Daria Potashnikova

Malaria-Infected Erythrocyte-Derived Microvesicles Mediate Cellular Communication within the Parasite Population and with the Host Immune System

Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system.

Cyclin D1 expression in B-cell lymphomas

OBJECTIVE Cyclin D1, an important component of cell cycle machinery and a protein with known oncogenic potential, is downregulated in normal mature B lymphocytes. Its expression detected in a number of malignancies, including B-cell lymphomas, may be important for oncogenesis. MATERIALS AND METHODS In our work, we determined the level of cyclin D1 expression in various B-cell lymphomas (i.e., mantle cell lymphoma, B-cell chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and compared it with normal B cells. For cyclin D1 level evaluation, the real-time quantitative polymerase chain reaction data was normalized. We tested five reference genes for stability on our sample set and using the three most stable ones (YWHAZ, ubiquitin c, and HPRT) obtained rather small intra-group variance for cyclin D1 expression in most lymphomas. This allowed their statistically significant ranking according to cyclin D1 expression level. RESULTS Median values of normalized cyclin D1 expression determined by real-time quantitative polymerase chain reaction were 1.32 for mantle cell lymphoma, 0.02 for B-cell chronic lymphocytic leukemia, 0.009 for diffuse large B-cell lymphoma, 0.004 for marginal zone lymphoma, 0.002 for follicular lymphoma compared with 0.0003 for reactive lymphoid tissue, and 0.00004 for sorted B cells of healthy donors. CONCLUSIONS Our data demonstrate that mantle cell lymphoma, a lymphoma with t(11;14)(q13;q32) translocation, has the level of cyclin D1 increased by four orders of magnitude, while other B-cell lymphomas without t(11;14)(q13;q32) translocation still have the level of cyclin D1 significantly elevated above that of normal lymphocytes (2 orders for B-cell chronic lymphocytic leukemia and an order for other lymphomas) and suggests more than one method of its upregulation in malignant B cells.

Cell sorting in cancer research--diminishing degree of cell heterogeneity.

Increasing evidence of intratumor heterogeneity and its augmentation due to selective pressure of microenvironment and recent achievements in cancer therapeutics lead to the need to investigate and track the tumor subclonal structure. Cell sorting of heterogeneous subpopulations of tumor and tumor-associated cells has been a long established strategy in cancer research. Advancement in lasers, computer technology and optics has led to a new generation of flow cytometers and cell sorters capable of high-speed processing of single cell suspensions. Over the last several years cell sorting was used in combination with molecular biological methods, imaging and proteomics to characterize primary and metastatic cancer cell populations, minimal residual disease and single tumor cells. It was the principal method for identification and characterization of cancer stem cells. Analysis of single cancer cells may improve early detection of tumors, monitoring of circulating tumor cells, evaluation of intratumor heterogeneity and chemotherapeutic treatments. The aim of this review is to provide an overview of major cell sorting applications and approaches with new prospective developments such as microfluidics and microchip technologies.

A charge-dependent mechanism is responsible for the dynamic accumulation of proteins inside nucleoli.

The majority of known nucleolar proteins are freely exchanged between the nucleolus and the surrounding nucleoplasm. One way proteins are retained in the nucleoli is by the presence of specific amino acid sequences, namely nucleolar localization signals (NoLSs). The mechanism by which NoLSs retain proteins inside the nucleoli is still unclear. Here, we present data showing that the charge-dependent (electrostatic) interactions of NoLSs with nucleolar components lead to nucleolar accumulation as follows: (i) known NoLSs are enriched in positively charged amino acids, but the NoLS structure is highly heterogeneous, and it is not possible to identify a consensus sequence for this type of signal; (ii) in two analyzed proteins (NF-κB-inducing kinase and HIV-1 Tat), the NoLS corresponds to a region that is enriched for positively charged amino acid residues; substituting charged amino acids with non-charged ones reduced the nucleolar accumulation in proportion to the charge reduction, and nucleolar accumulation efficiency was strongly correlated with the predicted charge of the tested sequences; and (iii) sequences containing only lysine or arginine residues (which were referred to as imitative NoLSs, or iNoLSs) are accumulated in the nucleoli in a charge-dependent manner. The results of experiments with iNoLSs suggested that charge-dependent accumulation inside the nucleoli was dependent on interactions with nucleolar RNAs. The results of this work are consistent with the hypothesis that nucleolar protein accumulation by NoLSs can be determined by the electrostatic interaction of positively charged regions with nucleolar RNAs rather than by any sequence-specific mechanism.

