CRISPR/Cas9 genome editing demonstrates functionality of the autoimmunity-associated SNP rs12946510.

Genome-wide association studies (GWAS) map genetic associations of complex traits with precision limited to a linkage disequilibrium group. To translate GWAS results into new understanding of disease mechanisms, individual causative polymorphisms and their target genes should be identified. CRISPR/Cas9 genome editing can be used to create isogenic cell lines bearing alternative genotypes of candidate single-nucleotide polymorphisms to test their causality and to reveal gene targets. An intergenic polymorphism rs12946510 is associated with multiple sclerosis, inflammatory bowel disease and asthma. We created sublines of the T-helper cell line bearing alternative genotypes of rs12946510 and showed that its risk ("T") allele is associated with lower expression of IKZF3 and ORMDL3 genes and reduced cell activation. Our editing procedure can become an effective tool for discovering new genes involved in pathogenesis of complex diseases.

Prenatal Stress and Adaptive Behavior of Offspring: The Role of Placental Serotonin.

The effect of mild prenatal stress in mice, leading to an increase in the placental serotonin level, on the formation of adaptive behavior in male offspring at the age of 35 days was studied. It was shown that, in BalbC mice, daily immobilization for 1 h during the period from 11 to 14 days of pregnancy led to an increase in placental and fetal serotonin levels on the 15th day of prenatal development. According to "resident-intruder" behavioral test, the prenatally stressed mice showed more reactive behavior in adulthood and low tendency to defend their territory. Thus, placental serotonin, formed under the stress condition, may act as a mediator between the environment and the fetuses and determine the adaptive behavior of offspring.

[The difficulties of cytologic diagnostic of breast diseases based on trepanobiopsy samples.]

The article analyses, on the basis of scientific publications and original data, the causes of erroneous cytological conclusion concerning samples of trepanobiopsies of mammary gland. The most prevalent benign affections that are characterized by most frequent hyper diagnostic of cancer are fibro-adenoma with proliferation of epithelium and sclerosing adenosis. At the same time, certain modifications of lobular carcinoma of mammary gland, tubular, papillary cancer as well as highly differentiated invasive carcinoma of nonspecific type from cells with ulterior nuclear atypism sometimes it is difficult to diagnose at the cellular level. The sensitivity of cytological diagnostic of pathology of mammary gland using samples of trepanobiopsies makes up 97.5%, specificity - 98.5%. The number of false positive cytological conclusions about presence of malignant tumor makes up to 0.6%, false negative - 1.5%. The reliability of cytological analysis makes up to 97.4%, efficiency - 96.3%.

Expression of components of the serotonergic system in the developing rat thymus.

The developing thymus of rat fetuses contains all components of the serotonergic system: receptors, enzymes of synthesis, and membrane transporters. The expression of receptors suggests the possibility of a direct influence of serotonin on thymic development. The presence of tryptophan hydroxylase (the key rate-limiting enzyme of serotonin synthesis) and aromatic l-amino acid decarboxylase indicates the ability of fetal thymic cells to synthesize serotonin. It was shown that the cells of a developing thymus can actively uptake extracellular monoamines. The results of this study suggest different functions of the intrathymic and circulating serotonin pools in the regulation of thymic development.

The inhibition of dopamine synthesis in fetuses changes the pattern of T-lymphocyte maturation in the thymus of adult rats.

The mRNA for dopamine receptors of type D1, D3, D5, but not type D2, was detected in the thymus of rats starting from day 16 of embryonic development (E16). Dopamine at concentrations of 10-8-10‒6 M inhibited fetus thymocyte response to mitogen, confirming the functionality of the receptors and the possibility of a direct effect of dopamine on the developing thymus. Pharmacological inhibition of catecholamine synthesis in the crucial period of thymus development leads to long-term changes in the T-system immunity due to increased production of natural regulatory T-lymphocytes. The presence and functional activity of dopamine receptors in the fetal thymus indicates its ability to influence the development of the immune system of rats during ontogeny.

[Reciprocal Humoral Regulation of Endocrine Noradrenaline Sources in Perinatal Development of Rats].

