Role of hyperpolarization-activated cyclic nucleotide-gated channel HCN2 in embryonic neural stem cell proliferation and differentiation

Funding Information: Supported by grants from the K. Albin Johansson foundation, the Magnus Ehrnrooth foundation, The Finnish Society of Sciences and Letters and the Medical Society of Finland. We are grateful for the laboratory assistance provided by Jarno Hörhä. Publisher Copyright: © 2022 The AuthorsHyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) are involved in spontaneous activity in many electrically active cell types such as cardiomyocytes and neurons. In this study, the role of HCN channels in proliferation and migration of Nestin and Sox2 expressing embryonic neural progenitor cells (NPC) originating from the subventricular zone (SVZ) was examined. Immunostaining and PCR data showed that the HCN2 subtype was highly expressed in these cells. Patch clamp recordings revealed a hyperpolarization-activated current, which was sensitive to inhibitors of HCN channels. Using the fluorescence dye bis-(1,3-dibutylbarbituric acid)-trimethineoxonol (DiBAC(4)(3)) we found that a prompt reduction of the extracellular K+ concentration, or exposing the cells to acute hypoxia, induced an instant hyperpolarization in the whole cell population. Recovery from low K+ induced hyperpolarization after extracellular calcium removal, or by re-oxygenation of hypoxic cells, was sensitive to ZD7288, a HCN channel inhibitor. Treatment of neurosphere cultures from the SVZ with ZD7288 caused a significant and reversible inhibition of neurosphere formation from single cells indicating that proliferation of progenitor cells was reduced. Furthermore, the migration of neuronal cells from neurospheres was considerably retarded in the presence of ZD7288. The results suggest that HCN2 channels are involved in controlling the proliferation of NPC and that HCN2 channel-induced spontaneous electrical activity may trigger the motility response of neurosphere-derived neurons in concert with other ion channels. Furthermore, the response to hypoxia suggests that HCN2 channels may trigger the chemotactic response of NPC to ischemic brain regions seen in many studies.Peer reviewe

Regulation of intracellular pH by electrogenic Na+/HCO3-co-transporters in embryonic neural stem cell-derived radial glia-like cells

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pH(i)) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extra cellular K+ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extra cellular calcium was removed, an instant depolarization and recovery of the pH(i), back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by 50859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pH(i) recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pH(i) regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pH(i) regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.Peer reviewe

Tannic acid inhibits electrogenic Na+/HCO3- co-transporter activity in embryonic neural stem cell-derived radial glial-like cells

Self-renewing neural stem cells and progenitor cells are cell populations that generate radial glial cells and neurons through asymmetric division. Regulation of intracellular pH in stem cells with high metabolic activity is critical for both cell signaling and proliferation. We have recently found that a S0859-inhibitable electrogenic Na+/HCO3- co-transporter (NBCe1, Slc4a4), is the primary pH(i) regulatory mechanism in stem cell-derived radial glial-like cells. Here we show, by using the voltage-sensitive fluorescent dye DiBAC 4(3) and BCECF, a pH-sensitive dye, that an antioxidant, tannic acid (100 mu M), can inhibit potassium- and calcium-dependent rapid changes in membrane potential and NBCe1 mediated pH i regulation in brain-derived glial-like cells in vitro. Furthermore, neural stem cell differentiation and neurosphere formation (proliferation) were completely inhibited by tannic acid. The present study provides evidence that tannic acid is a natural inhibitor of NBCe1. It is tempting to speculate that tannic acid or related compounds that inhibits NBCe1-mediated pH(i) regulation in glial-like cells may also have bearing on the treatment of glial neoplasms. NeuroReport 31: 57-63 Copyright (c) 2019 The Author(s). Published by Wolters Kluwer Health, Inc.Peer reviewe

Suppression of Calcium Entry Modulates the Expression of TRβ1 and Runx2 in Thyroid Cancer Cells, Two Transcription Factors That Regulate Invasion, Proliferation and Thyroid-Specific Protein Levels

