Analysis of circadian pattern reveals tissue-specific alternative transcription in leptin signaling pathway

*Background*
It has been previously reported that most mammalian genes display a circadian oscillation in their baseline expression. Consequently, the phase and amplitude of each component of a signal transduction cascade has downstream consequences. 

*Results*
We report our analysis of alternative transcripts in the leptin signaling pathway which is responsible for the systemic regulation of macronutrient storage and energy balance. We focused on the circadian expression pattern of a critical component of the leptin signaling system, suppressor of cytokine signaling 3 (SOCS3). On an Affymetrix GeneChip 430A2 microarray, this gene is represented by three probe sets targeting different regions within the 3’ end of the last exon. We demonstrate that in murine brown adipose tissue two downstream 3’ probe sets experience circadian baseline oscillation in counter-phase to the upstream probe set. Such differences in expression patterns are a telltale sign of alternative splicing within the last exon of SOCS3. In contrast, all three probe sets oscillated in a common phase in murine liver and white adipose tissue. This suggests that the regulation of SOCS3 expression in brown fat is tissue specific. Another component of the signaling pathway, Janus kinase (JAK), is directly regulated by SOCS and has alternative transcript probe sets oscillating in counter-phase in a white adipose tissue specific manner.
 
*Conclusion*
We hypothesize that differential oscillation of alternative transcripts may provide a mechanism to maintain steady levels of expression in spite of circadian baseline variation

Network Landscape from a Brownian Particle's Perspective

Given a complex biological or social network, how many clusters should it be decomposed into? We define the distance $d_{i,j}$ from node $i$ to node $j$ as the average number of steps a Brownian particle takes to reach $j$ from $i$. Node $j$ is a global attractor of $i$ if $d_{i,j}\leq d_{i,k}$ for any $k$ of the graph; it is a local attractor of $i$, if $j\in E_i$ (the set of nearest-neighbors of $i$) and $d_{i,j}\leq d_{i,l}$ for any $l\in E_i$. Based on the intuition that each node should have a high probability to be in the same community as its global (local) attractor on the global (local) scale, we present a simple method to uncover a network's community structure. This method is applied to several real networks and some discussion on its possible extensions is made.Comment: 5 pages, 4 color-figures. REVTeX 4 format. To appear in PR

Microvesicles derived from leukocytes in the peripheral blood of patients with external genital endometriosis

Endometriosis is a chronic gynecological disease, which poses a serious problem in terms of diagnosis and treatment. Despite decades of research, there are no specific signs and symptoms and no blood tests to clinically confirm the diagnosis, which makes timely diagnosis and treatment difficult. Therefore, the search for new markers for early non-invasive diagnosis of the disease remains relevant. Various subcellular structures involved in intercellular communication, in particular, microvesicles, can be considered promising biological markers for external genital endometriosis. The aim of this work was to assess the composition of microvesicles derived from leukocytes in the peripheral blood of patients with stage I-II of external genital endometriosis and the possibility of their use as markers of non-invasive diagnosis of peritoneal forms of endometriosis. The study involved 97 women aged 26-40 with stage I-II of external genital endometriosis, whose diagnosis was established intraoperatively and confirmed histologically. Pain syndrome was noted in all patients of the main group, with infertility also detected in 73.2% of the patients. The control group consisted of 20 patients, whose average age was 25.5±1.1 years, who were examined in connection with male infertility factor before the in vitro fertilization, and in whom, on the basis of intraoperative examination, presented no gynecological diseases, and no pain syndrome. Before the surgical intervention, peripheral blood was taken from all patients to determine the content of microvesicles derived from leukocytes. To isolate microvesicles, we used the previously described by M.P. Gelderman and J. Simak method. It was found that patients with stage I-II of external genital endometriosis experience an increase in the number of CD14+, CD16+ and CD54+CD14+ microvesicles in the peripheral blood by 1.1, 1.38 and 1.55 times, respectively, as well as a decrease in the number of CD45+CD4+, CD3+CD4+, CD3+CD8+ microvesicles by 1.2, 4 and 1.5 times, respectively, compared with patients from the control group. Therefore, in patients with stage I-II of external genital endometriosis, an increase in the relative number of CD54+CD14+ microvesicles in the peripheral blood above 5.22% can serve as a marker for early non-invasive diagnosis of the disease with sensitivity of 80.5% and specificity of 71%

Phenotypic and functional characteristics of endothelial cells: the <i>in vitro</i> effects of protein fractions from the lysate of natural killer-derived microvesicles

