Carboxyl-Modified Quantum Dots for NIR-IIb Bone Marrow Imaging

Real-time, noninvasive, and nonradiative bone imaging can directly visualize bone health but requires bone-targeted probes with high specificity. Herein, we propose that carboxyl-rich fluorescent nanoprobes are easily absorbed by macrophages in bone marrow during circulation, enabling optical bone marrow imaging in vivo. We used PbS/CdS core–shell quantum dots with NIR-IIb (1500–1700 nm) emission as substrates to prepare the carboxyl-rich nanoprobe. In vivo NIR-IIb fluorescence imaging with the nanoprobes showed high resolution and penetration depth in bone tissues and allowed for imaging-guided fracture diagnosis. Bone tissue slices showed substantial accumulation of carboxyl nanoprobes in the bone marrow and strong colocalization with macrophages. Similar results with CdSe quantum dots and an organic nanofluorophore suggest that carboxyl surface modification is effective to achieve bone marrow targeting, providing a novel strategy for developing bone/bone marrow imaging probes

Confocal microscopy of oocytes.

<p>A. An oocyte injected with phosphate-buffered saline. B. An oocyte injected with RNA encoding c-Myc-tagged DM2. Oocytes were treated with anti-cMyc antibodies (mouse 9E10) and then donkey anti-mouse Alexa Fluor 488 antibodies.</p

Amino acid sequences.

<p>A. Primary structures of influenza C virus M2 protein (CM2; Sequence ID, YP_089658.1 (C/Ann Arbor/1/1950)) and influenza D virus M2 protein (DM2; AFJ19025 (D/Swine/Oklahoma/1334/2011)). CM2 contains a 24-amino acid signal sequence (−24 to −1). Mature CM2 starts with Cys1 and contains extracellular, transmembrane (underlined), and internal domains in order from the amino to carboxyl terminals. Amino acid substitutions between Yamagata [BAA03793.1 (C/Yamagata/1/1988)] and Ann Arbor isolates are indicated in brackets. CM2 has disulfide-linked oligomerization sites (“*”; Cys1, Cys6, Cys20), a glycosylation site (Asn11, “<i>g</i>”), and a palmitoylation site (Cys65, “<i>p</i>”). A phosphorylation site involved in efficient virus replication is also indicated (Ser68, “<i>k</i>”) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199227#pone.0199227.ref011" target="_blank">11</a>]. In DM2, the predicted transmembrane domain is indicated by an underline. The YxxxK motif, Tyr72, and Lys76 in D is indicated by bold face. DM2 has a potential glycosylation site at Asn39 indicated by “<i>g</i>”. A C-terminal variant in D cMyc has a spacer GAG and a cMyc-tag EQKLISEEDL. A readthrough C-terminal variant of DM2, DM2 RT, has an extra peptide from the vector pNCB1. B. Structures of M2 proteins. AM2. The NMR structure (residues 22–62; PDB ID, 2L0J) and crystallographic structure (21–46; 4QKL) of influenza A virus M2 protein [AAA43303 [A/Udorn/1972(H3N2)]]. BM2. The NMR structure (1–33; 2KIX) of influenza B virus M2 protein [ACF54325.1 (B/Taiwan/70061/2006)]. CM2. The structure of influenza C virus M2 protein (27–46) was obtained by site-specific infrared dichroism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199227#pone.0199227.ref012" target="_blank">12</a>]. DM2. Influenza D virus M2 protein is focused on in this study. Solved or predicted structures are underlined. Amino acid residues providing experimental structure information are indicated by underlines. Primary structures of mutated DM2 are added with the isoelectric point (pI) and the hydrophobicity value (GRAVY) for the shaded helix domain. In GRAVY, greater positive values indicate higher hydrophobicity. C. Predicted secondary structure of carboxyl ends in DM2 constructs. Predicted secondary structures E and H correspond to β-strand and α-helix, respectively.</p

Activating and tail V_mid ^a values fitted by the charge–voltage Boltzmann distribution model (N = 5).

