A new device‑mediated miniprep method

Small-scale plasmid DNA preparation or miniprep is a fundamental technique in estimation cloning experiments and is widely used for DNA methylation analysis in epigenetic research. Current plasmid DNA minipreps use the alkali-SDS-based method in a three-solution format and require spin column-based purification steps. This procedure requires the vortexing or pipetting of pelleted bacteria by centrifugation and manual mixing of the solutions. Here, we describe a centrifuge/mixer-based instrument with the ability to perform centrifugation, vibration, and rotor oscillation in order to perform all steps of plasmid DNA isolation by device only. We found that by applying rotor oscillation-driven mixing of solutions added in the lysis and neutralization steps, homogeneous mixing was achieved within 5 s at a rotor oscillation amplitude of 45 degrees and oscillation frequency of 400 +/- 30 rpm, yielding the maximal quantity and quality of plasmid DNA. No increase in host chromosome presence purified by this approach occurs for high-copy-number plasmids compared to manually performed miniprep, and indeed, there is a significant decrease in the presence of the chromosomal fraction in low-copy-number plasmids. The supercoiled form of plasmid DNA purified at a rotor oscillation amplitude of 45 degrees does not turn into an open circular (OC) isoform when the plasmid is stored for 1 year at plus four degrees, in contrast to the plasmid purified with rotor oscillation amplitudes of 270 degrees, 180 degrees and 90 degrees. The programmed time-work-efficient protocol of plasmid miniprep installed in the device gives the extreme simplicity of plasmid minipreps speeding up and facilitating the isolation of plasmid DNAs. Keypoints New devise-mediated plasmid miniprep method (DM) performs all mixing steps without operator intervention. The DM method produces plasmid DNAs free of the dCCC form and significantly reduces the contamination with genomic DNA in the low-copy-number plasmid. DM miniprep plasmids are reliable templates for bisulfite PCR sequencing analysis.publishersversionPeer reviewe

The line-of-sight analysis of spatial distribution of galaxies in the COSMOS2015 catalogue

New observations of high-redshift objects are crucial for the improvement of the standard $\Lambda$CDM cosmological model and our understanding of the Universe. One of the main directions of modern observational cosmology is the analysis of the large-scale structure of Universe, in particular, in deep fields. We study the large-scale structure of the Universe along the line of sight using the latest version of the COSMOS2015 catalogue, which contains 518,404 high quality photometric redshifts of galaxies selected in the optical range of the COSMOS field ($2\times 2$ deg$^2$), with depth up to the redshift $z \sim 6$. We analyze large-scale fluctuations in the number of galaxies along the line of sight and provide an estimate of the average linear sizes of the self-correlating fluctuations (structures) in independent redshift bins of $\Delta z = 0.1$ along with the estimate of the standard deviation from homogeneity (the observed cosmic variance). We suggest a new method of the line-of-sight analysis based on previous works and formulate further prospects of method development. For the case of the theoretical form of approximation of homogeneity in the $\Lambda$CDM framework, the average standard deviation of detected structures from homogeneity is $\sigma_\text{mean}^{\Lambda \text{CDM}} = 0.09 \pm 0.02$, and the average characteristic size of structures is $R_\text{mean}^{\Lambda \text{CDM}} = 790 \pm 150$ Mpc. For the case of the empirical approximation of homogeneity, the average standard deviation of detected structures from homogeneity is $\sigma_\text{mean}^\text{empiric} = 0.08 \pm 0.01$, and the average characteristic size of structures is $R_\text{mean}^\text{empiric} = 640 \pm 140$ Mpc.Comment: 21 pages, 18 figures, Universe accepted 2020.11.1

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex

Copyright: Copyright 2008 Elsevier B.V., All rights reserved.Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated colocalization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.publishersversionPeer reviewe

Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program

Abstract CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments

ERp29 triggers a conformational change in polyomavirus to stimulate membrane binding

Funding Information: We thank Tom Rapoport and Kristen Verhey for critically reading the manuscript. B.T. is a Biological Scholar at the University of Michigan Medical School. E.K.R. is supported by a training grant from the National Science Foundation. S.M. was supported by the Swedish Medical Research Council and the Swedish Society of Medicine. M.B. received a scholarship from the Royal Swedish Academy. The work was supported in part by a grant to T.B. from the National Cancer Institute (CA 082395). Copyright: Copyright 2008 Elsevier B.V., All rights reserved.Membrane penetration of nonenveloped viruses is a poorly understood process. We have investigated early stages of this process by studying the conformational change experienced by polyomavirus (Py) in the lumen of the endoplasmic reticulum (ER), a step that precedes its transport into the cytosol. We show that a PDI-like protein, ERp29, exposes the C-terminal arm of Py's VP1 protein, leading to formation of a hydrophobic particle that binds to a lipid bilayer; this reaction likely mimics initiation of Py penetration across the ER membrane. Expression of a dominant-negative ERp29 decreases Py infection, indicating ERp29 facilitates viral infection. Interestingly, cholera toxin, another toxic agent that crosses the ER membrane into the cytosol, is unfolded by PDI in the ER. Our data thus identify an ER factor that mediates membrane penetration of a nonenveloped virus and suggest that PDI family members are generally involved in ER remodeling reactions.publishersversionPeer reviewe

