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).

[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.

[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.

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.