Towards an Electrochemical Immunosensor System with Temperature Control for Cytokine Detection.

The cytokine interleukin-13 (IL-13) plays a major role in airway inflammation and is a target of new anti-asthmatic drugs. Hence, IL-13 determination could be interesting in assessing therapy success. Thus, in this work an electrochemical immunosensor for IL-13 was developed and integrated into a fluidic system with temperature control for read-out. Therefore, two sets of results are presented. First, the sensor was set up in sandwich format on single-walled carbon nanotube electrodes and was read out by applying the hydrogen peroxide⁻hydroquinone⁻horseradish peroxidase (HRP) system. Second, a fluidic system was built up with an integrated heating function realized by Peltier elements that allowed a temperature-controlled read-out of the immunosensor in order to study the influence of temperature on the amperometric read-out. The sensor was characterized at the temperature optimum of HRP at 30 °C and at 12 °C as a reference for lower performance. These results were compared to a measurement without temperature control. At the optimum operation temperature of 30 °C, the highest sensitivity (slope) was obtained compared to lower temperatures and a limit of detection of 5.4 ng/mL of IL-13 was calculated. Taken together, this approach is a first step towards an automated electrochemical immunosensor platform and shows the potential of a temperature-controlled read-out.

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols.

Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3ß,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3ß,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.

Single-stranded DNA catalyzes hybridization of PCR-products to microarray capture probes.

Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays.

Dual control of seasonal time keeping in male and female juvenile European hamsters.

In contrast to photoperiodic rodent species, adult circannual European hamsters (Cricetus cricetus) do not rely on melatonin as transducer of the photoperiodic message. Instead, seasonal entrainment involves a special circadian organisation which characterizes a photoperiod-sensitive phase. When days shorten a precise activity pattern ("summer pattern") switches to a weak or arrhythmic "winter pattern". At the very same day gonadal regression is initiated and the circannual clock is reset. In contrast to this difference in photoperiodic time measurement, the broad time span in which offspring are born and the birth-season dependent timing of puberty is similar to photoperiodic rodents. We investigated how juvenile European hamsters measure photoperiod to situate themselves at the proper position in the annual cycle. Activity and 6-sulphatoxymelatonin (aMT6s) excretion were recorded in pups of five litters born at different seasons. Pups of all litters showed an activity pattern identical with the adults' summer pattern until postnatal day 78, suggesting that the pathway known to reset the circannual clock in adults is functional. The synchronous start of reproduction in yearlings supports this. However, since puberty and gonadal regression occurred before the switch in the activity pattern, the timing of reproduction in the birth year must be controlled by other means. As in photoperiodic species melatonin might be involved, since the aMT6s excretion showed daily and seasonal rhythms from early life on.

Simultaneous Detection of Different MicroRNA Types Using the ZIP-Code Array System.

MicroRNAs (miRNAs) are important negative regulators of gene expression. Their implication in tumorigenesis is based on their dysregulation in many human cancer diseases. Interestingly, in tumor cells, an altered ratio of precursor and mature miRNA levels has been described. Consequently, differences in miRNA type levels have a high potential as biomarkers and comparative high-throughput-based detection might permit a more accurate characterization of subtypes, especially in the case of very heterogeneous tumor entities. Several molecular methods exist for the detection of mature and precursor miRNAs. DNA microarrays are predestinated as a high-throughput method for comprehensive miRNA detection in tumors. However, the simultaneous array-based detection of both these miRNA types is limited because the mature miRNA sequence is identically present in both forms. Here we present a ZIP-code DNA microarray-based system in combination with a novel labeling approach, which enables the simultaneous detection of precursor and mature miRNAs in one single experiment. Using synthetic miRNA templates, we demonstrate the specificity of the method for the different miRNA types, as well as the detection range up to four orders of magnitude. Moreover, mature and precursor miRNAs were detected and validated in human tumor cells.

DNA microarray for genotyping antibiotic resistance determinants in Acinetobacter baumannii clinical isolates.