<p>Activating and tail V_mid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199227#t001fn001" target="_blank">^a</a> values fitted by the charge–voltage Boltzmann distribution model (N = 5).</p

Reversal potential (RP; N = 5).

<p>Reversal potential (RP; N = 5).</p

Western blot analysis.

<p>One each of the uninjected and the DM2 cMyc RNA injected oocyte was subjected to the Western Blot. The cMyc tag specific mouse monoclonal antibody 9E 10 was used to detect the expressed protein. The protein size is given in kDa.</p

Influenza D virus M2 protein exhibits ion channel activity in <i>Xenopus laevis</i> oocytes

<div><p>Background</p><p>A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a “mixing vessel” for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target.</p><p>Results</p><p>To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2–cMyc gene was synthesized and injected into <i>Xenopus</i> oocytes. The translation products of DM2–cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2–cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (V_mid) values in Boltzmann modeling for oocytes injected with DM2–cMyc RNA or a buffer were −152 and −200 mV, respectively. Assuming the same expression level in the <i>Xenopus</i> oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by V_mid for CM2 and DM2 were –141 and –146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were −21 and −22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower V_mid values while showing no change in reversal potential.</p><p>Conclusion</p><p>The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.</p></div

Induced current recorded by two-electrode voltage-clamp (TEVC) method at pH 8.5.

<p>A. Induced current and given membrane potential. An oocyte injected with either RNA or phosphate-buffered saline was subjected to a TEVC recording, and the current was measured for 2 s while the potential was kept constant at each of several membrane potentials with 10 mV intervals. A relaxation time of 5 s was set before the next measurement while the membrane was held at Vm = 0 mV. Short bars indicate values at -110 mV and the corresponding current for eye references. B-E. Overlapping traces for activating and tail current vs. holding potential. An average of 5 measurements were plotted with the SD. Solid bars indicate p<0.05 in the t-test. Cartoons represent the molecular organizations of M2 proteins. The green circle and D represents DIDS.</p

Mass spectrometry and peptide identification.

<p>Presented the MS/MS fragmentation spectrum of the 36 amino acids peptide identified, Arg123 –Lys158. The oocyte injected with RNA encoding DM2–cMyc was subjected to tryptic digestion followed by mass spectrometry using a nano-LC-MS/MS set-up. At 1255.91, a peptide of 36-amino acid long which includes the C-terminal region, the linker, and a portion of the cMyc sequence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199227#pone.0199227.g001" target="_blank">Fig 1</a>) was confidently identified with m/z value. The 20 and 17 successive peptide N-terminus retaining Y (blue) and peptide N-terminus retaining B fragment ions (red) matched the peptide sequence with a parent ion mass error of 1.5 ppm. The identified amino acid sequence for Y peptide shown is backward (blue). The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199227#pone.0199227.s001" target="_blank">S1 Table</a> shows the fragmentation table for the corresponding peptide Arg123 –Lys158 representing both b- and y-ions.</p

Deficiency in p16^Ink4a p19^Arf cooperates with oncogenic Kras^G12V to produce myeloid leukemia in NOD.SCID mice.

<p>(A) Kaplan-Meier survival curve for NOD.SCID recipients after transplantation with C57BL/6 bone marrow (BM) cells modified by the indicated oncogenetic lesions. (B, C) Body weight and spleen weight analyses for NOD.SCID mice receiving <i>p16p19^−/−; Kras(G12V)</i> BM cells via intra-venous transplantation at the time of sacrifice. Kras(G12V) and control groups were injected with cells and sacrificed for analysis at the same time points. (D) Hematoxylin & eosin stain of bone, spleen and liver from NOD.SCID leukemic mice, indicating metastasis of myeloid leukemia cells (60x). (E) Representative flow cytometry analysis of bone marrow from a NOD.SCID recipient of <i>p16p19^−/−; Kras(G12V)-GFP</i> expressing cells. Data are shown as 2-parameter contour plots for Forward Scatter (FSC) or for the indicated cell surface makers. Plots at right show data for cells previously gated for viability (propidium iodide-, not shown) and GFP expression (leftmost plot).</p