Replacing BACK36 in mECK36 cells for rKSHV.219 generates the tumorigenic cells, mECK^null.rK.

<p>(A) Graph depicting mECK36 tumor kinetics in athymic nu/nu mice. 3×10⁶ mECK36 cells were subcutaneously injected into the hind flanks of 3 athymic nu/nu mice. Concurrently, 3×10⁶ mECK^null cells were subcutaneously injected into another group of 10 athymic nu/nu mice. Within 4–6 weeks solid mECK36 tumors were palpable and growth was monitored by caliper measurement (open squares). Error bars represent the standard deviation between 3 different tumors. mECK^null cells did not form tumors (black dots). (B) Fluorescence microscopy of mECK^null.rK. <i>In vitro</i>, mECK^null.rK express GFP constitutively, indicating rKSHV.219 infection, and maintain tight latency as determined by the absence of RFP expression. (C) Cell cultures of mECK^null.rK were prepared for immunofluorescence for the KSHV LANA which exhibited the classic punctate nuclear pattern of the protein.</p

rKSHV.219 lytic replication <i>in vivo</i> results in productively infected tumors culminating in the formation of virus-like particles.

<p>(A) rKSHV.219 lytic gene expression increases <i>in vivo</i> relative to mECK^null.rK133 cells in culture during tumorigenesis. RNA was isolated from tumors and cells in culture for analysis of rKSHV.219 gene expression by qRT-PCR. A representative comparative analysis is shown; error bars represent the SD of experimental duplicates. (B) RT-PCR analysis of rKSHV.219 transcripts was performed as a preliminary confirmation that the virus was able to express genes representative of the entire viral replicative cycle. RNA was isolated from tumors, reverse transcribed and run on a 3% agarose gel. Gene expression <i>in vivo</i> reveals the presence of transcripts that span the entire KSHV genome and replicative potential. Reverse transcriptase negative and non-template controls were run to confirm the absence of contamination. (C) Transmission electron microscopy (TEM) analysis of tumors: Tumors were excised and fixed in gluteraldehyde. TEM revealed the presence of herpesvirus-like particles (100 nm–200 nm) <i>in vivo</i>.</p

rKSHV.219 infected cells and viral DNA can be detected throughout the murine host.

<p>(A) Graph depicts viral DNA copy number per 500 ng total DNA in 10×10⁶ cells purified from bone marrow and in 500 µl whole blood of tumor bearing mice. (B) rKSHV.219 infected cells were detected in murine lymph nodes. Cells from murine lymph nodes were dissociated into single cell suspensions and plated in chamber slides and fixed for fluorescence microscopy for GFP expression. The top panels depict IgG control antibody and native GFP expression, which is quite dim. GFP expression was enhanced with an antibody directed against GFP in the middle and bottom panels. (C) rKSHV.219 infected cells are present in murine spleen. Spleen from a tumor bearing mouse was excised, dissociated with collagenase IV and cultured in puromycin containing selective medium. GFP expressing LANA positive cells grew from the splenic cell culture.</p

Newly isolated murine bone marrow-derived cells infected with rKSHV.219 generate productively infected tumors in nude mice.

<p>(A) Graph depicting growth kinetics in athymic nu/nu mice. 3×10⁶ mECrK cells were subcutaneously injected into the hind flanks of 4 athymic nu/nu mice. Within 4–6 weeks solid tumors were palpable and growth was monitored by caliper measurements. (B) mECrK tumor cells express CD31. Cells from a dissociated tumor were immunostained for CD31, an angiogenic marker and analyzed by flow cytometry for GFP/CD31 co-expression. About half of the CD31+ cells are also rKSHV.219 infected as determined by GFP expression. The vast majority of rKSHV.219 infected cells also expressed CD31. (C) Tumors were excised and fixed in gluteraldehyde for TEM imaging. TEM revealed the presence of herpesvirus-like particles <i>in vivo</i>. Bar = 300 nm.</p

Microarray analysis rKSHV.219-infected murine cells confirms that the primary bone marrow-derived cells and mECK^null. rK133, are closely related cells of the endothelial lineage.

