Transcriptome and proteome dynamics during the embryonation of Fasciola hepatica eggs

Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite’s communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs at different ages and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Using these methods, we observed significant changes in the composition of peptidases during egg maturation.

Spatial patterns of ectoenzymatic kinetics in relation to biogeochemical properties in the Mediterranean Sea and the concentration of the fluorogenic substrate used

Ectoenzymatic activity, prokaryotic heterotrophic abundances and production were determined in the Mediterranean Sea. Sampling was carried out in the sub-surface, the deep chlorophyll maximum layer (DCM), the core of the Levantine intermediate waters and in the deeper part of the mesopelagic layers. Michaelis–Menten kinetics were assessed using a large range of concentrations of fluorogenic substrates (0.025 to 50 µM). As a consequence, Km (Michaelis–Menten half-saturation constant) and Vm (maximum hydrolysis velocity) parameters were determined for both low- and high-affinity enzymes for alkaline phosphatase, aminopeptidase (LAP) and β-glucosidase (βGLU). Based on the constant derived from the high-LAP-affinity enzyme (0.025–1 µM substrate concentration range), in situ hydrolysis of N proteins contributed 48 % ± 30 % to the heterotrophic bacterial nitrogen demand within the epipelagic layers and 180 % ± 154 % in the Levantine intermediate waters and the upper part of the mesopelagic layers. The LAP hydrolysis rate was higher than bacterial N demand only within the deeper layer and only when considering the high-affinity enzyme. Based on a 10 % bacterial growth efficiency, the cumulative hydrolysis rates of C proteins and C polysaccharides contributed on average 2.5 % ± 1.3 % to the heterotrophic bacterial carbon demand in the epipelagic layers sampled (sub-surface and DCM). This study clearly reveals potential biases in current and past interpretations of the kinetic parameters for the three enzymes tested based on the fluorogenic-substrate concentration used. In particular, the LAP / βGLU enzymatic ratios and some of the depth-related trends differed between the use of high and low concentrations of fluorogenic substrates.

Nigella sativa itself and as a part of a balm inhibits activity of purified 20S proteasome

Background. Ubiquitin-dependent proteasomal proteolysis is the perspective target for the therapy of the high amount of different diseases. the special attention is paid to the therapy of inflammatory processes of both origins: infection and aseptic. Besides proteolytic processing of the inhibitor of NFkappaB proteasome is important for activation of polymorphonuclear neutrophils (PMN) and neutrophil extrcellular traps (NETs) formation. Basing on the mentioned above information we have evaluated some plant extracts included in the medical compound «Healthy Tonsils» that is used for the treatment of the chronic tonsillitis for their ability to inhibit activity of 20S proteasome. Methods. For in vitro experiments the purified 20S proteasome was used. 20S proteasome was incubated for 30 minutes at 36 C degrees in a mixture with phytobalm components. After the next 30 minutes of incubation with 6 microM solution of corresponded fluorogenic substrates the measurement of fluorescence products was performed (exciting/emission wave length was 360/440) on the spectrofluorimeter Hitachi-4000 with the use of free 7-amido-4-methylcoumarine for a standard curve. Results. In the same concentrations the compounds investigated has demonstrated different ability to inhibit activity of purified 20S proteasome. The most effective compound was the extract of Nigella sativa that inhibits chemotrypsin-like and caspase-like activities of purified 20S proteasome on 69 % and 85 % correspondingly. Conclusions. Thus, our results allow to explaine the anti-inflammatory activity of phytobalm particularly by ability of some of its compounds to inhibit proteasomal proteolysis.

VEGF Detection via Simplified FLISA Using a 3D Microfluidic Disk Platform

Fluorescence-linked immunosorbent assay (FLISA) is a commonly used, quantitative technique for detecting biochemical based on antigen–antibody binding reactions using a well-plate platform. With the developments in the manufacturing technology of microfluidic systems, FLISA can be implemented onto microfluidic disk platforms, which allows the detection of trace biochemical with high resolutions. Apart from requiring a lower proportion of reagent (1/10), this method also reduces the time required for the entire process to less than an hour. The incubation process involves antigen–antibody binding reactions as well as the binding of fluorogenic substrates to target proteins. The protocol for FLISA on a microfluidic platform necessitates the appropriate execution of liquid reagent movements during each step in order to ensure sufficient binding reactions. Herein, we propose a novel microfluidic disk comprising a 3D incubation chamber. Vascular endothelial growth factor as concentration with ng mL-1 is detected sequentially using a benchtop process employing this 3D microfluidic disk. The 3D microfluidic disk is implemented without requiring manual intervention or additional procedures for liquid control. During the incubation process, microbead movement is controlled through centrifugal force, generated due to disk rotation, and gravitational force via bead sedimentation on the sloped floor of the chamber.

Synthesis and evaluation of sensitive coumarin-based fluorogenic substrates for discovery of α-N-acetyl galactosaminidases through droplet-based screening

Synthesis of sensitive coumarin α-GalNAc glycosides as substrates for droplet-based screening of GalNAcases.

From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology

Chemical probes have been instrumental in microbiology since its birth as a discipline in the 19th century when chemical dyes were used to visualize structural features of bacterial cells for the first time. In this review article we will illustrate the evolving design of chemical probes in modern chemical biology and their diverse applications in bacterial imaging and phenotypic analysis. We will introduce and discuss a variety of different probe types including fluorogenic substrates and activity-based probes that visualize metabolic and specific enzyme activities, metabolic labeling strategies to visualize structural features of bacterial cells, antibiotic-based probes as well as fluorescent conjugates to probe biomolecular uptake pathways.

Identification of Proteinase Activated Receptor (PAR) cleaving enzymes in human osteoarthritis knee joint synovial fluids

ABSTRACTObjectiveOsteoarthritis (OA) is the most prevalent joint disorder with incidence increasing worldwide. Mechanistic insights into OA pathophysiology are still evolving and there are currently no disease-modifying OA drugs available. It is well established that an increase in proteolytic enzyme activity is linked to progressive degradation of the cartilage in OA. Proteolytic enzymes can also trigger inflammation through activation of a family of G-protein coupled receptors (GPCRs) called the Proteinase Activated Receptors (PARs). Here we sought to characterize the PAR activating enzyme repertoire in human OA knee joint fluids.MethodsHuman knee joint synovial fluids derived from twenty-five OA patients and four healthy donors were screened for PAR cleavage activity using novel genetically encoded human PAR biosensor expressing cells. The class or type of enzymes cleaving the PARs was further characterized using enzyme-selective inhibitors and enzyme-specific fluorogenic substrates.ResultsActivity of PAR1, PAR2 and PAR4 activating enzymes were identified at substantially different levels in OA patients relative to healthy knee joint synovial fluids. Using enzyme class or type selective inhibitors and fluorogenic substrates we found that serine proteinases, including thrombin-like enzymes, trypsin-like enzymes, and matrix metalloproteinases are the major PAR activating enzymes present in the OA knee synovial fluids.ConclusionsMultiple enzymes activating PAR1, PAR2 and PAR4 are present in OA joint fluids. PAR signalling can trigger pro-inflammatory responses and targeting PARs has been proposed as a therapeutic approach in OA. Knowledge of the PAR activators present in the human knee joint will guide study of relevant signaling events and enable future development of novel PAR targeted therapies for OA and other inflammatory joint diseases.