Zeno Foldes-Papp

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Abstract: We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:10^5. Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~9 s during muscle contraction

Abstract: We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N <1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume �V at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ T(m) between the measurement time T and the meaningful time T(m), which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

Abstract: RNA interference (RNAi) is an evolutionary conserved mechanism by which small double-stranded RNA (dsRNA)--termed small interfering RNA (siRNA)--inhibit translation or degrade complementary mRNA sequences. Identifying features and enzymatic components of the RNAi pathway have led to the design of highly-effective siRNA molecules for laboratory and therapeutic application. RNA activation (RNAa) is a newly discovered mechanism of gene induction also triggered by dsRNAs termed small activating RNA (saRNA). It offers similar benefits as RNA interference (RNAi), while representing a new method of gene overexpression. In the present study, we identify features of RNAa and explore chemical modifications to saRNAs that improve the applicability of RNAa. We evaluate the rate of RNAa activity in order to define an optimal window of gene induction, while comparing the kinetic differences between RNAa and RNAi. We identify Ago2 as a conserved enzymatic component of both RNAa and RNAi implicating that saRNA may tolerate modification based on Ago2 function. As such, we define chemical modifications to saRNAs that manipulate RNAa activity, as well as exploit their effects to design saRNAs with enhanced medicinal properties. These findings reveal functional features of RNAa that may be utilized to augment saRNA function for mechanistic studies or the development of RNAa-based drugs.

Abstract: Currently, work with subnanomolar concentrations is routine while femtomolar and even single-molecule studies are possible with some efforts getting high on single-molecule biophysics and biochemistry. Methodological breakthroughs, such as reducing the background light contribution in single-molecule studies, which has plagued many studies of molecular fluorescence in dilute solution, and particularly in live cells, have recently described by us. We first demonstrated how optimized time-gating of the fluorescence signal, together with time-correlated, single-photon counting, can be used to substantially boost the experimental signal-to-noise ratio about 140-fold, making it possible to measure analyte concentrations that are as low as 15 pM. By detection of femtomolar bulk concentrations, confocal microsopy has the potential to address the observation of one and the same molecule in dilute solution without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than currently available. We present relevant physics. The equations are derived using Einstein's approach showing how it fits with Fick's law and the autocorrelation function. An improved technology is being developed at ISS for femtomolar microscopy. The general concepts and provided experimental examples should help to compare our approach to those used in conventional confocal microscopy.

Abstract: Based on classical mean-field approximation using the diffusion equation for ergodic normal motion of single 24-nm and 100-nm nanospheres, we simulated and measured molecule number counting in fluorescence fluctuation microscopy. The 3D-measurement set included a single molecule, or an ensemble average of single molecules, an observation volume DeltaV and a local environment, e.g. aqueous solution. For the molecule number N << 1 per DeltaV, there was only one molecule at a time inside DeltaV or no molecule. The mean rate k of re-entries defined by k = N / tau(dif) was independent of the geometry of DeltaV but depended on the size of DeltaV and the diffusive properties tau(dif). The length distribution ? of single-molecule trajectories inside DeltaV and the measured photon count rates I obeyed power laws with anomalous exponent kappa =-1.32 approximately -4/3.

Abstract: The novel utrashort femtosecond laser scanning microscope FemtOgene (JenLab GmbH, Germany) with 12 femtoseconds at the focal plane have been employed in marker-free imaging and optical manipulation of stem cells as well as for the non-contact introduction of microRNA in cancer cells. Human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells have been investigated by femtosecond laser multiphoton microscopy. Autofluorescence based on NAD(P)H and flavoproteins and second harmonic generation due to collagen have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. Major emission peaks at 460 nm and 530 nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured in a variety of stem cells using spectral imaging and time-correlated single photon counting. During differentiation, the ratios of bound to free NAD(P)H and NAD(P)H/flavoproteins changed. In addition, the biosynthesis of lipids and collagen was detected over a long period of time of up to 5 weeks. Nanoprocessing was performed with 12 femtosecond laser pulses and low picojoule pulse energies to realize targeted transfection and optical cleaning of human adult stem cell populations. Multiphoton sub-20fs microscopes may become novel non-invasive tools for marker-free optical stem cell characterization, for on-line monitoring of differentiation within a three-dimensional microenvironment, and for optical manipulation. ©2010 COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Abstract: In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent alpha also determines the resolution of differences in diffusion times between two components in addition to photophysical parameters well-known for normal motion in dilute solution. The resolution limit between two different kinds of single molecule species is also analyzed under translational anomalous motion with broken ergodicity. We apply our theoretical predictions of diffusion times and lower limits for the time resolution of two components to fluorescence images in human prostate cancer cells transfected with GFP-Ago2 and GFP-Ago1. In order to mimic heterogeneous behavior in crowded environments of living cells, we need to introduce so-called continuous time random walks (CTRW). CTRWs were originally performed on regular lattice. This purely stochastic molecule behavior leads to subdiffusive motion with broken ergodicity in our simulations. For the first time, we are able to quantitatively differentiate between anomalous motion without broken ergodicity and anomalous motion with broken ergodicity in time-dependent fluorescence microscopy data sets of living cells. Since the experimental conditions to measure a selfsame molecule over an extended period of time, at which biology is taken place, in living cells or even in dilute solution are very restrictive, we need to perform the time average over a subpopulation of different single molecules of the same kind. For time averages over subpopulations of single molecules, the temporal auto- and crosscorrelation functions are first found. Knowing the crowding parameter alpha for the cell type and cellular compartment type, respectively, the heterogeneous parameter gamma can be obtained from the measurements in the presence of the interacting reaction partner, e.g. ligand, with the same alpha value. The product alpha x gamma = gamma is not a simple fitting parameter in the temporal auto- and two-color crosscorrelation functions because it is related to the proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in cellular systems.We have already derived an analytical solution gamma for in the special case of gamma = 3/2. In the case of two-color crosscorrelation or/and two-color fluorescence imaging (co-localization experiments), the second component is also a two-color species gr, for example a different molecular complex with an additional ligand. Here, we first show that plausible biological mechanisms from FCS/ FCCS and fluorescence imaging in living cells are highly questionable without proper quantitative physical models of subdiffusive motion and temporal randomness. At best, such quantitative FCS/ FCCS and fluorescence imaging data are difficult to interpret under crowding and heterogeneous conditions. It is challenging to translate proper physical models of anomalous (spatial) and heterogeneous (temporal) randomness in living cells and their cellular compartments like the nucleus into biological models of the cell biological process under study testable by single-molecule approaches. Otherwise, quantitative FCS/FCCS and fluorescence imaging measurements in living cells are not well described and cannot be interpreted in a meaningful way.

Abstract: Indirect effects of 3,4-Methylenedioxy-N-methylamphetamine (MDMA) and metabolites on the cardiac cells are well-known, the mechanism(s) underlying direct MDMA-induced cardiotoxicity remaining to be clarified. To better understand the immuno-inflammatory phenomena accompanying the cardiac alterations during MDMA administration, we conducted a study in an in vivo animal model to evaluate the cellular morphological alterations related to the biological response between MDMA administration and inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6, 8, 10, and monocyte chemotactic protein-1). A total of 25 male rats were used. The effects were evaluated at 6, 16 and 24hours after a single dose MDMA administered (20 mg/kg i.p.). We found high levels of the cardioinhibitory cytokines in rat heart after 3 and 6hs from MDMA administration. Strongest reaction was observed at 24hs for TNF-alpha, IL-1beta, IL-6, 8, 10 and for MCP-1. Furthermore, we still determined the presence of MDMA and MDA in the plasma of rats treated with MDMA intra-peritoneal single injection; it was present as early at 6hs and still present 24hs after treatment. Western blot analysis in cardiac samples demonstrated the IL-1beta and IL-6 reactions in rats died spontaneously at fourth hour. The rise of the selective cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium to the cardiac dysfunction resulting from MDMA injection.

Abstract: Fluorescent beads (nanoparticles, nanospheres) are commonly used in fluorescence spectroscopy and microscopy. Due to the random distribution of dye and high dye to nanoparticle ratio, the fluorescence polarization observed from the beads is low. Therefore beads are not used for polarization study. We demonstrate that photoselective bleaching creates beads with highly polarized fluorescence. First, the beads were immobilized in a PVA polymer. Second, the beads-doped PVA film was exposed to the illumination within the dye absorption band. A progressive decrease of absorption was observed. Next, photophysical properties of photobleached and not bleached films dissolved in water were compared.

Abstract: We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.

Abstract: The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

Abstract: The objective of this study was to assess and to compare the time course of the immunological signaling marker IL-6 and the non-immunological marker PCT in surgical patients with local and systemic bacterial infections and with multiple trauma of well-defined severity. 45 surgical and 26 intensive care patients were enrolled in this collaborative study. We analyzed IL-6 and PCT blood concentrations under the different experimental conditions with Cochran�s Q -criterion for testing null hypotheses. For the surgical patients with local infections and with systemic infections, we did not find a significant decline of IL-6 in the time course at the confidence intervals 0.999, 0.990, and 0.950. No differences in PCT concentrations were found for the various experimental conditions. The time course of IL-6 did not change significantly with medium and high severity trauma, whereas PCT declined over time with significance levels 0.990 and 0.950. The measurement of IL-6 is predominantly a research tool at present. There are no absolute indications for routine measurements. Regarding PCT, our results do not point to this marker as a valid predictor of bacterial infections including sepsis during the early period after trauma.

Abstract: Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for all pathways of DNA metabolism. To study the function of RPA in living cells the second largest RPA subunit and an N-terminal deletion mutant thereof were fused to green fluorescent protein (GFP; GFP-RPA2 and GFP-RPA2deltaN, respectively) in a controlled, molecular biological way. These proteins were expressed in HeLa cells under the control of the inducible tetracycline expression system. GFP-RPA2 and GFP-RPA2deltaN are predominately nuclear proteins as determined by confocal laser scanning microscopy. Low basal expression of GFP-RPA2deltaN allowed the measurement of kinetic parameters of RPA. Using fluorescence correlation spectroscopy (FCS) two populations--a fast and a slow moving species--were detected in the nucleus and the cytosol of human cells. The translational diffusion rates of these two RPA populations were approximately 15 microm2/s and 1.8 microm2/s. This new finding reveals the existence of different multiprotein and ssDNA-protein complexes of RPA in both cellular compartments and opens the possibility for their analyses.

Abstract: Short report Exploring the biomedical applications of microscopy and spectroscopy Zeno Földes-Papp: Traditional imaging and spectroscopic approaches such as scanning electron microscopy, computed tomography, magnetic resonance imaging, single-photon emission tomography, Raman, bioluminescence and radioisotope imaging are based on physical properties of molecules. Molecular imaging and spectroscopy uses specific molecules as the source of contrast. The concept of smart labels with specific fluorescent molecules is exploring the next frontier in the life sciences: ultrasensitive detection at a single-molecule level to monitor biological interactions without disturbing the integrity of the sample. Lower detection limits have driven developments of novel approaches to molecular analytics and diagnostics. A challenge has been the achievement of higher-quality data and more accurate results. This special issue section presents original research articles by top researchers. The originality and innovative nature of the articles resides in the strict coupling of microscopic and spectroscopic developments with physiological relevance. For example, quantitative understanding of molecular interactions at the level of single molecules and single cells is the next step in basic and applied biomedical research for the analysis of the dynamics and localization of molecules in a variety of physiological and pathophysiological processes. The articles should provide the reader with a better understanding of some of the theoretical backgrounds of these types of analyses and biological or medical applications. We look forward to the exciting new discoveries that these methodologies promise in the years to come. Some of the contributions were presented at the 30th Annual Meeting of the Microscopical Society of Ireland, August 30th� September 1st, 2006, held in Galway, Ireland.

Abstract: Reentries of a single molecule in the confocal, femtoliter-sized probe region (about 10(-16) L and less) are significant because during measurement times they give rise to fluctuation phenomena such as molecule number fluctuations at the single-molecule level in solution without immobilization or hydrodynamic focusing. These fluctuations are the fundamental physical process on which, for example, fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy are based. The reentries of just one molecule in the confocal probe region are theoretically examined in this original article using a hidden, continuous-time Markov model. The system is not set up to have systemic drift or convection. It is found that the reentries obey certain conditions and analytical expressions for the reentry probabilities are obtained first. In particular, the time constant of the mean value and the variance of the reentry probabilities are obtained. The fractions of non-meaningful reentries and meaningful reentries are found for these experimental situations. Therewith, the concentration dependence of the meaningful time that one can study bimolecular reactions of the selfsame molecule in the confocal probe region is derived for the first time. The meaningful time in the probe volume is proportional to the diffusion time of the selfsame molecule and related inversely to the size of the given confocal probe volume. For small molecules, i.e. small diffusion times at a given size of the confocal probe region, one needs lower concentrations of molecules of the same kind in the bulk phase, whereas large molecules can be studied at higher concentrations. The selfsame molecule scenario is compared with the molecular scenario that a second molecule enters the probe volume at random as a function of the meaningful time. The analytical solutions of the physical reentry model (mechanism) hold for the one-, two- (membrane), or three- (solution, live cell) dimensional Brownian motion.

Abstract: Fluorescence correlation spectroscopy (FCS) and two-color fluorescence cross-correlation spectroscopy (FCCS) are a measure of fluctuations of detected light as a fluorescence molecule diffuses through a femtoliter detection volume caused by a tightly focused laser and confocal optics. Fluorescence from a single molecule can easily be distinguished from the slight background associated with a femtoliter of solvent. At a solution concentration of about 1 nM, the probability that there is an analyte molecule in the probe volume is less than one. Although fluorescence from individual molecules is collected, the data are analyzed by autocorrelation or two-color cross-correlation functions that are the average of thousands of molecules. Properties of single molecules are not obtained. I have been working on problems and opportunities associated with very dilute solutions. The molecule in the confocal probe volume is most probably the molecule that just diffused out, turned around, and diffused back in, i.e., reentered. For the first time, some theoretical results of the novel theory of the meaningful time are presented that enable study of just one single molecule over extended periods of times without immobilization or hydrodynamic focusing. Reentries that may also be called reoccurrences or encounters of a single molecule are significant because during measurement times they give rise to fluctuation phenomena such as molecule number fluctuations. Likewise, four criteria have been developed that can be used to verify that there is only one "selfsame" molecule in the laser probe volume during the experiment: (Földes-Papp, Z., 2006. What it means to measure a single molecule in a solution by fluorescence fluctuation spectroscopy. Exp. Mol. Pathol. 80 (3) 209-218).

Abstract: The literature suggests that autoantibody formations and disturbances in cellular or humoral immunities are relevant immunological events in lichen sclerosus (LS). We examined 39 patients (age range: 7-81 years) enrolled in this experimental immunopathology study and treated for vulvar LS. In the serum, we used 88 clinical immunology parameters to evaluate the immunological patterns, i.e., autoimmune phenomena, humoral immunity, cellular immunity, and inflammation. The analyses permitted direct comparison of the measured distributions of alternative data. We found that all pathological findings of single immunological events followed a random distribution without any positive or negative trend or a distribution with a negative trend. There was a lack of correlation between the majority of cases and the presence of pathological findings (confidence intervals 0.950 and 0.999). Combinations of two or more of the four patterns did not improve the outcomes (confidence intervals 0.950 and 0.999). However, abnormalities in systemic immune parameters implying system impairments might have occurred long before the patients with such a chronic disease presented to the clinic. This may be especially true of such diseases as vulvar LS, where local skin scarring might represent a local tissue response secondary to an initial insult by immune or other processes.

Abstract: Single-molecule fluorescence methods enable a new class of nucleic acid assays to be performed that are not possible with PCR-based methods. In this basic study, the methylene tetrahydrofolate reductase (MTHFR)-genotypes (normal, homozygous mutated, as well as heterozygous mutated) were directly detected for the first time onto unamplified double-stranded genomic DNA in solution down to femtomolar allele concentrations (10(-15) M) in a homogeneous assay format. This was accomplished by taking advantage of the decrease by a factor of 40 to 100 in fluorescence background signals of the non-bound nonlinear hybridization probes in two colors and two-color fluorescence cross-correlation spectroscopy. The designed 'intelligent' probes contained the built-in 5'-fluorescent dyes rhodamine green and Alexa633, respectively, and the 3'-non-fluorescent quenchers BHQ1 and BHQ3, respectively, with perfectly matched spectral overlaps for both dye-quencher combinations. Upon binding of two appropriate probes that were sequence-specific for the genotype, the steady-state fluorescence in two colors increased by about two orders of magnitude. The obtained allele sensitivity of femtomolar and the specificity of the described molecular interactions allow PCR-based allele distinction to be circumvented. Furthermore, the results present an alternative to existing hybridization approaches that are currently used with and without amplification at the 'many-molecule' level and the 'single-molecule' level.

