Diagnostic value of microbiome biomarkers of the periodonite microbiome in patients with the association of chronic periodontitis and diabetes mellitus type 2.

The place of high-tech methods of molecular biology in clinical laboratory diagnostics of various diseases and the development of a system of biomarkers as an important component of diagnostic research is currently attracting the closest attention of the scientific community. In this paper, an attempt is made to use high-tech metagenomic analysis to solve problems that arise due to the high frequency of association of periodontal diseases with systemic pathology, in particular, with type 2 diabetes mellitus. The aim of the study was to determine the taxonomic and metabolic features of the microbiome of periodontal tissues in periodontal diseases associated with type 2 diabetes mellitus, as a model of the ratio of local and systemic effects of periodontal pathogenic bacteria. The study included 16S shotgun sequencing of bacterial DNA as part of biological material from periodontal pockets/dentoalveolar furrows of 46 people - 15 patients with chronic periodontitis associated with type 2 diabetes mellitus, 15 patients with chronic periodontitis unrelated to systemic pathology, as well as 16 healthy people in the control group, followed by bioinformatic processing of the data obtained. The obtained data allowed us to establish the taxonomic features of the periodontal microbiome in the association of chronic periodontitis with type 2 diabetes mellitus, which included the predominance of representatives of the families Prevotellaceae and Spirochaetaceae in its composition. The features of metabolic processes in periodontal tissues with the participation of the microbiome were also revealed, which consisted in an increase in the exchange of cysteine and methionine against the background of a decrease in the metabolism of pyrimidine, methane, sphingolipids, and the synthesis of fatty acids, which are of diagnostic value in assessing the condition of patients with type 2 diabetes mellitus.

KrioBlast TM as a New Technology of Hyper-fast Cryopreservation of Cells and Tissues. Part I. Thermodynamic Aspects and Potential Applications in Reproductive and Regenerative Medicine.

Kinetic (dynamic) vitrification is a promising trend in cryopreservation of biological materials because it allows avoiding the formation of lethal intracellular ice and minimizes harmful effects of highly toxic penetrating cryoprotectants. A uniform cooling protocol and the same instruments can be used for practically all types of cells. In modern technologies, the rate of cooling is essentially limited by the Leidenfrost effect. We describe a novel platform for kinetic vitrification of biological materials KrioBlast TM that realizes hyper-fast cooling and allows overcoming the Leidenfrost effect. This opens prospects for creation of a novel technology of cell cryopreservation for reproductive and regenerative medicine.

KrioBlast® as a New Technology of Hyper-fast Cryopreservation of Cells and Tissues. Part II. 2. Kinetic Vitrification of Human Pluripotent Stem Cells and Spermatozoa [corrected].

Pilot experiments on kinetic vitrification of human pluripotent stem cells and spermatozoa using a KrioBlastTM-2 without penetrating cryoprotectants have shown high survival of cells (75-85% in both cases).

Correction to: KrioBlastTM as a New Technology of Hyper-fast Cryopreservation of Cells and Tissues. Part II. 2. Kinetic Vitrification of Human Pluripotent Stem Cells and Spermatozoa.

The title of the article should be «KrioBlastTM as a New Technology of Hyper-fast Cryopreservation of Cells and Tissues. Part II. 2. Kinetic Vitrification of Human Pluripotent Stem Cells and Spermatozoa¼.

Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report.

The aims of this investigation were to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryo-injuries. Spermatozoa were isolated and vitrified using three different media: Group 1: standard buffer for fish spermatozoa, Cortland(®) medium (CM, control); Group 2: CM + 1% BSA + 40% seminal plasma; and Group 3: CM + 1% BSA + 40% seminal plasma + 0.125 m sucrose. For cooling, 20-µl suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM + 1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility and cytoplasmic membrane integrity with SYBR-14/propidium iodide staining technique. Motility (86%, 81% and 82% for groups 1, 2 and 3, respectively) (P > 0.1) was not decreased significantly. At the same time, cytoplasmic membrane integrity of spermatozoa of Groups 1, 2 and 3 was changed significantly (30%, 87% and 76% respectively) (P < 0.05). All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility. However, cytoplasmic membrane integrity was maximal in Group 2 (CM + 1% BSA + 40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA + 40% seminal plasma.

Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose.

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mul suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 degrees C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1-1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1+/-3.2%, P<0.05) when compared with controls (19.4+/-1.9%). The combination of HSA and sucrose (65.2+/-2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6+/-4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.

Factors affecting yield and survival of cells when suspensions are subjected to centrifugation. Influence of centrifugal acceleration, time of centrifugation, and length of the suspension column in quasi-homogeneous centrifugal fields.

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.

Influence of centrifugation regimes on motility, yield, and cell associations of mouse spermatozoa.

Mouse sperm are exceptionally sensitive to mechanical forces associated with pipetting and mixing. This characteristic raised the question of the sensitivity of mouse sperm to centrifugation, a step necessary in the removal of cryoprotectants and a common component in the general manipulation of sperm suspensions for experimental purpose. Epididymal spermatozoa from ICR mice were isolated and manipulated to minimize pipetting and mixing damage. The centrifugal accelerations studied were 200, 400, 600, and 800 x g (measured with a stroboscope) for 5, 10, or 15 minutes of centrifugation time. The number of cells and the number of motile cells were counted. The percent motility and longevity, total yield, and motile yield were calculated. Centrifugation at 200 and 400 x g for short times (5 minutes) caused only a small loss in either immediate or 2.5-hour motility, but centrifugation at 600 and 800 x g for 15 minutes produced up to a fivefold loss. Low speed/short time centrifugation pelleted only about half of the cells; the others were lost when the supernatant was removed. The maximum number of motile sperm (motile yield) was obtained at intermediate centrifugal forces (approximately 400 x g for 10-12 minutes), and it is the total number of motile sperm (and not the percent motility) that is important in the use of cryopreserved sperm to regenerate cryopreserved mutant lines. Relative centrifugal force and centrifugation time exhibit reciprocity (e.g., 200 x g for 10 minutes produces similar results to 400 x g for 5 minutes). The spermatozoa must be centrifuged under carefully defined conditions to minimize the damage and to maximize the recovery of viable cells.

Do conventional CASA-parameters reflect recovery of kinematics after freezing? CASA paradox in the analysis of recovery of spermatozoa after cryopreservation.

Conventional kinematic parameters (KPs) are averages of values obtained from analysing the entire motile fraction of cells in a sample. Occasionally, in spite of overall deterioration of semen samples after cryopreservation and other 'damaging' manipulations, the average relative (derived) KPs such as linearity (LIN), dance (DNC), dance mean (DNM) etc., may show apparently elevated values. Similarly, the absolute (actual) KPs such as straight line velocity (VSL), curvilinear velocity (VCL), average velocity path (VAP), average lateral head displacement (ALH), beat-cross frequency (BCF), etc., may also be higher after damaging treatments depending on the tolerance of various sub-populations in a single sample. The conditions for this CASA-paradox are discussed. Simple modifications of actual CASA-parameters are proposed to correct the pseudo-enhancement of kinematics characteristics in processed semen samples

[Effect of cholesterol on the stability of human erythrocyte membranes to electric breakdown]. / Vliianie kholesterina na ustoichivost' membran éritrotsitov cheloveka k élektricheskomu proboiu.

Electrical stability of human erythrocyte membranes with different cholesterol content was studied. Breakdown in the cell membranes was generated by application of electric pulses with field strengths of 1.4-3.2 kV/cm. The share of perforated cells was registered by measuring hemolysis level. The red blood cells from patients with psoriasis and normal erythrocytes after incubation in the presence of liposomes were used as a model of cells with cholesterol-rich membranes. It was discovered that an increase of cholesterol content in the membranes moved the field-dependent curves to a higher field range. The obtained effect is attributed to the increase of the breakdown membrane potential. Application of high-pulse-electric-field technique for investigating the properties of cell membranes is discussed.