Design and Performance of a Sideband Separating SIS Mixer for 800-950 GHz

We present the design and results of characterization of a new sideband separating (2SB) mixer for 800-950GHz, based on superconductor-insulator-superconductor (SIS) junctions. This is the first waveguide 2SB SIS mixer demonstrated at such a high frequency. The design is following the classical quadrature hybrid architecture, meanwhile additional attention was put on the reduction of reflections in the RF structure in order to minimize the RF imbalance, to achieve a high image rejection ratio (IRR). The RF waveguide block was manufactured by micromilling and populated by single-ended SIS mixers developed earlier for upgrade of the CHAMP+ high-band array on the APEX telescope. These SIS mixers have double-sideband (DSB) noise temperatures from 210 to 400K. The assembled 2SB mixer yields a SSB noise temperature from 450 to 900K, with an IRR above 15dB in 95 of the band. Comparing the DSB and the SSB sensitivities, we find that the waveguide losses are as low as expected and do not exceed 0.6dB. The presented mixer is a prototype for use in a 2SB dual polarization receiver planned for deployment on the APEX telescope

Oligomerization properties of ERp29, an endoplasmic reticulum stress protein

Funding Information: This work was supported by the Swedish Medical Research Council. We thank Prof. H.F. Gilbert for helpful discussions and critical reading of the manuscript. Copyright: Copyright 2007 Elsevier B.V., All rights reserved. Correction(s) for this article: Oligomerization properties of ERp29, an endoplasmic reticulum stress protein. FEBS Letters,Vol.433, N.3, p.335-335. - First Published online: June 28, 1999.ERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress-inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross-linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution as well as in rat hepatoma cells self-associates predominantly into homodimers. Labeling of the cells with [35S]methionine with subsequent cross-linking and immunprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.publishersversionPeer reviewe

The Early Cell Cultures from Prostate Cancer Tissue Produce Tissue Specific Epithelial and Cancer Markers

Prostate cancer (PCa) is a widespread oncogenic disease which in majority of patients proceeds in an indolent form. However, in some cases the indolent form is transformed into an aggres-sive metastatic incurable cancer. The most important task of PCa diagnostics is the search for early markers that allow predicting the transition of indolent cancer to an aggressive disease. There are now two effective preclinical models to study the PCa pathogenesis: patients derived xenografts (PDX) and patients derived organoids (PDO). Both models have limitations and are not affordable for wide research usage. In this work we investigated the ability of the prostate primary 2D cell cultures (PCC) from prostate cancer patients to produce epithelial and cancer markers. We found that early PCC are formed initially by epithelial cells which are progres-sively replaced with fibroblast-like cells. Early PCC contain tissue-specific stem cells that grow in 3D culture and form PDO similar to those from the prostate tissue. Early PCC and corre-sponding PDO from prostate cancer patients express the prostate basal, luminal epithelial and cancer markers AMACR, TMPRSS2-ERG and EZH2 which is a promising candidate to mark the transition from indolent to aggressive PCa. We have identified a diversity of TMPRSS2-ERG fu-sion transcripts in PCC and PDO, including new chimeric variants resulting from intra- and in-terchromosomal translocation. The results obtained suggest that early PCC from cancer and normal tissue sustain maternal prostate phenotype and may be used as a preclinical model to study the pathogenesis of PCa.publishersversionPeer reviewe

Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter

Funding Information: We many thanks Dr. P. Hamel and Dr. R. Phillips, The Hospital for Sick Children, Toronto, Canada for generously presented plasmids. This work was supported by an internal grant from the Institute of Cytology RAS and in part by an internal grant from Rīga Stradiņš University under a cooperation agreement. Publisher Copyright: © 2020 Chongqing Medical University Copyright: Copyright 2020 Elsevier B.V., All rights reserved.The retinoblastoma gene product (pRb) is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation. We found that twelve weeks after transfection of the exogenous active (ΔB/X and Δр34) or inactive (ΔS/N) forms of pRb into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous pRb, despite being G-418 resistant. However, the consequences of the transient production of exogenous pRb had different effects on the cell fate. The ΔB/X and Δр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation (AD). On the contrary, the ΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2. Additionally, the PPARγ2 promoter in undifferentiated ΔS/N cells was hypermethylated, but all except −60 position CpG became mostly demethylated after cells exposure to AD. We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis, production of active exogenous RBs results in an AD-promoting epigenetic state. These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.publishersversionPeer reviewe

Deuterium-Depleted Water Influence on the Isotope 2H/1H Regulation in Body and Individual Adaptation

This review article presents data about the influence of deuterium-depleted water (DDW) on biological systems. It is known that the isotope abundances of natural and bottled waters are variable worldwide. That is why different drinking rations lead to changes of stable isotopes content in body water fluxes in human and animal organisms. Also, intracellular water isotope ratios in living systems depends on metabolic activity and food consumption. We found the 2H/1H gradient in human fluids (δ2H saliva >> δ2H blood plasma > δ2Hbreast milk), which decreases significantly during DDW intake. Moreover, DDW induces several important biological effects in organism (antioxidant, metabolic detoxification, anticancer, rejuvenation, behavior, etc.). Changing the isotope 2H/1H gradient from “2H blood plasma > δ2H visceral organs” to “δ2H blood plasma << δ2H visceral organs” via DDW drinking increases individual adaptation by isotopic shock. The other possible mechanisms of long-term adaptation is DDW influence on the growth rate of cells, enzyme activity and cellular energetics (e.g., stimulation of the mitochondrion activity). In addition, DDW reduces the number of single-stranded DNA breaks and modifies the miRNA profile