Improvement of HIV-1 and Human T Cell Lymphotropic Virus Type 1 Replication-Dependent Vectors via Optimization of Reporter Gene Reconstitution and Modification with Intronic Short Hairpin RNA

ABSTRACT Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in cocultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents expression of the reporter gene in the transfected cells but permits its expression in target cells after infection. However, the gamma-globin intron is not efficiently removed by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the intron-containing RNA via microRNA 30 (miR30)-based short hairpin RNAs. Using two optimized fluorescent reporter vectors and flow cytometry, we determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cell-free infection; furthermore, this increase was dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify the infection in lymphocytes and in primary cells with a higher sensitivity and allow the detection and quantitation of multiply infected cells, providing better tools to study retroviral cell-mediated infection. IMPORTANCE The human-pathogenic retroviruses HTLV-1 and HIV-1 can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current study, we generated vectors that are significantly improved over the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in cells in mixed culture. These luciferase- or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one-step transfection/infection assay.

RNA-dependent disassembly of nuclear bodies.

ABSTRACT Nuclear bodies are membraneless organelles that play important roles in genome functioning. A specific type of nuclear bodies known as interphase prenucleolar bodies (iPNBs) are formed in the nucleoplasm after hypotonic stress from partially disassembled nucleoli. iPNBs are then disassembled, and the nucleoli are reformed simultaneously. Here, we show that diffusion of B23 molecules (also known as nucleophosmin, NPM1) from iPNBs, but not fusion of iPNBs with the nucleoli, contributes to the transfer of B23 from iPNBs to the nucleoli. Maturation of pre-ribosomal RNAs (rRNAs) and the subsequent outflow of mature rRNAs from iPNBs led to the disassembly of iPNBs. We found that B23 transfer was dependent on the synthesis of pre-rRNA molecules in nucleoli; these pre-rRNA molecules interacted with B23 and led to its accumulation within nucleoli. The transfer of B23 between iPNBs and nucleoli was accomplished through a nucleoplasmic pool of B23, and increased nucleoplasmic B23 content retarded disassembly, whereas B23 depletion accelerated disassembly. Our results suggest that iPNB disassembly and nucleolus assembly might be coupled through RNA-dependent exchange of nucleolar proteins, creating a highly dynamic system with long-distance correlations between spatially distinct processes. Summary: Interphase prenucleolar body disassembly and nucleolus assembly might be coupled through RNA-dependent exchange of nucleolar proteins.

Dual Role of the Extracellular Domain of Human Mucin MUC1 in Metastasis: MUC1 DUAL ROLE IN METASTASIS

Human mucin MUC1 plays an important role in cancer development. The increased level of this molecule expression during cancer cell progression induces metastasis and is associated with poor prognosis for patients. There is a large body of experimental data on the role of various functional domains of human mucin MUC1 in metastasis. While, the cytoplasmic domain determined to play a definitive role, the influence of extracellular domain on cancer cell invasiveness still remains unclear. The present paper reveals that the extracellular domain of MUC1 molecule consists of two functional subdomains—the region of tandem repeats (TR) and the region of irregular repeats (IR). We demonstrate the ability of each of these subdomains to alter the invasiveness of cancer cells. The presence of the MUC1 molecules containing TR subdomain (MUC1‐TR) on the surface of low‐invasive cancer cells leads to the increase in their transendothelial migration potency, while the addition of the IR subdomain to the MUC1‐TR molecule (MUC1‐IR‐TR) restores their natural low invasiveness. J. Cell. Biochem. 118: 4002–4011, 2017.

