Preparation of a Single-Cell Suspension from Tumor Biopsy Samples for Single-Cell RNA Sequencing.

An essential requirement for single-cell RNA sequencing in cancer is the preparation of high-quality single-cell suspensions from the tumor tissue. In this work, various methods of dissociation of tumor biopsy specimens were analyzed and developed to obtain a cell suspension with at least 80% viability. It was found that the optimal conditions for sample preparation are mechanical dissociation followed by incubation with a collagenase/hyaluronidase mixture with addition of DNAase I for 60 min. Thus, we optimize the approach for preparing single-cell suspensions from the tumor biopsy tissue for single-cell RNA sequencing.

[Experimental studies of the biomechanical properties of the cornea]. / Eksperimental'nye issledovaniya biomekhanicheskikh svoistv rogovitsy.

The review presents the results of experimental studies of the biomechanical properties of the cornea. Selective evaluation of the individual corneal structures (for example, limiting membranes) using classical mechanical tests is to a certain extent limited due to the rather small thickness of these structures and the related difficulties in sample fixation. In real practice, the use of a method better adapted for conducting such studies - atomic force microscopy (AFM) - remains promising, since on the one hand it eliminates the need for mechanical capture and retention of the sample, and on the other - provides the capability for studying its segments separately.

Pseudodesulfovibrio alkaliphilus, sp. nov., an alkaliphilic sulfate-reducing bacterium isolated from a terrestrial mud volcano.

The diversity of anaerobic microorganisms in terrestrial mud volcanoes is largely unexplored. Here we report the isolation of a novel sulfate-reducing alkaliphilic bacterium (strain F-1T) from a terrestrial mud volcano located at the Taman peninsula, Russia. Cells of strain F-1T were Gram-negative motile vibrios with a single polar flagellum; 2.0-4.0 µm in length and 0.5 µm in diameter. The temperature range for growth was 6-37 °C, with an optimum at 24 °C. The pH range for growth was 7.0-10.5, with an optimum at pH 9.5. Strain F-1T utilized lactate, pyruvate, and molecular hydrogen as electron donors and sulfate, sulfite, thiosulfate, elemental sulfur, fumarate or arsenate as electron acceptors. In the presence of sulfate, the end products of lactate oxidation were acetate, H2S and CO2. Lactate and pyruvate could also be fermented. The major product of lactate fermentation was acetate. The main cellular fatty acids were anteiso-C15:0, C16:0, C18:0, and iso-C17:1ω8. Phylogenetic analysis revealed that strain F-1T was most closely related to Pseudodesulfovibrio aespoeensis (98.05% similarity). The total size of the genome of the novel isolate was 3.23 Mb and the genomic DNA G + C content was 61.93 mol%. The genome contained all genes essential for dissimilatory sulfate reduction. We propose to assign strain F-1T to the genus Pseudodesulfovibrio, as a new species, Pseudodesulfovibrio alkaliphilus sp. nov. The type strain is F-1T (= KCTC 15918T = VKM B-3405T).

[Research of quality indicators of confectionery paste with sunflower flour].

