Reciprocating Motion of a Self-Propelled Object on a Molecular Layer with a Local Minimum and a Local Maximum Isotherm

The mode change of a simple autonomous motor depending on the nature of an <i>N</i>-acyl-<i>p</i>-nitroaniline, (C_<i>n</i>ANA, CH₃(CH₂)_<i>n</i>−2CONHφNO₂, <i>n</i> = 8, 14, 16, 18, or 22) monolayer on water was investigated. A camphor disk was floated on a molecular layer of C_<i>n</i>ANA, which gave a characteristic surface pressure (Π) vs area (<i>A</i>) isotherm. The nature of the camphor motion changed depending on the Π vs <i>A</i> isotherm, and in particular reciprocating motion was observed for C₁₄ANA, C₁₆ANA, and C₁₈ANA, which gave a Π vs <i>A</i> isotherm with a local minimum and a local maximum. The characteristic motion of a camphor disk is discussed in relation to the Π vs <i>A</i> isotherm of C_<i>n</i>ANA and the influence of the interaction between molecules on the driving force of motion. Reciprocating motion was qualitatively reproduced by numerical calculation

Transient Reciprocating Motion of a Self-Propelled Object Controlled by a Molecular Layer of a <i>N</i>‑Stearoyl‑<i>p</i>‑nitroaniline: Dependence on the Temperature of an Aqueous Phase

The mode-bifurcation of a self-propelled system induced by the property of a <i>N</i>-stearoyl-<i>p</i>-nitroaniline (C₁₈ANA) monolayer developed on an aqueous phase was studied. A camphor disk was placed on a C₁₈ANA monolayer, which indicated a characteristic surface pressure–area (π–<i>A</i>) isotherm. A camphor disk transiently exhibited reciprocating motion at a higher surface density of C₁₈ANA. The amplitude of the reciprocating motion increased with an increase in the temperature of the aqueous phase below 290 K, but reciprocating motion varied to irregular motion over 290 K. The temperature-dependent reciprocating motion is discussed in terms of the π–<i>A</i> curve for C₁₈ANA depending on the temperature. The interaction between C₁₈ANA molecules was measured by Fourier transform IR spectrometry and Brewster-angle microscopy. As an extension of the study, the trajectory of reciprocating motion could be determined by writing with a camphor pen on the C₁₈ANA monolayer

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury

<div><p>Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.</p></div

ESAM deficiency caused reduced erythropoiesis potential in the spleen, but not in the BM.

<p>WT and ESAM-KO mice were sacrificed, and flow cytometry (FACS) analyses or methylcellulose colony cultures were performed. (A) The representative FACS profiles of the Lin^- IL-7Rα^- c-Kit⁺ Sca1^- fraction of WT and ESAM-KO BM cells are shown. Each number indicates the percentage of FCγR^Lo CD34⁺ CMP, FCγR^Hi CD34⁺ GMP, or FCγR^Lo CD34^- MEP population within the Lin^- IL-7Rα^- c-Kit⁺ Sca1^- fraction. (B) The numbers of CMPs, GMPs, and MEPs in the BM are shown (n = 5 in each). (C) The numbers of CMPs, GMPs, and MEPs in the spleen are shown (n = 4 in each). (D) The number of total mononuclear cells (MNC) and LSK cells in the spleen are shown (n = 4 in each). (E) BM cells and splenocytes were subjected to methylcellulose colony formation assays for counting BFU-E and CFU-E. Each bar represents the number of BFU-E (left panel) or CFU-E (right panel) in the BM and spleen (BM; n = 6 in each, spleen; n = 5 in each). (F-G) Representative FACS profiles of c-kit^- fractions of BM cells (F) and splenocytes (G) from WT and ESAM-KO mice are shown. In the panels, each number indicates the percentage of the Ter119⁺ CD71^Hi population in the c-Kit^- fraction. The numbers of c-Kit^- Ter119⁺ CD71^Hi cells in the BM and spleen are shown in the right graphs (n = 5 in each). (H) The relative numbers of BFU-E, CFU-E, and c-Kit^- Ter119⁺ CD71^Hi progenitors divided by the number of MEP in WT or ESAM-KO mice’s spleen are shown. (I) The number of BUF-E in nucleated cells derived from 100 μL of PB is shown (n = 4 in each). The blue bars represent the results for the WT mice, and the red bars represent those of ESAM-KO mice. Data are from one of two independent experiments that gave similar results. Data are shown as mean ± SEM. Statistically significant differences are represented by asterisks (*<i>P</i> < 0.05, **<i>P</i> < 0.01, *** <i>P</i> < 0.001).</p

Differential gene expression in WT and ESAM-KO pre CFU-E fraction after 5-FU treatment.