In recent decades, Acinetobacter baumannii has emerged as an organism of great concern due to its ability to accumulate antibiotic resistance. In order to improve the diagnosis of resistance determinants in A. baumannii in terms of lead time and accuracy, we developed a microarray that can be used to detect 91 target sequences associated with antibiotic resistance within 4 h from bacterial culture to result. The array was validated with 60 multidrug-resistant strains of A. baumannii in a blinded, prospective study. The results were compared to phenotype results determined by the automated susceptibility testing system VITEK2. Antibiotics considered were piperacillin-tazobactam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, amikacin, gentamicin, tobramycin, ciprofloxacin, and tigecycline. The average positive predictive value, negative predictive value, sensitivity, and specificity were 98, 98, 99, and 94%, respectively. For carbapenemase genes, the array results were compared to singleplex PCR results provided by the German National Reference Center for Gram-Negative Pathogens, and results were in complete concordance. The presented array is able to detect all relevant resistance determinants of A. baumannii in parallel. The short handling time of 4 h from culture to result helps to provide fast results in order to initiate adequate anti-infective therapy for critically ill patients. Another application would be data acquisition for epidemiologic surveillance.

A screening assay based on host-pathogen interaction models identifies a set of novel antifungal benzimidazole derivatives.

Fungal infections are a serious health problem in clinics, especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses, only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapy, significant side effects, and high costs for several antifungals, there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities, we screened a compound library including more than 35,000 individual compounds derived from organic synthesis as well as combinatorial compound collections representing mixtures of compounds for antimycotic activity. In total, more than 100,000 compounds were screened using a new type of activity-selectivity assay, analyzing both the antifungal activity and the compatibility with human cells at the same time. One promising hit, an (S)-2-aminoalkyl benzimidazole derivative, was developed among a series of lead compounds showing potent antifungal activity. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole showed the highest antifungal activity and the best compatibility with human cells in several cell culture models and against a number of clinical isolates of several species of pathogenic Candida yeasts. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol pathway, in contrast to other benzimidazole derivatives, which target microtubules.

Adaptation, adhesion and invasion during interaction of Candida albicans with the host--focus on the function of cell wall proteins.

Infectious diseases have long been regarded as losing their threat to mankind. However, in the recent decades infectious diseases have been regaining grounds and are back in the focus of research. This is also due to the fact that medical progress has enabled us to treat and cure a much higher fraction of severe diseases or trauma, resulting in a significant proportion of temporarily or constantly immune-suppressed patients. Infectious diseases result from the interplay between pathogenic microorganisms and the hosts they infect, especially their defense systems. Consequently, immune-suppressed patients are at high risk to succumb from opportunistic infections, like Candida infections. To study the balance between host and C. albicans with regard to the establishment of disease or asymptomatic, commensal colonisation, we developed host-pathogen interaction systems to study both the adaptation of C. albicans to different epithelia as well as to investigate the sensors of the innate immune system, the pattern recognition receptors. These host-pathogen interaction systems, as well as some of the results gained are described in this review.

Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin.

BACKGROUND: The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or ß-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of ß-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin. RESULTS: Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a ß-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces ß-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the ß-carotene-ketolase gene crtW148 (NpF4798) and the ß-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw). CONCLUSIONS: By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this E. coli strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.

Quantifying cytosolic messenger RNA concentrations in Escherichia coli using real-time polymerase chain reaction for a systems biology approach.

Current messenger RNA (mRNA) quantification methods are sophisticated tools for the analysis of gene regulation. However, these methods are not suitable for more complex quantitative approaches such as the mathematical modeling of the in vivo regulation of transcription where dynamic cytosolic mRNA concentrations need to be taken into consideration. In the current study, the "standard curve method" for quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) was extended by including an internal RNA standard. This standard enables transcript losses that occur during the process, as well as variations resulting from nonquantitative processes, to be accounted for. The use of an internal standard yielded transcript concentration estimates that were on average seven times higher than those in cases where an internal standard is omitted. Choosing the cra modulon in Escherichia coli as an example, the method applied shows that the regulation of the Cra protein, as well as the growth rate-dependent regulation, need to be taken into consideration. The new method, which enables the determination of cytosolic mRNA concentrations, allows the quantitative representation of transcriptional dynamics. This is an important aspect of the analysis of the complex interactions of metabolism and regulation and in the application of mathematical modeling for systems biology.

