Emission Control under Private Port Operator Duopoly

Recent trends in regulating maritime vessel emissions have negative effects on the competitiveness of many ports as regulations increase costs for shipping operators calling the ports. This paper develops analytical models to examine the emission standards set by governments for ports in their jurisdictions. Given the emission standards set by governments, which affects fuel cost experienced by shipping operators, ports determine charges for shipping operators. Unilateral, bilateral, and single-country regulation cases are investigated. Specifically, our analysis focuses on how increase in the maximum reservation price of shipping operators, port capacity, and environmental damage costs of ports affect optimal emission standards

<i>In Vivo</i> Detection of Macrophage Recruitment in Hind-Limb Ischemia Using a Targeted Near-Infrared Fluorophore

<div><p>Macrophages are an essential component of the immune system and have protective and pathogenic functions in various diseases. Imaging of macrophages <i>in vivo</i> could furnish new tools to advance evaluation of disease and therapies. Critical limb ischemia is a disease in which macrophages have considerable pathogenic roles, and are potential targets for cell-based immunotherapy. We sought to develop a new near-infrared fluorescence (NIRF) imaging probe to target macrophages specifically <i>in</i> <i>vivo</i> in various pathological states, including hind-limb ischemia. We rapidly screened the photostable cyanine-based NIRF library against different blood cell lines. The identified monocyte/macrophage-selective hit was tested <i>in</i> <i>vitro</i> in live-cell labeling assay. Non-invasive NIRF imaging was performed with murine models of paw inflammation by lipopolysaccharide challenge and hind-limb ischemia with femoral artery ligation. <i>in</i> <i>vivo</i> macrophage targeting was further evaluated using intravital microscopy with Csf1r-EGFP transgenic mice and immunofluorescent staining with macrophage-specific markers. We discovered MF800, a Macrophage-specific near-infrared Fluorophore, which showed selective live-cell imaging performance in a panel of cell lines and primary human blood samples. MF800 outperforms the clinically-available NIRF contrast agent ICG for <i>in</i> <i>vivo</i> specificity in paw inflammation and hind-limb ischemia models. We observed a marked overlap of MF800-labeled cells and EGFP-expressing macrophages in intravital imaging of Csf1r-EGFP transgenic mice. In the histologic analysis, MF800-positive cells also expressed the macrophage markers CD68 and CD169. NIRF imaging showcased the potential of using MF800 to understand macrophage behavior <i>in</i> <i>vivo</i>, characterize macrophage-associated diseases, and may help in assessing therapeutic responses in the clinic.</p></div

<i>In vivo</i> targeting of macrophage-rich inflamed paw with MF800.

<p>(<b>a</b>) Pictures of left control (top) and right LPS-injected (bottom) paws 24 hours after s.c. injection. Significant swelling of the ankle and the tarsus was observed in the right paw. (<b>b</b>) Flow cytometric dot plots of single-cell suspensions isolated from paw tissues of csf1r-EGFP mice in which macrophages express EGFP. Csf1r-EGFP-positive population constituted 10.8 % and 2.8 % of the total cells in the right and the left paws of LPS-injected transgenic mice, respectively, indicating recruitment of monocytes/macrophages to the inflamed right paw. Control paw-derived cells from non injected mice contained as 1.6 % and 0.7 % Csf1r-EGFP-positive cells from Csf1r-EGFP transgenic and naïve C57BL/6 mice, respectively. (<b>c–l</b>) <i>in</i> <i>vivo</i> NIRF imaging and signal quantifications of mice subcutaneously injected with LPS in the right paw, then i.v. injected with (<b>c, h</b>) MF800 (n = 6), (<b>d, i</b>) ICG (n = 2) or (<b>e, j</b>) vehicle control (n = 2). As controls, mice injected with (<b>f, k</b>) saline (n = 2) or (<b>g, l</b>) left uninjected (n = 2) were imaged after i.v. injection of MF800. Pictures shown are overlaid fluorescence and white light images, with corresponding color lookup tables 4 hours after i.v. injection. Note that MF800-mediated NIRF imaging highlights the macrophage-rich localized inflammation site (arrow) whereas ICG fails to resolve it (dotted arrow). The TBR value for the inflamed right paw in MF800-injected mice was higher than the contralateral paw, as well as ICG-injected and other control groups.</p

Intravital visualization of MF800 labeling to macrophages in csf1r-EGFP transgenic mice.

<p>(<b>a</b>) Flow cytometric plots of cells from the occluded artery tissues of csf1r-EGFP transgenic mice receiving an i.v. injection of MF800 3 days after ligation (right) and from healthy tissues of naïve C57BL/6 mice (left). The Csf1r-EGFP fluorescence (y axis) from macrophages correlated with MF800 intensity (x axis). Asterisk denotes co-localized cell population (8.36 % of total cells, 77.19% of EGFP⁺ cells). (<b>b</b>) A stereomicroscopic view of the ligated limb after removal of the skin and fascia, ventral aspect. (<b>c</b>) and (<b>d</b>) were taken from indicated regions. (<b>c</b>) Intravital NIRF imaging along the femoral artery displays MF800-labeled cells (pseudo-colored red), that co-localize with genetically-labeled macrophages (green) in csf1r-EGFP mice (merge). Scale bars are 200 µM. (<b>d</b>) A region of collateral arteries where arteriogenesis occurred shows prominent cellular infiltration of MF800⁺Csf1r⁺ cells, indicative of arterial growth and repair by macrophages. Scale bars are 1000 µM.</p

<i>In vivo</i> NIRF imaging of hind-limb ischemia with MF800.

