Fluorescent Imaging of Extracellular Fungal Enzymes Bound onto Plant Cell Walls

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries.     

Evolutionary Overview of Molecular Interactions and Enzymatic Activities in the Yeast Cell Walls

Fungal cell walls are composed of a polysaccharide network that serves as a scaffold in which different glycoproteins are embedded. Investigation of fungal cell walls, besides simple identification and characterization of the main cell wall building blocks, covers the pathways and regulations of synthesis of each individual component of the wall and biochemical reactions by which they are cross-linked and remodeled in response to different growth phase and environmental signals. In this review, a survey of composition and organization of so far identified and characterized cell wall components of different yeast genera including Saccharomyces, Candida, Kluyveromyces, Yarrowia, and Schizosaccharomyces are presented with the focus on their cell wall proteomes.     

Effective Release of Intracellular Enzymes by Permeating the Cell Membrane with Hydrophobic Deep Eutectic Solvents

An efficient and green method is crucial for the recovery of intracellular biological products. The major drawbacks of the conventional cell disruption method are nonselectivity and enzyme denaturation. The permeability of hydrophobic deep eutectic solvents (DESs) to the cell membrane was studied, for the first time, and then hydrophobic DESs were innovatively applied to release intracellular enzymes from recombinant Escherichia coli. After optimization, a DES suspension of l -menthol/oleic acid (0.5 %, v/v) showed the highest release yield of intracellular enzyme. Compared with that released by sonication, a release yield of phospholipase D (PLD) of up to 114.58 % was achieved, and the specific activity was increased by 1.96 times. The microstructure of the cell membrane under different treatments was observed by using an electron microscope to understand the permeation of DESs to the cell membrane. The feasibility and applicability of the proposed release method in industrial applications were also demonstrated. The effective and green release method of intracellular enzymes developed herein has bright prospects for industrial application to replace traditional cell disruption methods. A preliminary study on the permeability of hydrophobic DESs to the cell membrane showed that there would be a potential application prospect of hydrophobic DESs not only in releasing intracellular contents, but also in seeking new green penetrating agents.     

Size exclusion chromatographic approach for the evaluation of processes for upgrading heavy petroleum

A new application of size exclusion chromatography for the evaluation of processes for upgrading heavy crudes is described. The comparison of the elution curves of a feedstock of heavy crude, selected for an upgrading process, is made with the resulting products of the process. A quantitative assessment of the extent of the improvement as a result of the hydrogenation in the crude is presented by defining an algorithm which measures the conversion of material up to 550 °C. The defined conversion is correlated with conventional crude properties and there is a linear relation between the conversion obtained by s.e.c. and certain selected properties. The results for a number of products are included and the relation between conversion and process conditions is discussed.     

A Study on the Removal of Humic Acid Using Advanced Oxidation Processes

Abstract

Humic acid (HA) removal using advanced oxidation processes (AOPs) was investigated, particularly UVA/H₂O₂ and photo Fenton‐like process (UVA/Fe(III)/H₂O₂). Changes in the UV₂₅₄ absorbance, dissolved organic carbon (DOC), apparent molecular weight (AMW) distribution, and the Trihalomethane formation potential (THMFP) of the organics were monitored. UVA/Fe(III)/H₂O₂ based process was found to be effective in removing more than 80% DOC and 90% UV₂₅₄ absorbance. Differences in the reduction profiles of AMW distributions for UVA/Fe(III)/H₂O₂ based process and UVA/H₂O₂ process were observed, with the latter showing preferential removal of a certain molecular weight range. Selected samples were then fractionated into four components: very hydrophobic acids (VHA), slightly hydrophobic acids (SHA), hydrophilic charged (CHA), and hydrophilic neutral (NEU). The HA used is found to consist mostly of VHA fraction that is very susceptible to AOP treatments. The results illustrate that the degradation process occurred via the fragmentation of VHA fraction to form SHA, CHA, and NEU fractions.     

