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Semiconductor nanocrystals or quantum dots (QDs) have unique optical and physical properties that make them potential imaging tools in biological and medical applications. However, concerns over the aqueous dispersivity, toxicity to cells, and stability in biological environments may limit the use of QDs in such applications. Here, we report an investigation into the cytotoxicity of aqueously dispersed CdSe(S) and CdSe(S)/ZnO core/shell QDs in the presence of human colorectal carcinoma cells (HCT-116) and a human skin fibroblast cell line (WS1). The cytotoxicity of the precursor solutions used in the synthesis of the CdSe(S) QDs was also determined in the presence of HCT-116 cells. CdSe(S) QDs were found to have a low toxicity at concentrations up to 100 &micro, g/mL, with a decreased cell viability at higher concentrations, indicating a highly dose-dependent response. Meanwhile, CdSe(S)/ZnO core/shell QDs exhibited lower toxicity than uncoated QDs at higher concentrations. Confocal microscopy images of HCT-116 cells after incubation with CdSe(S) and CdSe(S)/ZnO QDs showed that the cells were stable in aqueous concentrations of 100 &micro, g of QDs per mL, with no sign of cell necrosis, confirming the cytotoxicity data.

Highly luminescent quantum dots (QDs) that emit in the visible spectrum are of interest to a number of imaging technologies, not least that of biological samples. One issue that hinders the application of luminescent markers in biology is the potential toxicity of the fluorophore. Here we show that hydrothermally synthesized ZnSe(S) QDs have low cytotoxicity to both human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1). The QDs exhibited a high degree of crystallinity, with a strong blue photoluminescence at up to 29% quantum yield relative to 4',6-diamidino-2-phenylindole (DAPI) without post-synthetic UV-irradiation. Confocal microscopy images obtained of HCT-116 cells after incubation with the QDs highlighted the stability of the particles in cell media. Cytotoxicity studies showed that both HCT-116 and WS1 cells retain 100% viability after treatment with the QDs at concentrations up to 0.5g/L, which makes them of potential use in biological imaging applications.

Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets.

Semiconductor nanocrystals or quantum dots (QDs), have unique optical and physical properties that make them potential imaging tools in biological and medical applications. However, concerns such as the aqueous dispersivity, toxicity to cells and stability in biological environments may limit the use of QDs in bioapplications. Here, we report an investigation into the cytotoxicity of aqueously dispersed CdSe(S) and CdSe(S)/ZnO core/shell QDs in the presence of human colorectal carcinoma cells (HCT-116) and a human skin fibroblast cell line (WS-1). The cytotoxicity of the precursor solutions used in the synthesis of the CdSe(S) QDs was also determined in the presence of HCT-116 cells and compared to that of the heat-shock protein (Hsp90) inhibitor, 17-AAG. CdSe(S) QDs were found to have a low toxicity at concentrations up to 100 µg/ml, with a decreased cell viability at higher concentrations, indicating a highly dose-dependent response. Meanwhile, CdSe(S)/ZnO core/shell QDs exhibited lower toxicity than uncoated QDs at higher concentrations. Confocal microscopy images of HCT-116 cells after incubation with CdSe(S) and CdSe(S)/ZnO QDs showed that the cells were stable in aqueous concentrations of 100 µg of QDs per ml, with no sign of cell necrosis, confirming the cytotoxicity data. Key words: HCT-116, WS1, water dispersive QDs, aqueous synthesis, cytotoxicity of QDs.

Described are mechanistic studies of two Sanguinamide B (San B) derivatives. These compounds were identified as eukaryotic ribosomal inhibitors. Two biotinylated San B derivatives were synthesized and used to capture protein targets in a pull-down assay. LC/MS/MS analysis of the San B-captured targets identified several proteins that comprise eukaryotic ribosomal subunits. The translation inhibitory effect of San B was confirmed using an in vitro translation assay. Moreover, an evaluation of cell death mechanisms is reported.

