Technology of Two-dimensional Bioimpedance Analysis of the Human Body Composition.

The BIA primary result sheets as a rule contain one-dimensional graphical scales with a selected area of normal values. In 1994, Piccoli et al. proposed BIVA, an alternative form of BIA data presentation, where two bioimpedance parameters are considered simultaneously as tolerance ellipses: resistance and reactance normalized to height. The purpose of this study is to develop an approach to data analysis in body composition bioimpedance research in two-dimensional representations. The data of 1.124.668 patients aged 5 to 85 years who underwent a bioimpedance study in Russian Health Centers from 2009 to 2015 were used. Statistical programming in the R Studio environment was carried out to estimate two-dimensional distribution densities of pairs of body composition parameters for each year of life. The non-Gaussian distribution is found in most parameters of bioimpedance analysis of body composition for most ages (Lilliefors test, p-value << 0.0001). The slices of the actual two-dimensional distribution pairs of body composition parameters had an irregular shape. The authors of the article propose using the actually observed distribution for populations where numerous bioimpedance studies have already been carried out. Such technology can be called two-dimensional bioimpedance analysis of human body composition (2DBIA). The 2DBIA approach is clearer for practitioners and their patients due to the use of body composition parameters in addition to electrical impedance parameters.

[Research of the inactivation of pathogens in water under exposure to low temperature plasma].

There was investigated the effect of barrier and spark discharge low temperature plasma on water containing the cells of Escherichia coli (Escherichia coli), hay bacillus (Bacillus subtilis) and yeast (Saccharomyces cerevisiae). There was shown a general decline in the concentration of viable microbial cells after the treatment of suspensions. There was especially marked the detrimental effect of the method on the viability of sanitary-indicative coliform bacteria in the water.

Age-dependent and 'pathologic' changes in ICG waveforms resulting from superposition of pre-ejection and ejection waves.

Impedance cardiography (ICG) is a popular bioimpedance application used for the non-invasive evaluation of the left ventricular stroke volume and contractility. It implies the correct determination of ejection start and end points and the amplitudes of certain peaks in a differentiated impedance cardiogram. An accurate identification of ejection onset by ICG is often problematic, especially in cardiologic patients, due to the peculiar character of the waveforms. A simple theoretical model was employed to test the consequences of the hypothesis that two major processes can contribute to the formation of an impedance systolic wave: (1) the pre-ejection changes in heart geometry and the surrounding vessels produced by ventricular contraction during the isovolumic phase, and (2) the expansion of aorta and adjacent arteries during the ejection per se. The former process initiates the pre-ejection wave while the latter triggers the ejection wave, both of which contribute to the impedance pulse waves associated with the heartbeats. A new two-bell model predicts a potential mechanism responsible for the abnormal shapes of ICG derivative dZ/dt due to the presence of the pre-ejection waves and explains the related errors in systolic time intervals and amplitude parameters derived from such ICG waveforms. It also advances an alternative viewpoint on the nature of the dZ/dt B-point notch. An appropriate decomposition method opens a promising way to avoid the masking effects of these waves and to correctly determine the onset of ejection as well as the corresponding peak amplitudes from the 'pathologically shaped' ICG signals.

[Oxidative activity of electrochemically synthesized sodium persulfate solutions against some psychotropic agents].

By using some psychotropic agents as an example, investigations of the oxidative activity of electrochemically synthesized sodium persulfate solutions were continued. The derivatives of phenothiazines, xanthene, and dibenzazepines were shown to be oxidized by synthesized sodium persulfate solution to low-toxic products. Oxidation products were ascertained to coincide with the known products of their biotransformation in the body.

Expression of the chimeric IgE gene in cell culture and in various mouse tissues.