<p>(A-E) WT and ESAM-KO mice were treated with a single 5-FU (120 mg/kg) injection and sacrificed 8 days after treatment. BM cells from each of the three mice were pooled and the cells in the pre CFU-E fraction were sorted. The extracted RNA samples were used to conduct microarray and bioinformatic analyses. Gene expression profiles of ESAM-KO pre CFU-E relative to its WT counterpart were evaluated. (A) As a result, a color-coded heat map analysis was obtained. This heat map allows for the visualization of the differential expression data of genes categorized by their functions using the Ingenuity Knowledge Base. The color bar indicates the <i>z</i>-score for each category: the strongest predicted increase (orange square) corresponds to <i>z</i>-score <i>></i> 2, the strongest predicted decrease (blue square) corresponds to <i>z</i>-score <i>< –</i>2. Gray and white colors indicate categories with a –2 <i>< z</i>-score <i><</i> 2 and without <i>z</i>-score, respectively. Larger squares indicate a more significant overlap among the genes altered in the dataset. In the heat map, the results of “hematological system development and function,” “inflammatory response”, “cell-to-cell signaling and interaction”, “immune cell trafficking”, “cellular movement”, and “cellular development” are shown. (B) The lists of differential expression data of genes categorized by their functions using the Ingenuity Knowledge Base. (C) Venn diagram of the 65 genes that are shared between the genes that have more than a 2-fold increased or decreased expression in ESAM-KO pre CFU-E compared to WT pre CFU-E, and the “erythropoiesis”-related genes. (D) The upstream regulator analyses were performed with respect to extracted genes in Fig 6C. The significantly altered upstream regulators are shown. (E) Genes downstream of <i>Gata1</i> are shown. Genes shown with green graphics are genes that are down-regulated, and those shown with red graphics are genes that are up-regulated in ESAM-KO mice. (F) A model of stress-induced erythropoiesis after 5-FU treatment is shown.</p

ESAM deficiency caused serious and prolonged anemia after 5-FU treatment.

<p>(A) WT or ESAM-KO mice were intravenously administered with a single 200 mg/kg dose of 5-FU to then examine the PB every 5 days (up to day 20) using a blood cell analyzer (KX-21, Sysmex) (n = 10 in each). WBC, Hb, and platelet counts were plotted. Two ESAM-KO mice died between day 10 and day 15 after 5-FU treatment. (B, C) WT or ESAM-KO mice were administered 150 mg/kg of 5-FU and PB analyses were performed at day 8. (B) The number of reticulocytes were quantified by visual counting (n = 3). (C) The mean percentage of neutrophil, monocyte, and lymphocyte subsets in WBC were quantified by visual counting (n = 3). Data are shown as mean ± SEM. Statistically significant differences are represented by asterisks (*<i>P</i> < 0.05, ** <i>P</i> < 0.01).</p

ESAM expression in hematopoietic cells is required for hematopoietic recovery after BM injury.