We report the synthesis of a novel heat shock protein 90 (hsp90) inhibitor conjugated to a star polymer. Using reversible addition-fragmentation chain-transfer (RAFT) polymerization, we prepared star polymers comprised of PEG attached to a predesigned functional core. The stars were cross-linked using disulfide linkers, and a tagged version of our hsp90 inhibitor was conjugated to the polymer core to generate nanoparticles (14 nM). Dynamic light scattering showed that the nanoparticles were stable in cell growth media for 5 days, and HPLC analysis of compound-release at 3 different pH values showed that release was pH dependent. Cell cytotoxicity studies and confocal microscopy verify that our hsp90 inhibitor was delivered to cells using this nanoparticle delivery system. Further, delivery of our hsp90 inhibitor using star polymer induces apoptosis by a caspase 3-dependent pathway. These studies show that we can deliver our hsp90 inhibitor effectively using star polymers, and induce apoptosis by the same pathway as the parent compound.

Approaches to safely induce tolerance in vascularized composite allotransplantation (VCA) with chimerism through bone marrow transplantation (BMT) are currently being pursued. However, VCA was historically performed sequentially after donor chimerism was established. Delayed VCA is not clinically applicable due to the time constraints associated with procurement from deceased donors. A more clinically relevant approach to perform both BMT and VCA simultaneously was evaluated.Wistar Furth (RT1A) rats were treated with a short course of immunosuppressive therapy (anti-αβ-TCR monoclonal antibody, FK-506, and anti-lymphocyte serum). One day before BMT, rats were treated with varying doses of total body irradiation (TBI) followed by transplantation of heterotopic osteomyocutaneous flaps from hindlimbs of August Copenhagen Irish (RT1A) rats.Eighty percent of rats conditioned with 300 cGy TBI and 40% of rats receiving 400 cGy TBI accepted the VCA. Mixed chimerism was detected in peripheral blood at 1 month after VCA, but chimerism was lost in all transplant recipients by 4 months. Most peripheral donor cells originated from the BMT and not from the VCA. Acceptors of VCA were tolerant of a donor skin graft challenge and no anti-donor antibodies were detectable, suggesting a central deletional mechanism for tolerance. Regulatory T cells (Treg) from spleens of acceptors more potently suppressed lymphocyte proliferation than Treg from rejectors in the presence of donor stimulator cells.These studies suggest that simultaneous BMT and VCA may establish indefinite allograft survival in rats through Treg-mediated suppression and thymic deletion of alloreactive T cells.

We report the synthesis, cytotoxicity, and phenotypic analysis of oxazole and thiazole containing fragments. Evaluating the optimal size and heterocycle for growth inhibition and apoptosis showed that activity required at least two thiazoles sequentially connected. This is the first detailed comparison of biological activity between multi-heterocyclic containing fragments.

We report the first synthesis of sanguinamide B analogues. Substituting N-methylated (N-Me) amino acids, glycine (Gly), and L- or D-phenylalanine (Phe) into the backbone of sanguinamide B showed that only l- and d-Phe residues controlled the macrocycle conformation. The N-methylated and glycine analogues all had multiple conformations, whereas the L- and D-Phe derivatives only had a single conformation. Testing of all conformer analogues showed that inclusion of an L- or D-Phe was a superior design element than incorporating the N-Me moiety that is often utilized to control macrocyclic conformation. Finally, we show that there is an ideal Phe residue (in this case L-Phe) for generating compounds that have the greatest inhibitory effect on bacterial motility. Our data support the hypothesis that the macrocyclic conformation is dictated by the benzyl moiety requiring a "pseudoequatorial" position, and all other energy considerations are secondary.

Described is the synthesis of two biotinylated derivatives of a cytotoxic macrocycle. Pull-down assays indicate that this macrocycle targets the N-middle domain of Hsp90. Untagged compound can effectively compete away tagged compound–Hsp90 protein complexes, confirming the binding specificity of the macrocycle for Hsp90. The macrocycle is similar in potency to other structurally-related analogs of Sansalvamide A (San A) and induces apoptosis via a caspase 3 mechanism. Unlike other San A derivatives, we show that the macrocycle does not inhibit binding between C-terminal client proteins and co-chaperones and Hsp90, suggesting that it has a unique mechanism of action.

