Effects of nanoparticles on murine macrophages

Metallic nanoparticles are more and more widely used in an increasing number of applications. Consequently, they are more and more present in the environment, and the risk that they may represent for human health must be evaluated. This requires to increase our knowledge of the cellular responses to nanoparticles. In this context, macrophages appear as an attractive system. They play a major role in eliminating foreign matter, e.g. pathogens or infectious agents, by phagocytosis and inflammatory responses, and are thus highly likely to react to nanoparticles. We have decided to study their responses to nanoparticles by a combination of classical and wide-scope approaches such as proteomics. The long term goal of this study is the better understanding of the responses of macrophages to nanoparticles, and thus to help to assess their possible impact on human health. We chose as a model system bone marrow-derived macrophages and studied the effect of commonly used nanoparticles such as TiO2 and Cu. Classical responses of macrophage were characterized and proteomic approaches based on 2D gels of whole cell extracts were used. Preliminary proteomic data resulting from whole cell extracts showed different effects for TiO2-NPs and Cu-NPs. Modifications of the expression of several proteins involved in different pathways such as, for example, signal transduction, endosome-lysosome pathway, Krebs cycle, oxidative stress response have been underscored. These first results validate our proteomics approach and open a new wide field of investigation for NPs impact on macrophagesComment: Nanosafe2010: International Conference on Safe Production and Use of Nanomaterials 16-18 November 2010, Grenoble, France, Grenoble : France (2010

Label-free isolation of prostate circulating tumor cells using Vortex microfluidic technology.

There has been increased interest in utilizing non-invasive "liquid biopsies" to identify biomarkers for cancer prognosis and monitoring, and to isolate genetic material that can predict response to targeted therapies. Circulating tumor cells (CTCs) have emerged as such a biomarker providing both genetic and phenotypic information about tumor evolution, potentially from both primary and metastatic sites. Currently, available CTC isolation approaches, including immunoaffinity and size-based filtration, have focused on high capture efficiency but with lower purity and often long and manual sample preparation, which limits the use of captured CTCs for downstream analyses. Here, we describe the use of the microfluidic Vortex Chip for size-based isolation of CTCs from 22 patients with advanced prostate cancer and, from an enumeration study on 18 of these patients, find that we can capture CTCs with high purity (from 1.74 to 37.59%) and efficiency (from 1.88 to 93.75 CTCs/7.5 mL) in less than 1 h. Interestingly, more atypical large circulating cells were identified in five age-matched healthy donors (46-77 years old; 1.25-2.50 CTCs/7.5 mL) than in five healthy donors <30 years old (21-27 years old; 0.00 CTC/7.5 mL). Using a threshold calculated from the five age-matched healthy donors (3.37 CTCs/mL), we identified CTCs in 80% of the prostate cancer patients. We also found that a fraction of the cells collected (11.5%) did not express epithelial prostate markers (cytokeratin and/or prostate-specific antigen) and that some instead expressed markers of epithelial-mesenchymal transition, i.e., vimentin and N-cadherin. We also show that the purity and DNA yield of isolated cells is amenable to targeted amplification and next-generation sequencing, without whole genome amplification, identifying unique mutations in 10 of 15 samples and 0 of 4 healthy samples

Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by l,d-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model l,d-transpeptidase (Ldt) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldt. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation

MicroRNA-221 Modulates RSV Replication in Human Bronchial Epithelium by Targeting NGF Expression

Background: Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells. Methodology: Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into noninfected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication. Principal Findings: RSV caused downregulation of 24 miRNAs and upregulation of 2 (p,0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75 NTR. Among the selected miRNAs, miR-221 was significantly downregulated by RSV and it

