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Small-angle X-ray scattering (SAXS) is used for structural determination of biomolecules in liquids. To prevent radiation damage during bio-SAXS at single-stranded DNA-binding proteins the OH-radical scavenger and compatible solute Ectoine is used.

Purpose of the study: Long-term graft survival rates after renal transplantation are still poor. We aimed to build an early predictor of an established long-term outcomes marker, the estimated glomerular filtration rate (eGFR) one year post-transplant (eGFR-1y). Materials and Methods: A large cohort of 376 patients was characterized for a multi-level bio-marker panel including gene expression, cytokines, metabolomics and antibody reactivity profiles. Almost one thousand samples from the pre-transplant and early post-transplant period were analysed and employed for machine learning-assisted prediction. Results: Pre-transplant data led to a prediction achieving a Pearson's correlation coefficient of r=0.38 between measured and predicted eGFR-1y. Two weeks post-transplant, the correlation was improved to r=0.63, and at the third month, to r=0.76. eGFR values were stable throughout the first post-transplant year. Several characteristics were predictive for eGFR, including age of donor and recipient, body mass index, HLA mismatch, cytomegalovirus mismatch and valganciclovir prophylaxis. Additionally, a subset of 19 nuclear magnetic resonance bins of the urine metabolome data was shown to have potential applications in non-invasive eGFR monitoring. Importantly, we identified the expression of the genes TMEM176B and HMMR as potential prognostic markers for changes in the eGFR after the second post-transplantation week. Conclusions: Our multi-center, multi-level data set represents a milestone in the efforts to predict transplant outcome. While an acceptable predictive capacity was achieved, we are still far from predicting changes in the eGFR precisely. Additional studies employing further marker panels are needed to establish predictors of eGFR-1y for clinical application; herein, gene expression markers seem to hold the most promise.

Epstein-Barr virus (EBV) reactivation is a very common and potentially lethal complication of renal transplantation. However, its risk factors and effects on transplant outcome are not well known. Here, we have analysed a large, multi-centre cohort (N = 512) in which 18.4% of the patients experienced EBV reactivation during the first post-transplant year. The patients were characterized pre-transplant and two weeks post-transplant by a multi-level biomarker panel. EBV reactivation was episodic for most patients, only 12 patients showed prolonged viraemia for over four months. Pre-transplant EBV shedding and male sex were associated with significantly increased incidence of post-transplant EBV reactivation. Importantly, we also identified a significant association of post-transplant EBV with acute rejection and with decreased haemoglobin levels. No further severe complications associated with EBV, either episodic or chronic, could be detected. Our data suggest that despite relatively frequent EBV reactivation, it had no association with serious complications during the first post-transplantation year. EBV shedding prior to transplantation could be employed as biomarkers for personalized immunosuppressive therapy. In summary, our results support the employed immunosuppressive regimes as relatively safe with regard to EBV. However, long-term studies are paramount to support these conclusions.

IntroductionLong-term graft survival rates after renal transplantation are still moderate. We aimed to build an early predictor of an established long-term outcomes marker, the glomerular filtration rate (eGFR) one year post-transplant (eGFR-1y).Materials and MethodsA large cohort of 376 patients was characterized for a multi-level bio-marker panel including gene expression, cytokines, metabolomics and antibody reactivity profiles. Almost one thousand samples from the pre-transplant and early post-transplant period were analysed. Machine learning-based predictors were built employing stacked generalization.ResultsPre-transplant data led to a prediction achieving a Pearson’s correlation coefficient of r=0.39 between measured and predicted eGFR-1y. Two weeks post-transplant, the correlation was improved to r=0.63, and at the third month, to r=0.76. eGFR values were remarkably stable throughout the first year post-transplant and were the best estimators of eGFR-1y already two weeks post-transplant. Several markers were associated with eGFR: The cytokine stem cell factor demonstrated a strong negative correlation; and a subset of 19 NMR bins of the urine metabolome data was shown to have potential applications in non-invasive eGFR monitoring. Importantly, we identified the expression of the genes TMEM176B and HMMR as potential prognostic markers for changes in the eGFR.DiscussionOur multi-centre, multi-level data set represents a milestone in the efforts to predict transplant outcome. While an acceptable predictive capacity was achieved, we are still very far from predicting changes in the eGFR precisely. Further studies employing further marker panels are needed in order to establish predictors of eGFR-1y for clinical application; herein, gene expression markers seem to hold the most promise.

Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine (THP(B)) and hydroxyectoine (THP(A)). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSBs), abasic sites and cyclobutane pyrimidine dimers (CPDs). A complex interplay between these factors was observed with respect to the nature and occurrence of DNA damage with 266 nm photons. In PBS, the cosolutes showed efficient protection against base damage, whilst in pure water, a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused by ectoine binding to DNA, further experiments were conducted: small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, a close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed that the alterations of UV damage to DNA are attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.