Transient expression of recombinant gene constructs is now more widely used in gene therapy as well as in DNA vaccination. In this study, the ability of one and the same genetic construct to drive gene expression both in cell culture and in tissues of the whole organism was demonstrated. Chinese hamster ovarian cells (CHO) were transfected in vitro with plasmids bearing the genes for chimeric IgE (mouse/human) antibodies under control of the human cytomegalovirus (hCMV) promoter. Secretion of recombinant IgE antibodies by transfected cells reached 60% of the intracellular concentration of antibodies. The same gene constructs were introduced into various mouse tissues using ballistic transfection in vivo. The IgE content in blood after transfection of cartilage was found to be several times lower than after transfection of the liver, spleen, or foot pad. At the same time, the content of antibodies to the xenogenous determinants of IgE was essentially independent of the tissue type. These data can be employed in selecting conditions for genetic immunization and gene therapy.

[The BCL-xL and ACR-1 genes promote differentiation and reduce apoptosis in muscle fibers of mdx mice]. / Geny BCL-xL i ACR-1 sposobstvuiut differentsirovke i umen'shaiut gibel' myshechnykh volokon myshei mdx.

The effects of the human BCL-xL and ACR-1 genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. Introduction of the dystrophin minigene (20 micrograms of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 +/- 1.4% and 4.5 +/- 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 micrograms of each plasmid per shot), the death of CSMF decreased to 3.7 +/- 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 +/- 2.2%. Contransfection with the dystrophin minigene and the BCL-xL gene at 20 micrograms of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 +/- 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 micrograms of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 +/- 0.5% and significantly reduced the proportion of dying CSMF to 0.9 +/- 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human-dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xL or ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.

[Gene hACR-1 suppresses apoptosis of striated muscle fibres of m. quadriceps femoris after ballistic transfection of mdx mice]. / Gen hACR-1 podavliaet apoptoz poperechnopolosatykh myshechnykh volokon m.quadriceps femoris myshei mdx pri ballisticheskoi transfektsii.

Human minidystrophin gene (pSG5dys plasmid) and hACR-1 gene (pRc-CMV-10.1 plasmid) were cotransfected by means of "gene-gun" to M. quadriceps femoris of mdx mice. Effects of transfection on dystrophin expression and survival of striated muscle fibres (SMF) were studied on the 21st day after shots. In the control mdx dystrophin-positive muscular fibers [D(+)] SMF and destroyed SMF made 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. In mice transfected with pSG5dys plasmid (20 mkg of DNA per mouse), the shares of D(+) SMF and dead SMF raised, respectively, up to 5.6 +/- 1.4 and 4.5 +/- 0.9%. Transfection of mice with pRc-CMV-10.1 (DNA dose is 20 mkg per mouse) reduced the levels of apoptosis in SMF and D(+) SMF level to 1.6 +/- 0.6 and 1.1 +/- 0.4%, respectively. Cotransfection by pSG5dys and pRc-CMV-10.1 plasmids (10 and 10 mkg of each plasmids DNA per mouse) reduced the share of D(+) SMF to 1.1 +/- 0.5% and SMF destruction to 0.9 +/- 0.3%. pSG5dys transfection considerably reduced the share of SMF having peripherally located nuclei, thus indicating a decrease in SMF differentiation level after transfection. Cotransfection of ACR-1 gene and a dystrophin minigene did not suppress further cytodifferentiation of mdx muscle fibers. A conclusion is made that ballistic transfection by hACR-1 gene reduces the level of apoptosis in mdx mice SMF without changing the level of SMF differentiation. The cotransfection of mdx mice muscle by hACR-1 and human minidystrophin gene reduces SMF destruction and supports SMF differentiation, too.

The quantitative characteristics of efficiency of ballistic transfection of chimeric antibody genes.

A quantitative approach was applied to the study of in vivo expression of foreign genes introduced into mice by ballistic transfection. Because in some cases one must take into account both the level of synthesized protein and that of antibodies to it, we derived the equation which allows to calculate the exact quantity of both proteins. This formula was applied to in vivo expression of a chimeric (human/mice) immunoglobulin E gene. The immunochemical analysis using this equation showed that the Ig concentration succeeded 4, 6, 12 IU/ml and undetectable level, respectively, upon transfection in mouse liver, spleen, foot pad and ear cartilage.