Jejunum free flap in hypopharynx reconstruction: Case series

BACKGROUND: Surgical treatment of hypopharyngeal cancers with extension to the retrocricoid region generally requires a circumferential pharyngolaryngectomy followed by a reconstruction of the removed segment of the upper digestive tract. Historically, many techniques have been used in order to achieve a safe and functional reconstruction. Jejunum interposition is generally considered the best reconstructive technique. METHODS: This study examines the details of the surgical technique, the complications, the oncological and the functional results in a series of 29 consecutive patients submitted to circumferential pharyngoesophageal resection for advanced hypopharyngeal cancer followed by reconstruction with a free flap of jejunum. RESULTS: Three of the transplants failed because of venous thrombosis. The overall success rate was 90%. There were no general complications. A good swallowing has been preserved in all our patients. All our patients where a phonatory prosthesis was positioned (20/29) were able to achieve speech following speech therapy and all were satisfied with their own capacity to communicate. CONCLUSIONS: The prognosis of hypopharyngeal tumours (18–40% at 5 years) remains poor, but jejunum autografts are being shown to be an excellent choice for the reconstruction of the cervical hypopharyngo-oesophagus offering the patient fast rehabilitation and a reasonable quality of survival. Our experience confirm that this kind of reconstruction is safe with a good results in improving oncologic controls and restoring a good quality of life

Early and long-term morbidity after total laryngopharyngectomy

To determine the early and long-term morbidity of patients treated with a total laryngopharyngectomy and reconstruction using a jejunum interposition or gastric pull-up procedure. It is a retrospective study; and it is conducted in tertiairy referral center. Sixty-three patients were included in whom 70 reconstructions were performed (51 jejunum interpositions and 19 gastric pull-up procedures) between 1990 and 2007. The studied parameters were success rate of the reconstruction, early and long-term complication rate, and functional outcome including quality of life. Subjective quality of life analysis was determined by two questionnaires: the EORTC Quality of Life Questionnaire (QLQ)-C30 Dutch version 3.0, and the EORTC-Head and Neck (H & N 35). The success rates were 84 and 74%, respectively. The procedures were associated with a high complication rate (63% after jejunum interposition and 89% after gastric pull-up), and a lengthy rehabilitation. Surviving patients were found to have a good long-term quality of life. Complete oral intake was achieved in 97%, and speech rehabilitation in 95%. These procedures are associated with significant morbidity, high complication rates, lengthy rehabilitation, but a good long-term quality of life

HIV-1 Tat protein alter the tight junction integrity and function of retinal pigment epithelium: an in vitro study

<p>Abstract</p> <p>Background</p> <p>How HIV-1 enter into the eyes remains obscure. We postulated that HIV-1 Tat protein can alter the expression of specific tight-junction proteins and disturb the blood retinal barrier, and contributes to HIV trafficking into the eyes. This study is to determine the effects of HIV-1 Tat proteins on the barrier function and tight-junction protein expression of retinal pigment epithelial cell (RPE).</p> <p>Methods</p> <p>A human RPE cell line (D407) cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudins were determined by real-time polymerase chain reaction, immunofluorescence, and Western blot analysis. Activation of ERK1/2 was detected by Western blot analysis with specific antiphospho protein antibodies. NF-κB DNA binding activity was determined by transcription factor assay. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in barrier destruction of RPE cells exposed to HIV-1 Tat.</p> <p>Results</p> <p>Treating cultured human retinal pigment epithelial cells with 100 nM Tat for 24 hours increased the permeability and decreased the TER of the epithelial monolayer. HIV-1 Tat also disrupted and downregulated the tight-junction proteins claudin-1, claudin-3, and claudin-4 in these cells, whereas claudin-2 was upregulated, and the expression of occludin was unaffected. HIV-1 Tat protein also induced activation of ERK1/2 and NF-κB. HIV-1 Tat protein induced barrier destruction, changes in expression of TJs, and activation of ERK1/2 and NF-κB were abrogated by inhibitor of ERK1/2 and NF-κB.</p> <p>Conclusion</p> <p>HIV-1 Tat protein causes increases in the paracellular permeability of RPE cells in vitro concomitant with changes in expression of certain transmembrane proteins associated with the tight junction. The effects of HIV-1 Tat on barrier function of the RPE may be mediated by ERK MAPK and NF-κB activation, which may represent potential targets for novel therapeutic approaches for the retinopathy induced by HIV infection.</p

LIN28 phosphorylation by MAPK/ERK couples signaling to the post-transcriptional control of pluripotency

Signaling and post-transcriptional gene control are both critical for the regulation of pluripotency1,2, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein (RBP), has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA (miRNA) biogenesis and direct modulation of mRNA translation3. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells (PSCs), which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced LIN28’s effect on its direct mRNA targets, revealing a mechanism that uncouples LIN28’s let-7-dependent and independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naïve to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signaling, post-transcriptional gene control, and cell fate regulation