Abstract: Fluorescence correlation spectroscopy (FCS) and two-color fluorescence cross-correlation spectroscopy (FCCS) are among the cutting-edge technologies for measuring molecule numbers at the single-molecule level in liquid phases. Yet, even after single molecule technologies caught up with theory, the techniques remained tools only for specialists able to navigate the formulas that give meaning to their observations. This original article aims at the derivations of relevant and useful quantification of molecule numbers for researchers with more diverse backgrounds who have begun probing questions previously unanswerable, except on the level of the molecule. The quantitation depends on the exact conditions of measurement. To some extent these are arbitrary, so that standard procedures are necessary in for valid comparisons of measurements among different data sets. To agree on and specify such procedures is one of the further aims here. No matter what fluorophores, which have, of course, to meet photophysical and photochemical requirements for FCS/FCCS, and optical setups/devices are used, the primary measurement signal arises from fluctuations of the mean molecule number in a confocal femtoliter or smaller probe region. Since FCS/FCCS relies on fluorescence emission measurements of rare events, one is looking for small signals on essentially zero background. Optical separation by FCCS setups is usually defined in terms of cross-talk and cross-excitation/cross-emission, respectively, which can be calculated and minimized by the experimenter from readily measurable quantities of the absorption/emission scenario for single labels and multiple labels n and m bound to or incorporated into the two-color molecules. Furthermore, this article derives relevant formulas for the quantification of molecule numbers under different experimental conditions with substantial quenching of the two-color molecules such as single labels and multiple labels n and m bound to or incorporated into the two-color molecules, high-density labeling of two-color molecules with multiple n green labels and one red label. Here, we summarize and extend the formulas to make them more generally applicable.

Abstract: Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

Abstract: Just because there is an average of one molecule in the observation volume of a solution or membrane (single-phase), one cannot say that this is an individual molecule since many different single molecules measured one by one or the same single, individual molecule not leaving the detection volume on time average can cause a single-molecule event. The latter case is of interest and allows the continuous observation of one and the same single molecule without averaging over many 'different' single molecules. For the first time a universal theoretical and experimental framework is presented for the continuous observation of the same single, individual molecule without immobilization, hydrodynamic flow, or burst size histograms of fluorescence intensity traces. In this original article, the stochastic approach is derived and its main characteristics are demonstrated with the free fluorophore rhodamine-green in solution for simpler experimental realization. Single (solution)-phase single-molecule fluorescence auto- (or two-color cross-) correlation spectroscopy (SPSM-FCS) is used as a specific application in order to count the absolute number of molecules in the observation volume. The absolute number of molecules, the diffusion coefficient of the single fluorescent molecule, the lower limit of distance, and the molar concentration of the bulk phase (solution) were directly obtained from the measured auto- or (cross)-correlation curves of the SPSM-FCS experiments. For this purpose, the detection volume that was measured was less then 1 fl (10(-15) l). Then, a concentration of the bulk solution was chosen in such a way that the probability of detecting more than one molecule in the detection volume was very small. The Poisson probability was experimentally determined for the absolute number of molecules depending upon a specified bulk concentration. From the diffusion coefficient of the molecule, it was found that the probability of the molecule diffusing out of the probe volume during the measurements was negligibly small.

Abstract: BACKGROUND: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample. METHODS: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures. RESULTS: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. CONCLUSIONS: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.

Abstract: Malignant cells in the peripheral blood of patients with solid tumours are of considerable importance for the prognosis and therapeutic correlation. Their detection however is difficult due to lack of sensitivity, specificity and technical problems in standardisation. In this original article we show a new sensitive method overcoming the hitherto known difficulties by combining traditional antibody-techniques with a RT-PCR. Due to this method 2 tumour cells within 5 ml of peripheral blood can be detected in spiking experiments.

Abstract: Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.

Abstract: Foreword When we started preparing the edition of the two theme issues � each divided in two parts (i.e., four issues in total) � we felt that the time had come to present and discuss recent achievements in biotechnology and bioanalysis that are creating a new array of opportunities for the pharmaceutical and biomedical sectors. The inter-disciplinary topics presented in these four issues stem from scientists in biology, chemistry, and biochemistry as well as molecular biology, immunology, physics, mathematics, engineering, computation and medicine. After studying the cell and its constituents and working the way down to the genome and the proteome, huge amount of data have been generated during the last decade and can now be deciphered to shed light on the basic principles of life. Among the new technologies that are making this progress possible, the DNA chip is only one example that has helped address many molecular biology problems, such as rapid identification of genetic disorders. The foundations of microarray and biochip technologies are firmly rooted in the rapid advances in nucleic acid chemistry and biochemistry. The overwhelming majority of the early-phase trials using these developments was not primarily aimed at clinical efficacy but instead at assessing the biological problems to be solved before genomics, epigenomics and proteomics can deliver useful information for clinical applications. In an attempt to better understand these problems, investigators returned to basic studies of gene expression, immunology and virology. Finally, the cloning and sequencing of the human genome has significantly expanded the range of actual and potential molecular drug targets. The discovery and optimization of molecular drug targets, e.g., disease specific proteins, receptors, enzymes, or genes, is a time-consuming and extremely expensive process. Therefore, in today's preclinical drug research, there is only one key to success: scientific teamwork and the use of the most advanced biotechnological approaches. The advances in biotechnology directly affect improvements in human health and outcomes. That especially holds true in our time of rapid changes. The title of the four special issues implies that their scope encompasses the fundamentals of the way down from single genes and proteins to single molecules. Should we have started this Edition from the top downward, beginning with medicine and clinical practice based on empirical ("complex") thinking, where the risk exists of serious error by failing to account accurately for gene or protein interactions? Alternatively, should we proceed from the bottom upward beginning with single molecules and their behavior, which often seems to be different from that of the ensemble in biological systems? The answer is probably a compromise, working in both directions from the middle. The contributions to these four Special Issues of Current Pharmaceutical Biotechnology are written by leading specialists in fields that cover a wide range of current topics on biotechnological and biomedical aspects of drug target screening and characterization. Several articles present overviews on the latest developments and future prospects. They are devoted to discussing multidisciplinary topics at a level that will be useful to the practicing scientist. The contributions are not simply reviews and mini-reviews on narrow subjects, but deal with focal areas at the frontier of science and are expected to be of large interest for many researchers working in medicinal chemistry, biochemistry, immunology, biophysics, and pharmacology of molecular drug targets. In presenting the different contributions in the four issues, we combine practical and theoretical aspects, allowing for design, characterization and application of rare molecular events at the many-molecule level (the first two-part special issue) and at the single-molecule level (the second two-part special issue). The second part of the first two-part special issue contains articles focused on nucleic acid and protein analyses in manymolecule systems: The article of J. Zhang and Zichun Hua entitled �Targeted gene silencing by small interfering RNA-based knock-down technology� presents a concise and balanced update of the field for a broad readership with many practical advises for design and applications. Small interfering RNA-based knock-down technology and its applications are a very important new area of research and a promising tool for new therapeutics. Sergey Y. Tetin and Stephen D. Stroupe provide a review on immunoassays and their configurations from classical studies to present entitled "Antibodies in diagnostics applications". Immunoassays made an extraordinary success story in biomedical and clinical analytics. They are among the mostly used analytical tools in chemical, biochemical and immunological analytics. The material is presented by discussing the pros and cons. The article "Combinatorial protein biochemistry for therapeutics and proteomics� of David Lowe and Lutz Jermutus is an overview about applications of in-vitro methods for the display of peptides and proteins that are of therapeutic and diagnostic interests. The authors belong to the leading research groups in this field, and thus give the information from first hand including latest developments. Thomas Bruckdorfer and Oleg Marder provide an overview on �From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future�. Peptides are key to modern drug discovery. The requirements are discussed to produce peptides, initially in millions in mg-scale for research purposes and then for some newly discovered sequences from the basis of modern drugs in multi-tons. The article addresses central prerequisites for success of high-throughput screening. Further it gives access to complex theoretical/mathematical analyses and relationships of multi-step syntheses in solution and on solid support. The review article of Andreas Wiesner entitled �Detection of tumor markers with the ProteinChip technology� provides an overview on tumor markers as diagnostic tools. The early diagnosis of cancer is one of the major challenges in modern, evidence-based medicine. Relevant cases are considered which are of high practical value. Thereby the article deals with up-todate protein mapping platforms for screening hundreds of samples a day. The contribution considers the most relevant literature in the field and provides an introduction into the ProteinChip Biomarker system. Steven Pelech�s paper �Tracking cell signaling protein expression and phosphorylation by innovative proteomic solutions� gives a broad overview that puts current proteomics approaches into perspective. Among the platforms currently used, the article highlights mass spectrometry and the multi-immunoblotting procedure to analyze cell-signaling proteins. Furthermore, the description of the peptide antibody mimetic (PAM) technology with actual working examples represents the first time that this idea with proof of concept has appeared in the scientific literature. PAM technology has the potential to completely replace antibodies in a wide range of applications. Martin Blueggel, Daniel Chanrad, and Helmut E. Meyer contribute the article �Bioinformatics in proteomics". It contains outstanding procedures of molecular sciences. Bioinformatics approaches and platforms, mainly for expression proteomics, are discussed with their pros and cons from mass spectrometric data, peptide mass fingerprinting, peptide fragmentation fingerprinting, peptide de-novo sequencing, protein sequence databases, and proteome databases to gel image analysis. The paper of Wolfgang Domej and Zeno Földes-Papp on �Clinical applicability of mass spectrometry for inhaled carbon compounds and the characterization of trace element patterns in body fluids� presents an overview on application of mass spectroscopy in the medical field with laser activated microprobe mass analysis (LAMMA) and inductively coupled argon plasma mass spectroscopy (ICP-MS). The work is an attempt to review characteristics of carbonaceous particles deposited in lung and of trace element analysis in body fluids. The paper may be of great interest for researchers who are planning to use mass spectroscopy in clinical medicine at the bed side. Katharina Schroecksnadel, B. Frick, B. Wirleitner, C. Winkler, H. Schennach, and Dietmar Fuchs provide the overview �Moderate hyperhomocysteinemia and immune activation" about the extended folate one-carbon metabolism and the folaterelated homocysteine metabolism. The multifactorial biology including immune activation and several disorders are presented. The paper demonstrates that folate supplementation lowers homocysteine concentrations in almost all subjects regardless of the cause. Susanne Brakmann�s article "Optimal enzymes for single-molecule sequencing" is about recent advances in single-molecule sequencing from the viewpoint of biochemistry. The author compares the synthesis of high-density labeled DNA strands and the digestion process of fluorophore-labeled strands with different combinations of enzymes and substrates. How DNA sequencing will work in future and how high-throughput genetic screeing may be performed in gene therapy is a very important research line. The first part of the second two-part special issue contains articles focused on fluorescence correlation spectroscopy (auto- and two-color cross-correlation mode) in single-molecule systems: The paper of Klaus Weisshart, Volker Jurgel and Stephen J. Briddon entitled �The LSM 510 META - ConfoCor 2 system: an integrated imaging and spectroscopic platform for single molecule detection� is a very nice and comprehensive survey of fluorescence correlation spectroscopy (FCS) and the ConfoCor2/LSM-system that is produced by the Zeiss company. The authors also deal with the basic theory of fluctuation analysis, calibration analysis and evaluation of FCS data, and describe technical advances and new improvements in the software for FCS and two-color fluorescence cross-correlation spectroscopy (FCCS) methods. FCS in auto- and two-color cross-correlation mode will be a powerful tool for in-vivo pharmacological investigations in near future. Jörg Enderlein, Ingo Gregor and Jörg Fitter present a balanced overview of some artifacts in fluorescence correlation spectroscopy measurements. Their article �Art and Artefacts of Fluorescence Correlation Spectroscopy� discusses optical and photophysical effects in the correlation function like optical saturation and triplet-states with fast regions. The article of Zeno Földes-Papp and Gerd Baumann entitled �Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics� is an exposition of ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- and two-color cross-Correlation Spectroscopy (SPSMFCS). The �algorithms� developed are called �the selfsame single-fluorescent-molecule regime� and permit the continuous measurement of one and the same single, individual molecule in solution or a membrane by fluorescence detection techniques from sub-milliseconds up to several hours without hydrodynamic flow, immobilization, or arrival time histograms. All time time-dependent processes that involve a sufficiently small number of molecules in the bulk phase like solution or membrane can be treated by the stochastic formulism. Antonie J.W.G.Visser, Beno H. Kunst, Hans Keller and Arjen Schots provide the mini-review �Towards sorting of biolibraries using single-molecule fluorescence detection techniques�. They discuss efforts and achievements in the generation of biolibraries, single-molecule fluorescence detection techniques, and microfluidic devices. The authors come up with the widely accepted standpoint that a combination of the disciplines is necessary for the sorting and selection of large biolibraries. The review of T. Wakatsuki, J.A. Fee and Elliot L. Elson on "Phenotypic screening for pharmaceuticals using tissue constructs" covers a hot and controversial topic in drug discovery screening: Should primary screening be performed in a molecular or cellular environment? The method presented in this article describes a very interesting approach for screening in the cellular / tissue environment. The article of Holger Winter, Kerstin Korn and Rudolf Rigler entitled �Direct gene expression analysis" presents a recently developed approach for two-color hybridization-based gene expression analysis avoiding amplification of the target or the signal. The authors utilize two-color fluorescence cross-correlation spectroscopy �at the single-molecule level� as singlemolecule fluorescence detection technique. Takuya Takagi, Hiroaki Kii and Masataka Kinjo provide the article �DNA measurements by using fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy�. It deals with an important aspect in two-color fluorescence cross-correlation measurements by presenting experimental results on diffusion times of long dsDNAs in the Kbp range. The long model DNAs contained fluorophores at different distances. Measured two-color cross-correlated diffusion times consisted of the �real diffusion time� and the �delay� between the red and green signals. Aladdin Pramanik�s contribution "Ligand-receptor interactions in live cells by fluorescence correlation spectroscopy� refers to original research papers of the author on C-peptide, EGF, and GAL. He also provides technical details of these methods at single-molecule detection sensitivity. The review of Anja Nohe and Nils O. Petersen on "Analyzing for co-localization of proteins at a cell membrane� presents techniques for determining how intramolecular interactions in cell membranes control the signal transduction process. First, the reader is given a short introduction including orientation on the present view of structure, organization and dynamics of cell membranes, their proteins, and their physiological role. Next, there is a survey of approaches to make binding and colocalization evident. Finally the article focuses on image cross-correlation spectroscopy as a relatively new quantitative tool developed by the authors. Michael Jahnz and Petra Schwille provide an overview on "Enzyme assays for confocal single molecule spectroscopy". The paper deals with the analysis of enzymatic reactions in solution using optical confocal techniques such as FCS, FCCS and FRET �at the single-molecule level�. The authors describe various types of FCS and FCCS methods based on principles, algorithms and data processing. The authors also mention strategic pathways of fluorophore labeling and conjugating methods for these approaches. Labeling probes and methods are of importance for fluorescent measurements in biological systems and samples. The contribution of Hans Blom and Michael Gösch focuses on "Parallel confocal detection of single biomolecules using diffractive optics and integrated detector units�. The authors present recent results of implementing parallel laser excitation and fluorescence detection for high-throughput miniaturized array-based analytical systems. They also report a multiplexing extension that permits multifocal laser excitation together with matched parallel fluorescence detection. The second part of the second two-part special issue contains articles focused on single molecules and should be of interest to anyone interested in applying single-molecule analyses. The article by Karl-Otto Greulich entitled "Single molecule techniques for biomedicine and pharmacology" is a comprehensive survey of single-molecule studies with relevance to applications for the biomedical and pharmaceutical sectors. First, the reader is given a general introduction to the materials and methods of singlemolecule spectroscopy. Next, the results section presents and discusses, in more detail, several interesting and important applications of single-molecule detection in molecular biology and cellular physiology. The article focuses on topics where the author has extensive first-hand expertise. H. Peter Lu�s article on "Single-molecule spectroscopy studies of conformational change dynamics in enzymatic reactions" deals with interesting data on the T4 lysozyme hydrolyzation reaction of a polysaccharide. The spectroscopic and theoretical results presented form a collection of the author's original work. The article by Peter M. Goodwin, Rhiannon L. Nolan and Hong Cai on "Single-molecule spectroscopy for nucleic acid analysis: a new approach for disease detection and genomic analysis" is a survey of single-molecule detection and identification for genomic analyses. The authors are among the pioneers in this field of research. With "Applications of single-molecule detection to the analysis of pathogenic DNA" Oana Marina and Alonso Castro provide an original and effective application of single-molecule fluorescence detection for the analysis of pathogenic DNA, particularly for ultrasensitive pathogen detection. This method is one of the rare cases were single-molecule detection is applied to an important bio-analytical task, the detection of small amounts of pathogenic DNA in a large sample. H. Neuweiler and Markus Sauer survey their recent work on "Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level". It deals with charge-transfer probes for monitoring and studying the conformational dynamics of peptides and DNA, as well as the application of these methods to the single-molecule sensitive detection of target molecules. The measurement techniques presented (direct inter-photon distance timing), novel data analysis, as well as the newly designed charge�transfer probes are a new and promising branch in single-molecule fluorescence spectroscopy. The work of Michael Wahl, F. Koberling, M. Patting, H. Rahm, and R. Erdmann on "Time-resolved confocal fluorescence imaging and spectroscopy system with single-molecule sensitivity and sub-micrometer resolution� describes the authors� recently developed confocal laser-scanning microscope. The paper presents a thorough introduction into the system and its performance in various applications, including lifetime imaging of surfaceadsorbed molecules and of live cells, or fluorescence-fluctuation and FRET measurements in solution. It should be of great interest for researchers who intend to start working in single-molecule spectroscopy and its applications. A survey by Jan Hesse, Max Sonnleitner, and Gerhard J. Schutz on "Ultra-sensitive fluorescence reader for bioanalysis" covers the group�s recent development and application of a new single-molecule sensitive highthroughput plate reader. This is the first attempt to apply a single-molecule sensitive imaging technology to the screening of large sample areas for bio-analytic and bio-diagnostic purposes. Of special interest is the application for the single-molecule sensitive screening of live cells on a surface. Zeno Foldes-Papp (Responsible Editor) Graz University Medical School and Hospital A-8036 Graz-LKH, Austria

Abstract: In the accompanying original article, the universal theoretical and experimental framework was developed for quantifying one and the same single (selfsame), individual fluorescent-tagged biological molecule without immobilization, hydrodynamic flow or photon burst analysis of fluorescence intensity traces. In the present original article, we describe an application to the detection and identification of circulating anti-glomerular basement membrane antibodies (BMAs) in Goodpasture syndrome. The same single, individual two-color molecule complex was observed among many other molecules. The molecule consisted of the green-tagged antigen, sandwiched autoantibody and red-tagged secondary (detecting) antibody. A 200-fold increase in sensitivity was obtained as compared to the conventional ELISAs on solid phase. This novel concept has several advantages, namely (i) the sensitivity to detect an individual molecule in solution; (ii) the association of the signal with the reaction event, independent of any immobilization procedure and the artifacts thereof; (iii) the assessment of the broad field of natural antibodies. The theoretical and experimental results obtained bring advanced ultrasensitive analytics to the direct investigation of one and the same single, individual immune molecules as exemplified by the experiments performed with Goodpasture antibody. The novel universal theoretical and experimental framework for continuous measuring the same single, individual immune molecule can be readily transferred to other applications.

