Designed Peptide Inhibitors of STEP Phosphatase-GluA2 AMPA Receptor Interaction Enhance the Cognitive Performance in Rats.

Cognitive impairment and learning ability of the brain are directly linked to synaptic plasticity as measured in changes of long-term potentiation (LTP) and long-term depression (LTD) in animal models of brain diseases. LTD reflects a sustained reduction of the synaptic AMPA receptor content based on targeted clathrin-mediated endocytosis. AMPA receptor endocytosis is initiated by dephosphorylation of Tyr876 on the C-terminus of the AMPAR subunit GluA2. The brain-specific striatal-enriched protein tyrosine phosphatase (STEP) is responsible for this process. To identify new, highly effective inhibitors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization, we performed structure-based design of peptides able to inhibit STEP-GluA2-CT complex formation. Two short peptide derivatives were found as efficient in vitro inhibitors. Our in vivo experiments evidenced that both peptides restore the memory deficits and display anxiolytic and antidepressant effects in a scopolamine-treated rat model. The interference peptides identified and characterized here represent promising lead compounds for novel cognitive enhancers and/or behavioral modulators.

Flow injection enzymatic biosensor for aldehydes based on a Meldola Blue-Ni complex electrochemical mediator.

Carbon nanofibers (CNF) are efficient electrode modifiers in electrochemical biosensors that enhance the electrochemical active area, induce electrocatalytic effect toward the oxidation of the enzymatic cofactor nicotinamide adenine dinucleotide (reduced form, NADH), and enable the quantitative immobilization of enzymes. Combining CNF with efficient and stable mediators radically augments the speed of electron transfer between NADH and solid electrodes and leads to electrochemical sensors characterized by high sensitivity and stability. The main aim of this work was to investigate the performance of a novel mediator for NADH with advantageously low solubility in an electrochemical detector based on a screen-printed CNF electrode as well as its potential in biosensing. Using a mediator, prepared from Meldola Blue and Ni hexamine chloride, a stable and sensitive electrochemical NADH sensor is provided with a detection limit of 0.5 µmol L-1. Further on, covalent immobilization of a recently described aldehyde dehydrogenase from the Antarctic Flavobacterium PL002 strain on the surface of the mediator-modified electrode produced a stable biosensor for the detection of aldehydes. When integrated in a flow injection analysis (FIA) setup with amperometric detection at 0.1 V vs. Ag/AgCl, the measurement of benzaldehyde with a detection limit of 10 µmol L-1 over a linear range of 30-300 µmol L-1 is possible. Determination of trace benzaldehyde impurities in a pharmaceutical excipient was also demonstrated and results compared with a chromatographic method. Graphical abstract.

Potential bacterial biomarkers for insect colonization in forensic cases: preliminary quantitative data on Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica dynamics.

For the last decades, forensic microbiology became an emerging complementary tool in criminalistics. Although the insect-microbe interactions regarding pathogen transmission were extensively studied, only scarce information is available on bacterial transfer from necrophagous insects to host tissues. Our data provides the first report on the occurrence of Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica in Lucilia illustris Meigen, 1826 (Diptera: Calliphoridae), and the quantitative dynamics of the two bacterial species along the insect life-stages and transfer to beef and pork host tissues using qPCR gyrase b specific primers. The content of both bacterial species increased along the insect life stages. W. chitiniclastica was detected in all developmental stages independent of the feeding substrate. I. indica was measurable with 102 gene copies ng-1 DNA threshold starting from the third instar larvae when feeding on beef, and from the egg stage with a 102× higher representation when using the pork substrate. The transfer of bacterial species to both tissues occurred after 3 colonization days except for I. indica that was visible in beef liver only during day 5. Considering the utilization of pork tissues as human analogues, these quantitative microbial dynamics data provides first insect-specific bacterial candidates as potential colonization biomarkers in forensic investigations.

Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation.

Eyes absent (EYA) are non-thiol-based protein tyrosine phosphatases (PTPs) that also have transcriptional co-activator functions. Their PTP activity is involved in various pathologies. Recently, we demonstrated that Src tyrosine kinase phosphorylates human EYA3 by controlling its subcellular localization. We also found EYA3's ability to autodephosphorylate, while raising the question if the two opposing processes could be involved in maintaining a physiologically adequate level of phosphorylation. Using native and bottom-up mass spectrometry, we performed detailed mapping and characterization of human EYA3 Src-phosphorylation sites. Thirteen tyrosine residues with different phosphorylation and autodephosphorylation kinetics were detected. Among these, Y77, 96, 237, and 508 displayed an increased resistance to autodephosphorylation. Y77 and Y96 were found to have the highest impact on the overall EYA3 phosphorylation. Using cell cycle analysis, we showed that Y77, Y96, and Y237 are involved in HEK293T proliferation. Mutation of the three tyrosine residues abolished the pro-proliferative effect of EYA3 overexpression. We have also identified a Src-induced phosphorylation pattern of EYA3 in these cells. These findings suggest that EYA3's tyrosine phosphorylation sites are non-equivalent with their phosphorylation levels being under the control of Src-kinase activity and of EYA3's autodephosphorylation.