For statistical evaluation a normal distribution of the data is often necessary and essential. Parametric tests require values like arithmetic mean and standard deviation (sd) and therefor a normal distribution. They are often way more precise compared to non-parametric ones. That's why one should always check whether the data features a normal distribution. If that is not the case a robust method is to use only non-parametric tests but there is also a way to make use of parametric tests. For this purpose, data transformation can be applied to achieve normality.

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (

SummaryAcute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach. The data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA

This review addresses up-to-date applications of Protein Microarrays. Protein Microarrays play a significant role in basic research as well as in clinical applications and are applicable in a lot of fields, e.g., DNA, proteins and small molecules. Additionally they are on the way to enter clinics in routine diagnostics. Protein Microarrays can be powerful tools to improve healthcare. An overview of basic characteristics to mediate essential knowledge of this technique is given. To reach this goal, some challenges still have to be addressed. A few applications of Protein Microarrays in a medical context are shown. Finally, an outlook, where the potential of Protein Microarrays is depicted and speculations how the future of Protein Microarrays will look like are made.

In fields involving irradiated aqueous solutions, such as radiotherapy and nuclear waste remediation, it is often unclear whether the principal reactive species are OH° radicals or secondary (low-energy) electrons. This is mostly because both are rapidly attenuated in water. Presently a large part of the evidence for the involvement of low-energy electrons in biological radiation damage is based on “dry” DNA samples. We demonstrate irradiation of DNA in solution by direct injection of electrons through a 40-nm thin SiO 2 membrane, followed by in-situ detection of the DNA damage by a fluorescence-based method. Corresponding Monte Carlo simulations show that the spatial distribution of ionizing events in water with respect to the membrane is controlled by the electron impact energy. By immobilizing DNA to the solution side of the membrane, and because dynamics and reaction ranges of OH° radicals and low-energy electrons are dramatically different, it is possible to tune into the OH° radical or into the electron “reaction modes” by simply changing the electron impact energy. Such experiments have the potential to provide important information on the radio-sensitivity at a level of a single biomolecule and to contribute to the development of new dosage concepts.

Due to insufficient biomarker validation and poor performances in diagnostic assays, the candidate biomarker verification process has to be improved. Multi-analyte immunoassays are the tool of choice for the identification and detailed validation of protein biomarkers in serum. The process of identification and validation of serum biomarkers, as well as their implementation in diagnostic routine requires an application of independent immunoassay platforms with the possibility of high-throughput. This review will focus on three main multi-analyte immunoassay platforms: planar microarrays, multiplex bead systems and, array-based surface plasmon resonance (SPR) chips. Recent developments of each platform will be discussed for application in clinical proteomics, principles, detection methods, and performance strength. The requirements for specific surface functionalization of assay platforms are continuously increasing. The reasons for this increase is the demand for highly sensitive assays, as well as the reduction of non-specific adsorption from complex samples, and with it high signal-to-noise-ratios. To achieve this, different support materials were adapted to the immobilized biomarker/ligand, allowing a high binding capacity and immobilization efficiency. In the case of immunoassays, the immobilized ligands are proteins, antibodies or peptides, which exhibit a diversity of chemical properties (acidic/alkaline; hydrophobic/hydrophilic; secondary or tertiary structure/linear). Consequently it is more challenging to develop immobilization strategies necessary to ensure a homogenous covered surface and reliable assay in comparison to DNA immobilization. New developments concerning material support for each platform are discussed especially with regard to increase the immobilization efficiency and reducing the non-specific adsorption from complex samples like serum and cell lysates.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4 061 296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-l-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production.

Antibodies are used as powerful tools in basic research, for example, in biomarker identification, and in various forms for diagnostics, for example, identification of allergies or autoimmune diseases. Due to their robustness and ease of handling, immunoassays are favourite methods for investigation of various biological or medical questions. Nevertheless in many cases, additional analyses such as mass spectrometry are used to validate or confirm the results of immunoassays. To minimize the workload and to increase confidence in immunoassays, there are urgent needs for antibodies which are both highly specific and well validated. Unfortunately many commercially available antibodies are neither well characterized nor fully tested for cross-reactivities. Adequate quality control and validation of an antibody is time-consuming and can be frustrating. Such validation needs to be performed for every assay/application. However, where an antibody validation is successful, a highly specific and stable reagent will be on hand. This article describes the validation processes of antibodies, including some often neglected factors, as well as unspecific binding to other sample compounds in a multiparameter diagnostic assay. The validation consists of different immunological methods, with important assay controls, and is performed in relation to the development of a diagnostic test.

This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-e (PKC-e) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future.