Abstract: DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5' ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.

Abstract:

Abstract: Degradation of double-stranded DNA by Exonuclease III in the presence of complementary anti-parallel PNA was studied. It was found for the first time that the PNA suppresses the degradation of dsDNA in a sequence-specific manner as well as inhibits the activity of Exonuclease III in a non-specific way.

Abstract: BACKGROUND: Cigarette smoke is a major anthropogenic pollutant and contributes to the permanent load of ambient particulate matter in the air, particularly indoors. It is the leading risk factor for premature loss of life due to chronic bronchitis, emphysema and lung cancer. Smoker's lung and graphite pneumoconiosis are pathological states characterized by the deposition of carbonaceous particles. METHODS: Mass spectrometry was used to evaluate unstained lung sections obtained in vivo from a heavy smoker and a patient with occupationally acquired graphite pneumoconiosis. RESULTS AND DISCUSSION: The composition of carbon compounds deposited in lung tissue samples is demonstrated here for the first time. Thirty carbonaceous-containing microareas from ten biopsies (three areas per biopsy) of lung tissues were analyzed mass-spectrometrically. In each case, the samples were taken from a smoker's lung or those demonstrating a graphite pneumoconiosis. The lung-tissue samples were selected by light microscopy before they were evaporated for mass spectrometry. First-order criteria were anionic and cationic mass peaks which occur within the mass patterns in lung tissues of smoker's lung, although not in graphite pneumoconiosis. Second-order criteria were mass peaks from smoker's lung with standard deviations SD < or = 14% of the mean value. First and second-order mass peaks matched the mass peaks of experimental cigarette-smoke condensate in 9 out of 11 peaks. A software program was developed that enabled fast, automated recognition of the typical mass peaks, and thereby confirmed the histological diagnosis of smoker's lung. CONCLUSIONS: The analysis of carbonaceous particles within lung biopsies from a heavy smoker corresponded to the spectra of tobacco condensate and not to the investigated biopsies of graphite peneumoconiosis. RECOMMENDATION AND OUTLOOK: The analyses were performed in order to find out whether mass-spectrometric criteria exist for the differentiation of carbonaceous lung-tissue deposits. Mass spectrometry may be a valuable tool in determining the composition of carbon compounds deposited in human lung tissue. So far, qualitative assessment of the composition of deposits in lung tissue is only possible after the patient is deceased (autopsy).

Abstract: Whereas the majority of microarray applications still deal with expression analysis for gathering information about levels of gene products at certain cell states, other approaches simply ask the question whether particular genes, which are usually indicative for particular microorganisms and pathogens, are present in a sample or not. Investigations that are more detailed try to evaluate the presence of particular subtypes of a given pathogen. The combination of microarray technology and virus diagnostics promises to generate an ideal platform for fast, sensitive, specific, and parallelized virus diagnostics. Performing virus diagnostics on microarrays, however, requires other basic techniques to be optimized. This is necessary in order to obtain unambiguous and reproducible results, which are compatible with the needs for clinical routine. Parameters that have to be considered include supports, coupling chemistry, chemical oligonucleotide synthesis, signal enhancement strategies, and optimal coordination of PCR reactions, hybridizations, and signal detection, as well as interpretation strategies. Finally, considerations should be given to economic aspects, one chip-one patient strategies and low integrated arrays as a custom-tailored way to fast and accurate diagnostic tools.

Abstract: BACKGROUND: A methylene tetrahydrofolate reductase (MTHFR) deficiency at site C677T renders the enzyme thermolabile and consequently represents a risk factor for vascular disease, neural tube defects, preeclampsia, and thrombosis. Highly specific identification techniques for genotyping are mandatory to give guidance for the diagnosis and monitoring of this deficiency. METHODS: A new approach for performing genotyping has been introduced with the identification of single nucleotide polymorphisms of the human MTHFR. It is based on PCR followed by two-color cross-correlation fluorescence spectroscopy (FCS). Experiments were carried out with green- and red-tagged allele-specific primers, which were fully compatible with the two-color fluorescence cross-correlation setup at 488 nm and 633 nm excitation wavelengths. RESULTS: The measured data of the amplification mixes (tubes) were normalized as the maximum correlation amplitude of each tube. Correlated and uncorrelated data were optically separated in the amplification mixes by their characteristic correlation times, which significantly differed from each other. The correlated data were generated in the presence of the proper mutated genotype template, whereas uncorrelated data were due to the absence of the proper genotype template. Furthermore, the specific association of the two-color fluorescence correlated signals with the target DNA was experimentally proven. Using this novel two-color cross-correlation approach, the MTHFR genotypes, which were determined in 21 clinical samples, showed concordance with methods involving a PCR-based assay with hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxy-fluorescein (FAM)-tagged allele-specific primers and a subsequent separation step with capillary electrophoresis, yet are simpler to perform. There was no evidence of a central trend of false-positive or false-negative results. We demonstrated how the novel, ultrasensitive typing system could be applied to studies where researchers are trying to perfect their assays and are often working with the unknown, or application to problematic assays in a clinical environment for those involved in molecular diagnosis. CONCLUSIONS: We present an alternative method to those commonly used in genotyping. Two-color cross-correlation FCS allows the detection of the fluorescence signals specifically associated with the heterozygous mutated, the homozygous mutated, and normal individuals, as exemplified in this study. The presence of nonspecific amplification products, which interfere with subsequent DNA analysis, could therefore highlight the need for two-color cross-correlation FCS as a means of discriminating between specific association of the fluorescence signals with the target DNA and DNA not related to the target.

Abstract: Foreword When we started preparing the edition of the two theme issues � each divided in two parts (i.e., four issues in total) � we felt that the time had come to present and discuss recent achievements in biotechnology and bioanalysis that are creating a new array of opportunities for the pharmaceutical and biomedical sectors. The inter-disciplinary topics presented in these four issues stem from scientists in biology, chemistry, and biochemistry as well as molecular biology, immunology, physics, mathematics, engineering, computation and medicine. After studying the cell and its constituents and working the way down to the genome and the proteome, huge amount of data have been generated during the last decade and can now be deciphered to shed light on the basic principles of life. Among the new technologies that are making this progress possible, the DNA chip is only one example that has helped address many molecular biology problems, such as rapid identification of genetic disorders. The foundations of microarray and biochip technologies are firmly rooted in the rapid advances in nucleic acid chemistry and biochemistry. The overwhelming majority of the early-phase trials using these developments was not primarily aimed at clinical efficacy but instead at assessing the biological problems to be solved before genomics, epigenomics and proteomics can deliver useful information for clinical applications. In an attempt to better understand these problems, investigators returned to basic studies of gene expression, immunology and virology. Finally, the cloning and sequencing of the human genome has significantly expanded the range of actual and potential molecular drug targets. The discovery and optimization of molecular drug targets, e.g., disease specific proteins, receptors, enzymes, or genes, is a time-consuming and extremely expensive process. Therefore, in today's preclinical drug research, there is only one key to success: scientific teamwork and the use of the most advanced biotechnological approaches. The advances in biotechnology directly affect improvements in human health and outcomes. That especially holds true in our time of rapid changes. The title of the four special issues implies that their scope encompasses the fundamentals of the way down from single genes and proteins to single molecules. Should we have started this Edition from the top downward, beginning with medicine and clinical practice based on empirical ("complex") thinking, where the risk exists of serious error by failing to account accurately for gene or protein interactions? Alternatively, should we proceed from the bottom upward beginning with single molecules and their behavior, which often seems to be different from that of the ensemble in biological systems? The answer is probably a compromise, working in both directions from the middle. The contributions to these four Special Issues of Current Pharmaceutical Biotechnology are written by leading specialists in fields that cover a wide range of current topics on biotechnological and biomedical aspects of drug target screening and characterization. Several articles present overviews on the latest developments and future prospects. They are devoted to discussing multidisciplinary topics at a level that will be useful to the practicing scientist. The contributions are not simply reviews and mini-reviews on narrow subjects, but deal with focal areas at the frontier of science and are expected to be of large interest for many researchers working in medicinal chemistry, biochemistry, immunology, biophysics, and pharmacology of molecular drug targets. In presenting the different contributions in the four issues, we combine practical and theoretical aspects, allowing for design, characterization and application of rare molecular events at the many-molecule level (the first two-part special issue) and at the single-molecule level (the second two-part special issue). The first part of the first two-part special issue contains articles mainly focused on nucleic acid analyses in many-molecule systems: Margret R. Hoehe, Bernd Timmermann, and Hans Lehrach provide an extensive survey on �Human inter-individual DNA sequence variation in candidate genes, drug targets, the importance of haplotypes and pharmacogenomics�. The review deals with the integration of global information on DNA, mRNA, and protein to identify �the� gene most probably underlying disease and indicates the end of Mendel�s world of the two-allele concept of �the� gene. A single SNP (Single Nucleotide Polymorphisms) reflecting an early stage of approximation to completeness represents no longer a marker of a disease in this concept. The paper of Hartmut Seliger, Michael Hinz, and Erwin Happ is devoted to a critical discussion of contributions to nucleic acids technology for arrays of immobilized oligonucleotides. Main problems of state-of-the-art developments are addressed from a chemical point of view. The review is comprehensive and extensively covers the literature in this field. Xiao Zeng�s article �The making of a portrait � bringing it into focus� is a succinct overview of the current developments of DNA arrays in biomedical research fields with an emphasis on the pathway-specific array design. Hans-Martin Striebel, Eckhard Birch-Hirschfeld, Renate Egerer, and Zeno Földes-Papp provide an overview on �Virus Diagnostics on Microarrays". It combines microarray technology and virus diagnostics from classical study to present. The authors also mention strategic pathway of the coupling chemistry, chemical synthesis of oligonucleotides and DNA, fluorophore labeling and hybridization methods for microchip technology. Sensitivity and reproducibility are dependent on these parameters and they are important for routine diagnostics. The article of Irina M. Gana Dresen, Johannes Hüsing, E. Kruse, T. Boes, and K.-H. Jöckel "Software packages for quantitative microarray-based gene expressions analysis" presents three software packages that can be used for the analysis of microarray-based gene expressions. It starts with the technical description of the programs, including hardware and software requirements, handbook quality, and finally compares the performance of the three programs. Gwendolyn A. Lawrie, Bronwyn J. Battersby, Lisbeth Grondahl, and Matt Trau provide an comprehensive summary on the various techniques of labeling large compound libraries, and then focus on the technique of using colloids for encoding purposes. Encoding is a topic of enormous significance when synthesizing and screening large libraries in genomics, proteomics and drug discovery. The contribution of Lloyd M. Davis, Peter E. Williams, David A. Ball, Kerry M. Swift, and Edmund D. Matayoshi on �Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery" summarizes the work of the authors about numerically simulating fluorescence correlation spectroscopy (FCS) experiments, and it presents a biologically interesting application of FCS. Stefan Jäger, Leif Brand, and Christian Eggeling extend the contents of the first issue to an extensive overview on the new techniques of fluorescence fluctuation spectroscopy such as fluorescence-intensity distribution analysis (FIDA), fluorescenceintensity multiple distribution analysis (FIMDA), fluorescence-intensity and lifetime distribution analysis (FILDA), and also the more standard fluorescence-correlation spectroscopy (FCS) in high-throughput analysis. Kunio Hori, W.S. Shin, C. Hemmi, T. Toyo-oka, and T. Makino demonstrate the analysis of single point mutation in a mitochondrial gene, i.e., SNP analysis, by using fluorescence correlation spectroscopy (FCS) at the very low concentration of products. The authors also describe two types of methods for FCS analysis based on primer extension in DNA polymerase reaction. Graz, October 2003 Zeno Foldes-Papp (Responsible Editor) Graz University Medical School and Hospital A-8036 Graz-LKH Austria

Abstract: BACKGROUND: Eosinophils contain granule proteins such as eosinophil cationic protein (ECP) that have proinflammatory effects on airways. ECP may be released on activation of eosinophils into the plasma and is widely used as a marker of bronchial hyperreactivity and allergic inflammation. Environmental factors as well as intense physical exertion may influence eosinophil-related bronchial hyperreactivity. STUDY OBJECTIVES: To investigate the effect of endurance exercise at moderate altitude on levels of circulating eosinophils, serum ECP, serum osmolality (sOS), and dynamic pulmonary function parameters in healthy mountaineers. SETTING: Alpine field study performed in the Alps of Upper Styria in Austria. Type of exercise: Ascent of a mountain at maximal speed. PARTICIPANTS: Thirty healthy male volunteers from a troop of military mountaineers. RESULTS: Mean ECP concentration increased by 66% at the summit checkpoint (H2) and remained at 63% above baseline (base checkpoint [H0]) after descent (H4), while the blood eosinophil count decreased concomitantly from 250/microL at H0 (preexercise) to 118/microL (53%) at H2 and to 22/microL (81%) at H4. The total serum ECP concentration adjusted to sOS correlated negatively with blood eosinophil count (r = - 0.37; p < 0.0001) and PaO(2) (r = - 0.34; p < 0.001), but positively with the peak expiratory flow (PEF) [r = 0.45; p < 0.0001]. Although sOS correlated with serum ECP at H2 (r = 0.47; p = 0.02) and at 12 h after the start of the experiment (H12) [r = 0.57; p = 0.003], the relationship between total ECP and sOS (r = 0.19; p = 0.034) was less pronounced. FEV(1) in percentage of FVC (%FEV(1)/FVC) [the Tiffenau test], forced expiratory flow rate at 25% of vital capacity, and PEF were significantly higher at H2 than at H0 and H4. %FEV(1)/FVC decreased to 88% (p < 0.01) and 83% (p < 0.001) predicted at H12 and 24 h after start of the experiment, respectively. CONCLUSION: Results provide strong evidence for nonspecific activation of blood eosinophils during prolonged intense aerobic exercise at moderate altitude, modifying both eosinophil dynamics and regulation of ECP release in healthy subjects.

Abstract: Interaction of a single-chain antibody fragment (scFv) with its cognate antigen while still attached to the ribosome was studied by fluorescence correlation spectroscopy (FCS). In experiments with purified scFv, FCS was capable of resolving the difference in diffusion time between free and antibody-bound labelled antigen. Ribosome-displayed antibody fragments generated by in vitro translation, in which neither the protein nor the mRNA leaves the ribosome owing to the absence of a stop codon and stabilizing buffer conditions, could be shown to specifically bind the antigen. The antibody-antigen interaction was specific, as shown by inhibition or displacement with unlabelled antigen and by control experiments with a non-cognate antibody fragment.

