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Signal transducer and activator of transcription 4 (STAT4) plays a crucial role in the host immune response against Mycobacterium tuberculosis. This study investigates the association between STAT4 gene polymorphisms and pulmonary tuberculosis (TB) risk in the Moldavian population. A total of 272 TB patients and 251 community-matched controls underwent screening for functional single-nucleotide polymorphisms (SNPs) rs897200 and rs7574865 in the STAT4 gene. The minor T allele and the TT/CT genotype of rs897200 demonstrated a significant association with reduced pulmonary TB risk (allelic model: adjusted OR = .74, p = .025; log-additive model: adjusted OR = .72, p = .02; and dominant model: adjusted OR = .65, p = .023), indicating a protective effect. Similar associations, characterized by an even more pronounced reduction in risk, were observed among females and late-onset TB patients (>44 years). No significant associations were found for rs7574865. In addition, a combined genotype analysis incorporating 43 SNPs from our previous studies revealed potential associations, such as STAT4 rs897200 CT with IFNG rs2430561 AA (adjusted OR = .36, p = .0025) and STAT4 rs897200 CT with TNFA rs1800629 GA (adjusted OR = .33, p = .0012). This study emphasizes the significant association of STAT4 rs897200 with pulmonary TB risk in the Moldavian population, underscoring its role in the disease development., (© 2024 John Wiley & Sons Ltd.)

The concept of cis-regulatory modules located in gene promoters represents today's vision of the organization of gene transcriptional regulation. Such modules are a combination of two or more single, short DNA motifs. The bioinformatic identification of such modules belongs to so-called NP-hard problems with extreme computational complexity, and therefore, simplifications, assumptions, and heuristics are usually deployed to tackle the problem. In practice, this requires, first, many parameters to be set before the search, and second, it leads to the identification of locally optimal results. Here, a novel method is presented, aimed at identifying the cis-regulatory elements in gene promoters based on an exhaustive search of all the feasible modules' configurations. All required parameters are automatically estimated using positive and negative datasets. To be computationally efficient, the search is accelerated using a multidimensional hash function, allowing the search to complete in a few hours on a regular laptop (for example, a CPU Intel i7, 3.2 GH, 32 Gb RAM). Tests on an established benchmark and real data show better performance of BestCRM compared to the available methods according to several metrics like specificity, sensitivity, AUC, etc. A great practical advantage of the method is its minimum number of input parameters-apart from positive and negative promoters, only a desired level of module presence in promoters is required.

Heterozygous deletions in the ANKS1B gene cause ANKS1B neurodevelopmental syndrome (ANDS), a rare genetic disease characterized by autism spectrum disorder (ASD), attention deficit/hyperactivity disorder, and speech and motor deficits. The ANKS1B gene encodes for AIDA-1, a protein that is enriched at neuronal synapses and regulates synaptic plasticity. Here we report an unexpected role for oligodendroglial deficits in ANDS pathophysiology. We show that Anks1b-deficient mouse models display deficits in oligodendrocyte maturation, myelination, and Rac1 function, and recapitulate white matter abnormalities observed in ANDS patients. Selective loss of Anks1b from the oligodendrocyte lineage, but not from neuronal populations, leads to deficits in social preference and sensory reactivity previously observed in a brain-wide Anks1b haploinsufficiency model. Furthermore, we find that clemastine, an antihistamine shown to increase oligodendrocyte precursor cell maturation and central nervous system myelination, rescues deficits in social preference in 7-month-old Anks1b-deficient mice. Our work shows that deficits in social behaviors present in ANDS may originate from abnormal Rac1 activity within oligodendrocytes., (© 2023. The Author(s).)

