Wolfgang Budach

Gender differences in kidney function

Sex hormones influence the development of female (F) and male (M) specific traits and primarily affect the structure and function of gender-specific organs. Recent studies also indicated their important roles in regulating structure and/or function of nearly every tissue and organ in the mammalian body, including the kidneys, causing gender differences in a variety of characteristics. Clinical observations in humans and studies in experimental animals in vivo and in models in vitro have shown that renal structure and functions under various physiological, pharmacological, and toxicological conditions are different in M and F, and that these differences may be related to the sex-hormone-regulated expression and action of transporters in the apical and basolateral membrane of nephron epithelial cells. In this review we have collected published data on gender differences in renal functions, transporters and other related parameters, and present our own microarray data on messenger RNA expression for various transporters in the kidney cortex of M and F rats. With these data we would like to emphasize the importance of sex hormones in regulation of a variety of renal transport functions and to initiate further studies of gender-related differences in kidney structure and functions, which would enable us to better understand occurrence and development of various renal diseases, pharmacotherapy, and drug-induced nephrotoxicity in humans and animals.

Novel bioaffinity sensors for trace analysis based on luminescence excitation by planar waveguides

Abstract We have developed a novel generation of optical bioaffinity sensors for ultra trace analysis. These sensors are based on luminescence generation in the evanescent field of high-refractive-index single-mode planar waveguides, With the waveguiding layers and the grating parameters chosen, very sharp discrimination of bulk against surface-confined excitation, in combination with high excitation intensities in the evanescent field, can be achieved, leading to unprecedented sensitivity. Experimental data of the optimization of the transducer parameters will be presented. Incoupling of excitation light is performed using diffractive gratings. Different methods for the detection of both transmitted and luminescence light will be presented. The transmitted excitation light can be detected either at the distal waveguide chip end or using a second outcoupling grating. Both isotropically emitted luminescence, collected by a lens located below the transducer substrate (‘volume detection’), and emission coupled back into the waveguiding layer can be monitored, the latter via a second outcoupling grating. First experimental results obtained in model bioaffinity assays will be presented, demonstrating the feasibility of the different detection methods mentioned above, as well as the superior sensitivity of our novel sensor configuration. In still preliminary experiments, 100 attomoles of fluorescently labelled DNA (16-mer oligonucleotide), applied at 100 femtomolar concentration, can be detected.

Evanescent resonator chips: a universal platform with superior sensitivity for fluorescence-based microarrays

In the present paper, we introduce for the first time a novel generation of a universal fluorescence transducer, the so-called evanescent resonator (ER) platform. The device comprises a transparent substrate and a thin dielectric surface layer containing sub-micron corrugated structures. The ER chip exhibits an inherent physical signal amplification due to confinement of excitation energy in the thin surface layer. Energy confinement is based on interference effects created by the abnormal reflection geometry and leads to efficient excitation of surface-bound fluorophores in the evanescent field of the chip. The evanescent resonator platform has the potential to increase the fluorescence yield of labelled biomolecules to more than 100-fold when compared with conventional microarray chips. The new ER device has been developed for analysis of nucleic acids from different species. However, it can be used with all kinds of biomolecular affinity systems. The platform combines superior sensitivity with exceptional reproducibility and ease of use. The chips are compatible with commercially available laser scanners, confocal microscopes, and portable or miniaturised CCD read-out equipment.

Development of a Scale-Down Model of hydrodynamic stress to study the performance of an industrial CHO cell line under simulated production scale bioreactor conditions.

