Locking Multi-Laser Frequencies to a Precision Wavelength Meter: Application to Cold Atoms.

We herein report a simultaneous frequency stabilization of two 780-nm external cavity diode lasers using a precision wavelength meter (WLM). The laser lock performance is characterized by the Allan deviation measurement in which we find σy=10-12 at an averaging time of 1000 s. We also obtain spectral profiles through a heterodyne spectroscopy, identifying the contribution of white and flicker noises to the laser linewidth. The frequency drift of the WLM is measured to be about 2.0(4) MHz over 36 h. Utilizing the two lasers as a cooling and repumping field, we demonstrate a magneto-optical trap of 87Rb atoms near a high-finesse optical cavity. Our laser stabilization technique operates at broad wavelength range without a radio frequency element.

Discovery of 2-iminobenzimidazoles as potent hepatitis C virus inhibitors with a novel mechanism of action.

In this report we describe 2-iminobenzimidazole (IBI) analogs, identified during the course of a phenotypic high-throughput screening campaign, as novel hepatitis C virus (HCV) inhibitors. A series of IBI derivatives was synthesized and evaluated for their inhibitory activity against infectious HCV. Among the IBIs derivatives studied in this work, we identified promising compounds with high antiviral efficacy, high selectivity index and good microsomal stability. Noteworthy, the IBI series exhibited inhibitory activity on early and late steps of the viral cycle, but not in the HCV replicon system demonstrating a mechanism of action distinct from clinical-stage and approved anti-HCV drugs. Overall, our results suggest that IBIs are predestinated for further exploration as lead compounds for novel HCV interventions.

Identification of a series of 1,3,4-trisubstituted pyrazoles as novel hepatitis C virus entry inhibitors.

In this report we describe the identification of novel pyrazole analogs as potent hepatitis C virus (HCV) entry inhibitor. The pyrazoles were identified by our phenotypic high-throughput screening using infectious HCV. A series of pyrazole derivatives was synthesized and evaluated for inhibitory activity against HCV in the infectious cell culture system. Through evaluation of selected compounds we observed that the pyrazoles did not interfere with HCV RNA replication but with viral entry as shown by experiments with HCV replicons and HCV pseudo particles, respectively.

Discovery and characterization of a novel 7-aminopyrazolo[1,5-a]pyrimidine analog as a potent hepatitis C virus inhibitor.

We describe a novel 7-aminopyrazolo[1,5-a]pyrimidine (7-APP) derivative as a potent hepatitis C virus (HCV) inhibitor. A series of 7-APPs was synthesized and evaluated for inhibitory activity against HCV in different cell culture systems. The synthesis and preliminary structure-activity relationship study of 7-APP are reported.

Magnetic bionanoparticle enhances homing of endothelial progenitor cells in mouse hindlimb ischemia.

BACKGROUND AND OBJECTIVES: Poor homing efficiency is one of the major limitations of current stem cell therapy. Magnetic bionanoparticles (MPs) obtained from Magnetospirillum sp. AMB-1 have a lipid bilayer membrane and ferromagnetic properties. We evaluated a novel priming strategy using MPs to enhance the homing of transplanted progenitor cells to target tissue. MATERIALS AND METHODS: Effects of MP on proliferation, viability, and migration of late human endothelial progenitor cells (EPCs) were examined in vitro. Additionally, effects of MP on gene and protein expression related to survival and adhesion were evaluated. Homing and angiogenic efficiency of MP transferred late EPCs was evaluated in nude mouse hindlimb ischemia model. RESULTS: Below threshold concentration, MP transfer did not influence proliferation or survival of late EPCs, but enhanced migration and trans-endothelial migration of late EPCs toward magnet. Below threshold concentration, MP transfer did not influence gene and protein expression related to survival. In the mouse hindlimb ischemia model, late EPCs treated with high dose MP (5 ug/mL) showed enhanced homing of injected late EPCs in the ischemic limb by magnet, compared to low dose MP (1 ug/mL) treated late EPCs. In addition, high dose MP transferred EPC showed significantly better improvement of perfusion in ischemic limb compared to untreated EPC. CONCLUSION: MP transfer with magnet application can be a promising novel strategy to enhance homing efficacy and outcomes of current stem cell therapy.

Quantum dot-based screening system for discovery of g protein-coupled receptor agonists.