Specific and effective treatments for autism spectrum disorder (ASD) are lacking due to a poor understanding of disease mechanisms. Here we test the idea that similarities between diverse ASD mouse models are caused by deficits in common molecular pathways at neuronal synapses. To do this, we leverage the availability of multiple genetic models of ASD that exhibit shared synaptic and behavioral deficits and use quantitative mass spectrometry with isobaric tandem mass tagging (TMT) to compare their hippocampal synaptic proteomes. Comparative analyses of mouse models for Fragile X syndrome ( Fmr1 knockout), cortical dysplasia focal epilepsy syndrome ( Cntnap2 knockout), PTEN hamartoma tumor syndrome ( Pten haploinsufficiency), ANKS1B syndrome ( Anks1b haploinsufficiency), and idiopathic autism (BTBR+) revealed several common altered cellular and molecular pathways at the synapse, including changes in oxidative phosphorylation, and Rho family small GTPase signaling. Functional validation of one of these aberrant pathways, Rac1 signaling, confirms that the ANKS1B model displays altered Rac1 activity counter to that observed in other models, as predicted by the bioinformatic analyses. Overall similarity analyses reveal clusters of synaptic profiles, which may form the basis for molecular subtypes that explain genetic heterogeneity in ASD despite a common clinical diagnosis. Our results suggest that ASD-linked susceptibility genes ultimately converge on common signaling pathways regulating synaptic function and propose that these points of convergence are key to understanding the pathogenesis of this disorder., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Carbonell, Freire-Cobo, Deyneko, Dobariya, Erdjument-Bromage, Clipperton-Allen, Page, Neubert and Jordan.)

Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background: RNA-seq has become a standard technology to quantify mRNA. The measured values usually vary by several orders of magnitude, and while the detection of differences at high values is statistically well grounded, the significance of the differences for rare mRNAs can be weakened by the presence of biological and technical noise., Results: We have developed a method for cleaning RNA-seq data, which improves the detection of differentially expressed genes and specifically genes with low to moderate transcription. Using a data modeling approach, parameters of randomly distributed mRNA counts are identified and reads, most probably originating from technical noise, are removed. We demonstrate that the removal of this random component leads to the significant increase in the number of detected differentially expressed genes, more significant pvalues and no bias towards low-count genes., Conclusion: Application of RNAdeNoise to our RNA-seq data on polysome profiling and several published RNA-seq datasets reveals its suitability for different organisms and sequencing technologies such as Illumina and BGI, shows improved detection of differentially expressed genes, and excludes the subjective setting of thresholds for minimal RNA counts. The program, RNA-seq data, resulted gene lists and examples of use are in the supplementary data and at https://github.com/Deyneko/RNAdeNoise ., (© 2022. The Author(s).)

Inborn errors of immunity are known to influence susceptibility to mycobacterial infections. The aim of this study was to characterize the genetic profile of nine patients with mycobacterial infections (eight with BCGitis and one with disseminated tuberculosis) from the Republic of Moldova using whole-exome sequencing. In total, 12 variants in eight genes known to be associated with Mendelian Susceptibility to Mycobacterial Disease (MSMD) were detected in six out of nine patients examined. In particular, a novel splice site mutation c.373-2A>C in STAT1 gene was found and functionally confirmed in a patient with disseminated tuberculosis. Trio analysis was possible for seven out of nine patients, and resulted in 23 candidate variants in 15 novel genes. Four of these genes - GBP2 , HEATR3 , PPP1R9B and KDM6A were further prioritized, considering their elevated expression in immune-related tissues. Compound heterozygosity was found in GBP2 in a single patient, comprising a maternally inherited missense variant c.412G>A/p.(Ala138Thr) predicted to be deleterious and a paternally inherited intronic mutation c.1149+14T>C. Functional studies demonstrated that the intronic mutation affects splicing and the level of transcript. Finally, we analyzed pathogenicity of variant combinations in gene pairs and identified five patients with putative oligogenic inheritance. In summary, our study expands the spectrum of genetic variation contributing to susceptibility to mycobacterial infections in children and provides insight into the complex/oligogenic disease-causing mode., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Varzari, Deyneko, Bruun, Dembic, Hofmann, Cebotari, Ginda, Andresen and Illig.)

Auxins and cytokinins create versatile regulatory network controlling virtually all aspects of plant growth and development. These hormonal systems act in close contact, synergistically or antagonistically, determining plant phenotype, resistance and productivity. However, the current knowledge about molecular interactions of these systems is still scarce. Our study with potato plants aimed at deciphering potential interactions between auxin and cytokinin signaling pathways at the level of respective gene expression. Potato plants grown on sterile medium with 1.5% (vegetation) or 5% (tuberization) sucrose were treated for 1 h with auxin or cytokinin. Effects of these two hormones on expression profiles of genes belonging to main signaling pathways of auxin and cytokinin were quantified by RT-qPCR. As a result, several signaling genes were found to respond to auxin and/or cytokinin by up- or down-regulation. The observed effects were largely organ-specific and depended on sucrose content. Auxin strongly reduced cytokinin perception apparatus while reciprocal cytokinin effect was ambiguous and sucrose-dependent. In many cases, functional clustering of genes of the same family was observed. Promoters in some clusters are enriched with canonic hormone-response cis -elements supporting their direct sensitivity to hormones. Collectively, our data shed new light on the crosstalk between auxin- and cytokinin signaling pathways.

