times, the yield of biomass per unit of the culture broth volume grew by ∼ 40%, and that of the protein of interest increased by ∼ 60%.

In the present study, we examined the ability of the recombinant spidroin to serve as a substrate for the cardiac tissue engineering. For this purpose, isolated neonatal rat cardiomyocytes were seeded on the electrospun spidroin fiber matrices and cultured to form the confluent cardiac monolayers. Besides the adhesion assay and immunostaining analysis, we tested the ability of the cultured cardiomyocytes to form a functional cardiac syncytium by studying excitation propagation in the cultured tissue with the aid of optical mapping. It was demonstrated that recombinant spidroin fiber meshes are directly suitable for the adherence and growth of the cardiomyocytes without additional coating with the attachment factors, such as fibronectin.

By means of terahertz and infrared (THz, IR) spectroscopies we have measured the dielectric response of nano-filaments of electrogenic bacteria Shewanella oneidensis MR-1 in a wide frequency and temperature ranges. THz-far-IR spectra are dominated by absorption due to bound water. At helium temperatures and at sub-THz frequencies a boson-peak-like excitation is detected that is typical for disordered materials. The observation is in agreement with the heat capacity measurements.