Confectionery products are in constant steady demand among various categories of consumers. This group of foodstuffs has a low nutritional and high energy value, contains a large amount of fats and carbohydrates with an insignificant content of essential micronutrients. In this regard, research on the modernization of recipes and technologies for confectionery products enriched with essential nutrients, especially with the use of secondary products of food processing, remains relevant. The aim of the research was to study quality indicators of sunflower flour and confectionery paste produced from it. Material and methods. The main objects of the study were flour obtained from sunflower oil cake after extracting oil by cold pressing, and confectionery paste based on it. Measurement of mass fractions of moisture, sugar, fat, protein and fiber, determination of amino acid composition and viscosity, organoleptic and safety indicators (the content of toxic elements, mycotoxins and pesticides, sanitary and microbiological indicators) have been carried out. A study of the viscosity of the confectionery paste and rheological parameters have been performed. The shelf life of the finished product was determined using the "accelerated aging" technique, which was confirmed by test data under standard conditions. Results. The study of the chemical composition of sunflower flour showed that the content of protein fluctuate from 39.0 to 45.0%, fiber - from 10.0 to 15.0%, fat - from 7.0 to 12.0%, mass the proportion of moisture wa from 2.0 to 6.0%. The total content of essential amino acids in sunflower flour (6.64 g/100 g) was 16% of the total protein content. Based on the analysis of variance, mathematical models were obtained that described the dependences of the acid and peroxide values of sunflower flour on the processing temperature and the duration of its exposure; it was found that the optimal conditions for heat treatment of flour were the processing temperature - 60 °C, the exposure time - 30 min. The optimal dosage of prescription components has been established based on the organoleptic and rheological characteristics of the confectionery paste. A commodity assessment of the developed confectionery paste was carried out in terms of organoleptic and physic-chemical quality indicators. The studies of safety indicators, including microbiological ones, made it possible to conclude that the paste fully meets the requirements of the Technical Regulations of the Customs Union TR CU 021/2011 "On food safety". To predict the shelf life of the paste in order to save time, the accelerated aging technique at elevated temperature was applied, which includes an analysis of the quality of the sample using the Van't Hoff rule and the Arrhenius equation. The study results were extrapolated to normal storage conditions. A study of the storage of confectionery paste under normal conditions at standard temperature regimes confirmed the results obtained using the accelerated aging technique. Studies of the chemical composition and nutritional value of the developed confectionery paste showed that it contained fats, proteins, which are a source of essential amino acids, and mineral substances. The shelf life of confectionery paste with sunflower flour at a temperature of 20±3 °C and a relative humidity of no more than 75% for no more than 10 months has been established. Conclusion. The complex research carried out has demonstrated that the use of sunflower flour in the production of confectionery paste makes it possible to obtain a product with high consumer characteristics and increased nutritional value.

[Assessment of age-related changes in lens capsule biomechanics using atomic force microscopy]. / Otsenka vozrastnykh izmenenii «biomekhaniki¼ kapsuly khrustalika na osnove atomno-silovoi mikroskopii.

Studies devoted to the assessment of lens capsule biomechanics can be divided into fundamental and applied. The former are oriented towards analysis of various indicators characterizing elasticity of the capsule as a basal membrane that maintains and changes the shape of the lens, and the latter deal with widespread introduction of modern microinvasive methods of phaco surgery into clinical practice. PURPOSE: To assess age-related changes in lens capsule biomechanics based on atomic force microscopy (AFM). MATERIAL AND METHODS: The study included 50 central fragments of the anterior capsule of the human lens obtained intraoperatively during ultrasonic phacoemulsification by continuous circular capsulorhexis. The measurements were carried out in the Fast Force Volume (FFV) mode. The force curves were processed in the Nanoscope Analysis software (Bruker, USA) using the Hertz model that allows calculating the Young's modulus of the capsule sample based on the dependence of the force on the puncture depth. RESULTS: There was no statistically significant difference in the «stiffness¼ of the inner and outer surfaces before and after removal of the subcapsular epithelium (p=0.25). In all cases, the inner surface of the capsule turned out to be «harder¼ than the outer one. In this case, the ratio of Young's modulus of the inner and outer surfaces has a significant dependence on age (p<0.001). With an increase in age from 50 to 90 years, this ratio decreased from ~7 to ~1.5. This was due to a simultaneous change in Young's modulus of the opposite nature: an increase in the stiffness of the outer surface and its decrease in the inner one. CONCLUSION: It is possible to assess lens capsule biomechanics using AFM if the subcapsular epithelium is present. In this case, the objects of study are the areas of the capsule free of epithelium, and the epithelial cells themselves can be used to identify the inner surface of the capsule. Regardless of age, the stiffness of the inner surface of the anterior lens capsule significantly exceeds that of the outer surface.

[Serum adipokines and their receptors in endometrial and colon cancer patients: relationship with tumor invasion and metastasis].