<p>(A-F) 1 × 10⁶ BM cells from CD45.1 WT or CD45.2 WT mice were transplanted to lethally irradiated CD45.2 ESAM-KO or CD45.1 WT mice, respectively (n = 8 in each group). Sixteen weeks after transplantation, half of each group was sacrificed at 5-FU day 0, and others were treated with 150 mg/kg of 5-FU and sacrificed at day 8. (A) A scheme of the transplantation protocol is shown. (B) PB chimerisms that were analyzed by FACS are shown. The blue bar represents the percentages of CD45.1^- CD45.2⁺ cell population among CD45⁺ cells in WT recipient mice, and the red bar represents the percentages of CD45.1⁺ CD45.2^- cell population in ESAM-KO recipient mice at stable state (5-FU day 0). (C-E) The number of BM cells from a pair of femora and tibiae was analyzed after 5-FU treatment (day 0 and day 8). The number of total BM MNC, CD3⁺ T cells, CD19⁺ B cells, MAC1⁺ myeloid cells (C), LSK cells (D), and c-Kit^- Ter119⁺ CD71^Hi erythroid progenitors (E) is shown. (F) PB Hb levels in 5-FU-treated WT and ESAM-KO mice recipients at day 8 are shown. (G, H) Equal amounts (2 × 10⁵) of BM cells from CD45.1 WT and CD45.2 ESAM-KO mice were mixed and transplanted to lethally irradiated CD45.1 WT mice (n = 6). Sixteen weeks after transplantation, half of them were sacrificed at 5-FU day 0, and others were treated with 150 mg/kg of 5-FU and sacrificed at day 8. (G) A scheme of the transplantation protocol is shown. (H) The percentages of CD45.1^- CD45.2⁺ cell population among the LSK fraction in the BM at 5-FU day 0 and day 8 are shown. Data are shown as mean ± SEM. Statistically significant differences are represented by an asterisk (*<i>P</i> < 0.05).</p

ESAM is up-regulated after 5-FU treatment in erythroid progenitors and macrophages.

<p>(A-E) C57BL/6J WT mice were treated with a single intravenous 5-FU (150 mg/kg) injection, and ESAM expression levels in myeloid or erythroid progenitors and macrophages in the BM were evaluated by FACS. In each histogram, the solid line and tinted line show ESAM levels after 5-FU treatment at day 0 (control) and day 5, respectively. The background level is added to each panel with an isotype control Ab (dashed line). (A) ESAM expression levels of CMP, GMP, and MEP fractions are shown. (B) Representative FACS profiles of pre CFU-E cells in BM after 5-FU treatment at day 0 and day 5 are shown. (C) ESAM expression levels in the pre CFU-E fraction are shown. (D) ESAM expression levels in the Ter119^- CD71^Hi fraction and the c-Kit^- Ter119⁺ CD71^Hi fraction are shown. (E) ESAM expression levels of Gr1^- F4/80⁺ CD115^Int SSC^Int/Lo macrophages are shown. (F) Representative FACS profiles of BM cells from 5-FU-treated WT and ESAM-KO mice (150 mg/kg) at day 8 are shown. Each number indicates the percentage of F4/80⁺ Ter119⁺ multiplets (erythroblastic islands). (G) In the left histogram, cell sizes determined by FSC in the erythroblastic island population of WT (blue) and ESAM-KO (red) mice are shown. In the right graph, the actual value of FSC is shown. Data are shown as mean ± SEM. Statistically significant differences are represented by an asterisk (*<i>P</i> < 0.05).</p

Erythroid progenitors in the BM and spleen are reduced in 5-FU-treated ESAM-KO mice.

<p>(A-I) WT and ESAM-KO mice were treated with 150 mg/kg of 5-FU, and FACS analyses or methylcellulose cultures were performed at day 8. Then, the number of myeloid and erythroid progenitors from BM, spleen, or PB was evaluated. (A) The number of CMPs, GMPs, and MEPs in BM is shown (n = 4 in each). (B-C) BM cells and splenocytes were subjected to methylcellulose colony formation assays for counting BFU-E and CFU-E. Each bar represents the number of BFU-E (left panel) or CFU-E (right panel) in BM (B) and spleen (C) (n = 6 in each). (D-E) In the left panels, representative FACS profiles of BM (D) and spleen (E). In the right panels, quantification of c-Kit^- Ter119⁺ CD71^Hi cells in BM (D) and spleen (E) (BM; n = 7 in each, spleen; n = 6 in each). (F-H) CMPs, GMPs, and MEPs were sorted from 5-FU-treated BM of WT or ESAM-KO mice. Then, 1 × 10³ MEPs, 2× 10² CMPs, or 1 × 10³ GMPs were subjected to methylcellulose colony formation assays for counting BFU-E (F), CFU-Mix (G), or CFU-G/M/GM (H), respectively (n = 3 in each). (I) The numbers of BUF-E in nucleated cells derived from 100 μL of PB are shown (n = 5 in each). (A-I) The blue bars represent the results for the WT mice, and the red bars represent those of ESAM-KO mice. All data are from one of two independent experiments that gave similar results. Data are shown as mean ± SEM. Statistically significant differences are represented by asterisks (*<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001).</p