Production of stably transfected cell lines using immunoporation

Previous work from this laboratory has shown that immunoporation has the potential for the selective transfection of a range of different animal cells based on their immunological identity. The unique ability of immunoporation to target cells for transfection combined with the high efficiency of transfection and the high viability of cells make this method extremely promising for scientific and medical research. The experiments reported here show that not only can immunoporation produce transient transfection but also stably transfected cells are produced and such types of cells will be essential for the use of this method for gene therapy.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

One Step forward: Benthic Pelagic Coupling and Indicators for Environmental Status

A large data set from the Eastern Mediterranean was analyzed to explore the relationship between seawater column variables and benthic community status. Our results showed a strong quantitative link between the seawater column variables (Chlorophyll a and Eutrophication Index) and various indicators describing benthic diversity and community composition. The percentage of benthic opportunistic species increased significantly in the stations with high trophic status of the seawater column and so did the strength of the coupling between values of seawater column and benthic indicators. The Eutrophication Index threshold level of 0.85, separating the “Bad and Poor” from “Moderate to High” conditions could serve as an acceptable critical value above which there is a readily observable change in benthic community composition.     

Relationship of plasma adiponectin with sex hormone and insulin-like growth factor levels

Objective: Recent studies have suggested that a relationship between adiponectin and sex hormone, prolactin, and insulin‐like growth factor levels could be important for breast cancer risk and insulin sensitivity. Therefore, we assessed the relationship of adiponectin with plasma concentrations of estrone; estradiol; estrone sulfate; testosterone; androstenedione; dehydroepiandrosterone (DHEA); dehydroepiandrosterone sulfate (DHEAS); sex hormone binding globulin (SHBG); prolactin; insulin‐like growth factor (IGF‐1); its binding protein, IGF binding protein 3 (IGFBP‐3); c‐peptide; and IGF binding protein 1 (IGFBP‐1) among 360 postmenopausal women not taking postmenopausal hormones from the Nurses’ Health Study. Research Methods and Procedures: Multivariate models were adjusted for physical activity, alcohol consumption, age at blood draw, age at first birth/parity, fasting status, and time of day of blood draw; a separate model was additionally adjusted for BMI at blood draw. Results: Estrogens were inversely associated with adiponectin levels; however, except for free estradiol, these associations were substantially attenuated after adjustment for BMI. Free estradiol levels were 27% lower among women in the top vs. bottom quartile of adiponectin levels. No consistent associations were observed for the androgens, prolactin, IGF‐1, and IGFBP‐3. However, SHBG, c‐peptide, and IGFBP‐1 were strongly and independently associated with adiponectin levels (r = 0.29, ?0.30, 0.24, respectively). Conclusion: With the exceptions of SHBG, c‐peptide, and IGFBP‐1, the studied analytes were modestly associated with adiponectin and the associations were, in large part, mediated by body fat.     

Low CD10 mRNA Expression Identifies High-Risk Ductal Carcinoma In Situ (DCIS)

Purpose

Optimal management of breast ductal carcinoma in situ (DCIS) is controversial, and many patients are still overtreated. The local death of myoepithelial cells (MECs) is believed to be a pre-requisite to tumor invasion. We thus hypothesized that loss of CD10 expression, a MEC surface peptidase, would signify basement membrane disruption and confer increased risk of relapse in DCIS. The aim of our study was to retrospectively evaluate the prognostic value of CD10 in DCIS.

Experimental Design

CD10 expression was evaluated by quantitative RT-PCR and immunohistochemistry using paraffin-embedded samples of normal breast tissue (n = 11); of morphologically normal ducts associated with DCIS (n = 10); and of DCIS without an invasive component (n = 154).

Results

CD10 immunostaining was only observed in MECs in normal tissue and in DCIS. Normal tissue showed high mRNA expression levels of CD10, whereas DCIS showed a variable range. After a median follow-up of 6 years, DCIS with CD10 expression below the levels observed in normal tissue (71%) demonstrated a higher risk of local relapse (HR = 1.88; [95CI:1.30–2.70], p = 0.001) in univariate analysis. No relapse was observed in patients expressing high CD10 mRNA levels (29%) similar to the ones observed in normal tissue. In multivariate analysis including known prognostic factors, low CD10 mRNA expression remained significant (HR = 2.25; [95%CI:1.24–4.09], p = 0.008), as did the recently revised Van Nuys Prognostic Index (VNPI) score (HR = 2.03; [95%CI:1.23–3.35], p = 0.006).

Conclusion

The decrease of CD10 expression in MECs is associated with a higher risk of relapse in DCIS; this knowledge has the potential to improve DCIS management.     

SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERK-Dependent Tumors

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Synthesis and biological evaluation of novel angular fused Pyrrolocoumarins

Angular pyrrolocoumarins were synthesized from the reaction of 4-hydroxyindole or 5-hydroxyindole with DMAD and PPh(3) and were tested for anti-inflammatory and antioxidant activity. These compounds significantly inhibited the carrageenin-induced paw edema (60.5%-73.4%) and have important scavenging activity. Although their interaction with the free stable radical DPPH is not high, compound 9 is the most potent (73.4%) in the in vivo experiment. Compound 7 seems to be a potent LOX inhibitor. An attempt was made to correlate the biological results with their structural characteristics and physicochemical parameters.     

Expansion of the BioCyc collection of pathway/genome databases to 160 genomes

The BioCyc database collection is a set of 160 pathway/genome databases (PGDBs) for most eukaryotic and prokaryotic species whose genomes have been completely sequenced to date. Each PGDB in the BioCyc collection describes the genome and predicted metabolic network of a single organism, inferred from the MetaCyc database, which is a reference source on metabolic pathways from multiple organisms. In addition, each bacterial PGDB includes predicted operons for the corresponding species. The BioCyc collection provides a unique resource for computational systems biology, namely global and comparative analyses of genomes and metabolic networks, and a supplement to the BioCyc resource of curated PGDBs. The Omics viewer available through the BioCyc website allows scientists to visualize combinations of gene expression, proteomics and metabolomics data on the metabolic maps of these organisms. This paper discusses the computational methodology by which the BioCyc collection has been expanded, and presents an aggregate analysis of the collection that includes the range of number of pathways present in these organisms, and the most frequently observed pathways. We seek scientists to adopt and curate individual PGDBs within the BioCyc collection. Only by harnessing the expertise of many scientists we can hope to produce biological databases, which accurately reflect the depth and breadth of knowledge that the biomedical research community is producing.