The aim of the study was to investigate the serum adipokine levels and expression of adipokine receptors (AdipoR1, AdipoR2) in patients with endometrial and colon cancer in relation with the main clinical morphological parameters (tumor invasion, lymph node involvement). The study included 60 endometrial cancer patients with I-II Stage and 31 patients with colon cancer (T2-4N0-2M0). Serum adipokine levels, the level of soluble form of the leptin receptor (sOb-R) and AdipoR1 and AdipoR2 expression were evaluated with ELISA. In endometrial cancer serum leptin and adiponectin levels were associated not only with metabolic disorders but also with cervical invasion. In colon cancer serum leptin level was associated with lymph node involvement. The data obtained showed the potential implication of serum adipokines into tumor invasion and metastasis. In both sites intratumoral levels of AdipoR1 H AdipoR2 were not associated with the presence of metabolic syndrome. The AdipoR1 level was related with myometrial invasion. In colon cancer patients, AdipoR1 and AdipoR2 expressions were associated with lymph node involvement, and AdipoR1 expression was correlated with tumor size. The obtained results demonstrated involvement of adipose tissue hormones (leptin and adiponectin) and adiponectin receptors (AdipoR1 and AdipoR2) in tumor growth, invasion and lymphogenic metastasis.

[Morphological identification of blastocystis].

The authors have attempted to systematize the currently known specific morphological features of the composition of Blastocystis spp. existing in different forms and to present this material as a reference table, by understanding the need for further data clarification. In addition, the paper describes observations of variations in the forms of human blastocysts. In particular, it depicts the species of multinucleated cysts, the identification of which may cause difficulties in diagnosing and differentiating these forms with some representative species of the genus Entamoeba.

[Pseudoinvasion and transient carriage of the larvae of arthropods and soil nematodes in humans: problems and consequences. The authors' observations and a literature review].

The paper describes cases of pseudoinvasion and transient carriageof the larvae of arthropods and geohelminths. It highlights the objective and subjective factors in the diagnosis of arthropod-borne diseases and geohelminthiasis, which give rise to misdiagnosis. Distinctions between real invasion, transient invasion, and pseudoinvasion are pointed out. The fact that there are special problems with the misdiagnosis of people with mental diseases is emphasized. The key factors for preventing and diagnosing pseudoinvasions and transient carriage are defined.

[Results of studies of human dirofilariasis in Russia].

Human dirofilariasis is a pressing health problem in Russia. By 2014, there have been as many as 850 Dirofilaria repens-infested people living in 42 subjects of the Russian Federation. One of the favorable factors for circulation and spread of invasion is a temperature of above +20-24 degrees C; when the latter is maintained during at least 20 days there may be 1.-1.5 circulations of invasion in the carrier and a 2.8-fold increase in transmission intensity. The border ofa dirofilariasis area with a temperate climate is southward to 58 degrees N in European Russia and West Siberia and southward to 50 degrees N in the Far East. The conditions in the human body have been found to be more favorable for the development of Dirofilaria than considered before and allow the helminth to achieve sexual maturity and to propagate. If man has microfilaremia, he may be a source of invasion. It is necessary to examine venous blood by the enrichment method and, if possible, polymerase chain reaction and enzyme immunoassay, which make it possible to establish a diagnosis in occult invasion and to identify a pathogen species.

BODIPY Dye Derivative for Irreversible Fluorescent Labeling of Eukaryotic Cells and Their Simultaneous Cytometric Analysis.

In this work, we synthesized a green fluorescent dye derivative, 1,3,5,7-tetramethyl-BODIPY, with a heptyl substituent at the 8-position. The obtained highly hydrophobic compound was able to rapidly and irreversibly bind to eukaryotic cells. Incubation of cells with the dye over different periods of time or at different concentrations allowed us to control the degree of cell labeling and the level of fluorescence. This made it possible to modulate the fluorescence level of different eukaryotic cell cultures and then distinguish them by their level of fluorescence signal in the green channel in cytometric experiments. The labeled cells can be combined and further analyzed in the same test tube under identical conditions using the channels in which the dye does not fluoresce. This approach has been tested on a number of tumor cell cultures containing the HER2 receptor on their surface. The representation of the receptor in these cells was analyzed in one test tube in one run using a HER2-specific ligand based on the hybrid protein DARPin9_29-mCherry, which fluoresces in the red region of the spectrum.