Polymorphisms in genes that control immune function and regulation may influence susceptibility to pulmonary tuberculosis (TB). In this study, 14 polymorphisms in 12 key genes involved in the immune response ( VDR , MR1 , TLR1 , TLR2 , TLR10 , SLC11A1 , IL1B , IL10 , IFNG , TNF , IRAK1 , and FOXP3 ) were tested for their association with pulmonary TB in 271 patients with TB and 251 community-matched controls from the Republic of Moldova. In addition, gene-gene interactions involved in TB susceptibility were analyzed for a total of 43 genetic loci. Single nucleotide polymorphism (SNP) analysis revealed a nominal association between TNF rs1800629 and pulmonary TB (Fisher exact test P  = 0.01843). In the pairwise interaction analysis, the combination of the genotypes TLR6 rs5743810 GA and TLR10 rs11096957 GT was significantly associated with an increased genetic risk of pulmonary TB (OR = 2.48, 95% CI = 1.62-3.85; Fisher exact test P value = 1.5 × 10 -5 , significant after Bonferroni correction). In conclusion, the TLR6 rs5743810 and TLR10 rs11096957 two-locus interaction confers a significantly higher risk for pulmonary TB; due to its high frequency in the population, this SNP combination may serve as a novel biomarker for predicting TB susceptibility.

The aryl hydrocarbon receptor (AhR) contributes to immune regulation in autoimmune diseases such as multiple sclerosis (MS). Analysis of selected polymorphisms in AhR pathway genes in 805 MS patients and 1023 controls revealed a modest association of a CYP1B1 polymorphism with secondary progressive MS that became more pronounced in combination with other SNPs in the pathway, suggesting interactive effects. Additionally, first evidence for an interaction with smoking was found, but due to small sample sizes statistical significance was only nominal. Confirmation of these results in independent cohorts is recommended, since targeting the AhR constitutes a therapeutic option for autoimmune diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Toll-like receptors (TLRs) play a critical role in initiating an immune response to infections. In this study, we examined whether single nucleotide polymorphisms (SNPs) in TLR pathway genes are associated with pulmonary tuberculosis (PTB) in a Moldavian population. Thirty-four SNPs in genes associated with the TLR pathway and two SNPs in ASAP1 gene identified by GWAS were selected for genotyping in 272 patients and 251 community-matched healthy controls. Twenty-nine SNPs passed quality control and were statistically evaluated. SNPs TLR9 rs352139, TLR2 rs3804099 and MYD88 rs4988453 were associated with PTB in females (OR = 0.49, p = 0.0009; OR = 0.51, p = 0.0008; OR = 0.33, p = 0.027; here and below log-additive model with minor alleles assumed as effect associated alleles), while SNP TLR8 rs3764880 showed a significant association in males (OR = 0.44, p = 0.0087). Furthermore, SNPs TLR9 rs352139 and TLR8 rs3764880 were associated with PTB in the late-onset (≥39-year-old) patient group (OR = 0.60, p = 0.0029 and OR = 0.70, p = 0.021, respectively) and SNPs TLR2 rs3804099, TLR4 rs4986790 and TLR4 rs1927906 in the early-onset (≤ 38-year-old) group (OR = 0.53, p = 0.0012; OR = 3.45, p = 0.013; OR = 2.31, p = 0.044, respectively). After correction for multiple testing, only SNPs TLR9 rs352139 and TLR2 rs3804099 in the female group and SNP TLR2 rs3804099 in the early-onset group remained significant. In summary, we show an association of SNP TLR8 rs3764880 with PTB in the Moldavian male population, providing support to previous studies conducted on other populations. Polymorphisms rs3804099 (TLR2) and rs352139 (TLR9) may also be associated with PTB risk in the Moldavian population but their effect is less consistent across different studies. Additional large-scale association studies along with functional tests are required to dissect the relevance of these associations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The control of translation in the course of gene expression regulation plays a crucial role in plants' cellular events and, particularly, in responses to environmental factors. The paradox of the great variance between levels of mRNAs and their protein products in eukaryotic cells, including plants, requires thorough investigation of the regulatory mechanisms of translation. A wide and amazingly complex network of mechanisms decoding the plant genome into proteome challenges researchers to design new methods for genome-wide analysis of translational control, develop computational algorithms detecting regulatory mRNA contexts, and to establish rules underlying differential translation. The aims of this review are to (i) describe the experimental approaches for investigation of differential translation in plants on a genome-wide scale; (ii) summarize the current data on computational algorithms for detection of specific structure⁻function features and key determinants in plant mRNAs and their correlation with translation efficiency; (iii) highlight the methods for experimental verification of existed and theoretically predicted features within plant mRNAs important for their differential translation; and finally (iv) to discuss the perspectives of discovering the specific structural features of plant mRNA that mediate differential translation control by the combination of computational and experimental approaches.