Cellular imaging has emerged as an important tool to unravel biological complexity and to accelerate the drug-discovery process, including cell-based screening, target identification, and mechanism of action studies. Recently, semiconductor nanoparticles known as quantum dots (QDs) have attracted great interest in cellular imaging applications due to their unique photophysical properties such as size, tunable optical property, multiplexing capability, and photostability. Herein, we show that QDs can also be applied to assay development and eventually to high-throughput/content screening (HTS/HCS) for drug discovery. We have synthesized QDs modified with PEG and primary antibodies to be used as fluorescent probes for a cell-based HTS system. The G protein-coupled receptor (GPCR) family is known to be involved in most major diseases. We therefore constructed human osteosarcoma (U2OS) cells that specifically overexpress two types of differently tagged GPCRs: influenza hemagglutinin (HA) peptide-tagged κ-opioid receptors (κ-ORs) and GFP-tagged A3 adenosine receptors (A3AR). In this study, we have demonstrated that 1) anti-HA antibody-conjugated QDs could specifically label HA-tagged κ-ORs, 2) subsequent treatment of QD-tagged GPCR agonists allowed agonist-induced translocation to be monitored in real time, 3) excellent emission spectral properties of QD permitted the simultaneous detection of two GPCRs in one cell, and 4) the robust imaging capabilities of the QD-antibody conjugates could lead to reproducible quantitative data from high-content cellular images. These results suggest that the present QD-based GPCR inhibitor screening system can be a promising platform for further drug screening applications.

N-cadherin determines individual variations in the therapeutic efficacy of human umbilical cord blood-derived mesenchymal stem cells in a rat model of myocardial infarction.

In this study, we established and characterized human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) from four different donors. However, the hUCB-MSCs showed remarkable variations in their therapeutic efficacy for repairing rat infarcted myocardium (including the process of angiogenesis) 8 weeks after transplantation. In addition, we observed that the level of vascular endothelial growth factor (VEGF) is correlated with the therapeutic efficacy of the four hUCB-MSCs. Next, to investigate the practical application of hUCB-MSCs, we searched for surface signature molecules that could serve as indicators of therapeutic efficacy. The gene for N-cadherin was the only cell surface gene that was highly expressed in the most effective hUCB-MSCs, both at the transcriptional and translational levels. We observed downregulation and upregulation of VEGF in response to N-cadherin blocking and N-cadherin overexpression, respectively. Activation of extracellular signal-regulated kinase (ERK), but not protein kinase B, was increased when N-cadherin expression was increased, whereas disruption of N-cadherin-mediated cell-cell contact induced suppression of ERK activation and led to VEGF downregulation. Moreover, by investigating hUCB-MSCs overexpressing N-cadherin or N-cadherin knockdown hUCB-MSCs, we confirmed the in vivo function of N-cadherin. In addition, we observed that DiI-labeled hUCB-MSCs express N-cadherin in the peri-infarct area and interact with cardiomyocytes.

New culture system for human embryonic stem cells: autologous mesenchymal stem cell feeder without exogenous fibroblast growth factor 2.

Human embryonic stem (hES) cells have been successfully maintained using human-cell feeder systems or feeder-free systems. However, despite advances in culture techniques, hES cells require supplementation with fibroblast growth factor 2 (FGF-2), an exogenous stemness factor, which is needed to sustain the authentic undifferentiated status. We developed a new culture system for hES cells; this system does not require supplementation with FGF-2 to obtain hES cells that are suitable for tissue engineering and regenerative medicine. This culture system employed mesenchymal stem cells derived from hES cells (hESC-MSCs) as autologous human feeder cells in the absence of FGF-2. The hES cell line SNUhES3 cultured in this new autologous feeder culture system maintained the typical morphology of hES cells and expression of pluripotency-related proteins, SSEA-4, TRA-1-60, OCT4, and alkaline phosphatase, without development of abnormal karyotypes after more than 30 passages. RNA expression of the pluripotency-related genes OCT4 and NANOG was similar to the expression in SNUhES3 cells maintained on xenofeeder STO cells. To identify the mechanism that enables the cells to be maintained without exogenous FGF-2, we checked the secretion of FGF-2 from the mitomycin-C treated autofeeder hESC-MSCs versus xenofeeder STO cells, and confirmed that hESC-MSCs secreted FGF-2 whereas STO cells did not. The level of FGF-2 in the media from the autofeeder system without exogenous FGF-2 was comparable to that from the xenofeeder system with addition of FGF-2. In conclusion, our new culture system for hES cells, which employs a feeder layer of autologous hESC-MSCs, supplies sufficient amounts of secreted FGF-2 to eliminate the requirement for exogenous FGF-2.