System for Self-excited Targeted Photodynamic Therapy Based on the Multimodal Protein DARP-NanoLuc-SOPP3.

Despite the significant potential of photodynamic therapy (PDT) as a minimally invasive treatment modality, the use of this method in oncology has remained limited due to two serious problems: 1) limited penetration of the excitation light in tissues, which makes it impossible to affect deep-seated tumors and 2) use of chemical photosensitizers that slowly degrade in the body and cause photodermatoses and hyperthermia in patients. To solve these problems, we propose a fully biocompatible targeted system for PDT that does not require an external light source. The proposed system is based on bioluminescent resonance energy transfer (BRET) from the oxidized form of the luciferase substrate to the photosensitizing protein SOPP3. The BRET-activated system is composed of the multimodal protein DARP-NanoLuc-SOPP3, which contains a BRET pair NanoLuc-SOPP3 and a targeting module DARPin. The latter provides the interaction of the multimodal protein with tumors overexpressing tumor-associated antigen HER2 (human epidermal growth factor receptor type II). In vitro experiments in a 2D monolayer cell culture and a 3D spheroid model have confirmed HER2-specific photo-induced cytotoxicity of the system without the use of an external light source; in addition, experiments in animals with subcutaneous HER2-positive tumors have shown selective accumulation of DARP-NanoLuc-SOPP3 on the tumor site. The fully biocompatible system for targeted BRET-induced therapy proposed in this work makes it possible to overcome the following limitations: 1) the need to use an external light source and 2) the side phototoxic effect from aberrant accumulation of chemical photosensitizers. The obtained results demonstrate that the fully protein-based self-excited BRET system has a high potential for targeted PDT.

Biomechanical properties of the lens capsule: A review.

The lens capsule, a thin specialized basement membrane that encloses the crystalline lens, is essential for both the structural and biomechanical integrity of the lens. Knowing the mechanical properties of the lens capsule is important for understanding its physiological functioning, role in accommodation, age-related changes, and for providing a better treatment of a cataract. In this review, we have described the techniques used for the lens capsule biomechanical testing on the macro- and microscale and summarized the current knowledge about its mechanical properties.

Digging deeper: structural background of PEGylated fibrin gels in cell migration and lumenogenesis.

Fibrin is a well-known tool in tissue engineering, but the structure of its modifications created to improve its properties remains undiscussed despite its importance, e.g. in designing biomaterials that ensure cell migration and lumenogenesis. We sought to uncover the structural aspects of PEGylated fibrin hydrogels shown to contribute to angiogenesis. The analysis of the small-angle X-ray scattering (SAXS) data and ab initio modeling revealed that the PEGylation of fibrinogen led to the formation of oligomeric species, which are larger at a higher PEG : fibrinogen molar ratio. The improvement of optical properties was provided by the decrease in aggregates' sizes and also by retaining the bound water. Compared to the native fibrin, the structure of the 5 : 1 PEGylated fibrin gel consisted of homogenously distributed flexible fibrils with a smaller space between them. Moreover, as arginylglycylaspartic acid (RGD) sites may be partly bound to PEG-NHS or masked because of the oligomerization, the number of adhesion sites may be slightly reduced that may provide the better cell migration and formation of continuous capillary-like structures.

[Methodology to forecast risk of toxic effects caused by contemporary household chemicals].

The article contains to forecast risk of toxic effects caused by contemporary household chemicals. The article contains data on evaluating potential risk of health disorders among individuals contacting synthetic detergents and cleansers. The authors summarized experience of Public Health Research center on toxicologic hygienic research of various household chemicals, conducted for safety certification of the end products household application.