No abstract

S emiconductor nanocrystals, also known as quantum dots (QDs), have emerged as a significant new class of materials over the past decade. The ability to synthesize highly monodisperse colloidal QDs, has paved the way for numerous spectroscopic studies assigning the QD electronic states and mapping out their evolution as function of size. 1,2 Fundamental understanding of these semiconductor nanocrystals has further stimulated the development of new materials, such as quantum nanorods, 3 magnetic nanocrystals, 4 QD-based solar cells, and light-emitting devices. 5 These related materials augment the large potential for using luminescent QDs as labeling reagents to empower numerous biotechnological applications.The unique optical and spectroscopic properties of QDs offer a compelling alternative to traditional fluorophores in almost all fluorescence-based applications. 6 The radii of QDs are smaller than the average physical separation between the electrons and holes (Exciton Bohr Radius), which results in a class of materials that dwell between the molecular and bulk forms of matter. Quantum confinement of both the electron and hole in all three dimensions leads to quantized energy levels or band gaps, which increase with decreasing crystal- ABSTRACT:Nanocrystalline semi-conductor materials, called quantum dots (QDs), exhibit unique optical and spectroscopic properties, which include broad absorption,

We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).

We describe a new family of discrete supramolecules comprising leucine-zipper peptides noncovalently assembled upon cognate leucine zippers fused to a dendrimer core. Circular dichroism and sedimentation equilibrium experiments clearly demonstrate that each leucine-zipper dendrimer (D-EZ4 or D-KZ4) can noncovalently display four leucine zippers on their surface that can be utilized for the multivalent display of protein cargo. Furthermore, we show that matched leucine-zipper dendrimers (D-EZ4/D-KZ4) can self-organize into fibers at neutral pH, providing a new scaffold for nanotechnology.

BackgroundRecent studies have shown that androgen displays an inhibitory effect on breast cancer cell lines that express androgen receptor (AR) but not estrogen receptor (ER) and progesterone receptor (PR). We have previously reported that approximately 1/3 of ER negative high grade invasive ductal carcinomas express AR. Thus, AR can serve as a potential therapeutic target for this group of patients.AimHere we investigated AR expression patterns in 980 consecutive breast carcinomas.ResultsWe found that (1) AR was expressed more frequently (77%) than ER (61%) and PR (60%) in breast carcinomas; (2) AR expression was associated with ER and PR expression (P < 0.0001), small tumor size (P = 0.0324) and lower Ki-67 expression (P = 0.0013); (3) AR expression was found in 65% of ER negative tumors; (4) AR expression was associated with PR and Ki-67 in ER negative tumors, but not in ER positive tumors; (5) AR expression was higher in ER positive subtypes (Luminal A, Luminal B and Luminal HER2 subtypes, 80%–86%) and lower in ER negative subtypes [HER2, triple negative (TN), and TN EFGR positive subtypes; 52%–66%], with over 50% of TN tumors expressing AR.ConclusionMore breast carcinomas express AR than ER and PR, including significant numbers of ER negative and TN tumors, for which AR could serve as a potential therapeutic target.