Intracellular protein target detection by quantum dots optimized for live cell imaging.

Imaging of specific intracellular target proteins in living cells has been of great challenge and importance for understanding intracellular events and elucidating various biological phenomena. Highly photoluminescent and water-soluble semiconductor nanocrystal quantum dots (QDs) have been extensively applied to various cellular imaging applications due to the long-term photostability and the tunable narrow emission spectra with broad excitation. Despite the great success of various bioimaging and diagnostic applications, visualization of intracellular targets in live cells still has been of great challenge. Nonspecific binding, difficulty of intracellular delivery, or endosomal trapping of nanosized QDs are the main reasons to hamper specific target binding in live cells. In this context, we prepared the polymer-coated QDs (pcQD) of which the surface was optimized for specific intracellular targeting in live cells. Efficient intracellular delivery was achieved through PEGylation and subsequent cell penetrating peptide (i.e., TAT) conjugation to the pcQD in order to avoid significant endosomal sequestration and to facilitate internalization of the QDs, respectively. In this study, we employed HEK293 cell line overexpressing endothelin A receptor (ET(A)R), a family of G-protein coupled receptor (GPCR), of which the cytosolic c-terminal site is genetically engineered to possess green fluorescent protein (GFP) as our intracellular protein target. The fluorescence signal of the target protein and the well-defined intracellular behavior of the GPCR help to evaluate the targeting specificity of QDs in living cells. To test the hypothesis that the TAT-QDs conjugated with antibody against intracellular target of interest can find the target, we conjugated anti-GFP antibody to TAT-PEG-pcQD using heterobifunctional linkers. Compared to the TAT-PEG-pcQD, which was distributed throughout the cytoplasm, the antiGFP-functionalized TAT-PEG-pcQD could penetrate the cell membrane and colocalize with the GFP. An agonist (endothelin-1, ET-1) treatment induced GFP-ET(A)R translocation into pericentriolar region, where the GFP also significantly colocalized with antiGFP-TAT-PEG-pcQD. These results demonstrate that stepwise optimization of PEG-pcQD conjugation with both a cell penetrating peptide and an antibody against a target of interest allows specific binding to the intracellular target protein with minimized nonspecific binding.

Fluorogenic assay and live cell imaging of HIV-1 protease activity using acid-stable quantum dot-peptide complex.

A novel QD-peptide complex for detecting HIV-1 protease activity was prepared from simple one step electrostatic interaction. Fluorescence recovery of the pre-quenched QD through fluorescence resonance energy transfer allowed for in vitro assay and live cell imaging of the protease activity in HIV-1 transfected cells, proving the potential for cell-based protease inhibitor screening.

Characterization and cancer cell specific binding properties of anti-EGFR antibody conjugated quantum dots.

Synthesis of biologically active antibody conjugated quantum dots (QDs) has been of great importance in cellular imaging and diagnostics. Cetuximab (or Erbitux) is the first monoclonal antibody drug which targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. In the present work, we investigated three different conjugation strategies to obtain the biologically functional QD-cetuximab conjugates for the tumor-specific imaging. Successful conjugation of cetuximab to QDs was achieved using PEG conjugated polymer-coated QDs and two long-chain heterobifunctional linkers, sulfo-LC-SPDP and sulfo-SMCC. The dissociation constant of the QD-cetuximab conjugates to EGFR was determined to be 0.61 +/- 0.28 nM. The cancer cell-specific binding ability of the QD-cetuximab conjugates was evaluated in vitro, and the cellular internalization of the QD-cetuximab conjugates was clearly demonstrated in live cells by confocal microscopy. The cellular imaging experiments using the QD-cetuximab conjugates showed a clear endocytosis pathway, which was evidenced by the colocalization of the QD-cetuximab conjugates with dye-labeled transferrin. These results suggest that the QD-cetuximab conjugates as an imaging modality for tumor EGFR overexpression can be expected to provide important information on the expression levels of EGFR on the cancer cells.