Abstract: Biopsy is onerous and, for this reason, immunodiagnostics in sera of celiac disease patients are an "additional diagnostic standard." The objective of the study was to investigate the variability in diagnostic specificity of ELISAs for the detection of IgA anti-tissue transglutaminase antibodies in serum of celiac disease patients who underwent biopsy. All patients were included in the study on the basis that they had a small intestinal biopsy. We studied 18 patients with histological proven celiac disease (7 male, 11 female, mean age +/- SD: 35+/-19 years) from Graz, Austria. Healthy control subjects were also entered into the study. The determinations of the anti-tissue transglutaminase antibodies were simultaneously performed together with the endomysium and gliadin antibody markers. We analysed the 216 serum values according to Cochran's non-parametric Q-test. The complexity to the analysis reflects the complexity of the diagnostic situation with the patients. No real differences were found in the reactions of the anti-human IgA-type anti-tissue transglutaminase ELISAs. Based on these results, an association was established between the outcomes of anti-human IgA-type ELISAs for the specific antigen and patients with histologically proven celiac disease, treated for celiac disease after histology was carried out and the diagnosis was made, and healthy controls. The detection of IgA anti-tissue transglutaminase antibodies in serum is a promising alternative to the indirect immunofluorescence determination of IgA-type endomysium antibodies. One ELISA for the specific antigen showed some advantage with respect to its extended scale of detection. Immunopathology of celiac disease can be based on the results of the appropriate IgA anti-tissue transglutaminase ELISAs under uncomplicated gastrointestinal conditions.

Abstract: This study first indicates that the serum trace element Zn tends to decrease in the course of sequential thoracenteses. Other selected essential elements such as copper (Cu), manganese (Mn), molybdenum (Mo), and cobalt (Co) do not reveal loss changes in their serum levels. Therefore, Zn should be monitored in patients who undergo repeated thoracentesis. To measure the magnitude of changes in serum trace elements and the clinical relevance of potential imbalances, concentrations of the essential elements are analyzed in 57 serum/effusion pairs obtained from 5 patients (4 male, 1 female; age 28-78 yr) who underwent repeated thoracenteses as a result of recurrent pleural effusion. All patients declined other therapeutic options such as chemical pleurodesis and/or chest tube placement. The total volumes of fluid removed ranged from 2.3 to 19.3 L and the frequency of thoracentesis ranged from 6 to 15 within a period of 102-174 days. Two patients had benign pleural disease and three had malignancies. Three patients suffered from pleural effusions resulting from exudates (total protein content > 3.0 g/dL, LDH > 200 U/L), and two resulting from transudates (total protein < 3.0 g/dL, LDH < 200 U/L). All trace elements were simultaneously determined by inductively coupled argon plasma-mass spectrometry. In addition, the concentrations of the following clinically relevant parameters were analyzed by standard methods: total protein, pH, leukocyte count, lactate dehydrogenase, and glucose.

Abstract: Inhalation of dust containing graphite can cause lung disease in foundry workers and workers in graphite mines or mills. Mixed dust pneumoconiosis caused by long-term occupational exposure to graphite dust is a rare disease. Only a few cases of graphite pneumoconiosis have been reported in literature, and these were usually diagnosed post mortem. Our report is of an 80-year-old male patient who had worked in an iron foundry for 20 years and whose work had entailed regular contact with ground graphite and foundry vapors. Chest x-rays revealed both a reticular and nodular pattern in the lung, moderate apical distractions and pleural scarring, all of which were confirmed by high-resolution computed tomography. Bronchoalveolar lavage and transbronchial biopsies were also consistent with mixed dust pneumoconiosis, and due to the long-term dust exposure, graphite pneumoconiosis was strongly suspected. To confirm this diagnosis, the chemical composition of the dark granules in the semi-thin histological sections of the transbronchial biopsies were analyzed using laser microprobe mass spectroscopy. The mass spectra of these black particles were consistent with those of natural graphite powder. Comparative analyses of normal lung tissue did not produce similar spectral patterns. We conclude that histology and cytology does not always suffice to confirm a diagnosis of graphite pneumoconiosis, because black particles are also found in conditions resulting from other exposures, such as heavy smoking or coal mining. Analysis of the composition of particles deposited in the lung tissue offers more precise information, which can be used as evidence in occupational and forensic medicine. Laser microprobe mass spectroscopy can assess the mineral dust load in lung samples.

Abstract: There has been considerable recent interest in the potential use of serum cystatin C as a diagnostic tool. Here we examined the hypothesis that the cystatin C level in the pleural effusion can differ from the corresponding serum level. We evacuated pleural effusion fluids from 47 patients by thoracentesis. Cystatin C, beta(2)-microglobulin, inorganic phosphate, creatinine and total protein were quantified in both pleural effusion fluids and corresponding sera. We determined cystatin C levels in pleural effusions and calculated the ratio of cystatin C levels in serum and effusion, to discriminate between effusions caused by severe renal impairment and other types of effusion. Extremely high concentrations of cystatin C in serum/effusion pairs were only measured in patients with renal failure (6.0 +/- 0.8/6.0 +/- 0.8 mg/l, means +/- S.D., n=11). A clearly defined region was found to correspond to pleural effusion caused by renal failure (r=0.954). The quantification of cystatin C in the effusion was justified by the discovery that there were some patients with a high serum cystatin C level but a low effusion concentration, or a low serum cystatin C but a high effusion concentration, indicating causes other than renal failure. In conclusion, the pilot data indicate a relationship between the cystatin C concentration in pleural fluid and the underlying disease. Thus cystatin C levels in pleural effusion and serum may be a valuable criterion for the differential diagnosis of pleural diseases of different aetiologies.

Abstract: More sensitive techniques in molecular and clinical immunology are essential for the development of reproducible profiles. We have developed a novel methodology named solution-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) that fulfils these demands. It is based on the quantification of the probability density of single molecule events in solution is necessary. For example, the Brownian motion of the fluorophore rhodamine-green is detected. Counting about 100,000 photon counts per second and per molecule permits the identification of one single compound. In order to study the applicability of SPSM-FCS in immunology, we have detected and identified a larger nonfluorescent substance in a very complex mixture. The 'unknown' molecules studied were the autoantibodies in serum samples directed against the antigen alpha 3 chain of type IV collagen. In both systems, we were able to characterize the probability density of single fluorescent molecules by means of the averaged absolute molecule number without any calibration. The specific molecules exhibited a Poisson distribution in solution in terms of their 'critical' bulk concentration below about 1 nM. This proof of principle indicates how the SPSM-FCS methodology could be used in immunoassays.

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Abstract: We report on the spectroscopy and enzymatic activity of horseradish peroxidase (HRP) coupled to Ag nanoparticles. We show that vibrational spectra of single HRP molecules can be detected by measuring surface-enhanced Raman scattering (SERS) from isolated and immobilized protein�nanoparticle aggregates. However, the nanoparticle�protein interaction results in decreased enzymatic activity, as shown by an ensemble-averaged HRP activity assay. We demonstrate that H2O2, one of the enzyme substrates in the assay, reacts with the Ag surface and that it is necessary to pretreat the Ag particles with H2O2 to retain full HRP activity in the Ag sol. However, the concomitant changes in the Ag surface adsorbate layer caused by this treatment prevent strong HRP adsorption and therefore also single-molecule SERS sensitivity. Layers of H2O2-treated Ag particles can instead be used to enhance the fluorescence signal from single functional HRP molecules.

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Abstract: Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.

Abstract: We present results of an approach in which low-density labeled DNA itself provides an amplification of the cross-correlated fluorescent signal in the two-color cross-correlation function. Tetramethylrhodamine-4-dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp target DNA. We call this novel approach the 'two-color FCS signal amplification'. The signal amplification is an example for interactions of two ligands with different colors at multiple positions of the same target.

Abstract: An experimental application of fluorescence correlation spectroscopy is presented for the detection and identification of fluorophores and auto-Abs in solution. The recording time is between 2 and 60 sec. Because the actual number of molecules in the unit volume (confocal detection volume of about 1 fl) is integer or zero, the fluorescence generated by the molecules is discontinuous when single-molecule sensitivity is achieved. We first show that the observable probability, N, to find a single fluorescent molecule in the very tiny space element of the unit volume is Poisson-distributed below a critical bulk concentration c*. The measured probability means we have traced, for example, 5 x 10(10) fluorophore molecules per ml of bulk solution. The probability is related to the average frequency, C, that the volume of detection contains a single fluorescent molecule and to the concentration, c, of the bulk solution. The analytical sensitivity of an assay is calculated from the average frequency C. In the Goodpasture experiment, we determined as analytical sensitivity a probability of 99.1% of identifying one single immune complex. Under these conditions, a single molecule event is proven. There exist no instrumental assumptions of our approach on which the experiment itself, the theoretical background, or the conclusion are based. Our results open up a broad field for analytics and diagnostics in solution, especially in immunology.

Abstract: In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

Abstract: The enzymatic incorporation of deoxyribonucleoside triphosphates by a thermostable, 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase was studied for PCR-based high-density labeling of 217-bp "natural" DNA in which fluorescent-dUTP was substituted completely for the normal dTTP. The amplified DNA carried two different sorts of tethered dye molecules. The rhodamine-green was used for internal tagging of the DNA. Since high-density incorporation of rhodamine-green-X-dUTP led to a substantial reduction (quenching) of the rhodamine-green fluorescence, a second "high" quantum yield label, Cy5, was inserted via a 5'-tagged primer in order to identify the two-color product. A theoretical concept of fluorescence auto- and cross-correlation spectroscopy developed here was applied to quantify the DNA sequence formed in terms of both the number of two-color fluorescent molecules and the number of covalently incorporated rhodamine-green-X-dUMP residues. The novel approach allowed to separate optically the specific DNA product. After complete, exonucleolytic degradation of the two-color DNA we determined 82-88 fluorescent U* labels incorporated covalently out of 92 maximum possible U* incorporations. The heavily green-labeled DNA was then isolated by preparative mobility-shift electrophoresis, re-amplified in a subsequent PCR with normal deoxyribonucleoside triphosphates, and re-sequenced. By means of this novel methodology for analyzing base-specific incorporations that was first developed here, we found that all fluorescent nucleotides and the normal nucleotides were incorporated at the correct positions. The determined labeling efficiency of 0.89-0.96 indicated that a fraction of the substrate analog was not bearing the fluorophore. The results were used to guide developments in single-molecule DNA sequencing. The labeling strategy (principal approach) for PCR-based high-density tagging of DNA, which included an appropriate thermostable DNA polymerase and a suitable fluorescent dye-dNTP, was developed here.

Abstract: We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent dyes. Firstly, for "natural" template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucleolytic degradation by the 3'-->5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s(-1) at 16 degrees C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently incorporated into the antisense strand of "synthetic" 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occurring dNTPs. The 218-b DNA constructs were labeled by PCR with a thermostable 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase which we have selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodology of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the paper Földes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing project.

Abstract: We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.

Abstract: We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.

Abstract: The excess of structural degrees of freedom in a protein enzyme opens questions about the conformational homogeneity. We studied single horseradish peroxidase enzyme turnovers by fluorescence spectroscopy. Application of a two-state dynamic model to the measured data shows exponential product dissociation kinetics, but a large distribution of rates for the enzyme to form the enzyme-product complex. The experiments show that in addition to the peroxidative cycle thermodynamic fluctuation phenomena on a wide range of time scales affect enzyme activity.

Abstract: We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.

Abstract: In this article we present a new concept for the detection of any specifically amplified target DNA sequences in multiple polymerase chain reactions (PCR) based on fluorescence correlation spectroscopy (FCS). The accumulation of double-stranded target DNA is monitored by the cross-correlated fluorescence signals provided by two amplification primers which are 5'-tagged with two different kinds of fluorophores (Rhodamine-Green and Cy5). Only the amplified target DNA sequence carrying both primers is observed. Its signal emerges from the background of non-incorporated or non-specifically incorporated primers. Down to 10-25 initial copy numbers of the template in the PCR compartment DNA can presently be detected. No external or internal standards are required for determining the size and the amplified copy number of specific DNA. The PCR amplification process is started with all ingredients in a single compartment (e.g. of a microtiter plate), in which amplification and measurement are performed. This eliminates the need for post-PCR purification steps. The homogeneous one-tube approach does not depend on fluorescence energy transfer between the fluorogenic dyes. Thus, it does not interfere with the enzymatic amplification reaction of PCR and allows the continued use of different conditions for amplifying DNA. The results exemplified by PCR-amplified 217-bp and 389-bp target DNA sequences demonstrate that the analysis based on two-color fluorescence cross-correlation is a powerful method for simplifying the identification of targets in PCR for medical use. For this purpose, an instrument optimized for two-color excitation and detection of two-color emission has been developed, incorporating the principle of confocal arrangement.

Abstract: In this paper we put forward improved mathematical methods for detecting synthesis parameters in connection with analyzing crude products of chemically synthesized oligonucleotides. The crude products experimentally sampled are separated by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography. The measured separation profiles of experimental syntheses can be expressed as target and nontarget yields; they are characterized by a few parameters. These parameters account for nonlinear synthesis equations that are solvable by employing iteration procedures. We provide here a theoretical as well as computational analysis based upon specific models for stepwise chain growth. Under nonconstant (nonuniform) conditions we use here an exponential form of growth, with different expressions for calculating the fractal dimension of the biochemical process under study. Step lengths of parameter variations in an interval of finite length have to be adjusted properly to find convergent solutions in a mathematical, regularly four-dimensional parameter space. It is conceivable to have most, if not all, of the calculating and plotting carefully done by a computer. This analysis represents the experimental situation up to 65-mer target oligonucleotides analyzed so far. We thus obtain the dynamics of the polymerization process limited in number by fractal models. The advantage, calculating these new methods as compared to qualitatively judged experimental methods, lies in the satisfactory evaluation of crude products, also of large amounts, of syntheses of these biopolymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 361-379, 1998

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Abstract: We have developed models of patterns for nucleotide chain growth. These patterns are measurable by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography in crude products of solid-phase synthesized 30mer and 65mer oligodeoxyribonucleotide target sequences N. We introduce mathematical methods for finding characteristic values d(o) and p(o) for constant chemical modes of growth as well as d and p for non-constant chemical modes of growth (d = probability of propagation, p = probability of termination). These methods are employed by presenting the accompanying computer software developed by us in C code, Mathematica R languages, and Fortran. Characteristic values of the parameters d, p, and the target nucleotide length N describe the complete composition of the crude product. From this we have developed the relation 2 - [N/(N - 1)]/Da, measurable(N,d) as a universal quantitative measure for multicyclic synthesis conditions (D, fractal dimension and similarity exponent, respectively). We use this mathematical treatment to compare the efficiency of oligodeoxyribonucleotide syntheses of different target length N on polymer support materials. Further, we analyze selected syntheses of short and long oligodeoxyribonucleotides as well as single-stranded DNA sequences by well-known empirical autocorrelation, fast Fourier transformation, and embedding dimension techniques.

Abstract: Oligonucleotides are becoming more and more important in molecular biomedicine; for example, they are used as defined primers in polymerase chain reaction and as antisense oligonucleotides in gene therapy. In this paper, we model the dynamics of polymer-supported oligonucleotide synthesis to an inverse power law of driven multi-cycle synthesis on fixed starting sites. The mathematical model is employed by presenting the accompanying view of error sequences dynamics. This model is a practical one, and is applicable beyond oligonucleotide synthesis to dynamics of biological diversity. Computer simulations show that the polymer support synthesis of oligonucleotides and single-stranded DNA sequences in iterated cyclic format can be assumed as scale-invariant. This synthesis is quantitatively described by nonlinear equations. From these the fractal dimension Da (N,d) is derived as the growth term (N = number of target nucleotides, d = coupling probability function). Da(N,d) is directly measurable from oligonucleotide yields via high-performance liquid chromatography or capillary electrophoresis, and quantitative gel electrophoresis. Different oligonucleotide syntheses, including those with large-scale products can be directly compared with regard to error sequences dynamics. In addition, for short sequences the fractal dimension Da (N,d) is characteristic for the efficiency with which a polymer support of a given load allows oligonucleotide chain growth. We analyze the results of separations of crude oligonucleotide product from the synthesis of a 30 mer. Preliminary analysis of a 238 mer single-stranded DNA sequence is consistent with a simulated estimate of crude synthesis product, although the target sequence itself is not detectable. We characterize the oligonucleotide support syntheses by simulated and experimentally determined values of the fractal dimension Da (N,d0) within limitations (d0 = constant (average) coupling probability).

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Abstract: The decomposition of tert-butyl hydroperoxide by photochemically induced reactions in DMSO[Note ][Abbreviations used: DMSO, dimethyl sulfoxide; DMSO-d6, completely deuterium labelled dimethyl sulfoxide; cw-ESR, continuous-wave Electron Spin Resonance; u. v., ultra violet; DMPO, 5, 5-dimethyl-I-pyrroline N-oxide; TMPO, 3, 3, 5, 5-tetramethyl-l-pyrroline N-oxide.] and water was investigated by cw-e.s.r. spectroscopy. The products tert-butylperoxyl, methyl and sulfur-centered free radicals were identified. The tert-butoxyl free radical is involved in the primary process as shown by time-resolved e.s.r. technique. On the basis of directly identified radical species, a mechanism for the photochemically induced reactions of tert-butyl hydroperoxide in DMSO is proposed. At concentrations below 0.8 mol · l-1 the radical formation from tert-butyl hydroperoxide proceeds by cleavage of the OO bond rather than by hydrogen abstraction.