Biosynthesis of the vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells.

The biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E. coli. Tocochromanols are synthesized solely in photosynthetic organisms (cyanobacteria, algae, and higher green plants). For recombinant tocochromanol biosynthesis, the genes encoding hydroxyphenylpyruvate dioxygenase (hpd), geranylgeranylpyrophosphate synthase (crtE), geranylgeranylpyrophosphate reductase (ggh), homogentisate phytyltransferase (hpt), and tocopherol-cyclase (cyc) were cloned in a stepwise fashion and expressed in E. coli. Recombinant E. coli cells were cultivated and analyzed for tocochromanol compounds and their biosynthesis precursors. The expression of only hpd from Pseudomonas putida or crtE from Pantoea ananatis resulted in the accumulation of 336 mg L(-1) homogentisate and 84 microg L(-1) geranylgeranylpyrophosphate in E. coli cultures. Simultaneous expression of hpd, crtE, and hpt from Synechocystis sp. under the control of single tac-promoter resulted in the production of methyl-6-geranylgeranyl-benzoquinol (67.9 microg g(-1)). Additional expression of the tocopherol cyclase gene vte1 from Arabidopsis thaliana resulted in the novel formation of a vitamin E compound-delta-tocotrienol (15 microg g(-1))-in E. coli.

Quantification of rRNA in Escherichia coli using capillary gel electrophoresis with laser-induced fluorescence detection.

Over the past 10 years, sophisticated powerful techniques have been developed for the quantification of messenger RNA (mRNA) and ribosomal RNA (rRNA), enabling researchers in science, industry, and molecular medicine to explore gene expression. These techniques require the (reverse) transcription of analyte RNA, hybridization with synthetic oligonucleotides, and other additional steps that make them costly, time-consuming, and quantitatively difficult to perform. The current work demonstrates how 16S and 23S rRNA can be quantified precisely using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) directly after the extraction of total RNA without requiring further reactions or calibration. CGE-LIF normally is used for the qualitative examination of RNA preparations. Its quantitative performance could be improved significantly using MS2 bacteriophage RNA as an internal standard. The entire analytical procedure was validated for linearity, repeatability, reproducibility, and recovery. This validation also included total RNA extraction from bacterial cells, an aspect examined for the first time in absolute RNA quantification. Recovery is close to 100%, and the analytical precision was increased 10-fold (CV<3%), as compared with similar approaches. The demonstrated method is simple and opens up new possibilities for the absolute quantification of not only rRNA but also individual mRNAs.

Topology of the global regulatory network of carbon limitation in Escherichia coli.

One fundamental shortcoming of biotechnological processes operating under carbon-limiting conditions is the high-energy demand (maintenance) of the cells. Although the function of the central carbon metabolism in supplying precursors and energy for biosynthesis has been thoroughly characterized, its regulation and dynamic behaviour during carbon-limited growth has not yet been revealed. The current work demonstrates a time series of metabolic flux distributions during fed-batch cultivation of Escherichia coli K-12 W3110 applying a constant feed rate. The fluxes in glycolysis, pentose phosphate pathway and biosynthesis fell significantly, whereas TCA cycle fluxes remained constant. The flux redistribution resulted in an enhanced energy generation in the TCA cycle and consequently, in a 20% lower biomass yield. The intracellular alarmones ppGpp and cAMP accumulated in large quantities after the onset of nutrient limitation, subsequently declining to basal levels. The network topology of the regulation of the central metabolic pathways was identified so that the observed metabolic and regulatory behaviour can be described. This provides novel aspects of global regulation of the metabolism by the cra, crp and relA/spoT modulons. The work constitutes an important step towards dynamic mathematical modelling of regulation and metabolism, which is needed for the rational optimization of biotechnological processes.