Objectives: Chemokines play a key role in immune regulation and response, and have been implicated in the pathogenesis of tuberculosis (TB). In this study, we investigated whether functional polymorphisms of the chemokines CCL5, CCL2, and CXCL8 are associated with pulmonary TB in a Moldavian population., Materials and Methods: A total of 250 patients with TB and 184 healthy controls were screened for CCL5 -403G/A (rs2107538), CCL5 In1.1T/C (rs2280789), CCL2 -2518A/G (rs1024611), and CXCL8 -251A/T (rs4073) polymorphisms using standard polymerase chain reaction techniques., Results: None of the analyzed variants were found to be significantly associated with overall pulmonary TB susceptibility. However, the CCL5 In1.1T/C polymorphism was significantly associated with early-onset TB in patients younger than 30 (dominant model, odds ratio [OR] = 3.01, p = 0.0046) or younger than 40 years (dominant model, OR = 2.17, p = 0.0099), and the conducted case-only analysis demonstrated that CCL5 In1.1T/C C-allele carriers exhibited an earlier TB onset than TT homozygotes (36.14 years vs. 40.13 years, p = 0.0065). In addition, nominal significance was observed for an association between TB incidence and both the eight paired genotypes in the overall patient cohort (0.017 < p < 0.05) and the CCL2 -2518A/G polymorphism among males (dominant model, OR = 0.55, p = 0.041; log-additive model, OR = 0.57, p = 0.018)., Conclusion: The CCL5 In1.1T/C polymorphism may modulate pulmonary early-onset TB risk.

Main Conclusion: The software FlowerMorphology is designed for automatic morphometry of actinomorphic flowers. The novel complex parameters of flowers calculated by FlowerMorphology allowed us to quantitatively characterize a polyploid series of tobacco. Morphological differences of plants representing closely related lineages or mutants are mostly quantitative. Very often, there are only very fine variations in plant morphology. Therefore, accurate and high-throughput methods are needed for their quantification. In addition, new characteristics are necessary for reliable detection of subtle changes in morphology. FlowerMorphology is an all-in-one software package to automatically image and analyze five-petal actinomorphic flowers of the dicotyledonous plants. Sixteen directly measured parameters and ten calculated complex parameters of a flower allow us to characterize variations with high accuracy. The program was developed for the needs of automatic characterization of Nicotiana tabacum flowers, but is applicable to many other plants with five-petal actinomorphic flowers and can be adopted for flowers of other merosity. A genetically similar polyploid series of N. tabacum plants was used to investigate differences in flower morphology. For the first time, we could quantify the dependence between ploidy and size and form of the tobacco flowers. We found that the radius of inner petal incisions shows a persistent positive correlation with the chromosome number. In contrast, a commonly used parameter-radius of outer corolla-does not discriminate 2n and 4n plants. Other parameters show that polyploidy leads to significant aberrations in flower symmetry and are also positively correlated with chromosome number. Executables of FlowerMorphology, source code, documentation, and examples are available at the program website: https://github.com/Deyneko/FlowerMorphology .