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Abstract: The relationship between nucleotide catabolism and generation of activated oxygen species was investigated in liver, hepatocytes and small intestine of rats. In severe hypoxia nucleotide degradation via xanthine oxidase and urate oxidase requires about half of the oxygen consumed. Data on the changes of nucleobase compounds in rat hepatocytes and small intestine during ischemia and reoxygenation and the effects of allopurinol and oxypurinol thereon are presented. From EPR measurements it is concluded that OH. radicals induce reactions of allopurinol yielding long-living products which are able to react with DMPO-OH with loss of its radical properties.

Abstract: In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.

Abstract: Membrane vesicles from human platelets were prepared by various disruption and isolation techniques reported in the literature to yield fractions of predominantly surface or intracellular membrane origin. ATP + Mg2+-dependent Ca2+ accumulation and the formation of acylphosphate intermediates of the calcium pump(s) were followed in parallel experiments, and the consequences of a limited proteolysis of the membranes examined. In all types of preparations active Ca2+ uptake had both oxalate-sensitive and insensitive fractions and calmodulin had no effect on the rate of Ca2+ uptake. Limited proteolysis by trypsin eliminated oxalate-sensitive Ca2+ uptake while it had no effect on the oxalate-insensitive fraction. The Ca2+-induced EP complex had an apparent molecular mass of 100-110 kDa in all of the preparations, the EP showing a broad or even duplicated line in most autoradiographies. Mild trypsin digestion resulted in the formation of 80-, 55-, and 35-kDa phosphorylated fragments. The 80-kDa fragment corresponded to the limit polypeptide found in the proteolyzed erythrocyte membrane Ca2+ pump, its phosphorylation was stimulated by lanthanum, and it appeared in a different time course than the smaller fragments. The molecular mass and the formation pattern of the latter species corresponded to the tryptic fragments in the sarcoplasmic reticulum Ca2+ pump. Based on these results we suggest that platelet membrane preparations contain two types of Ca2+ pump proteins, one similar to the sarcoplasmic reticulum-type and the other to the erythrocyte-type enzyme.

Abstract: A NADPH-dependent t-butyl hydroperoxide ( TBH )-reducing activity independent of glutathione was found in addition to glutathione peroxidase activity bound to erythrocyte membranes. In "hypotonic" and "isotonic" membranes the NADPH-dependent TBH -reducing activity amounted to about 0.34 mu kat /l red blood cells (RBC) and the glutathione peroxidase activity to 0.32 mu kat /l RBC. The activities do not appear to be additive. The membrane association of the enzymes is independent of ionic strength. Under hypotonic condition about 0.2% of the total cellular catalase activity were bound to the membrane but none in "isotonic" membranes. The bound catalase appears to exhibit a glutathione dependent peroxidase activity. Membrane-bound haemoglobin exhibited a quasi- TBH -reductase activity which was inhibited by azide and cyanide.

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Abstract: Data concerning membrane binding of glutathione reductase (GSSG-R) have been reexamined with respect to the following experimental conditions: 1. Haemolysis and membrane preparation; 2. influence of nonionic detergents (Triton X-100, Lubrol PX). Haemolysis and membrane preparations performed at physiological ionic strength, and reduced glutathione concentration do not give any evidence for binding of GSSG-R to the erythrocyte membrane. Under hypotonic conditions of ghost preparation about 2% of the total cellular GSSG-R activity are membrane bound, indicating weak enzyme association at low ionic strength only.

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Abstract: The respiratory tract as the main entrance for various inhalative substances has great potential to generate reactive species directly or indirectly in excess. Thus, heavy smokers are at high risk for development, impairment and failed response to treatment of chronic obstructive pulmonary disease (COPD). The article is an update regarding the influence of reactive oxygen (ROS) and nitrogen (RNS) species on COPD; however, we do not intend to describe ROS and RNS actions on the entire lung tissue. Here, we focus on the airways, because in human most of the described effects of ROS and RNS species are measured on respiratory epithelial cells obtained by bronchoscopy. ROS and RNS species are physiological compounds in cells and risk factors for several respiratory diseases. In general, both kinds of species are thermodynamically stabile, but their reaction behaviors in cellular environments are very different. For example, the life times of the superoxide anion radical range from micro/milliseconds up to minutes and even hours in in-vitro model systems. Oxidative stress by cigarette smoke was investigated in detail by the authors of this article. In addition, original studies by the authors on the amount of fine particulate matter and trace elements in lung biopsies after defined inhalation indicate a distortion of the equilibrium between oxidants and antioxidants. We also try to present some modern views with respect to genomic medicine for future therapeutic perspectives, although this is an upcoming sector of COPD therapy.

Abstract: Traditional methodologies in micro- and nanofluidics measure biological mechanisms as an average of a population of molecules as only their combined effect can be detected. Fluorescence fluctuation spectroscopy methods such as fluorescence correlation spectroscopy (FCS) and two-color fluorescence cross-correlation spectroscopy (FCCS) are used as alternative experimental approaches in ultrasensitive analytics at the single-molecule level. However, what is the measurement time in which one is able to study just one single molecule in solution without immobilizing it? Existing theories are inadequate since they do not predict the meaningful time as a function of the concentration of other molecules of the same kind in bulk solution. This situation produces considerable concern, and experimental hypotheses differ according to which single-molecule detection methods are thought to have greater validity. This subject is clearly at the forefront of research and should be of great interest to experimental medical scientists. As will be seen in this article, it is worthwhile to obtain a correct form of the meaningful-time relationship through theoretical means. The new ideas are comprehensively presented, and this relationship is a new concept at this time. The meaningful time for studying just one molecule without immobilization specifies the time parameter in the selfsame molecule likelihood estimator. Possible users for this concept are those working in biotechnological applications dealing with gene technology. Furthermore, the concept is of interest for a great number of medical, pharmaceutical and chemical laboratories. It may serve as a foundation for further work in single-cell biology. It is suspected that heterogeneities play a much larger role inside the cell than in free solution--a perfect opportunity for single-molecule studies and, thus, a novel hypothesis regarding structure and dynamics of cellular networks is first presented for the minimal neurotrophin network model.

Abstract: In this article, current views on cellular and molecular biology (biochemical) mechanisms are discussed under the aspect of altitude exposition. The Andean, Tibetan, and Ethiopian patterns of adaptation to high-altitude hypoxia are known [Beal et al. (2002) Proc Natl Acad Sci USA 99: 17215-17218]. The phylogenetic tree of the human species suggests that there are genetic differences in adaptation patterns to chronic hypoxic hypoxia. Five defense mechanisms are well established for lowlanders who are exposed to acute hypoxic hypoxia. Consequences of the cellular decrease in ATP are the formation of hypoxanthine and xanthine, which are the substrates for the massive formation of superoxide anion radicals and hydrogen peroxide via the oxidase activity of the xanthine oxidoreductase reaction. Under severe hypoxia, about 51 % of the total inhaled oxygen is used to form superoxide anion radicals in rat liver [Gerber et al. (1989) Adv Exp Med Biol 253B, Plenum Press, New York, 497-504]. The reactivity and selectivity of the superoxide anion radical are modified by specific interactions and electron exchange. It is commonly accepted that the superoxide anion radical in aqueous solutions has a lifetime in the millisecond range. However, electron spin resonance spectroscopy studies in a KO2/H2O/iron ion system revealed for the first time a stabilization of a part of the initially added superoxide anion radicals lasting up to hours at room temperature [Földes-Papp (1992) Gen Physiol Biophys 11: 3-38]. Superoxide anion radicals adsorbed on an oxidic iron hydrate phase in aqueous systems might function as a strong oxidant similar to that species which has been suggested to be a complex between oxygen and different valence states of iron in the initiation of lipid peroxidation by ferrous iron. There were serious doubts about the identity of alkoxy radicals. For the first time, alkoxy radicals were directly demonstrated in solution by electron spin resonance spectroscopy [Földes-Papp et al. (1991) Adv Synth Catal 333: 293-301]. The redox status in mammalian cells is mainly determined by the antioxidant glutathione, which is a key player in maintaining the intracellular redox equilibrium and in the metabolic regulation of the cellular defense against oxidative stress. As reactive oxygen species occupy an essential role in membrane damage, the idea of membrane-bound enzymatic defense mechanisms gets a new dimension [Földes-Papp et al. (1981) Acta Biol Med Ger 40: 1129-1132; Földes-Papp and Maretzki (1982) Acta Biol Med Ger 41: 1003-1008]. The steady-state between antioxidants and pro-oxidants affects the gene expression via hypoxia-induced transcription activities. The transcription factor hypoxia-inducible factor 1 (HIF-1) is a global regulator of oxygen homeostasis. As discussed in this article, hypoxia or 'oxidative stress' is accompanied by appropriate molecular adaptation mechanisms at the enzymatic or epigenetic level (enzymatic and non-enzymatic radical inhibitors, posttranslational modifications) and at the genetic level (transcription, translation).

Abstract: Alpinism in all its variations is a leading factor in tourism. Within a few decades, alpine sports, even at high altitudes, have become available to a wide range of people. Now, more people than ever before are hiking, trekking, climbing and skiing at moderate and high altitudes. Annually, 40 million people spend time in the Alps and 100 million visit high altitudes worldwide. However, alpine excursions may entail health problems and many aspects of impaired adaptation to altitude remain unstudied. High-altitude research has mainly been associated with expeditions, with moderate altitudes receiving far less attention, though most tourism takes place at that level. The overwhelming numbers of alpine tourists mean that there is urgent need for high- and moderate-altitude medical research, which would also be within the realm of political responsibility in mountainous countries. Research in mountain medicine and dissemination of relevant findings can show how to improve and conserve performance in healthy individuals and could point the way toward new, safe approaches in the rehabilitation of patients with chronic diseases. It is imperative that mountain medicine continues to develop on a scientific basis.

Abstract: So far, chemists, molecular biologists and biochemists have reaped the greatest benefits from mass spectrometry (Aebersold et al., 2003). This type of analysis could, however, be useful in many fields. Mass spectrometry is on its way to the doctor's office (Pusch et al., 2003; Földes-Papp et al., 2002; Henry 1999). The article is focused on laser-activated microprobe mass analysis (LAMMA) and inductively coupled argon plasma mass spectrometry (ICP-MS). Potential applications of the two types of mass spectrometry are demonstrated in clinical medicine. It is the first comprehensive review on qualitative characterization of carbonaceous compounds in lung tissue samples in situ and quantitative trace element determination in body fluids.

Abstract: Innovative and important aspects of laser scanning confocal fluorescence imaging (LSCFI) are presented here as a general overview. We have described and discussed the technology of the procedure in some detail. We also report some of our original work with transmembranous uptake of 5S gamma-globulin on living human leukocytes as an example of one specific application of LSCFI. These original data and results are presented, as well as citing other uses and applications, to show the power of LSCFI technique. The article will hopefully be useful for those not familiar with the methodology and utility of laser scanning confocal fluorescence microscopy. Applications of LSCFI are very diverse, and there are new applications of this technology constantly being developed. Interest is growing in LSCFI, particularly in the pharmacologic and therapeutic areas, as demonstrated in this article.

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Abstract: Ultrasensitive detection methods such as laser-induced fluorescence represent the current state-of-the-art in analytics. Single-molecule detection in solution has received a remarkable amount of attention in the last few years because of its applicability to life sciences. Studies have been performed on the fundamentals of the detection processes themselves and on some biological systems. Fluorescence correlation spectroscopy (FCS) is the link for ultrasensitive multicomponent analysis, showing possibilities for experiments on molecular interactions. Based on the theoretical background of FCS, this article gives full explanation of FCS and an update of highlights in experimental biology and medicine studied by FCS. We focus on a repertoire of diverse immunoglobulin specificities, a ribosome display system, single-molecule DNA sequencing, and a mutant enzyme generated by random mutagenesis of amino acids. We describe the usefulness and the enormous potential of the methodology. Further, this contribution clearly indicates that FCS is a valuable tool for solution-phase single-molecule (SPSM) experiments in immunobiology and medicine. In experiments with the Goodpasture autoantibody, we worked out conditions for the design of experiments on a complex single molecule in solution. The possibility to use SPSM-FCS as a quantitation methodology opens up other important applications beyond the scope of this article. Original results extending the published studies are presented for the rational foundation of SPSM-FCS. In this original contribution, we deal with experimental systems for biology and medicine where the number of molecules in solution is very small. This article is mandatory for gaining confidence in the interpretation of experimental SPSM-FCS results on the selfsame, individual single molecule in solution.

Abstract: The molecular structures of animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases are not completely understood in part due to the fact that no suitable single crystal is available. The elucidation of the two-dimensional structure is in progress. The amino acid sequences of human erythrocyte and rat plasma membrane Ca2+ pump isoforms as well as of the pig smooth muscle plasma membrane Ca2+ pump are already known. This article reviews the present state of the knowledge in (Ca(2+)+Mg2+)-ATPase research of animal and human plasma membranes performed in the past few years, concerning in particular arrangements of proteolytically cleaved fragments, and relations between the erythrocyte (Ca(2+)+Mg2+)-ATPase in situ and the purified red cell enzyme, oxidative changes. Results of different experimental approaches concerning the structure of (Ca(2+)+Mg2+)-ATPases rather than the applications of the methods used are emphasized.

Abstract: The other perspective, which takes the extreme other position, explains: 'Due to Brownian diffusion, single molecules are randomly transported through this small [femtoliter-sized] sampling volume in 3-dimensions. It's virtually impossible to study a specific "single molecule" under such conditions because of the random movement of molecules in and out of the sampling volume in solution' by Li, X., Hofmeister, W., Shen, G., Davis, L., Daniel, C., 2007: Fabrication and characterization of nanofluidics device using fused silica for single protein molecule detection, In: Proceedings of Materials and Processes for Medical Devices (MPMD), Conference & Exposition, Sept. 23-25, USA. Essentially, we can say that there are two different perspectives with opposite outcomes. This has to do with the burden of evidence in single-molecule measurements and the way we are looking at the theory. ALL OF THIS IS EXPLAINED BY THE THEORETICAL MODEL, WHICH IS THE STARTING POINT.

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Abstract: We present ideas for increasing the observation time of a single molecule free in solution without the need for immobilization or hydrodynamic/electrokinetic focusing [1, 2]. By using hidden, continuous-time numerical Markov models for evaluation of our new concepts, we compare our way with normal fluctuation experiments. We first give answers to the questions: (1) What is the time and average entering frequency C, respectively, for a molecule to find the probe volume DV at an initial molar concentration in the bulk? (2) What is the probability (d/dt)p>0(Dt) that the entering molecule is the original molecule as function of the time Dt from last entry and the concentration of other molecules of the same kind in the bulk? (3) What is the number of meaningful re-entries of the original molecule? Approaches (1) � (3) give the meaningful time Tm in the probe region DV allowing to study just a single molecule in solution or a live cell without immobilization or hydrodynamic/electrokinetic focusing. Tm is physically defined by the stochastic variable waiting time Dt for the next entry of another molecule than the original one. We discuss and describe how our work differs from the results of other groups that have so far addressed the reentry problem of a single molecule. [1] Földes-Papp, Z. (2007). Fluorescence fluctuation spectroscopic approaches to the study of a single molecule diffusing in solution and a live cell without systemic drift or convection: a theoretical study. Curr. Pharm. Biotechnol. 8, 261-273. [2] Földes-Papp, Z. (2007). �True� single-molecule observations by fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy. Exp. Mol. Pathol. 82, 147-155. (*Financial support: FWF Austrian Science Fund Research Project P20454-N13, Medical University of Graz, Austria.)

