A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

Radio-Optical Imaging of ATLBS Survey

We present the radio-optical imaging of ATLBS, a sensitive radio survey (Subrahmanyan et al. 2010). The primary aim of the ATLBS survey is to image low-power radio sources which form the bulk of the radio source population to moderately high red-shifts (z similar to 1.0). The accompanying multiband optical and near infra-red observations provide information about the hosts and environments of the radio sources. We give here details of the imaging of the radio data and optical data for the ATLBS survey.

Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair Implications for Fanconi Anemia and Breast Cancer Susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.

Studying the resistivity imaging of chicken tissue phantoms with different current patterns in Electrical Impedance Tomography (EIT)

A current injection pattern in Electrical Impedance Tomography (EIT) has its own current distribution profile within the domain under test. Hence, different current patterns have different sensitivity, spatial resolution and distinguishability. Image reconstruction studies with practical phantoms are essential to assess the performance of EIT systems for their validation, calibration and comparison purposes. Impedance imaging of real tissue phantoms with different current injection methods is also essential for better assessment of the biomedical EIT systems. Chicken tissue paste phantoms and chicken tissue block phantoms are developed and the resistivity image reconstruction is studied with different current injection methods. A 16-electrode array is placed inside the phantom tank and the tank is filled with chicken muscle tissue paste or chicken tissue blocks as the background mediums. Chicken fat tissue, chicken bone, air hole and nylon cylinders are used as the inhomogeneity to obtained different phantom configurations. A low magnitude low frequency constant sinusoidal current is injected at the phantom boundary with opposite and neighboring current patterns and the boundary potentials are measured. Resistivity images are reconstructed from the boundary data using EIDORS and the reconstructed images are analyzed with the contrast parameters calculated from their elemental resistivity profiles. Results show that the resistivity profiles of all the phantom domains are successfully reconstructed with a proper background resistivity and high inhomogeneity resistivity for both the current injection methods. Reconstructed images show that, for all the chicken tissue phantoms, the inhomogeneities are suitably reconstructed with both the current injection protocols though the chicken tissue block phantom and opposite method are found more suitable. It is observed that the boundary potentials of the chicken tissue block phantoms are higher than the chicken tissue paste phantom. SNR of the chicken tissue block phantoms are found comparatively more and hence the chicken tissue block phantom is found more suitable for its lower noise performance. The background noise is found less in opposite method for all the phantom configurations which yields the better resistivity images with high PCR and COC and proper IRMean and IRMax neighboring method showed higher noise level for both the chicken tissue paste phantoms and chicken tissue block phantoms with all the inhomogeneities. Opposite method is found more suitable for both the chicken tissue phantoms, and also, chicken tissue block phantoms are found more suitable compared to the chicken tissue paste phantom. (C) 2012 Elsevier Ltd. All rights reserved.

Ferrocene-Conjugated Copper(II) Complexes of L-Methionine and Phenanthroline Bases: Synthesis, Structure, and Photocytotoxic Activity

Ferrocene-conjugated reduced Schiff base (Fc-metH) copper(II) complexes of L-methionine and phenanthroline bases, namely, Cu(Fc-met)(B)](NO3), where B is 1,10-phenanthroline (phen in 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq in 2), dipyrido3,2-a:2',3'-c]phenazine (dppz in 3), and 2-(naphthalen-1-yl)-1H-imidazo4,5-f]1,10]phenanthroline (nip in 4), were prepared and characterized and their photocytotoxicity studied (Fc = ferrocenyl moiety). Complexes Cu(Ph-met)(B)](NO3) of the reduced Schiff base from benzaldehyde and L-methionine (Ph-metH) and B (phen in 5, dppz in 6) were prepared and used as control species. Complexes 1 and 5 were structurally characterized by X-ray crystallography. Complex 1 as a discrete monomer has a CuN3OS core with the thiomethyl group as the axial ligand. Complex 5 has a polymeric structure with a CuN3O2 core in the solid state. Complexes 5 and 6 are formulated as Cu(Ph-met)(B)(H2O)] (NO3) in an aqueous phase based on the mass spectral data. Complexes 1-4 showed the Cu(II)-Cu(I) and Fc(+)-Fc redox couples at similar to 0.0 and similar to 0.5 V vs SCE, respectively, in DMF-0.1 M (Bu4N)-N-n](ClO4). A Cu(II)-based weak d-d band near 600 nm and a relatively strong ferrocenyl band at similar to 450 nm were observed in DMF-Tris-HCl buffer (1:4 v/v). The complexes bind to calf thymus DNA, exhibit moderate chemical nuclease activity forming (OH)-O-center dot radical species, and are efficient photocleavers of pUC19 DNA in visible light of 454, 568, and 647 rim, forming (OH)-O-center dot radical as the reactive oxygen species. They are cytotoxic in HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells, showing an enhancement of cytotoxicity upon visible light irradiation. Significant change in the nuclear morphology of the HeLa cells was observed with 3 in visible light compared to the nonirradiated sample. Confocal imaging using 4 showed its nuclear localization within the HeLa cells.

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the `mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes. Oncogene (2012) 31, 1896-1909; doi:10.1038/onc.2011.378; published online 29 August 2011

Spatial Filter Based Bessel-Like Beam for Improved Penetration Depth Imaging in Fluorescence Microscopy

Monitoring and visualizing specimens at a large penetration depth is a challenge. At depths of hundreds of microns, several physical effects (such as, scattering, PSF distortion and noise) deteriorate the image quality and prohibit a detailed study of key biological phenomena. In this study, we use a Bessel-like beam in-conjugation with an orthogonal detection system to achieve depth imaging. A Bessel-like penetrating diffractionless beam is generated by engineering the back-aperture of the excitation objective. The proposed excitation scheme allows continuous scanning by simply translating the detection PSF. This type of imaging system is beneficial for obtaining depth information from any desired specimen layer, including nano-particle tracking in thick tissue. As demonstrated by imaging the fluorescent polymer-tagged-CaCO3 particles and yeast cells in a tissue-like gel-matrix, the system offers a penetration depth that extends up to 650 mu m. This achievement will advance the field of fluorescence imaging and deep nano-particle tracking.

A phenanthrene based highly selective fluorogenic and visual sensor for Cu2+ ion with nanomolar detection limit and its application in live cell imaging

A new phenanthrene based chemosensor has been synthesized and investigated to act as highly selective fluorescence and visual sensor for Cu2+ ion with very low detection limit of 1.58 nM: this has also been used to image Cu2+ in human cervical HeLa cancer cells. (C) 2012 Elsevier B.V. All rights reserved.