FACS Isolation of Viable Cells in Different Cell Cycle Stages from Asynchronous Culture for RNA Sequencing

Recently developed high-throughput analytical techniques (e.g., protein mass spectrometry and nucleic acid sequencing) allow unprecedentedly sensitive, in-depth studies in molecular biology of cell proliferation, differentiation, aging, and death. However, the initial population of asynchronous cultured cells is highly heterogeneous by cell cycle stage, which complicates immediate analysis of some biological processes. Widely used cell synchronization protocols are time-consuming and can affect the finely tuned biochemical pathways leading to biased results. Besides, certain cell lines cannot be effectively synchronized. The current methodological challenge is thus to provide an effective tool for cell cycle phase-based population enrichment compatible with other required experimental procedures. Here, we describe an optimized approach to live cell FACS based on Hoechst 33342 cell-permeable DNA-binding fluorochrome staining. The proposed protocol is fast compared to traditional synchronization methods and yields reasonably pure fractions of viable cells for further experimental studies including high-throughput RNA-seq analysis.

From an increase in the number of tandem repeats through the decrease of sialylation to the downregulation of MUC1 expression level: SYRKINA et al.

Enhanced glucose uptake by cancer cells was demonstrated in many studies in vitro and in vivo. Glycolysis is one of the main ways of obtaining energy in hypoxia conditions. However, in addition to energy exchange, carbohydrates are also necessary for the posttranslational modification of the protein molecules. Cancer cells are often characterized by an enhanced expression of different glycoproteides. Correct glycosylation defines the structure and activity of such molecules. We demonstrated that under the same cultivation conditions, the intensity of glycosylation does not depend on the total number of potential O‐glycosylation sites in one molecule. As a model for the investigation, the tandem repeat region (region with variable number of tandem repeats) of the human mucin MUC1, in which each of the repeats carries four potential O‐glycosylation sites, was used. An increase of the tandem repeat number in the recombinant protein did not lead to a proportional increase in the level of sLea glycosides. A consequence of this was a reduction in the number of recombinant proteins associated with the cytoplasmic membrane at an overall high expression level. Prolongation of the cultivation duration led to a reduction in the expression level of the recombinant proteins by up to 30% of the initial level, and the intensity of this reduction was in a direct ratio to the number of tandem repeats in the protein molecule.

Comparison of the mRNA expression profile of B‐cell receptor components in normal CD5‐high B‐lymphocytes and chronic lymphocytic leukemia: a key role of ZAP70

The B‐cell receptor (BCR) signaling pathway is of great importance for B‐cell survival and proliferation. The BCR expressed on malignant B‐CLL cells contributes to the disease pathogenesis, and its signaling pathway is currently the target of several therapeutic strategies. Although various BCR alterations have been described in B‐CLL at the protein level, the mRNA expression levels of tyrosine kinases in B‐CLL compared to that in normal CD5‐high and CD5‐low B‐lymphocytes remain unknown. In the current study, we measured the mRNA expression levels of CD79A, CD79B, LYN, SYK, SHP1, and ZAP70 in purified populations of CD5‐high B‐CLL cells, CD5‐low B‐cells from the peripheral blood of healthy donors, and CD5‐high B‐cells from human tonsils. Here, we report a clear separation in the B‐CLL dataset between the ZAP70‐high and ZAP70‐low subgroups. Each subgroup has a unique expression profile of BCR signaling components that might reflect the functional status of the BCR signaling pathway. Moreover, the ZAP70‐low subgroup does not resemble either CD5‐high B‐lymphocytes from the tonsils or CD5‐low lymphocytes from PBMC (P < 0.05). We also show that ZAP70 is the only gene that is differentially expressed in CD5‐high and CD5‐low normal B‐lymphocytes, confirming the key role of Zap‐70 tyrosine kinase in BCR signaling alterations in B‐CLL.