Abstract: ST-5 THEORETICAL TESTING OF SINGLE�MOLECULE FLUORESCENCE CORRELATION SPECTROSCOPY AND TWO�COLOR FLUORESCENCE CROSS�CORRELATION SPECTROSCOPY WITHOUT IMMOBILIZATION OR HYDRODYNAMIC FOCUSING Zeno Földes-Papp Medical University of Graz Riesstrasse 58a/5, A-8047 Graz, Austria Although fluorescence from individual molecules is collected at a solution concentration of about 1 nM and less, the data are analyzed by an auto-correlation or two-color crosscorrelation function that is the average of thousands of molecules. We want to open the door for measurements directly in solution and a live cell without immobilization or hydrodynamic focusing in order to measure the kinetics and dynamics of single molecules undergoing reactions on a time scale not previously available [1-6]. The following main idea is put forward: is it possible to recognize the same molecule multiple times as it diffuses through a small confocal probe volume? If so, the detection time of an individual molecule freely diffusing in solution could be extended. For the new analysis approach that is called �the meaningful-time concept� [1-4], it is crucial to understand the balance between the time scale and the frequency that a single molecule reenters the confocal probe volume and the overall time necessary for the measurement. [1] Földes-Papp Z. Viral chip technology for Genomic Medicine. In: HF Willard, GS Ginsburg (Eds.), Handbook of Genomic Medicine. Academic Press, New York, USA, in press, (2008). Publication of the National Center for Human Genome Sequencing and Personalized Medicine of the USA. (http://www.ybp.com/acad/ads/0707_elsev_genomic.html) [2] Földes-Papp Z. Fluorescence fluctuation spectroscopic approaches to the study of a single molecule diffusing in solution and a live cell without systemic drift or convection: a theoretical study. Curr. Pharm. Biotechnol. 8 (5), 261-273, (2007). [3] Földes-Papp Z. True single-molecule molecule observations by fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy. Exp. Mol. Pathol. 82 (2), 147-155, (2007). [4] Földes-Papp Z. What it means to measure a single molecule in a solution by fluorescence fluctuation spectroscopy. Exp. Mol. Pathol. 80 (3), 209-218, (2006). [5] Földes-Papp Z, Baumann G, Kinjo M, Tamura M. Single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS). Distinguished article entry. In: J Fuchs, M Podda (Eds.), Encyclopedia of Medical Genomics & Proteomics, Marcel Dekker, New York, USA, (2005). [6] Földes-Papp Z, Kinjo M, Tamura M, Birch-Hirschfeld E. A new ultrasensitive way to circumvent PCR-based allele distinction: direct probing of unamplified genomic DNA by solution-phase hybridization using two-color fluorescence cross-correlation spectroscopy. Mol. Exp. Pathol. 78 (3), 177-189, (2005).

Abstract: Fluorescence Correlation Spectroscopy (FCS) and two-color Fluorescence Cross-Correlation Spectroscopy (FCCS) are a measure of fluctuations of detected light as a fluorescence molecule diffuses through a femtoliter detection volume caused by a tightly focused laser and confocal optics. Fluorescence from a single molecule can be distinguished easily from the small background associated with a femtoliter of solvent. At a solution concentration of about 1 nM, the probability that there is an analyte molecule in the probe volume is less than one. Although fluorescence from individual molecules is collected, the data is analyzed by an autocorrelation or two-color crosscorrelation function that is the average of thousands of molecules. Properties of single molecules are not obtained! In collaboration with Richard A. Keller, Los Alamos, we have been working on problems and opportunities associated in the very dilute solutions. The molecule in the probe volume is most probably the molecule that just diffused out, turned around, and diffused back in, i.e. reentered. Most people consider reentries a major problem. For the first time, it is turned into a potential opportunity. Four criteria have been developed that can be used to verify that there is only one "self-same" molecule in the laser probe volume during the experiment: Z. Földes-Papp (2006) What it means to measure a single molecule in a solution by fluorescence fluctuation spectroscopy, Exp Mol Pathol 80 (3), 209-218; Z. Földes-Papp (2007) In: Handbook of Genomic Medicine, Part 1 - The Basics, Technology (H.F. Willard, G.S. Ginsburg, Eds), Elsevier, New York.

Abstract: When we started preparing the edition of the two theme issues � each divided in two parts (i.e., four issues in total) � we felt that the time had come to present and discuss recent achievements in biotechnology and bioanalysis that are creating a new array of opportunities for the pharmaceutical and biomedical sectors. The inter-disciplinary topics presented in these four issues stem from scientists in biology, chemistry, and biochemistry as well as molecular biology, immunology, physics, mathematics, engineering, computation and medicine. After studying the cell and its constituents and working the way down to the genome and the proteome, huge amount of data have been generated during the last decade and can now be deciphered to shed light on the basic principles of life. Among the new technologies that are making this progress possible, the DNA chip is only one example that has helped address many molecular biology problems, such as rapid identification of genetic disorders. The foundations of microarray and biochip technologies are firmly rooted in the rapid advances in nucleic acid chemistry and biochemistry. The overwhelming majority of the early-phase trials using these developments was not primarily aimed at clinical efficacy but instead at assessing the biological problems to be solved before genomics, epigenomics and proteomics can deliver useful information for clinical applications. In an attempt to better understand these problems, investigators returned to basic studies of gene expression, immunology and virology. Finally, the cloning and sequencing of the human genome has significantly expanded the range of actual and potential molecular drug targets. The discovery and optimization of molecular drug targets, e.g., disease specific proteins, receptors, enzymes, or genes, is a time-consuming and extremely expensive process. Therefore, in today's preclinical drug research, there is only one key to success: scientific teamwork and the use of the most advanced biotechnological approaches. The advances in biotechnology directly affect improvements in human health and outcomes. That especially holds true in our time of rapid changes. The title of the four special issues implies that their scope encompasses the fundamentals of the way down from single genes and proteins to single molecules. Should we have started this Edition from the top downward, beginning with medicine and clinical practice based on empirical ("complex") thinking, where the risk exists of serious error by failing to account accurately for gene or protein interactions? Alternatively, should we proceed from the bottom upward beginning with single molecules and their behavior, which often seems to be different from that of the ensemble in biological systems? The answer is probably a compromise, working in both directions from the middle. The contributions to these four Special Issues of Current Pharmaceutical Biotechnology are written by leading specialists in fields that cover a wide range of current topics on biotechnological and biomedical aspects of drug target screening and characterization. Several articles present overviews on the latest developments and future prospects. They are devoted to discussing multidisciplinary topics at a level that will be useful to the practicing scientist. The contributions are not simply reviews and mini-reviews on narrow subjects, but deal with focal areas at the frontier of science and are expected to be of large interest for many researchers working in medicinal chemistry, biochemistry, immunology, biophysics, and pharmacology of molecular drug targets. In presenting the different contributions in the four issues, we combine practical and theoretical aspects, allowing for design, characterization and application of rare molecular events at the many-molecule level (the first two-part special issue) and at the single-molecule level (the second two-part special issue). The second part of the first two-part special issue contains articles focused on nucleic acid and protein analyses in manymolecule systems: The article of J. Zhang and Zichun Hua entitled �Targeted gene silencing by small interfering RNA-based knock-down technology� presents a concise and balanced update of the field for a broad readership with many practical advises for design and applications. Small interfering RNA-based knock-down technology and its applications are a very important new area of research and a promising tool for new therapeutics. Sergey Y. Tetin and Stephen D. Stroupe provide a review on immunoassays and their configurations from classical studies to present entitled "Antibodies in diagnostics applications". Immunoassays made an extraordinary success story in biomedical and clinical analytics. They are among the mostly used analytical tools in chemical, biochemical and immunological analytics. The material is presented by discussing the pros and cons. The article "Combinatorial protein biochemistry for therapeutics and proteomics� of David Lowe and Lutz Jermutus is an overview about applications of in-vitro methods for the display of peptides and proteins that are of therapeutic and diagnostic interests. The authors belong to the leading research groups in this field, and thus give the information from first hand including latest developments. Thomas Bruckdorfer and Oleg Marder provide an overview on �From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future�. Peptides are key to modern drug discovery. The requirements are discussed to produce peptides, initially in millions in mg-scale for research purposes and then for some newly discovered sequences from the basis of modern drugs in multi-tons. The article addresses central prerequisites for success of high-throughput screening. Further it gives access to complex theoretical/mathematical analyses and relationships of multi-step syntheses in solution and on solid support. The review article of Andreas Wiesner entitled �Detection of tumor markers with the ProteinChip technology� provides an overview on tumor markers as diagnostic tools. The early diagnosis of cancer is one of the major challenges in modern, evidence-based medicine. Relevant cases are considered which are of high practical value. Thereby the article deals with up-todate protein mapping platforms for screening hundreds of samples a day. The contribution considers the most relevant literature in the field and provides an introduction into the ProteinChip Biomarker system. Steven Pelech�s paper �Tracking cell signaling protein expression and phosphorylation by innovative proteomic solutions� gives a broad overview that puts current proteomics approaches into perspective. Among the platforms currently used, the article highlights mass spectrometry and the multi-immunoblotting procedure to analyze cell-signaling proteins. Furthermore, the description of the peptide antibody mimetic (PAM) technology with actual working examples represents the first time that this idea with proof of concept has appeared in the scientific literature. PAM technology has the potential to completely replace antibodies in a wide range of applications. Martin Blueggel, Daniel Chanrad, and Helmut E. Meyer contribute the article �Bioinformatics in proteomics". It contains outstanding procedures of molecular sciences. Bioinformatics approaches and platforms, mainly for expression proteomics, are discussed with their pros and cons from mass spectrometric data, peptide mass fingerprinting, peptide fragmentation fingerprinting, peptide de-novo sequencing, protein sequence databases, and proteome databases to gel image analysis. The paper of Wolfgang Domej, Zeno Földes-Papp, Ulrike Demel, and Genot P. Tilz on �Clinical applicability of mass spectrometry for inhaled carbon compounds and the characterization of trace element patterns in body fluids� presents an overview on application of mass spectroscopy in the medical field with laser activated microprobe mass analysis (LAMMA) and inductively coupled argon plasma mass spectroscopy (ICP-MS). The work is an attempt to review characteristics of carbonaceous particles deposited in lung and of trace element analysis in body fluids. The paper may be of great interest for researchers who are planning to use mass spectroscopy in clinical medicine at the bed side. Katharina Schroecksnadel, B. Frick, B. Wirleitner, C. Winkler, H. Schennach, and Dietmar Fuchs provide the overview �Moderate hyperhomocysteinemia and immune activation" about the extended folate one-carbon metabolism and the folaterelated homocysteine metabolism. The multifactorial biology including immune activation and several disorders are presented. The paper demonstrates that folate supplementation lowers homocysteine concentrations in almost all subjects regardless of the cause. Susanne Brakmann�s article "Optimal enzymes for single-molecule sequencing" is about recent advances in single-molecule sequencing from the viewpoint of biochemistry. The author compares the synthesis of high-density labeled DNA strands and the digestion process of fluorophore-labeled strands with different combinations of enzymes and substrates. How DNA sequencing will work in future and how high-throughput genetic screeing may be performed in gene therapy is a very important research line. We would like to thank all authors who contributed to the four special issues, Dr Gernot P. Tilz, the Editor-in-Chief Dr Alain Rolland, and the production team of Bentham Science Ldt particularly Mrs Ambreen Wasim, Dr Mansoor Alam and Mr. Mahmood Alam for their support and efforts to get this Special Edition to fruition. Graz, October 2003 Zeno Földes-Papp (Responsible Editor) Clinical Immunology and Jean Dausset Laboratory Graz University Medical School and Hospital A-8036 Graz-LKH Austria E-mail: Zeno.Foldes-Papp@uni-graz.at

Abstract: When we started preparing the edition of the two theme issues � each divided in two parts (i.e., four issues in total) � we felt that the time had come to present and discuss recent achievements in biotechnology and bioanalysis that are creating a new array of opportunities for the pharmaceutical and biomedical sectors. The inter-disciplinary topics presented in these four issues stem from scientists in biology, chemistry, and biochemistry as well as molecular biology, immunology, physics, mathematics, engineering, computation and medicine. After studying the cell and its constituents and working the way down to the genome and the proteome, huge amount of data have been generated during the last decade and can now be deciphered to shed light on the basic principles of life. Among the new technologies that are making this progress possible, the DNA chip is only one example that has helped address many molecular biology problems, such as rapid identification of genetic disorders. The foundations of microarray and biochip technologies are firmly rooted in the rapid advances in nucleic acid chemistry and biochemistry. The overwhelming majority of the early-phase trials using these developments was not primarily aimed at clinical efficacy but instead at assessing the biological problems to be solved before genomics, epigenomics and proteomics can deliver useful information for clinical applications. In an attempt to better understand these problems, investigators returned to basic studies of gene expression, immunology and virology. Finally, the cloning and sequencing of the human genome has significantly expanded the range of actual and potential molecular drug targets. The discovery and optimization of molecular drug targets, e.g., disease specific proteins, receptors, enzymes, or genes, is a time-consuming and extremely expensive process. Therefore, in today's preclinical drug research, there is only one key to success: scientific teamwork and the use of the most advanced biotechnological approaches. The advances in biotechnology directly affect improvements in human health and outcomes. That especially holds true in our time of rapid changes. The title of the four special issues implies that their scope encompasses the fundamentals of the way down from single genes and proteins to single molecules. Should we have started this Edition from the top downward, beginning with medicine and clinical practice based on empirical ("complex") thinking, where the risk exists of serious error by failing to account accurately for gene or protein interactions? Alternatively, should we proceed from the bottom upward beginning with single molecules and their behavior, which often seems to be different from that of the ensemble in biological systems? The answer is probably a compromise, working in both directions from the middle. The contributions to these four Special Issues of Current Pharmaceutical Biotechnology are written by leading specialists in fields that cover a wide range of current topics on biotechnological and biomedical aspects of drug target screening and characterization. Several articles present overviews on the latest developments and future prospects. They are devoted to discussing multidisciplinary topics at a level that will be useful to the practicing scientist. The contributions are not simply reviews and mini-reviews on narrow subjects, but deal with focal areas at the frontier of science and are expected to be of large interest for many researchers working in medicinal chemistry, biochemistry, immunology, biophysics, and pharmacology of molecular drug targets. In presenting the different contributions in the four issues, we combine practical and theoretical aspects, allowing for design, characterization and application of rare molecular events at the many-molecule level (the first two-part special issue published in Curr. Pharm. Biotech (2003) vol 4, no. 6, and Curr. Pharm. Biotech (2004) vol. 5, no. 1) and at the single-molecule level (the second two-part special issue published in Curr. Pharm Biotech (2004) vol. 5, no. 2, and Curr. Pharm Biotech (2004) vol. 5, no.3). The first part of the second two-part special issue contains articles focused on fluorescence correlation spectroscopy (auto- and two-color cross-correlation mode) in single-molecule systems: The paper of Klaus Weisshart, Volker Jurgel and Stephen J. Briddon entitled �The LSM 510 META - ConfoCor 2 system: an integrated imaging and spectroscopic platform for single molecule detection� is a very nice and comprehensive survey of fluorescence correlation spectroscopy (FCS) and the ConfoCor2/LSM-system that is produced by the Zeiss company. The authors also deal with the basic theory of fluctuation analysis, calibration analysis and evaluation of FCS data, and describe technical advances and new improvements in the software for FCS and two-color fluorescence cross-correlation spectroscopy (FCCS) methods. FCS in auto- and two-color cross-correlation mode will be a powerful tool for in-vivo pharmacological investigations in near future. Jörg Enderlein, Ingo Gregor and Jörg Fitter present a balanced overview of some artifacts in fluorescence correlation spectroscopy measurements. Their article �Art and Artefacts of Fluorescence Correlation Spectroscopy� discusses optical and photophysical effects in the correlation function like optical saturation and triplet-states with fast regions. The article of Zeno Földes-Papp, Gerd Baumann, Ulrike Demel and Gernot P. Tilz entitled �Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics� is an exposition of ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- and two-color cross-Correlation Spectroscopy (SPSMFCS). The �algorithms� developed are called �the selfsame single-fluorescent-molecule regime� and permit the continuous measurement of one and the same single, individual molecule in solution or a membrane by fluorescence detection techniques from sub-milliseconds up to several hours without hydrodynamic flow, immobilization, or arrival time histograms. All time time-dependent processes that involve a sufficiently small number of molecules in the bulk phase like solution or membrane can be treated by the stochastic formulism. Antonie J.W.G.Visser, Beno H. Kunst, Hans Keller and Arjen Schots provide the mini-review �Towards sorting of biolibraries using single-molecule fluorescence detection techniques�. They discuss efforts and achievements in the generation of biolibraries, single-molecule fluorescence detection techniques, and microfluidic devices. The authors come up with the widely accepted standpoint that a combination of the disciplines is necessary for the sorting and selection of large biolibraries. The review of T. Wakatsuki, J.A. Fee and Elliot L. Elson on "Phenotypic screening for pharmaceuticals using tissue constructs" covers a hot and controversial topic in drug discovery screening: Should primary screening be performed in a molecular or cellular environment? The method presented in this article describes a very interesting approach for screening in the cellular / tissue environment. The article of Holger Winter, Kerstin Korn and Rudolf Rigler entitled �Direct gene expression analysis" presents a recently developed approach for two-color hybridization-based gene expression analysis avoiding amplification of the target or the signal. The authors utilize two-color fluorescence cross-correlation spectroscopy �at the single-molecule level� as singlemolecule fluorescence detection technique. Takuya Takagi, Hiroaki Kii and Masataka Kinjo provide the article �DNA measurements by using fluorescence correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy�. It deals with an important aspect in two-color fluorescence cross-correlation measurements by presenting experimental results on diffusion times of long dsDNAs in the Kbp range. The long model DNAs contained fluorophores at different distances. Measured two-color cross-correlated diffusion times consisted of the �real diffusion time� and the �delay� between the red and green signals. Aladdin Pramanik�s contribution "Ligand-receptor interactions in live cells by fluorescence correlation spectroscopy� refers to original research papers of the author on C-peptide, EGF, and GAL. He also provides technical details of these methods at single-molecule detection sensitivity. The review of Anja Nohe and Nils O. Petersen on "Analyzing for co-localization of proteins at a cell membrane� presents techniques for determining how intramolecular interactions in cell membranes control the signal transduction process. First, the reader is given a short introduction including orientation on the present view of structure, organization and dynamics of cell membranes, their proteins, and their physiological role. Next, there is a survey of approaches to make binding and colocalization evident. Finally the article focuses on image cross-correlation spectroscopy as a relatively new quantitative tool developed by the authors. Michael Jahnz and Petra Schwille provide an overview on "Enzyme assays for confocal single molecule spectroscopy". The paper deals with the analysis of enzymatic reactions in solution using optical confocal techniques such as FCS, FCCS and FRET �at the single-molecule level�. The authors describe various types of FCS and FCCS methods based on principles, algorithms and data processing. The authors also mention strategic pathways of fluorophore labeling and conjugating methods for these approaches. Labeling probes and methods are of importance for fluorescent measurements in biological systems and samples. The contribution of Hans Blom and Michael Gösch focuses on "Parallel confocal detection of single biomolecules using diffractive optics and integrated detector units�. The authors present recent results of implementing parallel laser excitation and fluorescence detection for high-throughput miniaturized array-based analytical systems. They also report a multiplexing extension that permits multifocal laser excitation together with matched parallel fluorescence detection. We would like to thank all authors who contributed to the four special issues, Dr Gernot P. Tilz, the Editor-in-Chief Dr Alain Rolland, and the production team of Bentham Science Publishers Ldt particularly Mrs. Ambreen Wasim, Dr. Mansoor Alam and Mr. Mahmood Alam for their support and efforts to get this Special Edition to fruition. Graz, October 2003 Zeno Földes-Papp (Responsible Editor) Clinical Immunology and Jean Dausset Laboratory Graz University Medical School and Hospital A-8036 Graz-LKH Austria E-mail: Zeno.Foldes-Papp@uni-graz.at Sapporo, Japan