The first total synthesis of Sanguinamide B is reported, prepared via an efficient synthetic strategy. The natural product, trans,trans-Sanguinamide B (1), was generated in a thermodynamic ratio with trans,cis-Sanguinamide B (2) and cis,cis-Sanguinamide B (3). Complete conversion of the cis,cis-Sanguinamide B conformer (3) to the natural product (1) and the trans,cis- conformer (2) was achieved by heating to 170 °C. Biological evaluation indicated that the Sanguinamide B conformers disrupted the activity of a virulence determinant in P. aeruginosa.

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ -null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection.

Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ ( a lginate and m otility r egulator Z ).

Described is the antibiotic activity of a marine natural product. Psammaplysin F (1) inhibited the growth of four Gram-positive strains by >80% at 50 μM, and the amine at position C-20 is responsible for the observed antibacterial activity. When tested against two strains of methicillin resistant Staphylococcus aureus (MRSA), the minimum inhibitory concentrations (MICs) for psammaplysin F (40-80 μM) were similar to the structurally-related alkaloid psammaplysin H (2). Psammaplysin F (1) increased membrane permeability by two to four-fold compared to psammaplysin H (2) or control-treated bacteria, respectively. Unlike psammaplysin H (2), we show that psammaplysin F (1) inhibits equal partitioning of DNA into each daughter cell, suggesting that this natural product is a unique prokaryotic cell division inhibitor. © 2013 Elsevier Ltd. All rights reserved.

Described is a novel organorhodium(I) complex that is cytotoxic to the colon cancer cell line HCT116 and alters cell migration, DNA replication, and DNA condensation. Most importantly, the mechanism observed is not seen for the parent organorhodium dimer complex [{RhCl(COD)}2], RhCl3, or the free ligand/proligands (COD and 1-(n)butyl-3-methylimidazolium chloride). Thus, the activity of this organorhodium complex is attributable to its unique structure.

Metastasis occurs when cancer cells leave the primary tumor site and migrate to distant parts of the body. The CXCR4–SDF-1 pathway facilitates this migration, and its expression has become the hallmark of several metastatic cancers. Targeted approaches are currently being developed to inhibit this pathway, and several candidates are now in clinical trials. Continued exploration of CXCR4 inhibitors will generate compounds that have improved activity over current candidates.

Mucoid variants of the opportunistic pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate and colonize the respiratory tracts of cystic fibrosis patients. The genes encoding the alginate biosynthetic enzymes are clustered in a single operon, which is under tight transcriptional control. One essential activator of the alginate operon is AlgZ, a proposed ribbon-helix-helix DNA binding protein that shares 30% amino acid identity with the Mnt repressor of Salmonella enterica serovar Typhimurium bacteriophage P22. In the current study, we examined the role of AlgZ as an autoregulator. Using single-copy algZ-lacZ transcription fusions, an increase in algZ transcription was observed in an algZ mutant compared to the isogenic wild-type strain, suggesting that AlgZ may have an additional role as a repressor. To identify the AlgZ binding site, overlapping regions upstream of algZ were incubated with AlgZ and analyzed by electrophoretic mobility shift assays. Specific binding activity was localized to a region spanning from 66 to 185 base pairs upstream of the algZ transcriptional start site. Two AlgZ binding sites were defined using copper-phenanthroline footprinting and deletion analyses, with one site centered at 93 base pairs and the other centered at 161 base pairs upstream of the algZ promoter. Deletion of both binding sites resulted in the loss of AlgZ binding. These results indicate that AlgZ represses algZ transcription, and this activity is mediated by multiple AlgZ-DNA interactions.

Decades of research have been dedicated to the study of the opportunistic pathogen Pseudomonas aeruginosa, a Gram-negative, environmental bacterium that secretes the exopolysaccharide alginate during chronic lung infection of cystic fibrosis (CF) patients. Although P. aeruginosa utilizes a variety of factors to establish a successful infection in the lungs of CF patients, alginate has stood out as one of the best-studied prognostic indicators of chronic lung infection. While the genetics, biosynthesis and regulation of alginate are well understood, questions still remain concerning its role in biofilm development and its potential as a therapeutic target. The purpose of this review is to provide a brief summary of alginate biosynthesis and regulation, and to highlight recent discoveries in the areas of alginate production, biofilm formation and vaccine design. This information is placed in context with a proposed P. aeruginosa infectious pathway, highlighting avenues for the use of existing therapies as well as the potential for novel agents to reduce or eliminate chronic infections in CF patients.