Objectives: 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., Methods: This study was part of the HaBIDS study (Hygiene and Behaviour Infectious Diseases Survey), which is an online panel established in March 2014 in Lower Saxony, Germany with males and females aged between 15 and 69 years (n = 2379). Every few months, participants completed questionnaires on different aspects of infectious diseases. In September 2014, knowledge of vaccination recommendations, attitudes towards vaccinations and information on uptake of vaccinations in the last 10 years (practice) were collected using a knowledge-attitude-practice (KAP) questionnaire. Multiple correspondence analysis was applied to identify underlying structures in each KAP domain and fractional polynomial regression analysis to examine the associations of knowledge and attitudes with vaccination uptake., Results: Of the 2379 panel members, 1698 (71%) completed the KAP questionnaire on vaccinations. The majority of participants (80%) knew that the vaccine against diphtheria and tetanus should be administered every 10 years. Regarding other recommendations, the proportion of correct answers varied between 35% and 60%. 82% of participants agreed that adult vaccinations should be mandatory for selected groups such as health care workers and 40% stated that vaccinations should be mandatory for all adults. For the different vaccines, the odds of being unvaccinated were 1.5- to 5-times higher among participants with poor knowledge of vaccination recommendations compared to participants with good knowledge. Participants with negative attitudes towards vaccinations were also more likely to be unvaccinated., Conclusions: Efforts should be undertaken to improve knowledge of official vaccination recommendations in the general population and reduce common misconceptions about vaccinations. This information can be provided during general practitioner visits or through media campaigns., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

Background: Glutathione S-transferases (GSTM1, GSTT1, and GSTP1) and methylenetetrahydrofolate reductase (MTHFR) are important enzymes for protection against oxidative stress. In addition, MTHFR has an essential role in DNA synthesis, repair, and methylation. Their polymorphisms have been implicated in the pathogenesis of ulcerative colitis (UC). The aim of the present study was to investigate the role of selected polymorphisms in these genes in the development of UC in the Moldavian population., Methods: In a case-control study including 128 UC patients and 136 healthy individuals, GSTM1 and GSTT1 genotypes (polymorphic deletions) were determined using multiplex polymerase chain reaction (PCR). The GSTP1 rs1695 (Ile105Val), MTHFR rs1801133 (C677T), and MTHFR rs1801131 (A1298C) polymorphisms were studied with restriction fragment length polymorphism (RFLP) analysis. Genotype-phenotype correlations were examined using logistic regression analysis., Results: None of the genotypes, either alone or in combination, showed a strong association with UC. The case-only sub-phenotypic association analysis showed an association of the MTHFR rs1801133 polymorphism with the extent of UC under co-dominant (p corrected = 0.040) and recessive (p corrected = 0.020; OR = 0.15; CI = 0.04-0.63) genetic models. Also, an association between the MTHFR rs1801131 polymorphism and the severity of UC was reported for the over-dominant model (p corrected = 0.023; coefficient = 0.32; 95% CI = 0.10-0.54)., Conclusion: The GST and MTHFR genotypes do not seem to be a relevant risk factor for UC in our sample. There was, however, evidence that variants in MTHFR may influence the clinical features in UC patients. Additional larger studies investigating the relationship between GST and MTHFR polymorphisms and UC are required.

Activated naive CD4(+) T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4(+) T cells, Th1 and Th17 cells. We could demonstrate that naive CD4(+) T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Variation in genes encoding retinoid acid-inducible gene I (RIG-I)-like receptors (RLRs) has been implicated in the pathogenesis of autoimmune disorders. We investigated if polymorphisms in the IFIH1, RIG-I, LGP2 and VISA genes influence the risk for multiple sclerosis (MS) in a German case-control cohort comprising 716 patients and 706 controls. Evaluation of 18 single nucleotide polymorphisms (SNPs) in the four genes did not reveal significant single-SNP associations with MS risk, but two VISA polymorphisms were modestly associated with age of onset. Further, we provide initial evidence for combinatorial effects of polymorphic variants in the RIG-I, LGP2 and IFIH1 genes on MS risk., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Accurate recognition of regulatory elements in promoters is an essential prerequisite for understanding the mechanisms of gene regulation at the level of transcription. Composite regulatory elements represent a particular type of such transcriptional regulatory elements consisting of pairs of individual DNA motifs. In contrast to the present approach, most available recognition techniques are based purely on statistical evaluation of the occurrence of single motifs. Such methods are limited in application, since the accuracy of recognition is greatly dependent on the size and quality of the sequence dataset. Methods that exploit available knowledge and have broad applicability are evidently needed., Results: We developed a novel method to identify composite regulatory elements in promoters using a library of known examples. In depth investigation of regularities encoded in known composite elements allowed us to introduce a new characteristic measure and to improve the specificity compared with other methods. Tests on an established benchmark and real genomic data show that our method outperforms other available methods based either on known examples or statistical evaluations. In addition to better recognition, a practical advantage of this method is first the ability to detect a high number of different types of composite elements, and second direct biological interpretation of the identified results. The program is available at http://gnaweb.helmholtz-hzi.de/cgi-bin/MCatch/MatrixCatch.pl and includes an option to extend the provided library by user supplied data., Conclusions: The novel algorithm for the identification of composite regulatory elements presented in this paper was proved to be superior to existing methods. Its application to tissue specific promoters identified several highly specific composite elements with relevance to their biological function. This approach together with other methods will further advance the understanding of transcriptional regulation of genes.

