Revisiting classification as well as assessment associated with colon transepithelial passage

This can be accomplished with the MALDI-TOF mass spectrometry-based assay we describe here.Many proteins are regulated post-translationally by proteolytic processing. This includes plant signaling peptides that are proteolytically released from larger precursor proteins. The proteases involved in the biogenesis of signaling peptides and in regulation of other proteins by limited proteolysis are largely unknown. Here we describe how protease inhibitors that are specific for a certain class of proteases can be employed for the identification of proteases that are responsible for the processing of a given target protein. After having identified the protease family to which the processing enzyme belongs, candidate proteases and the GFP-tagged target protein are agro-infiltrated for transient expression in N. benthamiana leaves. Cleavage products are analyzed on immuno-blots and specificity of cleavage is confirmed by co-expression of class-specific inhibitors. For the identification of processing sites within the target protein, cleavage product(s) are purified by immunoprecipitation followed by polyacrylamide gel electrophoresis and analyzed by mass spectrometry.Protein expression in plants by agroinfiltration and subsequent purification is increasingly used for the biochemical characterization of plant proteins. In this chapter we describe the purification of secreted, His-tagged proteases from the apoplast of agroinfiltrated Nicotiana benthamiana using immobilized metal affinity chromatography (IMAC). We show quality checks for the purified protease and discuss potential problems and ways to circumvent them. As a proof of concept, we produce and purify tomato immune protease Pip1 and demonstrate that the protein is active after purification.Plant proteases of the legumain-type are key players in many processes along the plant life cycle. In particular, legumains are especially important in plant programmed cell death and the processing and maturation of seed storage proteins within the vacuole. Plant legumains are therefore synonymously called vacuolar processing enzymes (VPEs). Because of their dual protease and cyclase activities, plant legumains are of great interest to biotechnological applications, e.g., for the development of cyclic peptides for drug design. Despite this high interest by the scientific community, the recombinant expression of plant legumains proved challenging due to several posttranslational modifications, including (1) the formation of structurally critical disulfide bonds, (2) activation via pH-dependent proteolytic processing, and (3) stabilization by varying degrees of glycosylation. Recently we could show that LEXSY is a robust expression system for the production of plant legumains. Here we provide a general protocol for the recombinant expression of plant legumains in Leishmania cells. We further included detailed procedures for legumain purification, activation and subsequent activity assays and additionally note specific considerations with regard to isoform specific activation intermediates. This protocol serves as a universal strategy for different legumain isoforms from different source organisms.Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.Type I metacaspases are the most ubiquitous of the three metacaspase types and are present in representatives of prokaryotes, unicellular eukaryotes including yeasts, algae, and protozoa, as well as land plants. They are composed of two structural units a catalytic so-called p20 domain with the His-Cys catalytic dyad and a regulatory p10 domain. Despite their structural homology to caspases, these proteases cleave their substrates after the positively charged amino acid residues at the P1 position, just like the metacaspases of type II and type III. We present a protocol for expression and purification of the only type I protease from a secondary endosymbiosis Guillardia theta , GtMCA-I by overexpression of its gene in BL21 (DE3) E. coli cells and one-day sequential purification using nickel-affinity, ion-exchange, and size-exclusion chromatography.With a series of merits, Prussian blue analogs (PBAs) have been considered as superior cathode materials for sodium-ion batteries (SIBs). Their commercialization, however, still suffers from inferior stability, considerable [Fe(CN)6 ] defects and interstitial water in the framework, which are related to the rapid crystal growth. Herein, a "water-in-salt" nanoreactor is proposed to synthesize highly crystallized PBAs with decreased defects and water, which show both superior specific capacity and rate capability in SIBs. The air-stability, all-climate, and full-cell properties of our PBA have also been evaluated, and it exhibits enhanced electrochemical performance and higher volume yield than its counterpart synthesized via the water-based co-precipitation method. Furthermore, their highly reversible sodium-ion storage behavior has been measured and identified via multiple in situ techniques. This work could pave the way for the PBA-based SIBs in grid-scale energy-storage systems.The family Pennellidae comprises ecto- and mesoparasitic copepods on marine fishes. Although a preliminary scheme of phylogenetic relationships of pennellids based on morphological characters exists, it is difficult to objectively define character states because of their highly modified bodies and reduced appendages. This molecule-based study analysed phylogenetic relationships among seven genera and 12 species of pennellids, using 18S and 28S ribosomal DNA sequences in order to infer evolutionary trends within the family. Our molecular analysis recovered three clades (Clade-I, Peniculus; Clade-II, Haemobaphes-Lernaeocera-Phrixocephalus-Exopenna-Lernaeenicus radiatus; and Clade-III, Pennella-Lernaeenicus spp.). This result was congruent with some of the morphology-based phylogenetic relationships previously proposed but did not support a sister group comprising Exopenna, Phrixocephalus and Pennella. The second and third offshoots after the divergence of Clade-I species are characterized by reduced body tagmosis and changes in lifestyle from ectoparasites to mesoparasites. In some gill parasites of Clade-II, their sigmoid-shaped bodies and coiled egg strings have likely evolved in adaptation to the limited available space within the gill cavities of the hosts. Phrixocephalus is an eye parasite in Clade-II, which also has coiled egg strings, may have descended from an ancestral gill parasite. All species of Clade-III are characterized by the possession of a head region with processes deeply embedded into the host tissues and functioning as an anchor.Cefazolin (CFZ) is a ubiquitous antibiotic in hospital settings and has been recognized as an emerging contaminant due to its ecotoxicity. Despite the growing concern around this compound, the literature addressing feasible advanced techniques for CFZ uptake from aqueous matrices is still scarce. Thus, the objective of this work was to evaluate the adsorption of cefazolin on Spectrogel® organoclay in a batch system as an efficient remediation method. The optimization of experimental conditions was determined by a central composite rotational design. A pH study, as well as equilibrium, kinetic, and thermodynamic assays, was performed to assess the adsorption of CFZ on Spectrogel®. The kinetic and equilibrium models that best described the system were the external mass transfer resistance and Sips models, respectively. A removal efficiency above 80% was achieved, and the maximum adsorption capacity at 25 °C was 398.6 mg g-1. The post-process contaminated organoclay was thermally regenerated. The outcomes of this work indicate that Spectrogel® is an environmentally friendly adsorbent for the removal of cefazolin from wastewater.To develop high-efficiency antibiotic adsorbents, β-cyclodextrin and dopamine hydrochloride were used to modify graphene oxide to prepare a new type of ternary composite material (β-cyclodextrin/dopamine hydrochloride-graphene oxide, CD-DGO). this website The material was characterized using scanning electron microscopy, Fourier infrared spectrometry, transmission electron microscopy, and specific surface area optical analysis. Two typical sulfonamides antibiotics (sulfamethoxazole, sulfadiazine) adsorption capacity were evaluated in terms of the dosage of composite materials, the ratio of each component, and the pH of the solution. We analyzed the adsorption characteristics via adsorption kinetics and adsorption isotherms, and then investigated the stability of the adsorbent through desorption and regeneration of the adsorbent. The results show that the adsorption effect of sulfonamides antibiotics is best at pH = 2; the adsorption kinetics conform to the pseudo-second-order kinetic model, and the adsorption equilibrium follows the Langmuir adsorption isotherm; the maximum adsorption capacity of CD-DGO for sulfamethoxazole and sulfadiazine is 144 mg·g-1 and 152 mg·g-1, respectively. The material has good reusability, and the dominant force in the adsorption process is the π-π electron conjugation effect with hydrogen bonding. This offers a theoretical basis for the treatment of sulfonamides antibiotics water pollution.