Microarray technology plays an increasing role in proteomic research. We give an overview about recent developments in this technology focusing on molecular interaction studies using protein and antibody microarrays. We report about technical aspects in the development of protein microarrays and describe different surfaces and detection modes. Furthermore, we review the applications of protein microarrays in different molecular interaction studies including interactions of proteins with antibodies, proteins, DNA, small molecules and enzymes. Advantages and limitations of the microarray-based methods with other in vitro methods have been compared. We present the increasing applications of protein and antibody microarrays in basic research, diagnostics, drug discovery, and in vitro-risk assessment of nutrients.

To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-affinity DnaA box (R4) and with a low-affinity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433- 442 of Escherichia coli DnaA. This fragment contains six residues that were identified as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.

We report surface plasmon resonance (spr) and confocal fluorescence results concerning the interaction of a gene-5-protein (g5p) with single-stranded DNA oligonucleotides (dT 25 ) tethered to a gold surface. The spr data show that a highly stable g5p–ssDNA complex is readily formed on a gold surface with k off = 1.7 × 10 −3 s −1 . The extent of the complexion indicated involvement of the cooperative protein–protein interactions within the binding to DNA. In the experiments where dT 25 coexist with g5p in the solution, the fluorescence data show that g5p also mediates the binding between the non-complementary oligonucleotides in the solution and those immobilised on the surface.

Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration- dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms.

We analysed the enzymatic activity (strand dis-placement) of the Escherichia coli DnaB helicase on a mirror-image pair of oligonucleotide-based substrates mimicking the unwound replication origin oriC. Loading of the helicase complex occurred exclusively to the single-stranded 'lower strand' part of the substrates. Full helicase activity required DnaA bound to the double-stranded part of the substrates (oriC DnaA box R1) and to their single-stranded 'upper strand' part. We assume that in vivo DnaA also loads the first of two helicase complexes - required for the assembly of two replication forks - to the lower strand of oriC during initiation of bidirectional chromosome replication in E. coli.

We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC. The AT-rich region could be unwound by E. coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble. The results show that species specificity, i.e. which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a β-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF , mycA , mycB , and mycC , with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: ( i ) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. ( ii ) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

The determination of the protein composition is a challenging task, yet a valuable endeavor. Information that lies within the composition can help predicting pathogenic mechanisms and diseases. Therefore, a mandatory requirement is the accurate quantification. Protein microarrays are a promising alternative to complement mass spectrometry (MS) techniques. Nevertheless, effects in microarray production and assay development may lead to altered signal intensities. Techniques like Norma Curve provide a more robust quantification by taking the immobilized protein content into account. Printing dilution series can be used to construct a non-linear standard curve to estimate the amount of bound IgG, which helps to overrule the detection range of one order of magnitude. Standard protocols based on projects like the 'minimum information about a proteomics experiment' will help to improve quality and overall comparability.

The proteome is highly variable and differs from cell to cell. The reasons are posttranslational modifications, splice variants, and polymorphisms. Techniques like next-generation sequencing can only give an inadequate picture of the protein status of a cell. Protein microarrays are able to track these changes on the level they occur: the proteomic level. Therefore, protein microarrays are powerful tools for relative protein quantification, to unveil new interaction partners and to track posttranslational modifications. This papers gives an overview on current protein microarray techniques and discusses recent advances in relative protein quantification.

Sensitive detection assays are a prerequisite for the analysis of small amounts of samples derived from biological material. There is a great demand for highly sensitive and robust detection techniques to analyze biomolecules. The combination of catalytic active DNA (DNAzyme) with a peroxidase activity with rolling circle amplification (RCA) is a promising alternative to common detection systems. The rolling circle amplification leads to a product with tandemly linked copies of DNAzymes. The continuous signal generation of the amplified DNAzymes results in an increased sensitivity. The combination of two amplification reactions, namely RCA and DNAzymes, results in increased signal intensity by a factor of 10(6). With this approach the labeling of samples can be avoided. The advantage of the introduced assay is the usage of nucleic acids as biosensors for the detection of biomolecules. Coupling of the analyte molecule to the detection molecules allows the direct detection of the analyte molecule. The described label-free hotpot assay has a broad potential field of applications. The hotpot assay can be adapted to detect and analyze RNA, DNA and proteins down to femtomolar concentrations in a miniaturized platform with a total reaction solution of 50 nl. The applicability of the assay for diagnostics and research will be shown with a focus on high throughput systems using a nano-well platform.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581(T) thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4,061,296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-L-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production.

Microarrays have become an increasingly important tool for biotechnology and molecular diagnostics. Despite many advantages, their sensitivity is still insufficient for such tasks as the analysis of small sample quantities and for the detection of alterations in gene expression of low-abundance genes. Accordingly, amplification strategies are necessary. Approaches to amplify the signal intensity include the increase of the number of dye molecules per target through either particle labels or rolling circle amplification, as used for this study.