Background: Transcriptional activity of genes depends on many factors like DNA motifs, conformational characteristics of DNA, melting etc. and there are computational approaches for their identification. However, in real applications, the number of predicted, for example, DNA motifs may be considerably large. In cases when various computational programs are applied, systematic experimental knock out of each of the potential elements obviously becomes nonproductive. Hence, one needs an approach that is able to integrate many heterogeneous computational methods and upon that suggest selected regulatory elements for experimental verification., Results: Here, we present an integrative bioinformatic approach aimed at the discovery of regulatory modules that can be effectively verified experimentally. It is based on combinatorial analysis of known and novel binding motifs, as well as of any other known features of promoters. The goal of this method is the identification of a collection of modules that are specific for an established dataset and at the same time are optimal for experimental verification. The method is particularly effective on small datasets, where most statistical approaches fail. We apply it to promoters that drive tumor-specific gene expression in tumor-colonizing Gram-negative bacteria. The method successfully identified a number of potential modules, which required only a few experiments to be verified. The resulting minimal functional bacterial promoter exhibited high specificity of expression in cancerous tissue., Conclusions: Experimental analysis of promoter structures guided by bioinformatics has proved to be efficient. The developed computational method is able to include heterogeneous features of promoters and suggest combinatorial modules for experimental testing. Expansibility and robustness of the methodology implemented in the approach ensures good results for a wide range of problems.

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.

Identification of different functional elements and their properties is a fundamental need in biomedical research and phylogenetic comparisons of a growing number of sequenced genomes form a solid basis for this task. Most available phylogenetic approaches are focused on searching for individual sequence alterations, responsible for the observed phenotype, or statistically evaluate observed mutations to infer general trends. However, being applied to close genomes such methods suffer from poor statistics of rare mutations and give only (at its best) coarse results concerning the potential functional importance of the nucleotide differences. However, quantifying the changes in physical properties of DNA allows to see the strength of introduced mutations and hence to classify them for further investigations. In this work we present the comparative sequence analysis of two evolutionarily close species-human and chimpanzee. In contrast to previous studies we evaluate changes in melting enthalpy of DNA rather than count nucleotide mismatches. We find that nucleotide mismatches in promoters were apparently introduced in a correlated manner during the course of evolution, so that, for example, the DNA property "melting enthalpy" was retained. Such property conservation of promoters is significantly different from nucleotide conservation, shows significant positional and functional biases, and seems to represent a novel feature of gene regulation.

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 http://genome.gbf.de/featurescan/. As part of the HOBIT initiative (http://hobit.sourceforge.net/) 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.

We present an implementation of the signal theory based approach for detection of novel types of DNA similarity which are based on physical properties of DNA. Systematic study of the sensitivity of the new similarity measure revealed qualitative differences to letter-based similarity. A variety of physical parameters of DNA double strands, which in a straightforward way reflect different kinds of information hidden behind the primary structure of DNA, showed a wide range of recognition power of the signal similarity measure. We applied the novel DNA similarity measure for the analysis of promoters of E.coli genes. We found that promoter similarities revealed by our approach correlate with their transcription regulatory responsivenesses to different antibiotic and osmotic treatments. Accelerated by special hardware for fast Fourier transformations, the method is easily applicable for the analysis of entire eukaryotic genomes in minutes.