Microvesicles are membrane-derived formations ranging in size from 100 to 1000 nm, being produced by a variety of resting and activated cells. They can transfer their cargo to target cells, regulate physiological processes, and participate in the development of clinical disorders. Among the microvesicles of different origin, natural killers are of special interest. They represent a subpopulation of lymphocytes that eliminate aberrant cells, including virally infected and malignant cells, and participate in regulation of angiogenesis. By producing various stimuli and inhibitors of the latter process, natural killers are able to change functional activity of endothelial cells by means of microvesicle-mediated contacts. There are only scarce literature data on ability of the extracellular vesicles to influence endothelial functions, depending on the intrinsic balance of pro- and anti-angiogenic factors. Therefore, the aim of our study was to evaluate the effect of protein fractions derived from microvesicle lysate of the NK-92 natural killer cell line upon phenotype and functional characteristics of EA.hy926 endothelial cell line under in vitro experimental conditions. Using chromatographic micro-preparatory separation, twelve protein fractions (inducers) were obtained from the lysate. It was found that proliferation and migration of EA.hy926 cells after their cultivation with 10 of 12 protein fractions, were changed in different directions. These effects were dose-dependent, or remained unchanged, at distinct concentrations of active components in the fractions. The inducing factors from these fractions exerted predominantly stimulating effects on proliferation of the target cells, thus suggesting presence of proteins which are able of regulating endothelial functions. However, the size of residual area free of migrating endothelial cells treated by the inducers did not always correlate with the migration intensity and did not inversely correlate with the number of migrating cells. Moreover, it was found that the obtained protein fractions had no effect upon expression of CD54 (ICAM-1), CD34, CD31 (PECAM-1) and CD119 (IFNγR1) receptors by EA.hy926 cells. The data obtained confirm an involvement of microvesicles in communications between natural killer cells and endothelial cells, and presume different participation modes of microvesicle-derived effector proteins in the angiogenesis machinery

MALDI-TOF mass spectrometric protein profiling of microvesicles produced by the NK-92 natural killer cell line

Extracellular vesicles that are shed from the plasma membrane contain a wide range of molecules, among  which  are proteins, lipids, nucleic  acids,  and sugars. The cytotoxic proteins of natural killer cells play a key role in the implementation of their cytolytic  functions. One of the important steps in understanding the distant  communication of cells is the determination of the proteome of microvesicles. This study was aimed at the protein profiling of the microvesicles produced by the NK-92 natural killer cell line. 986 proteins with a variety of functions were identified in the lysate of microvesicles using the MALDI-TOF mass spectrometric analysis.  With automated methods of functional analysis  applied, it has been  shown  that  the  largest  protein groups  are  hypothetical proteins, proteins with  unknown functions, and  domains. The  most  representative groups  are  also  comprised by  transcription  regulators; intracellular  signaling  proteins; RNA  translation, transcription, processing, and utilization regulators; receptors; protein processing  and proteolysis regulators; amino acid metabolism enzymes, as well as transport proteins and transport regulators. Minor functional groups are represented by vitamins and mineral metabolism enzymes, membrane and microdomain-forming proteins, hormones, hemostatic regulators, regulators of sensory  systems,  specific  mitochondrial and  Golgi  apparatus proteins, and extracellular signaling proteins. An intermediate position is occupied by various functional groups, including cytoskeleton and motor proteins; proteins of centrioles; ion channels and their regulators; proteins of the ubiquitin-proteasome pathway  of protein degradation; lipid,  steroid, and fatty acid metabolism enzymes; nucleic  acid  base and  carbohydrate metabolism enzymes, as well as energy  metabolism enzymes  and  other proteins involved  in intermediate metabolism; proteins of the immune response  and  inflammation; antigens and histocompatibility proteins; cytokines and growth factors; regulators of apoptosis, autophagy, endocytosis, and  exocytosis;  regulators of the  cell cycle and  division;  regulators of proliferation, cell differentiation, and morphogenesis; regulators of cell adhesion and  matrix  metabolism; nuclear transport proteins; transposition proteins; DNA  replication and  repair  proteins, as well as inactive  proteins. The  data  obtained expand  the existing knowledge of the distant  communication of cells and indicate new mechanisms of interaction between natural killer and target cells

Turing Patterns Inside Cells

Concentration gradients inside cells are involved in key processes such as cell division and morphogenesis. Here we show that a model of the enzymatic step catalized by phosphofructokinase (PFK), a step which is responsible for the appearance of homogeneous oscillations in the glycolytic pathway, displays Turing patterns with an intrinsic length-scale that is smaller than a typical cell size. All the parameter values are fully consistent with classic experiments on glycolytic oscillations and equal diffusion coefficients are assumed for ATP and ADP. We identify the enzyme concentration and the glycolytic flux as the possible regulators of the pattern. To the best of our knowledge, this is the first closed example of Turing pattern formation in a model of a vital step of the cell metabolism, with a built-in mechanism for changing the diffusion length of the reactants, and with parameter values that are compatible with experiments. Turing patterns inside cells could provide a check-point that combines mechanical and biochemical information to trigger events during the cell division process