The objective of this study was to develop a Scale-Down Model of a hydrodynamic stress present in large scale production bioreactors to investigate the performance of CHO cells under simulated production bioreactor conditions. Various levels of hydrodynamic stress were generated in 2L bioreactors mimicking those present in different locations of a large scale stirred tank bioreactor. In general, it was observed that tested cells are highly robust against the effect of hydrodynamic stress. However, at elevated hydrodynamic stress equivalent to an average energy dissipation rate, ε, equal to 0.4W/kg, the specific monoclonal antibody productivity, qmAb, decreased by 25% compared to the cultivation conditions corresponding to ε equal to 0.01W/kg. Even stronger decrease of qmAb, in the order of 30%, was observed when ε was periodically oscillating between 0.01 and 0.4W/kg to simulate the repeated passage of cells through the highly turbulent impeller discharge zone of a production scale bioreactor. Despite this effect, no changes in metabolite consumption or byproduct formation were observed. Furthermore, considering the experimental error product quality was independent of the applied ε. To achieve a molecular insight into the observed drop of cellular productivity, a transcriptome analysis using mRNA microarrays was performed. It was found that transcripts related to DNA damage and repair mechanisms were upregulated when high ε was applied for cultivation.

Adaptation for survival: Phenotype and transcriptome response of CHO cells to elevated stress induced by agitation and sparging

In this work, the response and adaption of CHO cells to hydrodynamic stress in laboratory scale bioreactors originating from agitation, sparging and their combination is studied experimentally. First, the maximum hydrodynamic stress, τ(max), is characterized over a broad range of operating conditions using a shear sensitive particulate system. Separate stress regimes are determined, where τ(max) is controlled either by sparging, agitation, or their combination. Such conditions are consequently applied during cultivations of an industrial CHO cell line to determine the cellular responses to corresponding stresses. Our results suggest that the studied CHO cell line has different threshold values and response mechanisms for hydrodynamic stress resulting from agitation or sparging, respectively. For agitation, a characteristic local minimum in viability was found after stress induction followed by viability recovery, while at highest sparging stress a monotonic decrease in viability was observed. If both stresses were combined, also both characteristic stress responses could be observed, amplifying each other. On the other hand, cellular metabolism, productivity and product quality did not change significantly. Transcriptome analysis using mRNA microarrays confirmed that separate adaptation mechanisms are activated in the different stress situations studied, allowing identification of these stresses using a transcriptome fingerprinting approach. Functional analysis of the transcripts was consequently used to improve our understanding of the molecular mechanisms of shear stress response and adaptation.

Novel generation of luminescence-based biosensors: single-mode planar waveguide sensors

First results with a new generation of bioaffinity sensors based on luminescence excitation using single-mode planar waveguides are presented. Planar waveguides show superior physical characteristics, concerning sensitivity and required sample volumes, and can principally be fabricated in low-costs mass production processes. Additionally, they allow for different detection geometries, e.g., different configurations of fluorescence detection and simultaneous determination of the transmitted excitation light. The transducer characteristics, possible detection geometries and our present experimental configuration are explained. Results with different bioaffinity systems applied on the novel transducer generation are presented, demonstrating the capability of detecting attomole amounts of analytes within a few minutes.

Planar waveguides as efficient transducers for bioaffinity sensors

Specific detection of extremely low amounts of antigens or disease markers allowing the diagnosis of diseases and infections at a very early stage has become a major driving force for the development of new generations of biochemical sensors. To match this goal, we propose the substitution of the widely used fiber-shaped evanescent field sensors by planar single-mode metal oxide waveguides. In combination with bioaffinity assays, this transducer geometry offers benefits such as enhanced sensitivity, ease of sensor handling and preparation, sample volume reduction, versatility, and low cost per test. Recently, planar waveguides have been used in sensor schemes exploiting the changes of the so-called effective refractive index (caused by changes in mass of surface-bound biomolecules): grating couplers, surface plasmon resonance, and interferometers. However, compared to luminescence-based sensor schemes, the sensitivities of these label-free methods are inferior. In this paper we report on a new generation of luminescence- based bioaffinity sensors for human diagnostics including optimization of the planar evanescent field transducers, the design of a compact sensor system, and a first study of the binding of fluorophore-labeled IgG to protein A immobilized on the transducer.