Rolandas Meškys

α-Synuclein Reactive Antibodies as Diagnostic Biomarkers in Blood Sera of Parkinson's Disease Patients

Background Auto-antibodies with specificity to self-antigens have been implicated in a wide variety of neurological diseases, including Parkinsons (PD) and Alzheimers diseases, being sensitive indicators of neurodegeneration and focus for disease prevention. Of particular interest are the studies focused on the auto-immune responses to amyloidogenic proteins associated with diseases and their applications in therapeutic treatments such as vaccination with amyloid antigens and antibodies in PD, Alzheimers disease and potentially other neurodegeneration ailments. Methodology/Principal Findings Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies – α-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance. We found significantly higher antibody levels towards monomeric α-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression (P<0.0001). This indicates potential protective role of autoimmunity in maintaining the body homeostasis and clearing protein species whose disbalance may lead to amyloid assembly. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards α-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression (P<0.0001). Pooled IgGs from PD patients and controls interacted also with the amyloid fibrils of Aβ (1–40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to the generic amyloid conformational epitope, displaying higher specificity towards human amyloid species associated with neurodegeneration. Conclusions/Significance Our findings may suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein – α-synuclein can be of value in the development of treatment and diagnostic strategies, especially during the early disease stages.

4-Ferrocenylphenol as an electron transfer mediator in PQQ-dependent alcohol and glucose dehydrogenase-catalyzed reactions

Abstract Enzyme electrodes containing pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) as a biological component in combination with 4-ferrocenylphenol ( 1 ) as an electron transfer mediator between PQQ and a carbon electrode were constructed and used for measurements of ethanol and d -glucose. Analysis of the current response of the carbon electrodes modified with 1 at different pH and potentials demonstrated that 1 participates in the bioelectrocatalytic oxidation of d -glucose or ethanol. The biosensors showed the highest response at pH 5.5 and the working potentials of 0.3 and 0.4 V (versus Ag|AgCl) for ADH and GDH, respectively. The electrocatalytic processes under such conditions at these electrodes are characterized by the apparent values of the Michaelis constants K M app of 7.1 and 13 mM and the maximal current density j max 40 and 26 μA cm −2 for ethanol and d -glucose, respectively. No electrocatalysis was found when glucose oxidase from Aspergillus niger was used instead of GDH.

Regulation of a Novel Acidithiobacillus caldus Gene Cluster Involved in Metabolism of Reduced Inorganic Sulfur Compounds

ABSTRACT Acidithiobacillus caldus has been proposed to play a role in the oxidation of reduced inorganic sulfur compounds (RISCs) produced in industrial biomining of sulfidic minerals. Here, we describe the regulation of a new cluster containing the gene encoding tetrathionate hydrolase (tetH), a key enzyme in the RISC metabolism of this bacterium. The cluster contains five cotranscribed genes, ISac1, rsrR, rsrS, tetH, and doxD, coding for a transposase, a two-component response regulator (RsrR and RsrS), tetrathionate hydrolase, and DoxD, respectively. As shown by quantitative PCR, rsrR, tetH, and doxD are upregulated to different degrees in the presence of tetrathionate. Western blot analysis also indicates upregulation of TetH in the presence of tetrathionate, thiosulfate, and pyrite. The tetH cluster is predicted to have two promoters, both of which are functional in Escherichia coli and one of which was mapped by primer extension. A pyrrolo-quinoline quinone binding domain in TetH was predicted by bioinformatic analysis, and the presence of an o-quinone moiety was experimentally verified, suggesting a mechanism for tetrathionate oxidation.

Oxygen Insensitive Glucose Biosensor Based on PQQ-Dependent Glucose Dehydrogenase

ABSTRACT Oxygen insensitive glucose biosensors based on PQQ-dependent glucose dehydrogenase from Erwinia sp. 34-1 and carbon paste have been designed. Without special precautions the sensitivity of the biosensors with the immobilized enzyme was low. Several methods of enzyme immobilization were compared. The enzyme was immobilized in the polylysine-albumin gel and in a paste consisting of water insoluble organic mediator, chemically modified carbon powder, fumed silica and binding material. Chemical modification of carbon with quinonic water insoluble mediators and application of fumed silica in the paste considerably improves the reproducibility, stability and sensitivity of the biosensors. The anodic response current of the biosensor on glucose was recorded at 0-200 mV vs Ag/AgCl reference electrode. Sensitivity can be increased more than 100 times in the presence of soluble mediators, such as phenazine methosulphate. Linearity of the biosensor can be extended by coating the enzymatic layer of the biose...

