In-situ sulphide control in the anaerobic co-digestion of residual biomass from the production of penicillin and cystine.

This study was focused on the anaerobic digestion of residual biomass from the production of penicillin and l-cystine. The biogas potential tests of individual substrates and their mixture showed good anaerobic biodegradability. The highest measured specific biogas production was 712 l/kg volatile solids (VS) for penicillin biomass and 676 l/ kg VS for cystine biomass. The biogas potential tests were performed at different inoculum-to-substrate ratios (ISR) and showed that high concentrations of nitrogen and sulphur present in residual biomass have a major impact on the anaerobic processes. The long-term operation of the laboratory anaerobic reactor showed that the mono-digestion of the penicillin biomass was stable at an OLR of 1 kg/(m3.d). When co-digestion of penicillin and cystine biomass at a ratio of 0.9:0.1 (on a VS basis) and at the same OLR was enhanced, the operation of the laboratory model turned unstable. During this phase of the operation, the course of anaerobic processes was affected by ammonia and especially sulphide inhibition. Sulphide inhibition was effectively reduced by direct dosing of FeCl2 (in-situ sulphide control), at a molar iron-to-sulphur ratio of ß = 1 (mol Fe/mol S). When suppressing sulphide inhibition, the operation of the laboratory model became stable even at a co-digestion ratio of 0.5:0.5 (VS basis). The results of this work open a new scope for future applications in the anaerobic digestion of waste biomass with high sulfur content, coming from industrial fermentation processes.

Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins.

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3ß were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni2+, or Co3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3ß and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

Difficulties associated with the structural analysis of proteins susceptible to form aggregates: The case of Tau protein as a biomarker of Alzheimer's disease.

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

Identification and characterization of natural antibodies against tau protein in an intravenous immunoglobulin product.

The latest therapeutic approaches to Alzheimer disease are using intravenous immunoglobulin (IVIG) products. Therefore, the detailed characterization of target-specific antibodies naturally occurring in IVIG products is beneficial. We have focused on characterization of antibodies isolated against tau protein, a biomarker of Alzheimer's disease, from Flebogamma IVIG product. The analysis of IgG subclass distribution indicated skewing toward IgG3 in anti-tau-enriched IgG fraction. The evaluation of their reactivity and avidity with several recombinant tau forms was performed by ELISA and blotting techniques. Truncated non-phosphorylated tau protein (amino acids 155-421) demonstrated the highest reactivity and avidity index. We provide the first detailed insight into the reactivity of isolated natural antibodies against tau protein.

Identification of Coxiella burnetii surface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy.

The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment protocols, we identified 196 nonredundant proteins, including 39 predicted OMP and/or LP, 32 unknown or poorly characterized proteins, and 17 effectors of the Type IV secretion system. We additionally identified eight proteins with moonlighting activities, and several proteins apparently peripherally associated with integral or anchored OMP and/or LP.

Comparison of inhibitory effects between acetaminophen-glutathione conjugate and reduced glutathione in human glutathione reductase.

Acetaminophen overdose is the most frequent cause of acute liver injury. The main mechanism of acetaminophen toxicity has been attributed to oxidation of acetaminophen. The oxidation product is very reactive and reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). Although this conjugate has been recognized to be generally nontoxic, we have found recently that APAP-SG could produce a toxic effect. Therefore, the aim of our study was to estimate the toxicity of purified APAP-SG by characterizing the inhibitory effect in human glutathione reductase (GR) and comparing that to the inhibitory effect of the natural inhibitor reduced glutathione. We used two types of human GR: recombinant and freshly purified from red blood cells. Our results show that GR was significantly inhibited in the presence of both APAP-SG and reduced glutathione. For example, the enzyme activity of recombinant and purified GR was reduced in the presence of 4 mm APAP-SG (with 0.5 mm glutathione disulfide) by 28% and 22%, respectively. The type of enzyme inhibition was observed to be competitive in the cases of both APAP-SG and glutathione. As glutathione inhibits GR activity in cells under physiological conditions, the rate of enzyme inhibition ought to be weaker in the case of glutathione depletion that is typical of acetaminophen overdose. Notably, however, enzyme activity likely remains inhibited due to the presence of APAP-SG, which might enhance the pro-oxidative status in the cell. We conclude that our finding could reflect some other pathological mechanism that may contribute to the toxicity of acetaminophen.

Overlap of epitopes recognized by anti-carbonic anhydrase I IgG in patients with malignancy-related aplastic anemia-like syndrome and in patients with aplastic anemia.

High titers of anti-carbonic anhydrase I (anti-CA I) autoantibodies were detected in the sera of patients with malignancies who developed an aplastic anemia-like (AA-like) syndrome after a high-dose therapy (HDT) and autologous stem cell transplantation (ASCT). It was found, that the presence of these anti-CA I autoantibodies is associated with spontaneous tumor regression. The main immunodominant epitopes of carbonic anhydrase isoform I (CA I) have previously been identified using epitope extraction technique in combination with mass spectrometric detection and bioinformatic verification. Similarly, the sera of patients with bona fide aplastic anemia (AA) who poorly responded to immunosuppressive treatment with anti-thymocyte globulin (ATG) demonstrated high titers of anti-CA I antibodies. In order to reveal differences between these antibodies, we applied the same methodology of epitope mapping procedure. Surprisingly, the anti-CA I antibodies from the both groups of patients compatibly recognized the same four candidate CA I epitopes--DGLAV, NVGHS, SLKPI, SSEQL. This finding may indicate common pathophysiological mechanisms in these two syndromes. However, at this moment it remains unresolved if anti-CA I antibodies are implicated in marrow or tumor suppression or are just an epi-phenomenon.