Phenotypic Profile of Peripheral Blood NK Cells under Culturing with Trophoblast Cells and IL-15 and IL-18 Cytokines

Natural killer cells (NK cells) are innate immunity lymphocytes. NK cell differentiation is controlled by the cellular microenvironment and locally produced cytokines, including IL-2, IL-15 and IL-18. NK cells are present in various tissues, forming pools of tissue-resident NK cells, e.g., decidual NK cell pool. Peripheral blood NK cells (pNK cells) are considered a supposed source of cells for decidual NK cell differentiation. In the uterus, NK cells contact with trophoblast cells, which can affect their phenotype. Contribution of trophoblast cells and IL-2, IL-15 and IL-18 cytokines to the pNK cell phenotype regulation is scarcely studied. In this regard, the aim of our research was to evaluate the effect of trophoblast cells on the phenotype of pNK cells when cultured in medium with IL-2, IL-15, and IL-18. We used mononuclear cells obtained from peripheral blood of healthy non-pregnant women at their reproductive age, with regular menstrual cycle (n = 21). Mononuclear cells were cultured in presence of IL-2, and either of cytokines regulating NK cell differentiation (IL-15, or IL-18). JEG-3 cells were used as trophoblast cells. We evaluated expression of CD45, CD3, CD56, CD14, KIR3DL1, KIR2DL3, KIR2DL4, KIR2DS4, NKp44, CD215, CD122, CD127, NKG2D, KIR2DL1, NKG2C receptors by pNK cells. It was found that pNK cells cultured in presence of trophoblast cells (JEG-3 cell line) were characterized by lower intensity of CD56 receptor expression, compared to pNK cells cultured without trophoblast cells. These changes were detected upon culturing both in medium supplied by IL-15, and with IL-18. A reduced number of NKG2C+ pNK cells was detected in presence of JEG-3 trophoblast cells, compared to NK cells cultured without trophoblast cells in medium with IL-15. The detected changes in the CD56 and NKG2C expression by pNK cells in presence of trophoblast cells proved to be opposite to those previously detected for NK cells derived from NK-92 cell line. Along with trophoblast cells, the monocytes isolated among mononuclear cells and being affected by cytokines, can apparently influence the phenotype of pNK cells in the model system used. Since monocytes/macrophages are present in decidua, further research is required to study the effect of cytokines and cellular microenvironment, including monocytes, on pNK cells

RANGING OF ANTIPHOSPOLIPID ANTIBODIES IN THE PATIENTS WITH THROMBOPHILIA AND RECURRENT MISCARRIAGE

Laboratory diagnosis of antiphospholipid syndrome (APS) is based on detection of antiphospholipid antibodies (aPLs). E.g., aPLs are directed against conformational epitopes of the so-called “co-factor” proteins: β2-gycoprotein 1 (β2-GP1), annexin V (An V) and prothrombin (Pt) that are formed during interaction with phospholipids – cardiolipin (CL), phosphatic acid (Pha), phosphatidylcholine (Pch), phosphatidylethanolamine (Pe), phosphatidylglycerol (Pg), phosphatidylinositol (Pi), phosphatidylserine (Ps). A routine methodology of detection based on ELISA testing is challenged by new tests when the antigen is absorbed on another kind of support like microbeads or membranes that can influence density of conformational epitopes for aPL’s binding. The aim of our study was to compare the results of aPLs detection by ELISA and multi-line immunodot assay (MLD). We collected blood serum samples from 45 patients with noncardioembolic ischemic strokes, 19 patients with recurrent deep vein thrombosis of lower limbs, 44 females with recurrent miscarriages, and 50 clinically healthy donors. To compare the results of aPL detection by ELISA and MLD kits, the test systems from different manufacturers were evaluated. We used an ELISA kits for detection of antibodies to CL IgG, aCL IgM, β2-GP1 produced by Euroimmun AG (Mr1) and Orgentec Diagnostica GmbH (Mr2) and MLD – for detection of antibodies to CL, β2-GP1, Pch, Pe, Pg, Pi, Ps, AnV and Pt (Medipan GmbH, Mr3). When a cut-off titer was used as the main index, 30.5% of patients were aPLs-positive with ELISA method by Mr1 and 38%, wiht Mr2. By MLD aPls were detected in 30% of patients. In the same cohort, medium and high aPLs titers (&gt; 40 U/mL) were determined in 12% of patients using ELISA kits. Positive and highly positive aPLs titers were determined in 16% when using a new method by Mr3. Medium and high titer were detected only for antibodies to β2-GP1, CL, An V, Pha and Phs. The use of ELISA approach for detection of aPLs in patients with thrombosis and obstetric pathology is associated with relatively high number of low-positive ELISA results. Due to higher sensitivity for medium and high aPLs titers, MLD testing may be used as a confirming method for APS diagnosis