Epithelial-to-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma and Pancreatic Tumor Cell Lines: The Role of Neutrophils and Neutrophil-Derived Elastase

Pancreatic ductal adenocarcinoma (PDAC) is frequently associated with fibrosis and a prominent inflammatory infiltrate in the desmoplastic stroma. Moreover, in PDAC, an epithelial-to-mesenchymal transition (EMT) is observed. To explore a possible connection between the infiltrating cells, particularly the polymorphonuclear neutrophils (PMN) and the tumor cell transition, biopsies of patients with PDAC (n=115) were analysed with regard to PMN infiltration and nuclear expression of β-catenin and of ZEB1, well-established indicators of EMT. In biopsies with a dense PMN infiltrate, a nuclear accumulation of β-catenin and of ZEB1 was observed. To address the question whether PMN could induce EMT, they were isolated from healthy donors and were cocultivated with pancreatic tumor cells grown as monolayers. Rapid dyshesion of the tumor cells was seen, most likely due to an elastase-mediated degradation of E-cadherin. In parallel, the transcription factor TWIST was upregulated, β-catenin translocated into the nucleus, ZEB1 appeared in the nucleus, and keratins were downregulated. EMT was also induced when the tumor cells were grown under conditions preventing attachment to the culture plates. Here, also in the absence of elastase, E-cadherin was downmodulated. PMN as well as prevention of adhesion induced EMT also in liver cancer cell line. In conclusion, PMN via elastase induce EMT in vitro, most likely due to the loss of cell-to-cell contact. Because in pancreatic cancers the transition to a mesenchymal phenotype coincides with the PMN infiltrate, a contribution of the inflammatory response to the induction of EMT and—by implication—to tumor progression is possible

Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in Aging Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans has emerged as an important model organism in the study of the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation, and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterization of different types of aggregates. Specifically, in longitudinal studies of C. elegans models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetized worms moving in specially designed agarose microchambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of protein aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity

Cloning of the Repertoire of Individual Plasmodium falciparum var Genes Using Transformation Associated Recombination (TAR)

One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member

Relativistic Brownian Motion

Stimulated by experimental progress in high energy physics and astrophysics, the unification of relativistic and stochastic concepts has re-attracted considerable interest during the past decade. Focusing on the framework of special relativity, we review, here, recent progress in the phenomenological description of relativistic diffusion processes. After a brief historical overview, we will summarize basic concepts from the Langevin theory of nonrelativistic Brownian motions and discuss relevant aspects of relativistic equilibrium thermostatistics. The introductory parts are followed by a detailed discussion of relativistic Langevin equations in phase space. We address the choice of time parameters, discretization rules, relativistic fluctuation-dissipation theorems, and Lorentz transformations of stochastic differential equations. The general theory is illustrated through analytical and numerical results for the diffusion of free relativistic Brownian particles. Subsequently, we discuss how Langevin-type equations can be obtained as approximations to microscopic models. The final part of the article is dedicated to relativistic diffusion processes in Minkowski spacetime. Due to the finiteness of velocities in relativity, nontrivial relativistic Markov processes in spacetime do not exist; i.e., relativistic generalizations of the nonrelativistic diffusion equation and its Gaussian solutions must necessarily be non-Markovian. We compare different proposals that were made in the literature and discuss their respective benefits and drawbacks. The review concludes with a summary of open questions, which may serve as a starting point for future investigations and extensions of the theory.Comment: review article, 159 pages, references updated, misprints corrected, App. A.4. correcte