Abstract 3709 CD8+/TCR− graft facilitating cells (FC) enhance engraftment of hematopoietic stem cells (HSC). They are distinct from HSC since they do not generate multilineage engraftment when infused alone, nor do they form colonies in vitro. The function of human FC remains to be defined. Here, we phenotypically characterized the human CD8+/TCR− FC subpopulations and evaluated their function in vivo and in vitro. Human CD8+/TCR− FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (PBMC). Approximately 55% of FC express CD56dim/−, and the remaining population is CD56bright. In the CD8+/TCR−/CD56bright cell population, approximately 65% of cells express CD11c+ and 67% of cells express CD11b+. In CD8+/TCR−/CD56dim/− cell population, the majority of cells express CD3ε+ (80%), 17% were CD11c+, 16% were CD19+, 14% were CD11b+, 11% were CD123+, and 30% were HLA-DR+. To evaluate whether human CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo, we transplanted 100,000 HSC alone or plus 300,000 CD8+/TCR−/CD56dim/− FC into NOD/SCID/IL2rγnull (NSG) recipient mice conditioned with 325 cGy of total body irradiation. At 30 days after transplantation, 8 of 21 (38%) recipients of HSC alone engrafted. In contrast, 81% of recipients (n = 16) receiving HSC plus CD8+/TCR−/CD56dim/− FC engrafted, and donor lymphocyte and monocyte chimerism in PB was 0.53% ± 0.16% and 3.93% ± 1.28%, respectively. At 6 months after transplantation, NSG recipients of HSC alone lost donor chimerism in PB and no donor cells were detected in spleen and BM. In contrast, NSG recipients of HSC + CD8+/TCR−CD56dim/− FC exhibited durable donor chimerism in PB and showed significantly higher levels of donor chimerism in spleen (13%) and BM (9.43%) compared to recipients of HSC alone. To evaluate the function of CD8+/TCR−/CD56dim/− FC in vitro, HSC were incubated with CD8+/TCR−CD56dim/− FC for 18 hrs and then cultured in methylcellulose for 14 days in colony-forming cell assay. HSC plus CD8+/TCR−CD56dim/− FC generated significantly more colonies compared with HSC alone (P = 0.0038), suggesting that human CD8+/TCR−/CD56dim/− FC have a direct effect on the clonogenicity of HSC (Figure). In summary, our data indicate that (1) human CD8+/TCR− FC are a heterogeneous cell population; (2) CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo and promote HSC clonogenicity in vitro; and (3) recipients of CD8+/TCR−/CD56dim/− FC maintain a durable donor chimerism in BM and spleen. Disclosures: Bozulic: Regenerex, LLC: Employment. King:Regenerex, LLC: Employment, Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.

Approaches to safely induce graft acceptance through bone marrow chimerism and limited immunosuppression are currently being pursued. Here we evaluated the effect of nonmyeloablative conditioning on the establishment of tolerance and mixed chimerism in recipients of bone marrow transplantation (BMT) and reconstructive tissue allotransplantation (RTA). Twenty Wistar-Firth rats were treated with a short course of immunosuppressive therapy (anti-ab-TCR mAb, tacrolimus, and anti-lymphocyte serum). One day prior to BMT, rats received total body irradiation followed by transplantation of heterotopic osteomyocutaneous flaps taken from hind limbs of ACI rats. Following BMT, rats were monitored for rejection and mixed chimerism (>2% donor cells). Fifteen rats rejected their RTA, and five rats accepted the graft. Mixed chimerism was detected in peripheral blood at one month after RTA, but chimerism was lost in all transplant recipients by four months. After 1.5 years post-RTA, the number of donor cells in spleen, mesenteric lymph node, recipient bone marrow and thymus was