Characterization of the extracts and residues from CS2-N-methyl-2-pyrrolidinone mixed solvent extraction

The extracts and residues obtained by extraction of five bituminous coals with CS₂-N-methyl-2-pyrrolidinone mixed solvent (1:1 by volume) were characterized at room temperature. The extraction yields were 31.1–63.0% (daf) and the extracts were fractionated into acetone soluble (AS), acetone insoluble-pyridine soluble (PS) and pyridine insoluble-mixed solvent soluble (MS) fractions. The MS fraction, which was the heaviest fraction examined, had higher values of % oxygen, f_a, molecular weight and spin concentration than the corresponding AS and PS fractions, but a similar degree of aromatic condensation. The quantities of volatile matter (daf) in the residues were similar or slightly less than those in the extracts.     

Exploring the Substrate Promiscuity of Drug‐Modifying Enzymes for the Chemoenzymatic Generation of N‐Acylated Aminoglycosides

Aminoglycosides are broad‐spectrum antibiotics commonly used for the treatment of serious bacterial infections. Decades of clinical use have led to the widespread emergence of bacterial resistance to this family of drugs limiting their efficacy in the clinic. Here, we report the development of a methodology that utilizes aminoglycoside acetyltransferases (AACs) and unnatural acyl coenzyme A analogues for the chemoenzymatic generation of N‐acylated aminoglycoside analogues. Generation of N‐acylated aminoglycosides is followed by a simple qualitative test to assess their potency as potential antibacterials. The studied AACs (AAC(6′)‐APH(2′′) and AAC(3)‐IV) show diverse substrate promiscuity towards a variety of aminoglycosides as well as acyl coenzyme A derivatives. The enzymes were also used for the sequential generation of homo‐ and hetero‐di‐N‐acylated aminoglycosides. Following the clinical success of the N‐acylated amikacin and arbekacin, our chemoenzymatic approach offers access to regioselectively N‐acylated aminoglycosides in quantities that allow testing of the antibacterial potential of the synthetic analogues making it possible to decide which molecules will be worth synthesizing on a larger scale.     

A comprehensive study on the size exclusion chromatography of kappa‐carrageenan for the identification of after‐peaks

Aqueous size exclusion chromatography (SEC) of polysaccharides in general and carrageenans in particular is complicated by a number of factors. The chromatograms of carrageenans which are sulfated anionic natural polymers contain a number of after‐peaks depending on the occlusion, adsorption, or association of various ionic species either naturally present or evolved during their processing. A systematic SEC analysis of after‐peaks appearing in the chromatograms was made to identify the species responsible for their formation. The five after‐peaks constantly appearing in the aqueous (0.1M NaNO₃) SEC of kappa‐carrageenan are attributed to sulfate, chloride, and nitrate anions whereas the first three and the fourth are due to divalent cations, mostly, and the fifth appears to result from the unknown impurities. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Antioxidant effects of protein hydrolysates in the reaction with glucose

Maillard reaction products (MRP) obtained by reaction between glucose and protein hydrolysates from casein and fish were investigated. The influence of parameters such as reaction time and glucose concentration was studied. The antioxidative activity of MRP was determined by using the β-carotene/linoleate model and the 1,1-diphenyl-2-pricrylhydrazyl method. All experiments showed that the antioxidative effect was improved by 20–30% when the hydrolysates were reached with glucose. A dramatic increase in antiradical efficiency of the MRP (up to 75%) was also observed. The study of the chromatographic profiles obtained before and after the Maillard reaction found changes in absorbance at 280 nm, indicating molecular rearrangements that could be involved in the improvement of the antioxidative and free radical-scavenging activities.     