Abstract: When we started preparing the edition of the two theme issues � each divided in two parts (i.e., four issues in total) � we felt that the time had come to present and discuss recent achievements in biotechnology and bioanalysis that are creating a new array of opportunities for the pharmaceutical and biomedical sectors. The inter-disciplinary topics presented in these four issues stem from scientists in biology, chemistry, and biochemistry as well as molecular biology, immunology, physics, mathematics, engineering, computation and medicine. After studying the cell and its constituents and working the way down to the genome and the proteome, huge amount of data have been generated during the last decade and can now be deciphered to shed light on the basic principles of life. Among the new technologies that are making this progress possible, the DNA chip is only one example that has helped address many molecular biology problems, such as rapid identification of genetic disorders. The foundations of microarray and biochip technologies are firmly rooted in the rapid advances in nucleic acid chemistry and biochemistry. The overwhelming majority of the early-phase trials using these developments was not primarily aimed at clinical efficacy but instead at assessing the biological problems to be solved before genomics, epigenomics and proteomics can deliver useful information for clinical applications. In an attempt to better understand these problems, investigators returned to basic studies of gene expression, immunology and virology. Finally, the cloning and sequencing of the human genome has significantly expanded the range of actual and potential molecular drug targets. The discovery and optimization of molecular drug targets, e.g., disease specific proteins, receptors, enzymes, or genes, is a time-consuming and extremely expensive process. Therefore, in today's preclinical drug research, there is only one key to success: scientific teamwork and the use of the most advanced biotechnological approaches. The advances in biotechnology directly affect improvements in human health and outcomes. That especially holds true in our time of rapid changes. The title of the four special issues implies that their scope encompasses the fundamentals of the way down from single genes and proteins to single molecules. Should we have started this Edition from the top downward, beginning with medicine and clinical practice based on empirical ("complex") thinking, where the risk exists of serious error by failing to account accurately for gene or protein interactions? Alternatively, should we proceed from the bottom upward beginning with single molecules and their behavior, which often seems to be different from that of the ensemble in biological systems? The answer is probably a compromise, working in both directions from the middle. The contributions to these four Special Issues of Current Pharmaceutical Biotechnology are written by leading specialists in fields that cover a wide range of current topics on biotechnological and biomedical aspects of drug target screening and characterization. Several articles present overviews on the latest developments and future prospects. They are devoted to discussing multidisciplinary topics at a level that will be useful to the practicing scientist. The contributions are not simply reviews and mini-reviews on narrow subjects, but deal with focal areas at the frontier of science and are expected to be of large interest for many researchers working in medicinal chemistry, biochemistry, immunology, biophysics, and pharmacology of molecular drug targets. In presenting the different contributions in the four issues, we combine practical and theoretical aspects, allowing for design, characterization and application of rare molecular events at the many-molecule level (the first two-part special issue published in Curr. Pharm. Biotech. (2003) vol 4, no. 6, and Curr. Pharm. Biotech. (2004) vol. 5, no. 1) and at the single-molecule level (the second two-part special issue published in Curr. Pharm. Biotech. (2004) vol. 5, no. 2, and Curr. Pharm. Biotech. (2004) vol. 5, no.3). The second part of the second two-part special issue contains articles focused on single molecules and should be of interest to anyone interested in applying single-molecule analyses. The article by Karl-Otto Greulich entitled "Single molecule techniques for biomedicine and pharmacology" is a comprehensive survey of single-molecule studies with relevance to applications for the biomedical and pharmaceutical sectors. First, the reader is given a general introduction to the materials and methods of singlemolecule spectroscopy. Next, the results section presents and discusses, in more detail, several interesting and important applications of single-molecule detection in molecular biology and cellular physiology. The article focuses on topics where the author has extensive first-hand expertise. H. Peter Lu�s article on "Single-molecule spectroscopy studies of conformational change dynamics in enzymatic reactions" deals with interesting data on the T4 lysozyme hydrolyzation reaction of a polysaccharide. The spectroscopic and theoretical results presented form a collection of the author's original work. The article by Peter M. Goodwin, Rhiannon L. Nolan and Hong Cai on "Single-molecule spectroscopy for nucleic acid analysis: a new approach for disease detection and genomic analysis" is a survey of single-molecule detection and identification for genomic analyses. The authors are among the pioneers in this field of research. With "Applications of single-molecule detection to the analysis of pathogenic DNA" Oana Marina and Alonso Castro provide an original and effective application of single-molecule fluorescence detection for the analysis of pathogenic DNA, particularly for ultrasensitive pathogen detection. This method is one of the rare cases were single-molecule detection is applied to an important bio-analytical task, the detection of small amounts of pathogenic DNA in a large sample. H. Neuweiler and Markus Sauer survey their recent work on "Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level". It deals with charge-transfer probes for monitoring and studying the conformational dynamics of peptides and DNA, as well as the application of these methods to the single-molecule sensitive detection of target molecules. The measurement techniques presented (direct inter-photon distance timing), novel data analysis, as well as the newly designed charge�transfer probes are a new and promising branch in single-molecule fluorescence spectroscopy. The work of Michael Wahl, F. Koberling, M. Patting, H. Rahm, and R. Erdmann on "Time-resolved confocal fluorescence imaging and spectroscopy system with single-molecule sensitivity and sub-micrometer resolution� describes the authors� recently developed confocal laser-scanning microscope. The paper presents a thorough introduction into the system and its performance in various applications, including lifetime imaging of surfaceadsorbed molecules and of live cells, or fluorescence-fluctuation and FRET measurements in solution. It should be of great interest for researchers who intend to start working in single-molecule spectroscopy and its applications. A survey by Jan Hesse, Max Sonnleitner, and Gerhard J. Schutz on "Ultra-sensitive fluorescence reader for bioanalysis" covers the group�s recent development and application of a new single-molecule sensitive highthroughput plate reader. This is the first attempt to apply a single-molecule sensitive imaging technology to the screening of large sample areas for bio-analytic and bio-diagnostic purposes. Of special interest is the application for the single-molecule sensitive screening of live cells on a surface. We would like to thank all authors who contributed to the four special issues, Dr Gernot P. Tilz, the Editor-in-Chief Dr Alain Rolland, and the production team of Bentham Science Publishers Ldt particularly Mrs Ambreen Wasim, Dr Mansoor Alam and Mr. Mahmood Alam for their support and efforts to get this Special Edition to fruition. Graz, October 2003 Zeno Földes-Papp (Responsible Editor) Clinical Immunology and Jean Dausset Laboratory Graz University Medical School and Hospital A-8036 Graz-LKH, Austria E-mail: Zeno.Foldes-Papp@meduni-graz.at

Abstract: When we started preparing the edition of the two theme issues � each divided in two parts (i.e., four issues in total) � we felt that the time had come to present and discuss recent achievements in biotechnology and bioanalysis that are creating a new array of opportunities for the pharmaceutical and biomedical sectors. The inter-disciplinary topics presented in these four issues stem from scientists in biology, chemistry, and biochemistry as well as molecular biology, immunology, physics, mathematics, engineering, computation and medicine. After studying the cell and its constituents and working the way down to the genome and the proteome, huge amount of data have been generated during the last decade and can now be deciphered to shed light on the basic principles of life. Among the new technologies that are making this progress possible, the DNA chip is only one example that has helped address many molecular biology problems, such as rapid identification of genetic disorders. The foundations of microarray and biochip technologies are firmly rooted in the rapid advances in nucleic acid chemistry and biochemistry. The overwhelming majority of the early-phase trials using these developments was not primarily aimed at clinical efficacy but instead at assessing the biological problems to be solved before genomics, epigenomics and proteomics can deliver useful information for clinical applications. In an attempt to better understand these problems, investigators returned to basic studies of gene expression, immunology and virology. Finally, the cloning and sequencing of the human genome has significantly expanded the range of actual and potential molecular drug targets. The discovery and optimization of molecular drug targets, e.g., disease specific proteins, receptors, enzymes, or genes, is a time-consuming and extremely expensive process. Therefore, in today's preclinical drug research, there is only one key to success: scientific teamwork and the use of the most advanced biotechnological approaches. The advances in biotechnology directly affect improvements in human health and outcomes. That especially holds true in our time of rapid changes. The title of the four special issues implies that their scope encompasses the fundamentals of the way down from single genes and proteins to single molecules. Should we have started this Edition from the top downward, beginning with medicine and clinical practice based on empirical ("complex") thinking, where the risk exists of serious error by failing to account accurately for gene or protein interactions? Alternatively, should we proceed from the bottom upward beginning with single molecules and their behavior, which often seems to be different from that of the ensemble in biological systems? The answer is probably a compromise, working in both directions from the middle. The contributions to these four Special Issues of Current Pharmaceutical Biotechnology are written by leading specialists in fields that cover a wide range of current topics on biotechnological and biomedical aspects of drug target screening and characterization. Several articles present overviews on the latest developments and future prospects. They are devoted to discussing multidisciplinary topics at a level that will be useful to the practicing scientist. The contributions are not simply reviews and mini-reviews on narrow subjects, but deal with focal areas at the frontier of science and are expected to be of large interest for many researchers working in medicinal chemistry, biochemistry, immunology, biophysics, and pharmacology of molecular drug targets. In presenting the different contributions in the four issues, we combine practical and theoretical aspects, allowing for design, characterization and application of rare molecular events at the many-molecule level (the first two-part special issue) and at the single-molecule level (the second two-part special issue). The first part of the first two-part special issue contains articles mainly focused on nucleic acid analyses in many-molecule systems: Margret R. Hoehe, Bernd Timmermann, and Hans Lehrach provide an extensive survey on �Human inter-individual DNA sequence variation in candidate genes, drug targets, the importance of haplotypes and pharmacogenomics�. The review deals with the integration of global information on DNA, mRNA, and protein to identify �the� gene most probably underlying disease and indicates the end of Mendel�s world of the two-allele concept of �the� gene. A single SNP (Single Nucleotide Polymorphisms) reflecting an early stage of approximation to completeness represents no longer a marker of a disease in this concept. The paper of Hartmut Seliger, Michael Hinz, and Erwin Happ is devoted to a critical discussion of contributions to nucleic acids technology for arrays of immobilized oligonucleotides. Main problems of state-of-the-art developments are addressed from a chemical point of view. The review is comprehensive and extensively covers the literature in this field. Xiao Zeng�s article �The making of a portrait � bringing it into focus� is a succinct overview of the current developments of DNA arrays in biomedical research fields with an emphasis on the pathway-specific array design. Hans-Martin Striebel, Eckhard Birch-Hirschfeld, Renate Egerer, and Zeno Földes-Papp provide an overview on �Virus Diagnostics on Microarrays". It combines microarray technology and virus diagnostics from classical study to present. The authors also mention strategic pathway of the coupling chemistry, chemical synthesis of oligonucleotides and DNA, fluorophore labeling and hybridization methods for microchip technology. Sensitivity and reproducibility are dependent on these parameters and they are important for routine diagnostics. The article of Irina M. Gana Dresen, Johannes Hüsing, E. Kruse, T. Boes, and K.-H. Jöckel "Software packages for quantitative microarray-based gene expressions analysis" presents three software packages that can be used for the analysis of microarray-based gene expressions. It starts with the technical description of the programs, including hardware and software requirements, handbook quality, and finally compares the performance of the three programs. Gwendolyn A. Lawrie, Bronwyn J. Battersby, Lisbeth Grondahl, and Matt Trau provide an comprehensive summary on the various techniques of labeling large compound libraries, and then focus on the technique of using colloids for encoding purposes. Encoding is a topic of enormous significance when synthesizing and screening large libraries in genomics, proteomics and drug discovery. The contribution of Lloyd M. Davis, Peter E. Williams, David A. Ball, Kerry M. Swift, and Edmund D. Matayoshi on �Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery" summarizes the work of the authors about numerically simulating fluorescence correlation spectroscopy (FCS) experiments, and it presents a biologically interesting application of FCS. Stefan Jäger, Leif Brand, and Christian Eggeling extend the contents of the first issue to an extensive overview on the new techniques of fluorescence fluctuation spectroscopy such as fluorescence-intensity distribution analysis (FIDA), fluorescenceintensity multiple distribution analysis (FIMDA), fluorescence-intensity and lifetime distribution analysis (FILDA), and also the more standard fluorescence-correlation spectroscopy (FCS) in high-throughput analysis. Kunio Hori, W.S. Shin, C. Hemmi, T. Toyo-oka, and T. Makino demonstrate the analysis of single point mutation in a mitochondrial gene, i.e., SNP analysis, by using fluorescence correlation spectroscopy (FCS) at the very low concentration of products. The authors also describe two types of methods for FCS analysis based on primer extension in DNA polymerase reaction. We would like to thank all authors who contributed to the four special issues, Dr Gernot P. Tilz, the Editor-in-Chief Dr Alain Rolland, and the production team of Bentham Science Ldt particularly Mrs Ambreen Wasim, Mr. Mansoor Alam and Mr. Mahmood Alam for their support and efforts to get this Special Edition to fruition. Graz, October 2003 Zeno Földes-Papp (Responsible Editor) Clinical Immunology and Jean Dausset Laboratory Graz University Medical School and Hospital A-8036 Graz-LKH Austria E-mail: Zeno.Foldes-Papp@uni-graz.at

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Abstract: 1. Das Werden der Klinischen Immunologie Die Klinische Immunologie hat sich in Graz als eigene Disziplin, als neues Fach seit 1968 entwickelt und Praxis und Forschung befruchtet. Beispiele sind das Transplantationswesen mit Pionierleistungen, z.B. durch unseren Präsidenten Rigler, der die erste Lebertransplantation vorgenommen hat. In Eigenentwicklung haben wir das erste Antilymphozytenglobulin, Antithymozytenglobulin, aber auch monoklonale Marker wie Anti-CD3 hergestellt. 2. Das Wirken der Klinischen Immunologie Für Patienten mit Immundefekten und Autoimmunerkrankungen sowie für die Entwicklung von neuen immuntherapeutischen Ma�nahmen, hat die Klinische Immunologie in Graz Neuland eröffnet. Patienten mit schweren Immundefekt waren bis zur Gründung der Klinischen Immunologie nicht therapierbar, Gleiches gilt für einige Patienten mit Autoimmunerkrankungen. Vor diesem Hintergrund sehen wir uns nunmehr in der Lage, pro Jahr etwa 2.000 Patienten zu untersuchen, 20.000 Einsendungen zu bearbeiten und rund 200.000 Befunde zu erstellen. 3. Visionen der Klinischen Immunologie Durch die stark forschungsorientierte Klinische Immunologie konnten wir neue diagnostische und therapeutische Verfahren bzw. Korrelationen aufbauen, die der Zeit des gegenwärtigen medizinischen Wissensstandes vorauseilen. Damit haben wir die Empfindlichkeit erreicht, einzelne Moleküle zu messen, darüber hinaus an soliden Oberflächen unmarkierte Laserspektren zu entwickeln. Diese Kenntnisse führen uns zur visionären Aussage, da� eine Klinische Immunologie von Heute einer Medizin von Morgen entspricht.

