Fate of Hydrophilic Nanoparticles in Biological Environments

The nanoparticles developed are based on chitosan, a biocompatible and biodegradable polysaccharide. The chitosan nanoparticles are formed in an entirely water-based process by electrostatic interactions with other biocompatible molecules. As a prerequisite to understand the fate of such nanoparticles in cells, comprehensive characterization and stability studies serve to identify quantitatively the impact of the raw material characteristics and preparation conditions on the nanoparticle characteristics. Methods included H-1 NMR spectroscopy, dilution viscometry, particle size analysis and electron microscopy. Cytotoxicity and cell uptake experiments on RAW 264.7 murine macrophages and p23 murine endothelial cells were performed to investigate the correlation with nanoparticle characteristics and effect of surface decoration with alginate. Cytotoxicity was assessed by the MTT survival test; cell uptake was monitored by fluorescent microscopy using labeled polymers.

Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells.

Direct identification as well as isolation of antigen-specific T cells became possible since the development of "tetramers" based on avidin-fluorochrome conjugates associated with mono-biotinylated class I MHC-peptide monomeric complexes. In principle, a series of distinct class I MHC-peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8(+) T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8(+) T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application.

Mites as biological tags of their hosts.

Movements and spatial distribution of host populations are expected to shape the genetic structure of their parasite populations. Comparing the genetic patterns of both interacting species may improve our understanding of their evolutionary history. Moreover, genetic analyses of parasites with horizontal transmission may serve as indicators of historical events or current demographic processes that are not apparent in the genetic signature of their hosts. Here, we compared mitochondrial variation in populations of the ectoparasitic mite Spinturnix myoti with the genetic pattern of its host, the Maghrebian bat Myotis punicus in North Africa and in the islands of Corsica and Sardinia. Mite mitochondrial differentiation among populations was correlated with both host mitochondrial and nuclear differentiation, suggesting spatial co-differentiation of the lineages of the two interacting species. Therefore our results suggest that parasite dispersal is exclusively mediated by host movements, with open water between landmasses as a main barrier for host and parasite dispersal. Surprisingly the unique presence of a continental European mite lineage in Corsica was inconsistent with host phylogeographical history and strongly suggests the former presence of European mouse-eared bats on this island. Parasites may thus act as biological tags to reveal the presence of their now locally extinct host.

Regulation of mucoidy in the biological control agent Pseudomonas fluorescens CHA0 and construction of an exopolysaccharide-overproducing variant

Pseudomonas fluorescens CHA0, an effective biological control agent of soilborne plant diseases, is naturally non-mucoid. We have isolated a highly mucoid Tn5 insertion mutant of strain CHA0. The mucoid phenotype was found to be due to the overproduction of exopolysaccharide (EPS), as a result of a mutation in the mucA gene. The wild-type mucA gene was cloned by a two-step, Tn5-dependent cloning procedure previously described and the deduced amino acid sequence showed 71% identity with MucA of P. aeruginosa, a negative regulator of the alternative sigma factor AlgU (=s22, sE). As in P. aeruginosa, mucA is preceded by the algU gene encoding s22 (91% identity at the amino acid sequence level). A mucA in-frame deletion mutant of CHA0 overproduced EPS and formed mucoid colonies, whereas an algU in-frame deletion mutant showed a non-mucoid phenotype. Pyoluteorin, an antibiotic produced by P. fluorescens, was found to be entrapped in EPS of a mucoid mutant. In natural soil, mucoidy negatively affected survival of the bacteria, suggesting that under these conditions the potential to produce abundant EPS does not confer a selective advantage on the bacteria.

Regulation of antibiotic production in root-colonizing Peudomonas spp. and relevance for biological control of plant disease.

Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants. The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics. Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots. Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide. In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level. Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway. One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor α/γ ligand LT175.

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARα and fine-tuned moderate activity on PPARγ. For the most interesting compound (S)-3, docking studies in PPARα and PPARγ provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARα/γ activity which probably involves a synergistic effect on both receptor subtypes.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Exploring neural cell dynamics with digital holographic microscopy.

In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological information provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach.

Mining the human phenome using allelic scores that index biological intermediates.