The thyroid hormone receptor beta 1 (TRβ1) is downregulated in several human cancer cell types, which has been associated with development of an aggressive tumor phenotype and the upregulation of Runt-related transcription factor 2 (Runx2). In this study, we show that the expression of TRβ1 protein is downregulated in human thyroid cancer tissues and cell lines compared with the normal thyroid tissues and primary cell line, whilst Runx2 is upregulated under the same conditions. In contrast, the expression of TRβ1 is upregulated, whereas Runx2 is downregulated, in STIM1, Orai1 and TRPC1 knockdown cells, compared to mock transfected cells. To study the functional significance of Runx2 in follicular thyroid cancer ML-1 cells, we downregulated it by siRNA. This increased store-operated calcium entry (SOCE), but decreased cell proliferation and invasion. Moreover, restoring TRβ1 expression in ML-1 cells decreased SOCE, basal and sphingosine 1-phosphate (S1P)-evoked invasion, the expression of the promigratory S1P3 receptor and pERK1/2, and at the same time increased the expression of the thyroid specific proteins thyroglobulin, thyroperoxidase, and thyroid transcription factor-1. In conclusion, we show that TRβ1 is downregulated in thyroid cancer cells and that restoration of its expression can reverse the cancer cell phenotype towards a normal thyroid cell phenotype

Suppression of Calcium Entry Modulates the Expression of TRβ1 and Runx2 in Thyroid Cancer Cells, Two Transcription Factors That Regulate Invasion, Proliferation and Thyroid-Specific Protein Levels

Peer reviewe

SPRIT: Identifying horizontal gene transfer in rooted phylogenetic trees

<p>Abstract</p> <p>Background</p> <p>Phylogenetic trees based on sequences from a set of taxa can be incongruent due to horizontal gene transfer (HGT). By identifying the HGT events, we can reconcile the gene trees and derive a taxon tree that adequately represents the species' evolutionary history. One HGT can be represented by a rooted Subtree Prune and Regraft (<smcaps>R</smcaps>SPR) operation and the number of <smcaps>R</smcaps>SPRs separating two trees corresponds to the minimum number of HGT events. Identifying the minimum number of <smcaps>R</smcaps>SPRs separating two trees is NP-hard, but the problem can be reduced to fixed parameter tractable. A number of heuristic and two exact approaches to identifying the minimum number of <smcaps>R</smcaps>SPRs have been proposed. This is the first implementation delivering an exact solution as well as the intermediate trees connecting the input trees.</p> <p>Results</p> <p>We present the SPR Identification Tool (SPRIT), a novel algorithm that solves the fixed parameter tractable minimum <smcaps>R</smcaps>SPR problem and its GPL licensed Java implementation. The algorithm can be used in two ways, exhaustive search that guarantees the minimum <smcaps>R</smcaps>SPR distance and a heuristic approach that guarantees finding a solution, but not necessarily the minimum one. We benchmarked SPRIT against other software in two different settings, small to medium sized trees i.e. five to one hundred taxa and large trees i.e. thousands of taxa. In the small to medium tree size setting with random artificial incongruence, SPRIT's heuristic mode outperforms the other software by always delivering a solution with a low overestimation of the <smcaps>R</smcaps>SPR distance. In the large tree setting SPRIT compares well to the alternatives when benchmarked on finding a minimum solution within a reasonable time. SPRIT presents both the minimum <smcaps>R</smcaps>SPR distance and the intermediate trees.</p> <p>Conclusions</p> <p>When used in exhaustive search mode, SPRIT identifies the minimum number of <smcaps>R</smcaps>SPRs needed to reconcile two incongruent rooted trees. SPRIT also performs quick approximations of the minimum <smcaps>R</smcaps>SPR distance, which are comparable to, and often better than, purely heuristic solutions. Put together, SPRIT is an excellent tool for identification of HGT events and pinpointing which taxa have been involved in HGT.</p

Sustained benefits from previous physical activity on bone mineral density in males