The goal of the present study was to verify our hypothesis of humoral interaction between the norepinephrine secreting organs in the perinatal period of ontogenesis that is aimed at the sustaining of physiologically active concentration of norepinephrine in blood. The objects of the study were the transitory organs, such as brain, organ of Zuckerkandl, and adrenals, the permanent endocrine organ of rats that releases norepinephrine into the bloodstream. To reach this goal, we assessed the adrenal secretory activity (norepinephrine level) and activity of the Zuckerkandl's organ under the conditions of destructed noradrenergic neurons of brain caused by (1) their selective death induced by introduction of a hybrid molecular complex, which consisted of antibodies against dopamine-ß-hydroxylase (DBH) conjugated with saporin cytotoxin (anti-DBH-saporin) into the lateral brain ventricles of neonatal rats; and (2) microsurgical in utero destruction of embryo's brain (in utero encephalectomy). It was observed that 72 h after either pharmacological or microsurgical norepinephrine synthesis deprivation in the newborn rat's brain, the level of norepinephrine was increased in adrenals and, conversely, decreased in the Zuckerkandl's organ. Therefore, the experiments with models of chronical inhibition of norepinephrine synthesis in prenatal and early postnatal rat's brain revealed changes in the secretory activity of peripheral norepinephrine sources. This, apparently, favors the sustaining of physiologically active norepinephrine level in the bloodstream.

Vital function of PRELI and essential requirement of its LEA motif.

Proteins containing the late embryogenesis abundant (LEA) motif comprise a conserved family, postulated to act as cell protectors. However, their function and mechanisms of action remain unclear. Here we show that PRELI, a mammalian LEA-containing homolog of yeast Ups1p, can associate with dynamin-like GTPase Optic Atrophy-1 (OPA1) and contribute to the maintenance of mitochondrial morphology. Accordingly, PRELI can uphold mitochondrial membrane potential (ΔΨ(m)) and enhance respiratory chain (RC) function, shown by its capacity to induce complex-I/NADH dehydrogenase and ATP synthase expression, increase oxygen consumption and reduce reactive oxygen species (ROS) production. PRELI can also inhibit cell death induced by STS, TNF-α or UV irradiation. Moreover, in vitro and in vivo dominant-negative overexpression of mutant PRELI/LEA(-) (lacking the LEA motif) and transient in vitro PRELI-specific knockdown can render lymphocytes vulnerable to apoptosis, cause mouse embryo lethality and revert the resistance of lymphoma cells to induced death. Collectively, these data support the long-presumed notion of LEA protein-dependent mechanisms of cytoprotection and suggest that PRELI interacts with OPA1 to maintain mitochondria structures intact, sustain balanced ion(-)/proton(+) gradients, promote oxidative phosphorylation reactions, regulate pro- and antiapoptotic protein traffic and enable cell responses to induced death. These findings may help to understand how bioenergetics is mechanistically connected with cell survival cues.

Immune proteasomes in the developing rat thymus.

The age dynamics of the content of the immune proteasome subunits LMP2 and LMP7 in rat thymus during prenatal and early postnatal ontogeny was studied. The LMP2 and LMP7 immune subunits were detected by Western blotting already by the 18th day of embryonic development, their amount increased to the 21st day to the level characteristic of the postnatal state. Double immunofluorescent labeling showed that in the thymus tissue the largest amount of LMP2 and LMP7 is localized in epithelial cells, whereas the level of their expression in thymocytes is lower. The results suggest that the establishment in thymus of selection processes, which depend on activity of immune proteasomes, can take place already in prenatal ontogeny. Analysis of age dynamics of the natural apoptosis level in thymocytes also favors this supposition. The presence of immune proteasomes in thymocytes during perinatal ontogeny suggests that, besides the antigen presentation, immunoproteasomes may possess other important functions.

Loss of AP-2alpha results in deregulation of E-cadherin and MMP-9 and an increase in tumorigenicity of colon cancer cells in vivo.

