Influence of human peripheral blood samples preprocessing on the quality of Hi-C libraries.

The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

Antibodies Against Unusual Forms of Sialylated Glycans.

Previous studies have shown that in the blood of healthy donors (1) there are no natural antibodies against sialylated glycoproteins, which contain Neu5Acα (N-acetylneuraminic acid) as the most widespread form of human sialic acid, and (2) there is a moderate level of antibodies capable of binding unnatural oligosaccharides, where Neu5Ac is beta-linked to a typical mammalian glycan core. In the present study, we investigated antibodies against ßNeu5Ac in more detail and verified the presence of Kdn (2-keto-3-deoxy- D-glycero-D-galacto-nonulosonic acid) as a possible cause behind their appearance in humans, taking into account the expected cross-reactivity to Kdn glycans, which are found in bacterial glycoconjugates in both the α- and ß-forms. We observed the binding of peripheral blood immunoglobulins to sialyllactosamines (where "sialyl" is Kdn or neuraminic acid) in only a very limited number of donors, while the binding to monosaccharide Kdn occurred in all samples, regardless of the configuration of the glycosidic bond of the Kdn moiety. In some individuals, the binding level of some of the immunoglobulins was high. This means that bacterial Kdn glycoconjugates are very unlikely to induce antibodies to ßNeu5Ac glycans in humans. To determine the reason for the presence of these antibodies, we focused on noninfectious pathologies, as well as on a normal state in which a significant change in the immune system occurs: namely, pregnancy. As a result, we found that 2/3 of pregnant women have IgM in the blood against Neu5Acß2-3Galß1-4GlcNAcß. Moreover, IgG class antibodies against Neu5Acß2-3Galß1-4GlcNAcß and Neu5Acß2-6Galß1-4GlcNAcß were also detected in eluates from the placenta. Presumably, these antibodies block fetal antigens.

Optimization of the sperm processing protocol for subsequent molecular cytogenetic studies.

One of the causes of spontaneous pregnancy termination, infertility, and birth of children with development delay and malformations are chromosomal abnormalities (CA) as well as spontaneous aneuploidies in gametes of phenotypically normal parents. Often couples with reproductive problems, as well as spouses one of whom is a carrier of CA, turn to the programs of assisted reproductive technologies (ART) for preimplantation evaluation of the zygote chromosomal status. As part of ART programs, parental gametes are examined to assess the level of spontaneous aneuploidy. As a rule, the most accessible material for analysis is the ejaculate. Fluorescent in situ hybridization (FISH) is used to examine male gametes obtained from the ejaculate. However, this FISH-analysis has a number of limitations and difficulties because of the peculiarities of the sperm head structure, namely the supercondensed state of chromosome chromatin. In order to optimize the FISH protocol, five different protocols were used for pre-hybridization processing of ejaculate samples obtained from nine phenotypically normal men. A comparative analysis of hybridization efficiency showed that the protocol using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a decondensation agent was the most effective for subsequent molecular cytogenetic studies. The developed hybrid protocol combining proteolytic pretreatment, TCEP and thermal decondensation can be used when other protocols for pre-hybridization treatment of ejaculate preparations are not effective.

SNP-Based Chromosomal Microarray Analysis for Detecting DNA Copy Number Variations in Fetuses with a Thickened Nuchal Fold.

The aim of the study was to assess the diagnostic potential of SNP-based chromosomal microarray analysis for detecting pathogenic copies number variations (CNVs) in fetuses with a normal karyotype, in which an increase in the nuchal translucence of >2.5 mm was detected by ultrasound at a gestational age of 11 weeks to 13 weeks 6 days. Materials and Methods: The study included 225 pregnant women who underwent invasive prenatal diagnostic procedures following the detection of an isolated thickening of the fetal nuchal fold. The fetal material obtained was examined using a cytogenetic test; if a normal karyotype was confirmed, chromosomal microarray analysis was performed as a second-line test. Results: Pathogenic CNVs were detected in 22 of 225 fetuses (9.8%) with a normal karyotype. Of these 22 fetuses, pathogenic CNVs not classified as syndromes were detected in 14 cases (63.6%), and those previously described as syndromes - in 8 cases (36.4%). In 9 fetuses (41%), CNVs in two non-homologous chromosomes were determined; these findings indicated a high likelihood of carrying balanced translocations in the parents. Indeed, when analyzing the parent's karyotype, in 8 out of 9 couples, balanced translocations were found in one of the parents. Conclusion: Using chromosomal microarray analysis in fetuses with a thickened nuchal fold makes it possible to increase the ability to detect chromosomal imbalances, including those caused by pathological meiotic segregation of parental reciprocal translocation.

Expression of fucosylated glycans in endothelial glycocalyces of placental villi at early and late fetal growth restriction.