A two-parameter model of cell membrane permeability for multisolute systems.

A two-parameter model of cell osmotic response (F. W. Kleinhans, 1998, Cryobiology 37, 271-289) is expanded for multisolute systems. The cell water volume W and intracellular osmolalities of N solutes are related as W[1 + L(p)RTSigma(N)(i=1)(M(i)/P(i))] = W(0)[1 + L(p)RTSigma(N)(i=1)(M(0)i)/P(i))], where M(i) is the intracellular osmolality of the ith solute (i = 1 ellipsis N), P(i) is the membrane permeability of the ith solute, L(p) is the membrane hydraulic conductivity, R is the gas constant, T is the absolute temperature, and the subscript "0" denotes the initial values at time zero. The above formula allows calculating the final (equilibrium) volume when all entities are permeable. Simple algebraic expressions for calculation of the number and magnitude of transient maximum volume excursions are presented. These simple expressions can all be calculated by hand on a pocket calculator. Practical examples of one-, two-, and three-solute systems are discussed. Special attention has been given to situations when systems contain an impermeable component. All formulas are simple to use for optimization of variety of cryobiological protocols. Application of the theory for optimization of addition and dilution of a permeable cryoprotectant is also discussed.

Mouse spermatozoa in high concentrations of glycerol: chemical toxicity vs osmotic shock at normal and reduced oxygen concentrations.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have identified several likely underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (0.3 M) than the approximately 1 M concentrations that are optimal for most cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have identified two contributors to damage from higher concentrations of glycerol, namely, chemical toxicity proportional to concentration and exposure time and osmotic damage arising from too rapid an addition and removal of the glycerol. When toxicity is minimized by restricting the exposure time to 1 or 5 min and osmotic shock is minimized by adding and removing the glycerol stepwise, relatively high percentages of the sperm survive contact with 0.8 M glycerol. Free-radical damage in mouse sperm is known to be proportional to the oxygen concentration. We have determined the consequences of reducing the oxygen to <3% of atmospheric by the use of a bacterial membrane preparation, Oxyrase. Oxyrase reduced damage from centrifugation and substantially reduced damage from osmotic shock; however, it did not significantly reduce glycerol toxicity.

The enhancement of the ability of mouse sperm to survive freezing and thawing by the use of high concentrations of glycerol and the presence of an Escherichia coli membrane preparation (Oxyrase) to lower the oxygen concentration.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have hypothesized several underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (<0.3 M) than the approximately 1 M concentrations that are optimal for most mammalian cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have been able to obtain high motilities in 0.8 M glycerol provided that the exposure time is held to approximately 5 min to minimize toxicity and provided that the glycerol is added and removed stepwise to minimize osmotic shock. Since free radical damage in mouse sperm is proportional to the oxygen concentrations, we have determined the consequences of reducing the oxygen to <3% of atmospheric by maintaining the sperm in contact with an Escherichia coli membrane preparation, Oxyrase, from the moment of collection throughout the assessment of motility. Prior studies have shown that the procedure significantly reduces damage from centrifugation and osmotic shock. In the experiments reported here we obtained approximately 50% motility relative to untreated controls when suspensions containing 3.8% Oxyrase were exposed approximately 5 min to a solution of 0.8 M glycerol and 0.17 M (10%) raffinose in a supplemented PBS and then frozen at approximately 25 degrees C/min to -75 degrees C. In the absence of Oxyrase, the normalized motility dropped to 31%. The protection by Oxyrase was in part a consequence of minimizing centrifugation damage, but in part it reflected a reduction in freeze-thaw damage. Preliminary experiments indicate that the number of motile sperm after cryopreservation in Oxyrase is higher when the sperm are collected without swim-up than when they are collected by swim-up. This is in part due to the fact that more cells are collected in the absence of swim-up and in part due to a greater protective effect of Oxyrase on those cells. The minimum temperature in these initial experiments was limited to -75 degrees C to avoid the potential contribution of other injurious factors between -75 and -196 degrees C.

DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification.

BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000 degrees K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.