Ultrathin silver nanowires produced by amyloid biotemplating.

By using a self‐assembled amyloid from lysozyme as biotemplate we produced an ultrathin silver wire of 1 nm diameter and up to 2 μm in length, which is at the limit attainable in nanobiotechnological manufacturing. We showed that 2,2,2‐trifluoroethanol produces a dual effect: it reduces ionic silver to colloidal nanoparticles with a regular size, depending on the length of incubation, and induces fibrillar assembly into the amyloid scaffold, forming the hollow channel filled with silver.

Modified graphitized carbon black as transducing material for reagentless H2O2 and enzyme sensors

Direct electron transfer between redox enzymes and electrodes is the basis for the third generation biosensors. We established direct electron transfer between quinohemoprotein alcohol dehydrogenase (PQQ-ADH) and modified carbon black (CBs) electrodes. Furthermore, for the first time, this phenomenon was observed for pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH). Reagentless enzyme biosensors suitable for the determination of ethanol, glucose and sensors for hydrogen peroxide were designed using CB electrodes and screen-printing technique. Aiming to create an optimal transducing material for biosensors, a set of CB batches was synthesized using the matrix of Plackett-Burman experimental design. Depending on the obtained surface functional groups as well as the nano-scale carbon structures in CBs batches, the maximal direct electron transfer current of glucose and ethanol biosensors can vary from 20 to 300 nA and from 30 to 6300 nA for glucose and ethanol, respectively. Using modified CB electrodes, an electrocatalytic oxidation of H(2)O(2) takes place at more negative potentials (0.1-0.4V versus Ag/AgCl). Moreover, H(2)O(2) oxidation efficiency depends on the amount and morphology of fine fraction in the modified CBs.

Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae

At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family.

Improvement of screen-printed carbon electrodes by modification with ferrocene derivative

Abstract 4-(4-Ferrocenephenyliminomethyl)phenol (FP1) was used to modify screen-printed carbon electrodes (CEs). It was shown that FP1 efficiently mediates the electron flow from pyrroloquinoline quinone (PQQ)-dependent alcohol (ADH) or glucose dehydrogenase (GDH) to the electrode surface. This CE modification increased the stability of immobilized enzymes as well. Moreover, FP1 sufficiently lowered the oxidation/reduction potential of H 2 O 2 . The FP1-modified CEs were integrated into the flow-through amperometric cell and applied for determination of H 2 O 2 , ethanol and glucose in beverages. Good correlations ( r =0.9778 and r =0.9204 for glucose and ethanol, respectively) between results obtained by these biosensors and by refractometric or hydrometric methods were observed.

An oxygen-independent ethanol sensor based on quinohemoprotein alcohol dehydrogenase covalently bound to a functionalized polypyrrole film

In the present work the characteristics of a phenazine methosulphate mediated alcohol biosensor based on a newly isolated quinohemoprotein alcohol dehydrogenase are described. The enzyme was covalently linked at a functionalized polypyrrole film which had been electrochemically deposited on the surface of a platinum-black electrode. The biosensor architecture developed was characterized with regard to sensitivity, selectivity, and long-term operational stability. Owing to the inherent properties of the new enzyme the related biosensors are oxygen-independent and exhibit improved selectivity to ethanol in contrast to alcohol biosensors based on alcohol oxidase or on cationic nicotinamide adenine dinucleotide dependent alcohol dehydrogenase.

Oxygen electroreduction catalysed by laccase wired to gold nanoparticles via the trinuclear copper cluster

Specific wiring of biocatalysts par excellence, viz. redox enzymes, to an electrode can be exploited in the fabrication of high-performance bioelectronic devices. Here we report oxygen electroreduction catalysed by Didymocrea sp. J6 laccase wired to gold nanoparticles via the trinuclear copper cluster. Bypassing the intramolecular electron transfer, which under certain conditions is the rate-limiting step of oxygen bioelectroreduction, has resulted in the fabrication of a high current density biocathode based on high-redox-potential laccase, which is able to operate in electrolytes with a broad pH range in the presence of high fluoride concentrations.