Antibodies against carbonic anhydrase in patients with aplastic anemia.

BACKGROUND/AIMS: Antibodies against carbonic anhydrase (CA) have been detected in patients with an aplastic anemia (AA)-like syndrome after autologous stem cell transplantation. METHODS: We analyzed sera of 53 bona fide AA patients before and after treatment with anti-thymocyte globulin (ATG) or bone marrow transplantation for the presence of anti-CA antibodies. RESULTS: Anti-CA antibodies were detected in 20 patients (38%) and were associated with older age at diagnosis of AA. Antibody-positive patients showed poor response to ATG treatment (complete response 14%) and inferior long-term survival (36% at 10 years), when compared to antibody-negative patients (complete response and 10-year survival both 64%). Two thirds of patients with antibodies at diagnosis of AA became antibody negative after treatment with ATG. Clearance of the antibody did not appear to be associated with hematological improvement. CONCLUSION: Antibodies against CA are detected frequently at diagnosis of AA, and their presence identifies a subset of patients with poor response to immunosuppressive treatment.

Development of a magnetic immunosorbent for on-chip preconcentration of amyloid ß isoforms: Representatives of Alzheimer's disease biomarkers.

Determination of amyloid ß (Aß) isoforms and in particular the proportion of the Aß 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer's disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Aß peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (µIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Aß antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Aß antibodies, as well as optimizing the immobilization technique and µIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Aß IS for µIP and specific capture of 5 Aß peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Aß peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Aß peptides in human CSF sample was performed.

New monodisperse magnetic polymer microspheres biofunctionalized for enzyme catalysis and bioaffinity separations.

Magnetic macroporous PGMA and PHEMA microspheres containing carboxyl groups are synthesized by multi-step swelling and polymerization followed by precipitation of iron oxide inside the pores. The microspheres are characterized by SEM, IR spectroscopy, AAS, and zeta-potential measurements. Their functional groups enable bioactive ligands of various sizes and chemical structures to couple covalently. The applicability of these monodisperse magnetic microspheres in biospecific catalysis and bioaffinity separation is confirmed by coupling with the enzyme trypsin and huIgG. Trypsin-modified magnetic PGMA-COOH and PHEMA-COOH microspheres are investigated in terms of their enzyme activity, operational and storage stability. The presence of IgG molecules on microspheres is confirmed.

Identification of carbonic anhydrase I immunodominant epitopes recognized by specific autoantibodies which indicate an improved prognosis in patients with malignancy after autologous stem cell transplantation.

This work employs an epitope mapping of carbonic anhydrase (CA), isoform I (CA I), for detection of the main immunodominant epitopes. Our interest has arisen from an observed spontaneous tumor regression in patients who developed an aplastic anemia type syndrome after a high-dose therapy with autologous stem cell transplantation and whose sera contained high titer of anti carbonic anhydrase (anti-CA) autoantibodies. There are many indications that the presence of these autoantibodies may provide significant survival benefit for the patients. Western blot analysis confirmed strong immunoreactivity of the patients' sera with several CA isoforms and the CA I has been selected for our study as a highly abundant and widely distributed isoform. The applied analytical approach consists of specific fragmentation of CA I protein followed by immunospecific isolation of peptides reacting with polyclonal anti-CA I autoantibodies of patients in spontaneous remission. We improved the standard epitope mapping schema by incorporating the benefits of magnetic carriers and biomagnetic separation techniques. Mass spectrometry has been applied for detection and identification of epitopes and the acquired results were verified by bioinformatic tools. The candidate epitopes of CA I (NVGHS, DGLAV, SSEQL, and SLKPI) are discussed herein as potential therapeutic targets. This work highlights the usefulness of the epitope mapping technique based on magnetic microspheres for effective and rapid determination of immunodominant epitopes of the target protein.

Epitope mapping of allergen ovalbumin using biofunctionalized magnetic beads packed in microfluidic channels The first step towards epitope-based vaccines.

Specific allergen immunotherapy is frequently associated with adverse reactions. Several strategies are being developed to reduce the allergenicity while maintaining the therapeutic benefits. Peptide immunotherapy is one such approach. Methods for the simple and rapid identification of immunogenic epitopes of allergens (i.e. allergenic epitopes) are ongoing and could potentially lead to peptide-based vaccines. An epitope extraction technique, based on biofunctionalized magnetic microspheres self-organized under a magnetic field in a channel of a simple microfluidic device fabricated from polydimethylsiloxane, was applied in the isolation and identification of prospective allergenic epitopes. Similarly to chromatographic column separations, the easily replaceable plug of self-organized beads in the channel benefits especially from an even larger surface-to-volume ratio and an enhanced interaction of the surfaces with passing samples. Ovalbumin, the major protein of egg white and a typical representative of food allergens, was selected as the model molecule. Highly resistant ovalbumin was at first efficiently digested by a magnetic proteolytic reactor with trypsin treated with l-1-tosylamido-2-phenylethyl chloromethyl ketone and the second step, i.e. capture of allergenic epitopes from the mixture of peptides, was performed by a magnetic immunoaffinity carrier with orientedly immobilized rabbit anti-ovalbumin IgG molecules. Captured peptides were released with 0.05% trifluoroacetic acid. The elution fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The peptide fragment of ovalbumin HIATNAVLFFGR (m/z: 1345.75, position: 371-382) was identified as a relevant allergenic epitope in this way. Such a microfluidic magnetic force-based epitope extraction technique applied in the epitope mapping of ovalbumin has the potential to be a significant step towards developing safe and cost-effective epitope-based vaccines.

Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategy.

We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.