It is common practice in genome-wide association studies (GWAS) to focus on the relationship between disease risk and genetic variants one marker at a time. When relevant genes are identified it is often possible to implicate biological intermediates and pathways likely to be involved in disease aetiology. However, single genetic variants typically explain small amounts of disease risk. Our idea is to construct allelic scores that explain greater proportions of the variance in biological intermediates, and subsequently use these scores to data mine GWAS. To investigate the approach's properties, we indexed three biological intermediates where the results of large GWAS meta-analyses were available: body mass index, C-reactive protein and low density lipoprotein levels. We generated allelic scores in the Avon Longitudinal Study of Parents and Children, and in publicly available data from the first Wellcome Trust Case Control Consortium. We compared the explanatory ability of allelic scores in terms of their capacity to proxy for the intermediate of interest, and the extent to which they associated with disease. We found that allelic scores derived from known variants and allelic scores derived from hundreds of thousands of genetic markers explained significant portions of the variance in biological intermediates of interest, and many of these scores showed expected correlations with disease. Genome-wide allelic scores however tended to lack specificity suggesting that they should be used with caution and perhaps only to proxy biological intermediates for which there are no known individual variants. Power calculations confirm the feasibility of extending our strategy to the analysis of tens of thousands of molecular phenotypes in large genome-wide meta-analyses. We conclude that our method represents a simple way in which potentially tens of thousands of molecular phenotypes could be screened for causal relationships with disease without having to expensively measure these variables in individual disease collections.

Post-mortem cardiac 3-T magnetic resonance imaging: visualization of sudden cardiac death?

OBJECTIVES: This study aimed to investigate post-mortem magnetic resonance imaging (pmMRI) for the assessment of myocardial infarction and hypointensities on post-mortem T2-weighted images as a possible method for visualizing the myocardial origin of arrhythmic sudden cardiac death. BACKGROUND: Sudden cardiac death has challenged clinical and forensic pathologists for decades because verification on post-mortem autopsy is not possible. pmMRI as an autopsy-supporting examination technique has been shown to visualize different stages of myocardial infarction. METHODS: In 136 human forensic corpses, a post-mortem cardiac MR examination was carried out prior to forensic autopsy. Short-axis and horizontal long-axis images were acquired in situ on a 3-T system. RESULTS: In 76 cases, myocardial findings could be documented and correlated to the autopsy findings. Within these 76 study cases, a total of 124 myocardial lesions were detected on pmMRI (chronic: 25; subacute: 16; acute: 30; and peracute: 53). Chronic, subacute, and acute infarction cases correlated excellently to the myocardial findings on autopsy. Peracute infarctions (age range: minutes to approximately 1 h) were not visible on macroscopic autopsy or histological examination. Peracute infarction areas detected on pmMRI could be verified in targeted histological investigations in 62.3% of cases and could be related to a matching coronary finding in 84.9%. A total of 15.1% of peracute lesions on pmMRI lacked a matching coronary finding but presented with severe myocardial hypertrophy or cocaine intoxication facilitating a cardiac death without verifiable coronary stenosis. CONCLUSIONS: 3-T pmMRI visualizes chronic, subacute, and acute myocardial infarction in situ. In peracute infarction as a possible cause of sudden cardiac death, it demonstrates affected myocardial areas not visible on autopsy. pmMRI should be considered as a feasible post-mortem investigation technique for the deceased patient if no consent for a clinical autopsy is obtained.

Electron microscopic visualization of protein-DNA interactions at the estrogen responsive element and in the first intron of the Xenopus laevis vitellogenin gene.

Stable protein-DNA complexes can be assembled in vitro at the 5' end of Xenopus laevis vitellogenin genes using extracts of nuclei from estrogen-induced frog liver and visualized by electron microscopy. Complexes at the three following sites can be identified on the gene B2: the transcription initiation site, the estrogen responsive element (ERE) and in the first intron. The complex at the transcription initiation site is stabilized by dinucleotides and thus represents a ternary transcription complex. The formation of the complexes at the two other sites is enhanced by estrogen and is reduced by tamoxifen, an antagonist of estrogen, while this latter effect is reversed by adding an excess of hormone. No sequence homology is apparent between the site containing the ERE and the binding site in intron I and functional tests in MCF-7 cells suggest that these two sites are not equivalent. Finally, we made use of previously characterized deletion mutants of the 5' flanking region of the gene B1, a close relative of the gene B2, to demonstrate that the 13-bp palindromic core element of the ERE is involved in the formation of the complexes observed upstream of the transcription initiation site.