A high throughput, interactive imaging, bright-field wound healing assay

The wound healing assay is a commonly used technique to measure cell motility and migration. Traditional methods of performing the wound healing assay suffer from low throughput and a lack of quantitative data analysis. We have developed a new method to perform a high-throughput wound healing assay that produces quantitative data using the LEAP? instrument. The LEAP? instrument is used to create reproducible wounds in each well of a 96-well plate by laser ablation. The LEAP? then records bright field images of each well at several time points. A custom texture segmentation algorithm is used to determine the wound area of each well at each time point. This texture segmentation analysis can provide faster and more accurate image analysis than traditional methods. Experimental results show that reproducible wounds are created by laser ablation with a wound area that varies by less than 10%. This method was tested by confirming that neuregulin-2β increases the rate of wound healing by MCF7 cells in a dose dependent manner. This automated wound healing assay has greatly improved the speed and accuracy, making it a suitable high-throughput method for drug screening.     

Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.     

Marmoset phylogenetics, conservation perspectives, and evolution of the mtDNA control region

Marmosets (genus Callithrix) are a diverse group of platyrrhine primates with 13-15 purported taxa, many of them considered endangered. Morphological analyses constitute most of the basis for recognition of these forms as distinct taxa. The purpose of this study was to provide a molecular view, based on mitochondrial control region sequences, of the evolutionary history of the marmosets, concomitant with a molecular phylogenetic perspective on species diversity within the group. An additional purpose was to provide the first comparative examination of a complete New World monkey control region sequence with those of other mammals. The phylogenetic analyses provide convincing support for a split between the Atlantic forest and Amazonian marmosets, with the inclusion of the pygmy marmoset (Cebuella pygmaea) at the base of the Amazonian clade. The earliest branch of the Atlantic forest group was C. aurita. In the Amazonian group, the analyses do not support the recognition of C. humeralifer and the recently described C mauesi as distinct taxa. They do, however, support a clear distinction between C. argentata and a strongly supported mixed clade of C. humeralifer and C. mauesi. In the Atlantic forest group, the phylogenetic tree suggests mixing between C. penicillata, C. kuhli, and possibly C. jacchus. Most of the sequence features characteristic of other mammal control regions were also evident in marmosets, with the exception that conserved sequence blocks (CSBs) 2 and 3 were not clearly identifiable. Tandem repeat units often associated with heteroplasmy in a variety of other mammals were not evident in the marmoset sequences.      

Memory and cellular immunity induced by a DNA vaccine encoding self antigen TPD52 administered with soluble GM-CSF

Tumor protein D52 (TPD52) is involved in cellular transformation, proliferation and metastasis. TPD52 over expression has been demonstrated in several cancers including prostate, breast, and ovarian carcinomas. Murine TPD52 (mD52) has been shown to induce anchorage independent growth in vitro and metastasis in vivo, and mirrors the function and normal tissue expression patterns of the human orthologue of TPD52. We believe TPD52 represents a self, non-mutated tumor associated antigen (TAA) important for maintaining a transformed and metastatic cellular phenotype. The transgenic adeno-carcinoma of the mouse prostate (TRAMP) model was employed to study mD52 as a vaccine antigen. Naïve mice were immunized with either recombinant mD52 protein or plasmid DNA encoding the full-length cDNA of mD52. Following immunization, mice were challenged with a subcutaneous, tumorigenic dose of mD52 positive, autochthonous TRAMP-C1 tumor cells. Sixty percent of mice were tumor free 85 days post challenge with TRAMP-C1 when immunized with mD52 as a DNA-based vaccine admixed with soluble granulocyte-macrophage colony stimulating factor (GM-CSF). Survivors of the initial tumor challenge rejected a second tumor challenge given in the opposite flank approximately 150 days after the first challenge, and remained tumor free for more than an additional 100 days. The T cell cytokine secretion patterns from tumor challenge survivors indicated that a T_H1-type cellular immune response was involved in tumor protection. These data suggest that mD52 vaccination induced a memory, cellular immune response that resulted in protection from murine prostate tumors that naturally over express mD52 protein.     

Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop

The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse-chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.     

Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Electronic nose for estimation of product concentration in mammalian cell cultivation

The off-gas composition from perfusion cultivation of a CHO-cell line producing recombinant human blood coagulation Factor VIII is monitored with an electronic nose. It is shown that the electronic nose in combination with an artificial neural network can be used for on-line estimation of the Factor VIII concentration in production-scale cultivations. The obtained prediction error (1†) for the Factor VIII concentration was 1.1 IU/ml. The potential of the electronic nose for estimation of viable cell count is outlined in laboratory-scale Factor VIII cultivations. The obtained prediction error (1†) for the viable cell count was 0.4᎒⁶ cells/ml. The results show that this non-invasive method is potentially useful for on-line bioprocess monitoring.