[Activity of exogenous genetic designs, introduced into cells by a ballistic transfection method, in murine ontogenesis]. / Aktivnost' ékzogennykh geneticheskikh konstruktsii, vvedennykh v kletki metodom ballisticheskoi transfektsii, v ontogeneze myshi.

We used the method of particle bombardment (ballistic transfection) to introduce beta-galactosidase and human dystrophin genes into mouse embryos and skeletal muscles of adult mice. We examined the mechanisms of DNA transfer into skeletal muscle cells, the biological processes accompanying and following this transfer, the susceptibility of various types of muscle cells to transfection, and the duration of expression of and conditions affecting the introduced genes. We have also developed an effective, convenient, and practical methods of skeletal muscles transfection.

[Expression of the human dystrophin gene in mdx mouse skeletal muscles after ballistic transfection]. / Ekspressiia gena distrofina cheloveka v skeletnykh myshtsakh myshei mdx posle ballisticheskoi transfektsii.

"Gene-gun" ballistic transfection (BT) was used to deliver genetic constructs pMLVDy and pHSADy containing full-length cDNA of the dystrophin gene to musculus quadriceps remoris and musculus gluteus of mdx mice, which represent a natural model of Duchenne muscular dystrophy. Clusters of dystrophin-positive muscular fibers (DPMF) were immunocytochemically detected in sites exposed to BT. The average number of DPMF was 2% by the 17th day and 3% by the 60th day after BT with pMLVDy, whereas the number of revertant DPMF was 0.2% in control mice (without BT). When pHSADy was used, the average number of DPMF was 3% 20 days after BT. In this case, dystrophin was uniformly spread though the myoplasm in 3% of cells and produced a slight signal in separate regions under the sarcolemma in 10% of muscle fibers. The number of revertant DPMF increased to 0.6% after BT with naked particles and to 2.8% after BT with the marker lacZ gene, in both bombarded and contralateral legs. The number of DPMF in the corresponding muscles of the contralateral leg significantly increased and reached 2.8% by the 60th day after BT with pMLVDy and 6.7% by the 20th day after BT with pHSADy. Human dystrophin gene cDNA was detected in all skeletal muscles, heart, intestine, tongue, and brain by polymerase chain reaction (PCR) three weeks after BT. Immunoblot analysis showed that normal 427-kDa human dystrophin was synthesized in muscles of mdx mice. The results suggest applicability of BT for delivery of dystrophin constructs into muscles.

[Differentiation of muscle fibers in mdx mice after ballistic transfection of cDNA of the human dystrophin gene]. / Differentsirovka myshechnykh volokon myshei MDX posle ballisticheskoi transfektsii kDNK gena distrofina cheloveka.

Changes in morphological dimensions of MDX mouse myofibres in M. rectus femoris were recorded after ballistic transfection (BT) with pHSADys and pVMMDys plasmids containing cDNA of the full-length human dystrophin gene. The dystrophin expression was observed by an immunomorphological procedure with P6 antibody and PAP method. Dystrophin positive (dyst+) myofibres were divided into two types, with a typical dystrophin expression under sarcoplasma membrane and an atypical expression through the whole sarcoplasm, respectively. The share of atypical dyst+ myofibres was seen to rise during the experiment from 27%, at 2-3 weeks after BT, up 84% by 2 months after BT. The atypical dyst+ myofibres usually underwent destruction. At the same time, the share of entire dyst+ myofibres decreased from 17 to 2-5% by 2 months. Morphological dimensions of the myofibres (square in mkm2, perimeter, smallest and largest diameters) were calculated with the help of computer analyser. The middle square of both types of dyst+ myofibres was larger than that of dyst- myofibres, both in BT target M. rectus femoris and in the same contralateral muscle, but never exceeded the value of middle square of C57B1 mouse myofibres in the same muscle. The form of dyst+ myofibres was not modified by the dystrophin expression. The nuclei of dyst+ myofibres remained in the central region of sarcoplasm. A conclusion is made that BT of human dystrophin gene inside MDX mouse myofibres allows dystrophin gene expression and enlargement of the dyst+ myofibres. Dystrophin expression is not able to induce a complete and stable differentiation of striated muscle of adult MDX mice.

Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection.

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.

[The ballistic transfection of mammalian cells in vivo]. / Ballisticheskaia transfektsiia kletok mlekopitaiushchikh in vivo.

The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Having penetrated in the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized and this allowed to study mammalian cells in situ. The studies performed allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ and in developing mouse embryos at the stages of two-cell embryo, morula and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was found within two-three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail and the published data on transfection and genetic transformation of mammalian cells are discussed.

[Case of hemangioendothelioma of peritoneal cavity organs]. / Sluchai gemangioéndoteliomy organov briushnoi polosti.

A 47-year-old male has been suffering for a long time from abdominal hemangioendothelioma diagnosed postmortally. The tumor is described clinically and morphologically.

Transfer of foreign DNA into the cells of developing mouse embryos by microprojectile bombardment.

Mouse cells of developing embryos at the 2-4 cell, morula and blastocyst stages, were bombarded by high velocity tungsten microprojectiles. About 70% of developing embryos survived the bombardment. The general embryo structure did not change as a result of the bombardment. Penetration of the tungsten microparticles into the embryo cell nuclei was found at all stages being investigated, and tungsten particle localization on mitotic chromosomes was demonstrated. The total DNA of the mice born from the bombarded embryos was analyzed by dot-blot hybridization and PCR with post-hybridization. The most important results were obtained in experiments with blastocysts. In three cases of blastocyst bombardment, the presence of transferred plasmid DNA (pSV3-neo) was revealed. Transfected cells were shown to be located in the fetal membrane as well as in the embryo. The bombardment of mouse culture cells resulted in their transfection and the production of G418-resistant clones.

[Use of high velocity mechanical injection for the transfer of foreign DNA into early mouse embryos]. / Ispol'zovanie vysokoskorostnoi mekhanicheskoi in"ektsii dlia perenosa chuzherodnoi DNK v rannie émbriony myshei.

The possibility of high velocity mechanical transfer of foreign DNA into inner cell mass of mouse blastocyst was shown. Penetration of tungsten microparticles into early embryo cell nuclei and their localization on mitotic chromosomes was demonstrated. About 70% of developing embryos survived the bombardment. Total DNA of the mice born from bombarded embryos was analyzed by blot-hybridization and PCR with Southern hybridization. In three cases, the presence of the transferred plasmid DNA (pSV3-neo) was revealed.

The delivery of foreign genes into fertilized fish eggs using high-velocity microprojectiles.

Fertilized eggs of loach (Misgurnus fossilis), rainbow trout (Salmo gairdneri) and zebrafish (Brachydanio rerio) were bombarded with high-velocity tungsten microprojectiles covered with plasmid DNA containing sequences of beta-galactosidase and neomycin phosphotransferase genes. About 70% of the eggs survived the bombardment. The activity of both transferred genes was revealed in the fish developed from the bombarded eggs. Neomycin phosphotransferase gene sequences were detected by means of PCR amplification and Southern hybridization in the total DNA of zebrafish that survived after G418 treatment.

High-velocity mechanical DNA transfer of the chloramphenicolacetyl transferase gene into rodent liver, kidney and mammary gland cells in organ explants and in vivo.

Mouse and rat liver, kidney and mammary gland explants were bombarded with high-velocity microprojectiles carrying a chloramphenicolacetyl transferase gene under different promoters (pTAT-cat, p chi-Casein-cat, p beta-Casein-cat). The expression of a CAT gene was revealed in all organ explants 24 h after transfection. The most pronounced expression was found when a TAT-CAT construction was used. In experiments in vivo rat liver was bombarded in situ with microprojectiles carrying pTAT-cat DNA. A marked activity of the CAT gene was detected 24 h after the bombardment.