<p>Dendrogram and heatmap of microarray data comparing the host transcriptome of mECK^null. rK133 cells (denoted as mEC36nullrK.219 in the figure) and mECrK.219 (denoted as mECrK.219 in the figure) to other murine cells of various lineages: ESC, embryonic stem cells; BM, total bone marrow; FLE, fetal liver erythroblasts; EC_NCadh, N-cadherin expressing endothelial cells; EC_VE+NCadh, VE+N-cadherin expressing endothelial cells; EC_VECadh, VE-cadherin expressing endothelial cells; BM_MSC, bone marrow derived mesenchymal stem cells; AEC, aortic endothelial cells; mECrK.219, primary bone marrow-derived mEC infected with rKSHV.219; mECK^null.rK133, mECK^null cells infected with rKSHV.219 and then enriched for Prominin-1; BM_Macro, bone marrow-derived macrophages; Macro, peripheral macrophages; HSC, hematopoietic stem cells; LSC, leukemia stem cells; GEC, glomerular endothelial cells; SubQ, subcutaneous tissue. Similar to the mECK36 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087324#pone.0087324-Cancian1" target="_blank">[9]</a>, the mECK^null.rK133 are of the endothelial lineage and cluster with murine aortic endothelial cells.</p

mECK^null. rK133 cells consistently form KS-like tumors in immunocompromised mice.

<p>(A) Graph depicting tumor kinetics in athymic nu/nu mice. 3×10⁶ mECK^null. rK133 cells were subcutaneously injected into the hind flanks of 5 athymic nu/nu mice. Concurrently, 3×10⁶ mECK^null cells were subcutaneously injected into another group of 10 athymic nu/nu mice. Within 4–6 weeks solid mECK^null. rK133 tumors were palpable and growth was monitored by caliper measurements. mECK^null cells did not form tumors (red line, x-axis). (B) Dissection site showing a GFP+ tumor indicating the presence of rKSHV.219. The subcutaneous tumor was visualized under UV. (C) Tumor morphology was analyzed by H&E staining of paraffin embedded sections. Pathologically, they are composed of spindle cells arranged in bundles with RBCs in slit-like vasculature (top panels, black arrows). Frozen sections were prepared for immunofluorescence for KSHV LANA which showed that the spindle cells of the tumor express the viral latent nuclear antigen, LANA (bottom panels). (D) Immunofluorescence analysis of angiogenic protein expression <i>in vivo</i> reveals that the tumor cells express VEGF-R2 and podoplanin. A representative tumor is shown.</p

CD133/Prominin-1 enrichment of the mECKnull.rK results in a lytically-inducible population that harbors KSHV as an episome.

<p>(A) mECK^null.rK were harvested from cell culture and CD133-expressing cells were positively selected using immunomagnetic beads. The histogram shows the parental population (green), the CD133 enriched (mECK^null.rK133) (blue) and the CD133-depleted populations (red) were analyzed by flow cytometry to confirm CD133 enrichment. A representative histogram is shown. (B) Graph depicting the percentage of CD133 expression in the mECK^null.rK, prior to CD133-enrichment, the percentage of CD133 in the enriched population of mECK^null.rK133 immediately post-enrichment, and the mECK^null.rK133 one month after CD133 enrichment. Error bars represent standard deviation between triplicate wells. (C) <i>In vitro</i>, mECK^null.rK133 express GFP, indicating rKSHV.219 infection, and maintain tight latency as determined by the absence of RFP expression (top panels). When treated with TSA lytic replication is induced as determined by the expression of RFP (bottom panels). (D) RFP induction after lytic reactivation is concurrent with the upregulation of KSHV lytic gene expression as determined by qRT-PCR for RTA, K8, vIRF-1 and K8.1. Error bars represent the SD of duplicate wells. Data are representative of three independent experiments. (E) mECK^null.rK133 cells are episomally-infected with KSHV. mECK^null.rK133 cells were serially passaged in the absence of hygromycin and GFP was measured by flow cytometry. While almost 100% of cells are GFP positive at day 0, by day 70, they are 100% GFP negative, suggesting that the virus exists as an episome in the murine cells.</p