<p>(<b>a</b>) To generate hind-limb ischemia, an incision was made in the skin, surgical thread was inserted underneath the femoral artery (left); then the right femoral artery was ligated by a triple surgical knot (right). (<b>b</b>) Flow cytometry analysis of digested tissues 3 days after ligation shows a higher proportion of csf1r-EGFP-positive macrophages in the ischemic right hind-limb (11.5 %, left) compared with the normoxic left hind-limb (2.7 %, middle) in csf1r-EGFP transgenic mice. Cells from the normoxic hind-limbs of naïve C57BL/6 mice were used for control (0.3 %, right). (<b>c–l</b>) <i>in</i> <i>vivo</i> NIRF imaging and signal quantifications of mouse models of hind-limb ischemia receiving (<b>c, h</b>) MF800 (n = 4), (<b>d, i</b>) ICG (n = 2) or (<b>e, j</b>) vehicle control (n = 2) intravenously. For negative controls, (<b>f, k</b>) sham-operated (n = 2) and (<b>g, l</b>) non-occluded (n = 2) groups were imaged after i.v. injection of MF800. Pictures shown are merges of pseudocolored fluorescence and white light images with corresponding color lookup tables 4 hours after i.v. injection. MF800 illuminated ischemic regions with significant macrophage recruitment (arrow) whereas ICGs were deposited nonspecifically. The TBR value for the ischemic right hind-limb in the MF800-injected mice was higher than that of the contralateral paw, as well as ICG-injected and other control groups.</p

Fluorescent Dye Cocktail for Multiplex Drug-Site Mapping on Human Serum Albumin

Elucidating how molecules bind to HSA is fundamental for predicting drug incompatibilities. Through combinatorial screening, we identified a novel fluorescent dye (<b>BD140</b>) with turn-on fluorescence emission and specific binding at HSA drug site 2. We further combined it with dansylamide to develop a fluorescent dye cocktail for high-throughput mapping of the interaction between therapeutics at HSA drug-binding sites

Synthesis of a Novel BODIPY Library and Its Application in the Discovery of a Fructose Sensor

We prepared a new library of 160 compounds by conjugation of a BODIPY core to a collection of aldehydes. This library was screened against 52 biologically relevant analytes and we identified one fluorescent sensor of fructose (Fructose Orange). Fructose Orange showed a 24-fold fluorescence increase upon recognition of fructose and an outstanding selectivity among 24 different saccharides. NMR studies confirmed that five different binding interactions were formed between the sensor and fructose. Furthermore, Fructose Orange was applied to the quantification of fructose in soft drinks, being the most selective fluorescent sensor for fructose reported to date

Focused Fluorescent Probe Library for Metal Cations and Biological Anions

A focused fluorescent probe library for metal cations was developed by combining metal chelators and picolinium/quinolinium moieties as combinatorial blocks connected through a styryl group. Furthermore, metal complexes derived from metal chelators having high binding affinities for metal cations were used to construct a focused probe library for phosphorylated biomolecules. More than 250 fluorescent probes were screened for identifying an ultraselective probe for dTTP

Correction to Focused Fluorescent Probe Library for Metal Cations and Biological Anions

Correction to Focused Fluorescent Probe Library for Metal Cations and Biological Anion

Selective Visualization of the Endogenous Peroxynitrite in an Inflamed Mouse Model by a Mitochondria-Targetable Two-Photon Ratiometric Fluorescent Probe

Peroxynitrite (ONOO^–) is a kind of reactive oxygen species (ROS) with super activity of oxidization and nitration, and overproduction of ONOO^– is associated with pathogenesis of many diseases. Thus, accurate detection of ONOO^– with high sensitivity and selectivity is imperative for elucidating its functions in health or disease states. Herein we for the first time present a new two-photon ratiometric fluorescent ONOO^– probe (<b>MITO</b>-<b>CC</b>) based on FRET mechanism by combining rational design strategy and dye-screening approach. <b>MITO</b>-<b>CC</b>, with fast response rate (within 20 s), excellent sensitivity (detection limit = 11.30 nM) and outstanding selectivity toward ONOO^–, was successfully applied to ratiometric detection of endogenous ONOO^– produced by HepG2/RAW264.7 cells and further employed for imaging oxidative stress in an inflamed mouse model. Therefore, probe <b>MITO</b>-<b>CC</b> could be a potential biological tool to explore the roles of ONOO^– under different physiological and pathological settings