Characterizing forensically important insect and microbial community colonization patterns in buried remains.

During violent criminal actions in which the perpetrator disposes of the victim's remains by burial, the analysis of insects and bacterial colonization patterns could be necessary for postmortem interval (PMI) estimation. Our research aimed to assess the decomposition process of buried rat carcasses from shallow graves (40 cm), the diversity and dynamics of insects and bacteria throughout the decomposition stages, and the environmental parameters' influence on these variations. The results provide further insight on decomposition in soil and contribute to a broader understanding of the factors involved in decomposition by qualitatively and quantitatively analysing the decomposer community (bacteria and insects). Additionally, two bacterial taxa, Enterococcus faecalis and Clostridium paraputrificum that were investigated for the first time as PMI indicators using quantitative polymerase chain reaction (qPCR) showed differential abundance over time, promising data for PMI estimation. The current study on the decomposition of buried rat carcasses in a natural environment will strengthen the current knowledge on decomposed remains from shallow graves and represents an effort to quantify insect and bacterial taxa as PMI estimators.

Collagen regulates the ability of endothelial progenitor cells to protect hypoxic myocardium through a mechanism involving miR-377/VE-PTP axis.

The possibility to employ stem/progenitor cells in the cardiovascular remodelling after myocardial infarction is one of the main queries of regenerative medicine. To investigate whether endothelial progenitor cells (EPCs) participate in the restoration of hypoxia-affected myocardium, we used a co-culture model that allowed the intimate interaction between EPCs and myocardial slices, mimicking stem cell transplantation into the ischaemic heart. On this model, we showed that EPCs engrafted to some extent and only transiently survived into the host tissue, yet produced visible protective effects, in terms of angiogenesis and protection against apoptosis and identified miR-377-VE-PTP axis as being involved in the protective effects of EPCs in hypoxic myocardium. We also showed that collagen, the main component of the myocardial scar, was important for these protective effects by preserving VE-PTP levels, which were otherwise diminished by miR-377. By this, a good face of the scar is revealed, which was so far perceived as having only detrimental impact on the exogenously delivered stem/progenitor cells by affecting not only the engraftment, but also the general protective effects of stem cells.

Expression, Purification, and Kinetic Analysis of PTP Domains.

Protein tyrosine phosphatases (PTP) are a large group of enzymes which work together with protein tyrosine kinases to control the tyrosine phosphorylation of proteins, thus playing a major role in cellular signaling. Here, we provide detailed protocols for expression and purification of the catalytic domain of RPTPµ and full length Eya3 as well as the extracellular region of PTPBR7. Methods are described for evaluation of the purity of the recombinant proteins thus obtained. For the purified Eya3 phosphatase we provide protocols for enzyme activity assay using either chromogenic, fluorescent, or peptide substrates. Determination of kinetic parameters by different graphical and computer-based procedures is also described.

Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics.

Phosphoketolases (PKs) are large thiamine pyrophosphate (TPP)-dependent enzymes playing key roles in a number of essential pathways of carbohydrate metabolism. The putative PK genes of Lactococcus lactis (Ll) and Leuconostoc mesenteroides (Lm) were cloned in a prokaryotic vector, and the encoded proteins were expressed and purified yielding high purity proteins termed PK-Ll and PK-Lm, respectively. Similarly, the PK gene of Pseudomonas aeruginosa was expressed, and the corresponding protein (PK-Pa) was purified to homogeneity. The amino acid sequences predicted on the basis of genes' nucleotide sequences were confirmed by mass spectrometry and display low relative similarities. Circular dichroism (CD) spectra of these proteins predict higher α-helix than ß-strand contents. In addition, it is predicted that PK-Ll contains tightly packed domains. Enzymatic analysis showed that all three recombinant proteins, despite their dissimilar amino acid sequences, are active PKs and accept both xylulose 5-phosphate (X5P) and fructose 6-phosphate (F6P) as substrates. However, they display substantially higher preference for X5P than for F6P. Kinetic measurements indicated that PK-Pa has the lowest Km values for X5P and F6P suggesting the highest capacity for substrate binding. PK-Ll has the largest kcat values for both substrates. Nevertheless, in terms of substrate specificity constant, PK-Pa has been found to be the most active PK against X5P. Structural models for all three analysed PKs predict similar folds in spite of amino acid sequence dissimilarities and contribute to understanding the enzymatic peculiarities of PK-Pa compared to PK-Ll and PK-Lm.

Preliminary X-ray crystallographic analysis of the D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis.

Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon-carbon bond of the specific substrate D-xylulose 5-phosphate (or D-fructose 6-phosphate) to give acetyl phosphate and D-glyceraldehyde 3-phosphate (or D-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P2(1). Diffraction data were obtained to a resolution of 2.2 A.