Self-Assembled Zn(II) Coordination Complexes Based on Mixed V‑Shaped Asymmetric Multicarboxylate and N‑Donor Ligands

Hydrothermal reaction between Zn­(OAc)₂·2H₂O and three asymmetric semirigid V-shaped multicarboxylate ligands H₃L^1–3 with the help of a 4,4'-bipyridine (4,4'-bpy) or 1,4-bis­(imidazol-1-ylmethyl)­benzene (bix) linker led to the isolation of six new coordination polymers, including [Zn₃(L¹)₂(4,4′-bpy)₂]_<i>n</i>·(H₂O)_2<i>n</i> (<b>1</b>), [Zn₃(L²)₂­(4,4′-bpy)­(H₂O)₂]_<i>n</i>·(H₂O)_2<i>n</i> (<b>2</b>), [Zn₃(L³)₂­(4,4′-bpy)₂­(H₂O)₄]_<i>n</i>·(H₂O)_6<i>n</i> (<b>3</b>), [Zn₃(L¹)₂­(bix)₃]_<i>n</i>·(H₂O)_7<i>n</i> (<b>4</b>), [Zn₃(L²)₂­(bix)₃]_<i>n</i>·(H₂O)_4<i>n</i> (<b>5</b>), and [Zn₃(HL³)₂­(bix)₂]_<i>n</i> (<b>6</b>), where H₃L¹, H₃L², H₃L³ ligands represent 3-(2-carboxyphenoxy)­phthalic acid, 4-(2-carboxyphenoxy)­phthalic acid, 3-(4-carboxyphenoxy)­phthalic acid, respectively. Single crystal X-ray diffraction analysis reveals a three-dimensional (3D) network for <b>1</b> and <b>3</b>–<b>5</b> but a two-dimensional (2D) structure for <b>2</b> and <b>6</b>. Despite the construction from the polymetallic chains connected by the 4,4′-bpy ligands for both compounds <b>1</b> and <b>2</b>, a 3D architecture was revealed for the former species while a 2D configuration for the latter one. Complex <b>3</b> contains open nanotube building units composed of sole 44-numbered metallomacrocycles. For <b>4</b>, the 20-numbered metallomacrocycle subunits linked by Zn ions give a 1D chain, which further form a 3D polymeric structure with the help of the other cyclic-shaped subunits made from the bix ligands and Zn ions. A 3D framework of <b>5</b> is generated from the 2D sheets simplified as a (6,3) net bound by the bix ligands. Compound <b>6</b> shows a 2D corrugated framework simplified as a (4,4) net assembled by the bix ligand and dinuclear zinc unit as node. These results seem to suggest that the diversity in the building subunits formed in <b>1</b>–<b>6</b> actually originates from the intrinsic nature of the three asymmetric V-shaped tricarboxylate ligands together with the tunable coordination geometry and molecular configurations of ligands by the N-donor ligand employed. In addition, the thermal stability and luminescence properties for the series of six complexes have also been investigated

Comparison of five statistics for proposed models.

<p>Models are ranked in order of their AIC (Akaike Information Criterion) scores, and other ranking are given along with values for corresponding statistics.</p

Effect of behavior on group–size distribution.

<p>Group-size distribution for individuals who are fighting (number of observations, <i>n</i> = 50) (A), flying (<i>n</i> = 942) (B), foraging (<i>n</i> = 199) (C) and perching (<i>n</i> = 4797) (D). Fighting distributions are adjusted to account for the fact that group sizes must be equal to or greater than 2.</p