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Abstract: Habilitation Papers: (i) ref. 49, Foldes-Papp, Z; Demel, U; Tilz, GP. Ultrasensitive detection and identification of fluorescent molecules by FCS: impact for immunobiology. Proc Natl Acad Sci U S A. 2001; 98(20):11509-11514 [OPEN ACCESS] PubMed FullText FullText_MUG Abstract: An experimental application of fluorescence correlation spectroscopy is presented for the detection and identification of fluorophores and auto-Abs in solution. The recording time is between 2 and 60 sec. Because the actual number of molecules in the unit volume (confocal detection volume of about 1 fl) is integer or zero, the fluorescence generated by the molecules is discontinuous when single-molecule sensitivity is achieved. We first show that the observable probability, N, to find a single fluorescent molecule in the very tiny space element of the unit volume is Poisson-distributed below a critical bulk concentration c*. The measured probability means we have traced, for example, 5 x 10(10) fluorophore molecules per ml of bulk solution. The probability is related to the average frequency, C, that the volume of detection contains a single fluorescent molecule and to the concentration, c, of the bulk solution. The analytical sensitivity of an assay is calculated from the average frequency C. In the Goodpasture experiment, we determined as analytical sensitivity a probability of 99.1% of identifying one single immune complex. Under these conditions, a single molecule event is proven. There exist no instrumental assumptions of our approach on which the experiment itself, the theoretical background, or the conclusion are based. Our results open up a broad field for analytics and diagnostics in solution, especially in immunology. ----------------------------------------------------------------------------------------- (ii) ref. 73, Foldes-Papp, Z; Demel, U; Tilz, GP. A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 1. Theory and a first application. J Immunol Methods. 2004; 286(1-2):1-11 PubMed FullText FullText_MUG Abstract: Just because there is an average of one molecule in the observation volume of a solution or membrane (single-phase), one cannot say that this is an individual molecule since many different single molecules measured one by one or the same single, individual molecule not leaving the detection volume on time average can cause a single-molecule event. The latter case is of interest and allows the continuous observation of one and the same single molecule without averaging over many 'different' single molecules. For the first time a universal theoretical and experimental framework is presented for the continuous observation of the same single, individual molecule without immobilization, hydrodynamic flow, or burst size histograms of fluorescence intensity traces. In this original article, the stochastic approach is derived and its main characteristics are demonstrated with the free fluorophore rhodamine-green in solution for simpler experimental realization. Single (solution)-phase single-molecule fluorescence auto- (or two-color cross-) correlation spectroscopy (SPSM-FCS) is used as a specific application in order to count the absolute number of molecules in the observation volume. The absolute number of molecules, the diffusion coefficient of the single fluorescent molecule, the lower limit of distance, and the molar concentration of the bulk phase (solution) were directly obtained from the measured auto- or (cross)-correlation curves of the SPSM-FCS experiments. For this purpose, the detection volume that was measured was less then 1 fl (10(-15) l). Then, a concentration of the bulk solution was chosen in such a way that the probability of detecting more than one molecule in the detection volume was very small. The Poisson probability was experimentally determined for the absolute number of molecules depending upon a specified bulk concentration. From the diffusion coefficient of the molecule, it was found that the probability of the molecule diffusing out of the probe volume during the measurements was negligibly small. ----------------------------------------------------------------------------------------- (iii) ref.74, Foldes-Papp, Z; Demel, U; Tilz, GP. A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 2. The individual immune molecule. J Immunol Methods. 2004; 286(1-2):13-20 PubMed FullText FullText_MUG Abstract: In the accompanying original article, the universal theoretical and experimental framework was developed for quantifying one and the same single (selfsame), individual fluorescent-tagged biological molecule without immobilization, hydrodynamic flow or photon burst analysis of fluorescence intensity traces. In the present original article, we describe an application to the detection and identification of circulating anti-glomerular basement membrane antibodies (BMAs) in Goodpasture syndrome. The same single, individual two-color molecule complex was observed among many other molecules. The molecule consisted of the green-tagged antigen, sandwiched autoantibody and red-tagged secondary (detecting) antibody. A 200-fold increase in sensitivity was obtained as compared to the conventional ELISAs on solid phase. This novel concept has several advantages, namely (i) the sensitivity to detect an individual molecule in solution; (ii) the association of the signal with the reaction event, independent of any immobilization procedure and the artifacts thereof; (iii) the assessment of the broad field of natural antibodies. The theoretical and experimental results obtained bring advanced ultrasensitive analytics to the direct investigation of one and the same single, individual immune molecules as exemplified by the experiments performed with Goodpasture antibody. The novel universal theoretical and experimental framework for continuous measuring the same single, individual immune molecule can be readily transferred to other applications. Authors Med Uni Graz: ----------------------------------------------------------------------------------------- (iv) ref. 80, Foldes-Papp, Z; Kinjo, M; Tamura, M; Birch-Hirschfeld, E; Demel, U; Tilz, GP. A new ultrasensitive way to circumvent PCR-based allele distinction: direct probing of unamplified genomic DNA by solution-phase hybridization using two-color fluorescence cross-correlation spectroscopy. Exp Mol Pathol. 2005; 78(3):177-189 PubMed FullText FullText_MUG Abstract: Single-molecule fluorescence methods enable a new class of nucleic acid assays to be performed that are not possible with PCR-based methods. In this basic study, the methylene tetrahydrofolate reductase (MTHFR)-genotypes (normal, homozygous mutated, as well as heterozygous mutated) were directly detected for the first time onto unamplified double-stranded genomic DNA in solution down to femtomolar allele concentrations (10(-15) M) in a homogeneous assay format. This was accomplished by taking advantage of the decrease by a factor of 40 to 100 in fluorescence background signals of the non-bound nonlinear hybridization probes in two colors and two-color fluorescence cross-correlation spectroscopy. The designed 'intelligent' probes contained the built-in 5'-fluorescent dyes rhodamine green and Alexa633, respectively, and the 3'-non-fluorescent quenchers BHQ1 and BHQ3, respectively, with perfectly matched spectral overlaps for both dye-quencher combinations. Upon binding of two appropriate probes that were sequence-specific for the genotype, the steady-state fluorescence in two colors increased by about two orders of magnitude. The obtained allele sensitivity of femtomolar and the specificity of the described molecular interactions allow PCR-based allele distinction to be circumvented. Furthermore, the results present an alternative to existing hybridization approaches that are currently used with and without amplification at the 'many-molecule' level and the 'single-molecule' level. ===================================================================================================================================================== And ten additional papers of mine after MD and PhD graduations: ref. paper 37, Foldes-Papp, Z; Angerer, B; Ankenbauer, W; Rigler, R. Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide. J Biotechnol. 2001; 86(3):237-253 PubMed FullText FullText_MUG Abstract: The enzymatic incorporation of deoxyribonucleoside triphosphates by a thermostable, 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase was studied for PCR-based high-density labeling of 217-bp "natural" DNA in which fluorescent-dUTP was substituted completely for the normal dTTP. The amplified DNA carried two different sorts of tethered dye molecules. The rhodamine-green was used for internal tagging of the DNA. Since high-density incorporation of rhodamine-green-X-dUTP led to a substantial reduction (quenching) of the rhodamine-green fluorescence, a second "high" quantum yield label, Cy5, was inserted via a 5'-tagged primer in order to identify the two-color product. A theoretical concept of fluorescence auto- and cross-correlation spectroscopy developed here was applied to quantify the DNA sequence formed in terms of both the number of two-color fluorescent molecules and the number of covalently incorporated rhodamine-green-X-dUMP residues. The novel approach allowed to separate optically the specific DNA product. After complete, exonucleolytic degradation of the two-color DNA we determined 82-88 fluorescent U* labels incorporated covalently out of 92 maximum possible U* incorporations. The heavily green-labeled DNA was then isolated by preparative mobility-shift electrophoresis, re-amplified in a subsequent PCR with normal deoxyribonucleoside triphosphates, and re-sequenced. By means of this novel methodology for analyzing base-specific incorporations that was first developed here, we found that all fluorescent nucleotides and the normal nucleotides were incorporated at the correct positions. The determined labeling efficiency of 0.89-0.96 indicated that a fraction of the substrate analog was not bearing the fluorophore. The results were used to guide developments in single-molecule DNA sequencing. The labeling strategy (principal approach) for PCR-based high-density tagging of DNA, which included an appropriate thermostable DNA polymerase and a suitable fluorescent dye-dNTP, was developed here. ----------------------------------------------------------------------------------------- ref. paper 38, Foldes-Papp, Z; Angerer, B; Thyberg, P; Hinz, M; Wennmalm, S; Ankenbauer, W; Seliger, H; Holmgren, A; Rigler, R. Fluorescently labeled model DNA sequences for exonucleolytic sequencing. J Biotechnol. 2001; 86(3):203-224 PubMed FullText FullText_MUG Abstract: We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent dyes. Firstly, for "natural" template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucleolytic degradation by the 3'-->5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s(-1) at 16 degrees C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently incorporated into the antisense strand of "synthetic" 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occurring dNTPs. The 218-b DNA constructs were labeled by PCR with a thermostable 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase which we have selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodology of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the paper Földes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing project. ----------------------------------------------------------------------------------------- ref. paper 42, Foldes-Papp, Z; Rigler, R. Quantitative two-color fluorescence cross-correlation spectroscopy in the analysis of polymerase chain reaction. Biol Chem. 2001; 382(3): 473-478. PubMed Abstract: We present results of an approach in which low-density labeled DNA itself provides an amplification of the cross-correlated fluorescent signal in the two-color cross-correlation function. Tetramethylrhodamine-4-dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp target DNA. We call this novel approach the 'two-color FCS signal amplification'. The signal amplification is an example for interactions of two ligands with different colors at multiple positions of the same target. ----------------------------------------------------------------------------------------- ref. paper 65, Foldes-Papp, Z; Kinjo, M; Saito, K; Kii, H; Takagi, T; Tamura, M; Costa, JM; Birch-Hirschfeld, E; Demel, U; Thyberg, P; Tilz, GP. C677T single nucleotide polymorphisms of the human methylene tetrahydrofolate reductase and specific identification : a novel strategy using two-color cross-correlation fluorescence spectroscopy. Mol Diagn. 2003; 7(2): 99-111. PubMed Abstract: BACKGROUND: A methylene tetrahydrofolate reductase (MTHFR) deficiency at site C677T renders the enzyme thermolabile and consequently represents a risk factor for vascular disease, neural tube defects, preeclampsia, and thrombosis. Highly specific identification techniques for genotyping are mandatory to give guidance for the diagnosis and monitoring of this deficiency. METHODS: A new approach for performing genotyping has been introduced with the identification of single nucleotide polymorphisms of the human MTHFR. It is based on PCR followed by two-color cross-correlation fluorescence spectroscopy (FCS). Experiments were carried out with green- and red-tagged allele-specific primers, which were fully compatible with the two-color fluorescence cross-correlation setup at 488 nm and 633 nm excitation wavelengths. RESULTS: The measured data of the amplification mixes (tubes) were normalized as the maximum correlation amplitude of each tube. Correlated and uncorrelated data were optically separated in the amplification mixes by their characteristic correlation times, which significantly differed from each other. The correlated data were generated in the presence of the proper mutated genotype template, whereas uncorrelated data were due to the absence of the proper genotype template. Furthermore, the specific association of the two-color fluorescence correlated signals with the target DNA was experimentally proven. Using this novel two-color cross-correlation approach, the MTHFR genotypes, which were determined in 21 clinical samples, showed concordance with methods involving a PCR-based assay with hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxy-fluorescein (FAM)-tagged allele-specific primers and a subsequent separation step with capillary electrophoresis, yet are simpler to perform. There was no evidence of a central trend of false-positive or false-negative results. We demonstrated how the novel, ultrasensitive typing system could be applied to studies where researchers are trying to perfect their assays and are often working with the unknown, or application to problematic assays in a clinical environment for those involved in molecular diagnosis. CONCLUSIONS: We present an alternative method to those commonly used in genotyping. Two-color cross-correlation FCS allows the detection of the fluorescence signals specifically associated with the heterozygous mutated, the homozygous mutated, and normal individuals, as exemplified in this study. The presence of nonspecific amplification products, which interfere with subsequent DNA analysis, could therefore highlight the need for two-color cross-correlation FCS as a means of discriminating between specific association of the fluorescence signals with the target DNA and DNA not related to the target. ----------------------------------------------------------------------------------------- ref. paper 75, Foldes-Papp, Z; Costa, JM; Demel, U; Tilz, GP; Kinjo, M; Saito, K; Kii, H; Takagi, T; Tamura, M; Thyberg, P; Birch-Hirschfeld, E. Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis. Exp Mol Pathol. 2004; 76(3):212-218 PubMed FullText FullText_MUG Abstract: Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born. ----------------------------------------------------------------------------------------- ref. paper 71, Foldes-Papp, Z; Baumann, G; Demel, U; Tilz, GP. Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics. Curr Pharm Biotechnol. 2004; 5(2):163-172 PubMed FullText Abstract: Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed. ----------------------------------------------------------------------------------------- ref. paper 60, Foldes-Papp, Z. Theory of measuring the selfsame single fluorescent molecule in solution suited for studying individual molecular interactions by SPSM-FCS PTERIDINES 2002 13: 73-82. Abstract: Three exact criteria are first derived for the probabilities that determine single molecule sensitivity in single-phases, for example in solutions or membranes. It is shown how the criteria can be used to decide whether single-molecule sensitivity is obtained. Most straightforwardly, all we require is that we experimentally determine the Poisson probability for the absolute number of fluorescent molecules in the volume of observation. This is achieved by fluorescence correlation spectroscopy and allows identifying the selfsame, one single fluorescent molecule in single-phases. Further, we first provide the new and powerful analytical capability to fluorescence-tagged individual molecules and reactions in in-vitro and living systems to follow their molecular interactions and function by means of spatial stochastic behavior. This fully stochastic modeling is derived which describes the influence of spatial separation on the reaction coordinate of one single individual molecule in single-phases within the observation volume. The theoretical concept is applied to experimental data 'at the single-molecule level' of photosensitive riboflavin and riboflavin-containing blue fluorescence protein. The data were taken from the literature. As simulation results of the elucidative examples, experiments are suggested that would measure individual, single molecules of flavins and flavoproteins in solution. They are of importance for experimentalists. ----------------------------------------------------------------------------------------- ref. paper 53, Foldes-Papp, Z; Demel, U; Domej, W; Tilz, GP. A new dimension for the development of fluorescence-based assays in solution: from physical principles of FCS detection to biological applications. Exp Biol Med (Maywood). 2002; 227(5):291-300 [OPEN ACCESS] PubMed FullText Abstract: Ultrasensitive detection methods such as laser-induced fluorescence represent the current state-of-the-art in analytics. Single-molecule detection in solution has received a remarkable amount of attention in the last few years because of its applicability to life sciences. Studies have been performed on the fundamentals of the detection processes themselves and on some biological systems. Fluorescence correlation spectroscopy (FCS) is the link for ultrasensitive multicomponent analysis, showing possibilities for experiments on molecular interactions. Based on the theoretical background of FCS, this article gives full explanation of FCS and an update of highlights in experimental biology and medicine studied by FCS. We focus on a repertoire of diverse immunoglobulin specificities, a ribosome display system, single-molecule DNA sequencing, and a mutant enzyme generated by random mutagenesis of amino acids. We describe the usefulness and the enormous potential of the methodology. Further, this contribution clearly indicates that FCS is a valuable tool for solution-phase single-molecule (SPSM) experiments in immunobiology and medicine. In experiments with the Goodpasture autoantibody, we worked out conditions for the design of experiments on a complex single molecule in solution. The possibility to use SPSM-FCS as a quantitation methodology opens up other important applications beyond the scope of this article. Original results extending the published studies are presented for the rational foundation of SPSM-FCS. In this original contribution, we deal with experimental systems for biology and medicine where the number of molecules in solution is very small. This article is mandatory for gaining confidence in the interpretation of experimental SPSM-FCS results on the selfsame, individual single molecule in solution. ----------------------------------------------------------------------------------------- ref. paper 61, Foldes-Papp, Z. Where is the field of Clinical Immunology going to go: assays to rapidly detect and assess human mutations. Mod. Asp. Immunobiol. 2 (5), 224-227. 2002 ----------------------------------------------------------------------------------------- ref. paper 83, Foldes-Papp, Z; Baumann, G; Kinjo, M; Tamura, M . Single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS). Distinguished article entry. In: Fuchs, J; Podda, M; editors(s). Encyclopedia of Medical Genomics & Proteomics. New York: Marcel Dekker, 2005.

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Abstract: SFI encourages the efficient use, renewal and development of existing national research infrastructure. Earlier this year, Science Foundation Ireland launched a �Call for Research Infrastructure 2012� in recognition of the need for continued investment in cutting edge research equipment and infrastructure, not otherwise reasonably accessible, in areas of national priority of particular relevance to SFI�s prior and future investments. Emphasis is being placed on collaborative efforts, shared facilities and on liaison with industry with the goal of strengthening and sustaining Ireland�s research base for all stakeholders.