Identification of tumor-specific Salmonella Typhimurium promoters and their regulatory logic

Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasi

FeatureScan: revealing property-dependent similarity of nucleotide sequences

FeatureScan is a software package aiming to reveal novel types of DNA sequence similarity by comparing physico-chemical properties. Thirty-eight different parameters of DNA double strands such as charge, melting enthalpy, conformational parameters and the like are provided. As input FeatureScan requires two sequences, a pattern sequence and a target sequence, search conditions are set by selecting a specific DNA parameter and a threshold value. Search results are displayed in FASTA format and directly linked to external genome databases/browsers (ENSEMBL, NCBI, UCSC). An Internet version of FeatureScan is accessible at . As part of the HOBIT initiative () FeatureScan is also accessible as a web service at its above home page. Currently, several preloaded genomes are provided at this Internet website (Homo sapiens, Mus musculus, Rattus norvegicus and four strains of Escherichia coli) as target sequences. Standalone executables of FeatureScan are available on request

Identification of tumor-specific Salmonella Typhimurium promoters and their regulatory logic

Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasia

FlowerMorphology: fully automatic flower morphometry software

Main conclusio

Composing a Tumor Specific Bacterial Promoter.

Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Specificity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling elements are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy

Poor knowledge of vaccination recommendations and negative attitudes towards vaccinations are independently associated with poor vaccination uptake among adults - Findings of a population-based panel study in Lower Saxony, Germany.

The aims of this study were to (a) assess knowledge of official vaccination recommendations and attitudes towards vaccinations among adults and (b) examine their association with vaccination uptake among adults

Identification and analysis steps of tumor specific promoters.

<p>A schematic overview. Two programs for basal promoter recognition, one based on sequence alignment kernel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.ref011" target="_blank">11</a>] and another on Hidden Markov Model (HMM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.ref012" target="_blank">12</a>] were applied to the TSP set (DNA sequences are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s006" target="_blank">S1 Text</a>). As a negative control, a set of DNA fragments that do not initiate expression either in tumor or in spleen was selected (negative promoters, NP). Both programs recognize potential promoters in either dataset and the number of predictions greatly depends on user-defined threshold parameters. To evaluate the specificity of predictions, it was assumed that a basal promoter should be recognized in at least 75% of TSP and at most 50% of NPs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#sec014" target="_blank">Methods</a>). This will ensure the generality and specificity of the recognized feature.</p

Schematic representation of promoter structure.

<p>Left: binding motifs for factors TGIF, FNR and NagC are shown in green, yellow and brown, respectively. Basal promoter is shown as a directed arrow. Knockout of essential nucleotides within motifs were according to the literature and are represented by crossing lines. Mutation of non-essential nucleotides within motifs was random and is represented by dashed crossing lines. Nucleotides outside motifs were mutated randomly. Right: representative flow-cytometric analyses of GFP-expression in tumor and spleen. Each green point on the blots represents GFP expression levels of an individual bacterial cell. Displayed values of expression are relative to the expression values of the original promoter P0.212_1. All nucleotide substitutions are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s002" target="_blank">S2 Fig</a>.</p

Expression of promoters P1.6 and P0.212_1 in tumor, spleen and liver.

<p>Homogenates were analyzed via two color flow cytometry and plating to allow normalization. Given are mean and SD. Expression of P1.6 in spleen and liver can be considered negligible compared to expression in tumor. Therefore expression of promoter P1.6 was accepted as tumor specific.</p