H. Guan, E. Kiss-Toth: Advanced Technologies for Studies on Protein Interactomes.- S.-Y. Shin, S.-M. Choo, S.-H. Woo, K.-H. Cho: Cardiac Systems Biology and Parameter Sensitivity Analysis: Intracellular Ca2+ Regulatory Mechanisms in Mouse Ventricular Myocytes.- T.G. Chappell, P.N. Gray: Protein Interactions: Analysis Using Allele Libraries.- M. Abu-farha, F. Elisma, D. Figeys: Identification of Protein-Protein Interactions by Mass Spectrometry Coupled Techniques.- Y. Zhu, B.E. Power: Lab-on-a-chip in vitro Compartmentalisation Technologies for Protein Studies.- U. Beutling, K. Stading, T. Stradal, R. Frank: Large-scale Analysis of Protein Protein Interactions Using Cellulose-bound Peptide Arrays.- U. Korf, F. Henjes, C. Schmidt, A. Tresch, H. Mannsperger, C. Lobke, T. Beissbarth, A. Poustka: Antibody Microarrays as Experimental Platform for the Analysis of Signal Transduction Networks.- P. Parekh, J. Martin, Y. Chen, D. Colon, H. Wang, W. Tan: Using Aptamers to Study Protein - Protein Interactions.- C.S. Chan, T.M.L. Winstone, R.J. Turner: Investigating Protein-Protein Interactions by far-Westerns.- S. Martin, W.M. Brown, J.-L. Faulon: Using Product Kernels to Predict Protein Interactions.- S. Pitre, M. Alamgir, J.R. Green, M. Dumontier, F. Dehne, A. Golshani: Computational Methods For Predicting Protein-Protein Interactions.

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His 6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.

Bacteriophages (prokaryotic viruses) are favourite model systems to study DNA replication in prokaryotes, and provide examples for every theoretically possible replication mechanism. In addition, the elucidation of the intricate interplay of phage-encoded replication factors with 'host' factors has always advanced the understanding of DNA replication in general. Here we review bacteriophage replication based on the long-standing observation that in most known phage genomes the replication genes are arranged as modules. This allows us to discuss established model systems--f1/fd, phiX174, P2, P4, lambda, SPP1, N15, phi29, T7 and T4--along with those numerous phages that have been sequenced but not studied experimentally. The review of bacteriophage replication mechanisms and modules is accompanied by a compendium of replication origins and replication/recombination proteins (available as supplementary material online).

V. Beier, C. Mund, J.D. Hoheisel: Monitoring Methylation Changes in Cancer.- J. Majka, C. Speck: Analysis of Protein - DNA Interactions Using Surface Plasmon Resonance.- D.F. Fischer, C. Backendorf: Identification of Regulatory Elements by Gene Family Footprinting and In Vivo Analysis.- M.L. Bulyk: Protein Binding Microarrays for the Characterization of DNA-Protein Interactions.- S. Field, I. Udalova, J. Ragoussis: Accuracy and Reproducibility of Protein-DNA Microarray Technology.- E. Nordhoff, H. Lehrach: Identification and Characterization of DNA-Binding Proteins by Mass Spectrometry.

Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss recent technological achievements and efforts on standardization.

A systematic approach for identifying human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli is described., We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.

A new strategy for identifying proteins by MALDI-TOF-MS peptide mapping is reported. In contrast to current approaches, the strategy does not rely on a good relative or absolute mass accuracy as the criterion that discriminates false positive results. The protein sequence database is first searched for all proteins that match a minimum five of the submitted masses within the maximum expected relative errors when the default or externally determined calibration constants are used, for instance, +/-500 ppm. Typically, this search retrieves many thousand candidate sequences. Assuming initially that each of these is the correct protein, the relative errors of the matching peptide masses are calculated for each candidate sequence. Linear regression analysis is then performed of the calculated relative errors as a function of m/z for each candidate sequence, and the standard deviation to the regression is used to distinguish the correct sequence among the candidates. We show that this parameter is independent of whether the mass spectrometric data were internally or externally calibrated. The result is a search engine that renders internal spectrum calibration unnecessary and adapts to the quality of the raw data without user interference. This is made possible by a dynamic scoring algorithm, which takes into account the number of matching peptide masses, the percentage of the protein's sequence covered by these peptides and, as new parameter, the determined standard deviation. The lower the standard deviation, the less cleavage peptides are required for identification and vice versa. Performance of the new strategy is demonstrated and discussed. All necessary computing has been implemented in a computer program, free access to which is provided in the Internet.

We describe the technical feasibility and methodology to characterize a protein by a minimal set of structural information generated by matrix assisted laser desorption/ionization (MALDI)-mass spectrometry, termed a "minimal protein Identifier" (MPI). MPIs can be determined for proteins from two-dimensional gels and recombinant proteins and can be used to compare and identify proteins from these sources.