Interferons (IFNs) play pivotal roles in host innate immunity to protect against various virus infections. In respiratory virus infection, a major population of cells responsible for production of alpha IFN (IFN-␣) includes alveolar macrophages, conventional dendritic cells (cDCs), and plasmacytoid DCs (pDCs) (1). Of these cell types, pDCs are unique in their capacity to secrete large amounts of IFN-␣ (2). However, pDCs are not recruited unless viruses overcome the first defense line to cause systemic infection or can effectively prevent cDCs and alveolar macrophages from producing IFN-␣ (1). Since the signaling pathway leading to IFN-␣ production in pDCs is different from that in cDCs and alveolar macrophages, pDCs can combat even viruses that have acquired the ability to successfully limit IFN-␣ production of alveolar macrophages and cDCs during evolution. Whereas cDCs and alveolar macrophages employ the signaling pathway dependent on retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated protein 5 (MDA5), pDCs utilize the Toll-like receptor 7 (TLR7)-and TLR9-dependent signaling pathway to produce IFN-␣. It was found that most paramyxoviruses, including parainfluenza viruses, have evolved the V protein that blocks the TLR7/9-dependent pathway as well as the RLR-dependent pathway to counteract such a highly organized IFN production system (3-12). Studies on the blocking mechanisms for the RLR-dependent pathway uncovered that the paramyxovirus V protein physically associated with MDA5 and prevented double-stranded RNA from binding to MDA5, thereby inhibiting MDA5-dependent signaling (13,14). In addition to this common mechanism targeting MDA5, some paramyxovirus V proteins have acquired an additional mechanism that targets a downstream step. The V protein of the human parainfluenza virus type 2 (HPIV2), mumps virus, and parainfluenza virus 5 in the genus Rubulavirus interacts with inducible I-kappa B (IB) kinase (IKKi)/TANK-binding kinase 1 (TBK1) and inhibits activation of interferon regulatory factor 3 (IRF3) by acting as alternative substrates for IKKi/TBK1 (15). On the other hand, the V proteins of Sendai virus (SeV), measles virus (MeV), and Newcastle disease virus interact with IRF3 and suppress the transcriptional activity of IRF3 (16). In contrast to these extensive studies on the blockade of RLR-dependent signaling, the molecular basis for the blockade of TLR7/9-dependent signaling has not been well characterized. This study was thus intended to elucidate how the paramyxovirus V proteins block TLR7/9-dependent signaling leading to IFN-␣.pDCs, unlike other cell types, express a definite amount of cytosolic latent IRF7 in addition to endosomal TLR7 and TLR9 (17,18). Engagement of TLR7 and TLR9 with guanosine-rich and uridine-rich single-stranded RNA and unmethylated DNA with CpG motifs, respectively, induces complex formation of IRF7 with myeloid differentiation factor 88 (MyD88), interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK4), tum...

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-␣) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages.We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)-and TLR9-dependent IFN-␣ induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X 3 -Trp-X 9 -Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.Plasmacytoid dendritic cells (pDCs) are unique in their capacity to rapidly secrete vast amounts of alpha interferon (IFN-␣) via the Toll-like receptor 7 (TLR7)-and TLR9-dependent signaling pathway (2,5,11,12,15). The TLR7/9-dependent signaling pathway is specific to pDCs. This specificity relies on the constitutive expression of latent IFN regulatory factor 7 (IRF7) (6) and endosomal TLR7/9 (7). It was reported that measles virus (MeV) infection inhibited IFN synthesis of pDCs stimulated by the TLR7/9 agonist (29), and this inhibitory effect was exerted by viral V protein (26). Since pDCs produce IFN-␣ only when viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs (cDCs) and macrophages (14), it is reasonable for MeV to have a strategy that antagonizes the function of pDCs. However, when virus infection was restricted in the lung, it was found that the major source of IFN-␣ was limited to cDCs and alveolar macrophages and that pDCs did not produce . Thus, we seek to determine whether paramyxoviruses, whose infections are restricted to the respiratory tract, can block TLR7/9-dependent signaling.To examine the effect of V proteins from various paramyxoviruses on TLR7/9-dependent signaling, we employed a reconstitution system in 293T cells according to the procedure of Pfaller and Conzelmann (26). 293T cells were transfected with a PGV-B2 (Wako)-based luciferase reporter plasmid under the control of the mouse IFN-␣6 promoter together with expression plasmids encoding a mouse TLR7/9 (mTLR7/9) downstream signaling molecule, MyD88, TRAF6, IB kinase ␣ (IKK␣), or IRF7 in various combinations. To create these expression plasmids, each gene was subcloned into pCA7, which carries the cytomegalovirus enhancer chicken ␤-actin hybrid promoter (25,30). Expression of IRF7 alone resulted in apparent induction of luciferase activity (Fig. 1A). It was enhanced 3-to 4-fold when upstream signaling molecules, MyD88, TRAF6, and IKK␣, were coexpressed. This enhanced activation was significantly suppressed in the presence of any V protein of the paramyxoviruses, causing a local infection (human parainfluenza virus type 2 [hPIV2], Sendai virus [SeV], and bovine PIV3 [bPIV3]) or a systemic infection (MeV and Nipah virus [NiV]) (Fig. 1A). Essentially the same results were obtained with an established cell line, 293XL-mTLR7 (...

a b s t r a c tParamyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN-a promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll-like receptor (TLR) 7-and TLR9-dependent IFN-a induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKa and inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion. Structured summary of protein interactions:SeV C physically interacts with IKKalpha by anti tag coimmunoprecipitation (View interaction) bPIV3 C physically interacts with IKKalpha by anti tag coimmunoprecipitation (1, 2) NiV C physically interacts with IKKalpha by anti tag coimmunoprecipitation (1, 2) MeV C physically interacts with IKKalpha by anti tag coimmunoprecipitation (1, 2)