The aim of the study was to investigate the content and distribution of fucosylated sugar residues and Lewis Y (LeY) in the endothelial glycocalyx (eGC) in placental tissue at early and late onset fetal growth restriction (FGR). Our findings demonstrated that the changes of the fucosylated glycans of type 2 (H2)/LeY in the vascular endothelium of the villi may reflect alteration of villi maturation, or adaptation to hypoxia through the change of cell proliferation potential and induction angiogenesis. Early onset FGR differs from late onset FGR by a markedly increased LeY expression, being associated with more severe pathological state.

Isolation of IgG Associated with Human Placenta.

We optimized the method of isolation of antibodies from placental tissue of a conventionally healthy patient. Four protocols of antibody isolation were evaluated and a protocol with tissue grinding (without homogenization) and successive elution of the antibodies with acidic and alkaline buffers was recommended for use. The repertoire of the isolated antibodies was characterized using a glycan array. Partial coincidence of the specificity of the isolated antibodies with antibodies in the peripheral blood was demonstrated, which indicates their possible association with carbohydrate antigens in the placenta. Identification of potential molecular targets of resident antibodies in the placenta is necessary for understanding the mechanisms of formation of immunological tolerance to the fetus.

The Repertoire of Human Antiglycan Antibodies and Its Dynamics in the First Year of Life.

The repertoire of antiglycan antibodies of peripheral blood was studied using a microarray containing 487 glycan antigens: fragments of mammalian glycans (N- and O-chains of glycoproteins, as well as glycolipids) and also bacterial polysaccharides. The sera samples correspond to the third, sixth, and twelfth months of life. The infants were divided into four groups according to their nutrition type: breast milk, standard formula, and partially or extensively hydrolyzed formula. During the first year of life, the total amount of IgG decreased; presumably, the lifetime of maternal IgG in the newborns' bloodstream is much greater than is generally assumed. At the same time, the IgM content was low during the first six months and increased significantly by the twelfth month. The antiglycan IgM repertoire of one-year-old infants was still different from that of their mothers, as well as from the repertoire of unrelated donors, in particular, by the absence of antibodies against the Galß1-3GlcNAc (LeC) disaccharide, which is found in almost all healthy humans. It is noteworthy that the level of IgM of breast-fed infants was significantly lower than that of formula-fed by the twelfth month.

Induced pluripotent stem cell line, ICAGi001-A, derived from human skin fibroblasts of a patient with 2p25.3 deletion and 2p25.3-p23.3 inverted duplication.

Skin fibroblasts from a patient with developmental delay and chromosome 2p25.3 deletion syndrome were reprogrammed into induced pluripotent stem cells (iPSCs) and the clonal stem cell line ICAGi001-A (iTAF9-11) was established. ICAGi001-A pluripotency was demonstrated in vitro by three germ layer differentiation capacity. This line is a good model for studying of the developmental delay and brain disorder.

Human antibodies eluted from ligand-free Sepharose capable of binding bacterial polysaccharides and sulfated glycans.

Sepharose matrix without immobilized ligands binds antibodies from human blood serum or immunoglobulin preparations. The eluted antibodies bind bacterial polysaccharides having no structural similarity to agarose (Sepharose is a cross-linked polysaccharide agarose) with a high affinity. It is concluded that the identified antibodies are capable of recognizing spatial rather than linear epitopes of bacterial polysaccharides. This side activity of Sepharose matrix should be taken into account in isolating target antibodies and other proteins from human blood.

Effect of Lithium Preparations on Cerebral Electrophysiological Activity in Rats.

The study examined the effects of a novel neurotropic medication based on a lithium complex composed of lithium citrate, polymethylsiloxane, and aluminum oxide on electrophysiological parameters of the rat brain. In contrast to lithium carbonate (the reference drug), the novel preparation resulted in a wave-like dynamics of electrical activity in the visual cortex. Rhythmic photic stimulation of the rats treated with lithium carbonate resulted in appearance of the signs attesting to up-regulation of excitability of cerebral cortex in all examined ranges. In contrast, the complex lithium preparation diminished the delta power spectrum, which was the only affected frequency band. It is hypothesized that the complex lithium medication induces milder activation of the cerebral cortex in comparison with lithium carbonate. The novel medication composed of lithium citrate, aluminum oxide, and polymethylsiloxane, is characterized by greater efficacy and safety than the preparation based on inorganic lithium salt (lithium carbonate).

Human tandem-repeat-type galectins bind bacterial non-ßGal polysaccharides.

Galectins are multifunctional effectors, for example acting as regulators of cell growth via protein-glycan interactions. The observation of capacity to kill bacteria for two tandem-repeat-type galectins, which target histo-blood epitopes toward this end (Stowell et al. Nat. Med. 16:295-301, 2010), prompted us to establish an array with bacterial polysaccharides. We addressed the question whether sugar determinants other than ß-galactosides may be docking sites, using human galectins-4, -8, and -9. Positive controls with histo-blood group ABH-epitopes and the E. coli 086 polysaccharide ascertained the suitability of the set-up. Significant signal generation, depending on type of galectin and polysacchride, was obtained. Presence of cognate ß-galactoside-related epitopes within a polysaccharide chain or its branch will not automatically establish binding properties, and structural constellations lacking galactosides, like rhamnan, were found to be active. These data establish the array as valuable screening tool, giving direction to further functional and structural studies.