Activator protein-2 (AP-2) is a transcription factor that regulates proliferation and differentiation in mammalian cells and has been implicated in the acquisition of the metastatic phenotype in several types of cancer. Herein, we examine the role of AP-2alpha in colon cancer progression. We provide evidence for the lack of AP-2alpha expression in the late stages of colon cancer cells. Re-expression of the AP-2alpha gene in the AP-2alpha-negative SW480 colon cancer cells suppressed their tumorigenicity following orthotopic injection into the cecal wall of nude mice. The inhibition of tumor growth could be attributed to the increased expression of E-cadherin and decreased expression and activity of matrix-metalloproteinase-9 (MMP-9) in the transfected cells, as well as a substantial loss of their in vitro invasive properties. Conversely, targeting constitutive expression of AP-2alpha in AP-2-positive KM12C colon cancer cells with small interfering RNA resulted in an increase in their invasive potential, downregulation of E-cadherin and increased expression of MMP-9. In SW480 cells, re-expression of AP-2alpha resulted in a fourfold increase in the activity of E-cadherin promoter, and a 5-14-fold decrease in the activity of MMP-9 promoter, indicating transcriptional regulation of these genes by AP-2alpha. Chromatin immunoprecipitation assay showed that re-expressed AP-2alpha directly binds to the promoter of E-cadherin, where it has been previously reported to act as a transcriptional activator. Furthermore, chromatin immunoprecipitation assay revealed AP-2alpha binding to the MMP-9 promoter, which ensued by decreased binding of transcription factor Sp-1 and changes in the recruitment of transcription factors to a distal AP-1 element, thus, contributing to the overall downregulation of MMP-9 promoter activity. Collectively, our data provide evidence that AP-2alpha acts as a tumor suppressor gene in colon cancer..

Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells.

One of the major targets for breast cancer therapy is the epidermal growth factor receptor (EGFR) and related receptors, which signal via different signal transduction pathways including the mitogen-activated protein kinase (MAPK) pathway. This study determined whether there is a correlation between EGFR/HER2 status and MAPK (ERK1/2) phosphorylation in breast cancer cells, and how this affects the response to an inhibitor of the receptors. Expression of EGFR, HER2 and phosphorylated ERK1/2 were measured by immunoblotting in a panel of breast cancer cell lines. Several lines expressed high levels of pERK1/2, with no obvious correlation with the level of EGFR/HER2. The EGFR tyrosine kinase inhibitor PKI166 inhibited growth and induced apoptosis in some cells with high levels of growth factor receptors (MDA-MB-468, SUM149, SKBR3), but was less effective in cells that also had high basal ERK1/2 activity (MDA-MB-231). The combination of an inhibitor of MAPK signalling (U0126) and PKI166 produced significantly more inhibition and apoptosis than either agent alone. This suggests that constitutive activation of the MAPK pathway may bypass inhibition of EGFR/HER2 tyrosine kinases, and lead to insensitivity to agents targeting the receptors. However, inhibiting both EGFR/HER2 and MAPK signalling can result in significant growth inhibition and apoptosis of EGFR-expressing breast cancer cells.

Dopamine synthesis by non-dopaminergic neurons expressing individual complementary enzymes of the dopamine synthetic pathway in the arcuate nucleus of fetal rats.

This study was aimed to test our hypothesis about dopamine (DA) synthesis by non-DAergic neurons expressing individual complementary enzymes of the DA synthetic pathway in cooperation, i.e. L-dihydroxyphenylalanine (L-DOPA) synthesized in tyrosine hydroxylase (TH)-expressing neurons is transported to aromatic L-amino acid decarboxylase (AADC)-expressing neurons for conversion to DA. The mediobasal hypothalamus of rats at the 21st embryonic day was used as an experimental model because it contains mainly monoenzymatic TH neurons and AADC neurons (>99%) whereas the fraction of bienzymatic (DAergic) neurons does not exceed 1%. The fetal substantia nigra containing DAergic neurons served as a control. DA and L-DOPA were measured by high performance liquid chromatography in: (1) cell extracts of the cell suspension prepared ex tempora; (2) cell extracts and incubation medium after the static incubation of the cell suspension with, or without exogenous L-tyrosine; (3) effluents of the incubation medium during perifusion of the cell suspension in the presence, or the absence of L-tyrosine. Total amounts of DA and L-DOPA in the incubation medium and cell extracts after the static incubation were considered as the indexes of the rates of their syntheses. L-Tyrosine administration caused the increased L-DOPA synthesis in the mediobasal hypothalamus and substantia nigra. Moreover, L-tyrosine provoked an increase of DA synthesis in the substantia nigra and its decrease in the mediobasal hypothalamus. This contradiction is most probably explained by the L-tyrosine-induced competitive inhibition of the L-DOPA transport to the monoenzymatic AADC-neurons after its release from the monoenzymatic TH neurons. Thus, this study provides convincing evidence of cooperative DA synthesis by non-DAergic neurons expressing TH or AADC in fetal rats at the end of the intrauterine development.