Biosensor approaches for the detection of autoantibodies in human serum

The analysis of autoantibodies in human serum plays a crucial role in the diagnosis and follow-up of autoimmune diseases. The analytical tools available for the determination of these analytes, however, are still far from mature, lack standardization, and give low negative predictive values. Approaches using biosensor technology for analysis are an attractive alternative to classical techniques. In special applications, biosensors already have been proven to be effective for clinical diagnostics. This is due to the fact that real-time monitoring of the antigen/antibody interaction gives valuable information on autoantibody affinity to the respective antigenic structure. The potentials of biosensors for the serological analysis of autoantibodies are evident from the increasing number of publications on the subject. Thus, this review focuses on underlying biosensor techniques and published clinical trials. The advantages of multiplexed analyses of autoantibodies by use of microarrays are also emphasized. This promising bioanalytical technique is also particularly important for the structural identification of novel antigens.     

Measurement of mRNA specific for preprocholecystokinin in rat caudatoputamen and areas projecting to it

The concentrations of mRNA specific for preprocholecystokinin were measured with a hybridisation procedure after its extraction from rat brain samples. mRNA specific for preprocholecystokinin was not consistently found in the caudatoputamen. In contrast, its concentrations were quite high in the cingulate, subcallosal and parietal cortical areas, which are assumed to send cholecystokinin-containing axons to the caudatoputamen. Also in other areas in which cholecystokinin-pathways to the caudatoputamen may originate, such as the amygdaloid complex, the piriform cortex and the ventral tegmental area/substantia nigra, mRNA specific for preprocholecystokinin could be demonstrated. It is concluded that the caudatoputamen contains no or very few perikarya which synthetize cholecystokinin and that cholecystokinin-peptides which are found in high concentrations in this part of the basal ganglia are located in afferents.     

Laser‐induced liquid bead ion desorption‐MS of protein complexes from blue‐native gels,a sensitive top‐down proteomic approach

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.     

In vivo cervical cancer growth inhibition by genetically engineered cytotoxic T cells

Purpose: The CD44 v7/8 splice variant that is frequently expressed in cervical carcinoma and rarely expressed in normal tissues displays promising properties as a target antigen for cancer immune therapy. In this study, cytotoxic T lymphocytes (CTLs) were genetically engineered to gain CD44v7/8 target specificity. Methods: Clone 96 (CI96), an established murine cytotoxic T-cell line, and naïve murine T cells were retrovirally transduced with a fusion gene construct encoding for the single chain fragment scFv of the monoclonal antibody VFF17 and for the chain of the T-cell receptor (TCR). The therapeutic potential of genetically engineered T cells was tested in vitro and in vivo. Results: Surface expression of the chimeric TCR on infected Cl96 and naïve T cells was shown by FACS analysis. CD44v7/8-positive target cells were efficiently lysed by transduced Cl96 and naïve T cells, demonstrating the functionality and specificity of the chimeric TCR. In a xenograft BALB/c mouse model, efficient growth retardation of CD44v7/8-positive tumours was mediated by genetically engineered Cl96(VFF17)cyYZ cells. Conclusions: We were able to reprogramme the target specificity of recombinant Cl96 and naïve CTLs resulting in efficient cytolysis of CD44v7/8-positive cervical cancer cells. High transduction rates and the specific cytolysis of CD44v7/8-redirected CTLs are promising tools for an immune gene therapy approach for advanced cervical cancer.Abbreviations Ab Antibody - CTL Cytolytic T lymphocyte - mAb Monoclonal antibody - TCR T-cell receptor     

Functional gene polymorphisms in the serotonin system and traumatic life events modulate the neural basis of fear acquisition and extinction

Fear acquisition and extinction are crucial mechanisms in the etiology and maintenance of anxiety disorders. Moreover, they might play a pivotal role in conveying the influence of genetic and environmental factors on the development of a (more or less) stronger proneness for, or resilience against psychopathology. There are only few insights in the neurobiology of genetically and environmentally based individual differences in fear learning and extinction. In this functional magnetic resonance imaging study, 74 healthy subjects were investigated. These were invited according to 5-HTTLPR/rs25531 (S+ vs. L(A)L(A); triallelic classification) and TPH2 (G(-703)T) (T+ vs. T-) genotype. The aim was to investigate the influence of genetic factors and traumatic life events on skin conductance responses (SCRs) and neural responses (amygdala, insula, dorsal anterior cingulate cortex (dACC) and ventromedial prefrontal cortex (vmPFC)) during acquisition and extinction learning in a differential fear conditioning paradigm. Fear acquisition was characterized by stronger late conditioned and unconditioned responses in the right insula in 5-HTTLPR S-allele carriers. During extinction traumatic life events were associated with reduced amygdala activation in S-allele carriers vs. non-carriers. Beyond that, T-allele carriers of the TPH2 (G(-703)T) polymorphism with a higher number of traumatic life events showed enhanced responsiveness in the amygdala during acquisition and in the vmPFC during extinction learning compared with non-carriers. Finally, a combined effect of the two polymorphisms with higher responses in S- and T-allele carriers was found in the dACC during extinction. The results indicate an increased expression of conditioned, but also unconditioned fear responses in the insula in 5-HTTLPR S-allele carriers. A combined effect of the two polymorphisms on dACC activation during extinction might be associated with prolonged fear expression. Gene-by-environment interactions in amygdala and vmPFC activation may reflect a neural endophenotype translating genetic and adverse environmental influences into vulnerability for or resilience against developing affective psychopathology.     

Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by ¹H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-¹³C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with ¹³C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-¹³C]cysteine. The presence of [1-¹³C]hypotaurine and [1-¹³C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-¹³C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-¹³C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.     

Cytoprotection against lipid hydroperoxides correlates with increased glutathione peroxidase activities, but not selenium uptake from different selenocompounds

Cells cultivated under standard conditions were highly deficient in tocopherol, selenium, and glutathione peroxidase (GPx) activities. We investigated whether and to what extent the addition of different selenocompounds to growth media would alter biochemical, physiological, and pathophysiological parameters of cultured liver cells. Cellular uptake of selenium, GPx activities, and cytoprotection were measured and compared in human hepatoma cells (HepG2). Selenite and selenocystine were Se donors of high bioavailability (i.e., with these culture supplements, the increased Se uptake, induction of GPx isoenzymes, and protection of treated cells from lipid hydroperoxides were well correlated). In contrast, selenium from selenomethionine was incorporated into cellular proteins but had no effect on GPx activities or cytoprotection. The data show that not all selenium donors provide selenium, which is bioactivated to act as antioxidant. Thus, cellular selenium content, in general, did not correlate with cytoprotective activity of this trace element. However, cellular GPx activities at different times, with different concentrations, and with different Se donors always correlated with protection from lipid hydroperoxides and may, thus, represent a more reliable parameter to define adequate Se supply.     

Description and recognition of regular and distorted secondary structures in proteins using the automated protein structure analysis method

The Automated Protein Structure Analysis (APSA) method, which describes the protein backbone as a smooth line in three‐dimensional space and characterizes it by curvature κ and torsion τ as a function of arc length s, was applied on 77 proteins to determine all secondary structural units via specific κ(s) and τ(s) patterns. A total of 533 α‐helices and 644 β‐strands were recognized by APSA, whereas DSSP gives 536 and 651 units, respectively. Kinks and distortions were quantified and the boundaries (entry and exit) of secondary structures were classified. Similarity between proteins can be easily quantified using APSA, as was demonstrated for the roll architecture of proteins ubiquitin and spinach ferridoxin. A twenty‐by‐twenty comparison of all α domains showed that the curvature‐torsion patterns generated by APSA provide an accurate and meaningful similarity measurement for secondary, super secondary, and tertiary protein structure. APSA is shown to accurately reflect the conformation of the backbone effectively reducing three‐dimensional structure information to two‐dimensional representations that are easy to interpret and understand. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/β-catenin/ROS signaling dependent manner

Background

Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis.

Methods

Effects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET).

Results

In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation.

Conclusions

Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.

     

From Recombination Dynamics to Device Performance: Quantifying the Efficiency of Exciton Dissociation,Charge Separation,and Extraction in Bulk Heterojunction Solar Cells with Fluorine‐Substituted Polymer Donors

An original set of experimental and modeling tools is used to quantify the yield of each of the physical processes leading to photocurrent generation in organic bulk heterojunction solar cells, enabling evaluation of materials and processing condition beyond the trivial comparison of device performances. Transient absorption spectroscopy, “the” technique to monitor all intermediate states over the entire relevant timescale, is combined with time‐delayed collection field experiments, transfer matrix simulations, spectral deconvolution, and parametrization of the charge carrier recombination by a two‐pool model, allowing quantification of densities of excitons and charges and extrapolation of their kinetics to device‐relevant conditions. Photon absorption, charge transfer, charge separation, and charge extraction are all quantified for two recently developed wide‐bandgap donor polymers: poly(4,8‐bis((2‐ethylhexyl)oxy)benzo[1,2‐b:4,5‐b′]dithiophene‐3,4‐difluorothiophene) (PBDT[2F]T) and its nonfluorinated counterpart poly(4,8‐bis((2‐ethylhexyl)oxy)benzo[1,2‐b:4,5‐b′]dithiophene‐3,4‐thiophene) (PBDT[2H]T) combined with PC₇₁BM in bulk heterojunctions. The product of these yields is shown to agree well with the devices' external quantum efficiency. This methodology elucidates in the specific case studied here the origin of improved photocurrents obtained when using PBDT[2F]T instead of PBDT[2H]T as well as upon using solvent additives. Furthermore, a higher charge transfer (CT)‐state energy is shown to lead to significantly lower energy losses (resulting in higher V_OC) during charge generation compared to P3HT:PCBM.