[Genetic and clinical characteristics of 22q11.2 deletion syndrome].

In a group of 140 patients with typical phenotype, the 22q11.2 microdeletion was detected in 43 patients (32%) using FISH and MLPA methods. There were no deletions of other chromosomal loci leading to phenotypes similar to the 22q11.2 deletion syndrome (22q11.2DS). Sequencing of the TBX1 gene did not detect any mutations, except for some common neutral polymorphisms. For the first time in the Russian Federation, the diagnostic efficiency of 22q11.2DS appeared to be 32%, as a result of the application of a combination of genetic approaches for a large group of patients with suspected 22q11.2DS.

[Generation and application of dynamic standard reference intervals for analyzing results of comparative genomic hybridization].

The present work was aimed at generating the dynamic standard reference intervals (DSRI) and their application for chromosomal-aberration (CA) analysis. The evaluation of the generated DSRI was performed using the DNA samples from four patients with already known CA. High-resolution comparative genomic hybridization analysis (HR-CGH) allowed us to not only identify all of the CAs, that were not revealed by CGH, but also to detect the breakpoints and to determine the size of chromosomal imbalance.

Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens.

The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization.

Hidden X chromosomal mosaicism in a 46,XX male.

We report on a 37-year-old XX male with complex hidden X chromosomal mosaicism. The patient had fully mature male genitalia with hypoplastic testes descended in the scrotum and no sign of undervirilization. Hormonal examination demonstrated hypergonadotropic hypogonadism, semen analysis showed severe oligoasthenoteratozoospermia. In situ hybridization revealed the presence of 3 SRY-positive cell lines bearing 1, 2 or 3 X chromosomes. Skewed inactivation of the paternal SRY-bearing X chromosome was detected by molecular analysis of the androgen receptor gene.

Unique mosaic X/Y translocation/insertion in infant 45,X male.

We report on a 45,X male with hydrocephaly, lobar holoprosencephaly and ichthyosis. In situ hybridization and molecular analysis have demonstrated the presence of a mosaic SRY-bearing derivative X chromosome that included Yp and heterochromatic Yq fragments.

2-Aminopyridine--a label for bridging of oligosaccharides HPLC profiling and glycoarray printing.

2-Aminopyridine derivatives of oligosaccharides (OS-AP) were printed onto microchips by two different ways. The first method is based on direct covalent insertion of OS-AP in polyacrylamide gel 3D chip. The second method is based on conversion of OS-AP into more reactive OS-aminoalditol followed by covalent printing onto NHS-activated glass slides. This approach extends the range of saccharides suitable for covalent printing due to availability of commercial OS-AP and easy high-performance liquid chromatography separation of glycoprotein N-chains in form of AP derivatives.

Design of carbohydrate multiarrays.

Recently, microarray technology has increasingly been widely applied in glycobiology. This technology has rather evident potential advantages: unlimited number of carbohydrate ligands coated onto one small sized chip, enormously low consumption of both carbohydrate ligands and carbohydrate-binding proteins to be tested, etc. Literature data demonstrate that three approaches are used for glycoarray design. The first one is based on the physical adsorption of glycomolecules on a surface (as in a common ELISA), the second one-on covalent immobilization, and the third one-on a streptavidin-biotin system. In all of the described methods, carbohydrate ligands were placed on chips as a 2D monolayer and high sensitivity was achieved due to fluorescent detection. Notably, a tendency of stepping from model chips toward real multiarrays, where the number of carbohydrate ligands can be up to two hundred, has been observed the last 2 years, this already producing a number of interesting findings when studying carbohydrate-binding proteins. In 2005 new construction, 3D glycochip was described, where 150 mum diameter polyacrylamide gel elements serve as microreactors instead of 2D dots. As a result of the 3D placement of a ligand, two orders of magnitude increase of its density is possible, this providing principal signal improvement during fluorescent detection and increasing method sensitivity. At the same time, carbohydrate consumption is low, i.e., approximately 1 pmol per gel element. Copolymerization chemistry enables the immobilization of several glycomolecule classes to the gel, in particular, aminospacered oligosaccharides, polyacrylamide conjugates, and even 2-aminopyridine derivatives of oligosaccharides, which are widely used in the structural analysis of glycoprotein N-chains.

Non-starch polysaccharides as correctors in cytostatic therapy of experimental tumors.

We studied the effects of low-esterified pectin, calcium pectate, and alginate on the development of transplanted Ehrlich adenocarcinoma and Lewis pulmonary carcinoma and on the efficiency of cyclophosphamide treatment in mice.

Analysis of cell-free fetal DNA in plasma and serum of pregnant women.

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.