Novel embryoid body-based method to derive mesenchymal stem cells from human embryonic stem cells.

Application of human embryonic stem cells (hESCs) to stem-cell therapy is not feasible because of the risk of tumorigenicity and rejection. In contrast, human mesenchymal stem cells (hMSCs) are free from the risk of tumorigenicity and also have immune privilege. However, hMSCs obtained from adults have infinite variety in terms of the biological characteristics and functionality. We report here a new derivation method of hMSCs from hESCs. The derivation of hMSCs from three different hESC lines (SNUhES3, CHA3-hESC, and H9) was performed by embryoid bodies formation and subsequent culture with stage-different media without using inductive xenogenic feeder and mechanical selection procedure. The derived cells were morphologically similar to the unique fingerprint-like pattern of hMSCs and grew stably for at least 35 passages in vitro. These cells had hMSCs-like immunophenotypes: negative for CD34 and CD45; positive for CD29, CD44, CD73, CD90, and CD105. They could be differentiated into multiple lineages including osteocytes, chondrocytes, adipocytes, and myocytes. They maintained normal karyotype during the long-term cultivation and did not show tumorigenicity when transplanted into the immunodeficient mice. In conclusion, the new embryoid body-based derivation method of hMSCs from hESCs is simple, safe, and reproducible in three different hESC lines. We expect that this method will provide a more effective and powerful tool to derive hMSCs from various hESC lines.

Compact and versatile nickel-nitrilotriacetate-modified quantum dots for protein imaging and Förster resonance energy transfer based assay.

The generation of compact quantum dots (QDs) probes is of critical importance for visualizing molecular interaction occurring in biological context, particularly when using the Förster resonance energy transfer (FRET) approach. This Article reports novel water-soluble compact CdSe/ZnS QDs prepared by ligand exchange reaction using thiolated nitrilotriacetate (NTA). The resulting NTA-QDs revealed higher stability and remarkable conjugation efficiency compared to the other QDs prepared with different ligands by using the ligand exchange method. The Ni-NTA group is a well-known binding moiety for the detection and purification of oligohistidine-tagged recombinant proteins. We demonstrated that NiNTA-QDs prepared by Ni(2+) complexation exhibited highly specific binding ability toward 6-histidine (His)-tagged peptides present in various experimental conditions (buffer solution, agarose beads, and HEK cells). Importantly, the compact NiNTA-QDs serve as an efficient FRET donor. These results suggest that the stable and highly selective multifunctional NTA-QDs can be useful for labeling and tracking molecular interactions within biological context.

Impact of myocardial infarct proteins and oscillating pressure on the differentiation of mesenchymal stem cells: effect of acute myocardial infarction on stem cell differentiation.

Stem cell transplantation in acute myocardial infarction (AMI) has emerged as a promising therapeutic option. We evaluated the impact of AMI on mesenchymal stem cell (MSC) differentiation into cardiomyocyte lineage. Cord blood-derived human MSCs were exposed to in vitro conditions simulating in vivo environments of the beating heart with acute ischemia, as follows: (a) myocardial proteins or serum obtained from sham-operated rats, and (b) myocardial proteins or serum from AMI rats, with or without application of oscillating pressure. Expression of cardiac-specific markers on MSCs was greatly induced by the infarcted myocardial proteins, compared with the normal proteins. It was also induced by application of oscillating pressure to MSCs. Treatment of MSCs with infarcted myocardial proteins and oscillating pressure greatly augmented expression of cardiac-specific genes. Such expression was blocked by inhibitor of transforming growth factor beta(1) (TGF-beta(1)) or bone morphogenetic protein-2 (BMP-2). In vitro cellular and electrophysiologic experiments showed that these differentiated MSCs expressing cardiomyocyte-specific markers were able to make a coupling with cardiomyocytes but not to selfbeat. The pathophysiologic significance of in vitro results was confirmed using the rat AMI model. The protein amount of TGF-beta(1) and BMP-2 in myocardium of AMI was significantly higher than that in normal myocardium. When MSCs were transplanted to the heart and analyzed 8 weeks later, they expressed cardiomyocyte-specific markers, leading to improved cardiac function. These in vitro and in vivo results suggest that infarct-related biological and physical factors in AMI induce commitment of MSCs to cardiomyocyte-like cells through TGF-beta/BMP-2 pathways.