Context: The effect of physical activity on bone mineral density (BMD) is not well investigated longitudinally after puberty in men.Objective: Our objective was to evaluate the effect of exercise and reduced exercise on BMD after puberty in men.Design: We conducted a longitudinal study.Participants: Sixty-three healthy young athletes and 27 male controls, both with a mean age of 17 yr at baseline, participated. Also, 136 of the participants’ parents were investigated to evaluate heritable influences.Main Outcome Measures: Total body, total hip, femoral neck, and humerus BMD (grams per square centimeter) were measured at baseline and after mean periods of 27, 68, and 94 months in the young cohort.Results: BMDs of control parents and athlete parents were equal, suggesting absence of selection bias. The 23 athletes that remained active throughout the study increased BMD at all sites when compared with controls (mean difference, 0.04–0.12 g/cm2; P &lt; 0.05) during the study period. After an average of 3 yr, 27 athletes ended their active careers. Although this group initially lost BMD at the hip compared with active athletes, the former athletes still had higher BMD than controls at the femoral neck (0.12 g/cm2; P = 0.007), total hip (0.11 g/cm2; P = 0.02), and humerus (0.10 g/cm2; P = 0.02) at the final follow-up.Conclusions: High sensitivity to physical loading persists after puberty in men. Reduced physical activity is associated with BMD loss in the first 3 yr in weight-bearing bone. Sustained benefits in BMD are preserved 5 yr after intensive training ends.</p

Business Oriented Systems Maintenance Management

In the introduction to this chapter, we discuss some of the common problems in maintenance. In order to solve these problems, we find it necessary to think in a new way, including the relationship of businesses to the system maintenance. The world outside organisations changes continuously, and the business processes and functions must change with it. However, if we only maintain information technology (IT) and do not co-manage the business changes accordingly, IT will not change at the same pace as the business changes. It would result a gap between the business needs and services provided by the IT product. In that case, IT systems would not be able to provide sufficient business value. The main part of this chapter contains a management model for solving these problems, based on theoretical research and practical experience. The central theme of the model is the connection between the business needs and systems maintenance. This is achieved by maintaining maintenance objects rather than the systems, establishing micro-organisations for each maintenance object where business processes as well as the system are represented. Our proposed model is widely used in more than 50 organisations in Sweden. In conclusion, some future trends and central concepts of the model are discussed.</p

Vad har hänt sedan sist?

Hur styr man systemförvaltning? Hur får man verksamhetoch teknik att samarbeta? Och vad är det egentligen som ska förvaltas? Det kan tyckas vara enkla frågor, men faktum är att många fortfarande letar efter svar på dem. I denna dokumentationger vi svaren - både på de här frågorna och många andra. Det vi presenterar här är en förkortad version av vår modell, Affärsmässig Förvaltningsstyrning. Sammanfattningsvis kanman säga att den bygger på fem framgångsfaktorer som alla genomsyras av ett affärsmässigt perspektiv. När modellen etableras säkerställs framgångsfaktorerna med hjälp av en etableringsmetod som innehåller ett antal verktyg. Resultatet beskrivs i en förvaltningsplan som utgör förvaltningsorganisationens styrinstrument för den kommande perioden

Method enabling detection of single-stranded DNA ligation activity [Elektronisk resurs]

The enzyme DNA ligase is an essential tool in many different applications in the field of biotechnology. For these applications the DNA quality can be crucial for the final result. In this paper we present a new method that can be used for specific studies of DNA ligases and for simultaneous analyzes of DNA quality. More specifically, a simple, fast and sensitive method for detection of single-stranded DNA (ssDNA) ligase activity is presented. The new method is based on real-time detection of nucleotide incorporation over the ligation site catalyzed by a DNA polymerase. The ratio between the incorporation signal after and before the ligation site gives an estimate of the ligation efficiency. To further increase the robustness of the assay the exact amount of unligated product can be easily estimated in a separate assay using a 3’-end blocked oligonucleotide functioning as template. Produced circular DNA can be cleaned from unligated DNA by exonuclease I treatment and the quality of the circle can be easily analyzed by a simple sequencing procedure. If the ssDNA used for the ligation reaction contains truncated fragments this will be observed by uneven signals during the analysis. The new method can be a useful tool for researchers working with both basic and applied projects in the field of biotechnology.</p