Plant Cell Wall Proteomes: The Core of Conserved Protein Families and the Case of Non-Canonical Proteins

Plant cell wall proteins (CWPs) play critical roles during plant development and in response to stresses. Proteomics has revealed their great diversity. With nearly 1000 identified CWPs, the Arabidopsis thaliana cell wall proteome is the best described to date and it covers the main plant organs and cell suspension cultures. Other monocot and dicot plants have been studied as well as bryophytes, such as Physcomitrella patens and Marchantia polymorpha. Although these proteomes were obtained using various flowcharts, they can be searched for the presence of members of a given protein family. Thereby, a core cell wall proteome which does not pretend to be exhaustive, yet could be defined. It comprises: (i) glycoside hydrolases and pectin methyl esterases, (ii) class III peroxidases, (iii) Asp, Ser and Cys proteases, (iv) non-specific lipid transfer proteins, (v) fasciclin arabinogalactan proteins, (vi) purple acid phosphatases and (vii) thaumatins. All the conserved CWP families could represent a set of house-keeping CWPs critical for either the maintenance of the basic cell wall functions, allowing immediate response to environmental stresses or both. Besides, the presence of non-canonical proteins devoid of a predicted signal peptide in cell wall proteomes is discussed in relation to the possible existence of alternative secretion pathways.     

Deciphering the Nature of Enzymatic Modifications of Bacterial Cell Walls

The major constituent of bacterial cell walls is peptidoglycan, which, in its crosslinked form, is a polymer of considerable complexity that encases the entire bacterium. A functional cell wall is indispensable for survival of the organism. There are several dozen enzymes that assemble and disassemble the peptidoglycan dynamically within each bacterial generation. Understanding of the nature of these transformations is critical knowledge for these events. Octasaccharide peptidoglycans were prepared and studied with seven recombinant cell‐wall‐active enzymes (SltB1, MltB, RlpA, mutanolysin, AmpDh2, AmpDh3, and PBP5). With the use of highly sensitive mass spectrometry methods, we described the breadth of reactions that these enzymes catalyzed with peptidoglycan and shed light on the nature of the cell wall alteration performed by these enzymes. The enzymes exhibit broadly distinct preferences for their substrate peptidoglycans in the reactions that they catalyze.     

Micropatterned Surfaces for the Study of Cancer and Endothelial Cell Interactions with Hyaluronic Acid

Hyaluronic acid (HA) is a glycosaminoglycan component of the extracellular matrix. Studies have shown that various cancers exhibit high levels of HA content, and that an increased amount of HA corresponds to poor patient prognosis. HA has been implicated in cellular interactions that are associated with cancer progression, including cell adhesion, motility, and differentiation. Micropatterned functional HA surfaces were developed to study interactions between cancer cells and HA. The adhesion and migration of cancer cells representing different stages of tumorigenesis were examined. A similar surface patterning approach was used to create HA regions next to fibronectin in two- and three-dimensional settings to visualize and study the interactions between cancer and endothelial cells. The ability to observe the dynamic interactions of cancer cells and angiogenesis within a HA-rich microenvironment will improve the fundamental understanding of cancer progression and contribute to the development of advanced therapeutic targets.     

Programmed Editing of Rice (Oryza sativa L.) OsSPL16 Gene Using CRISPR/Cas9 Improves Grain Yield by Modulating the Expression of Pyruvate Enzymes and Cell Cycle Proteins

Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T₀, T₁, and T₂ generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations.     

Log‐normal distribution for a description of the molecular weight distribution of N‐acetylated chitosans depolymerized with hydrolytic enzyme

N‐acetylated chitosans (NACs) with different degrees of N‐acetylation (DAs) were enzymatically depolymerized at pH 5.1 and 40 °C, and the molecular weight distributions (MWDs) of the depolymerized NACs were then measured by size exclusion chromatography and were fitted by the log‐normal distribution function with two distribution parameters, β and M₀. We discuss also the time‐evolution of the distribution parameters derived from the experimentally obtained MWD as well as the effects of experimental conditions, such as DA and initial NAC concentration (S₀), on the distribution parameters. Copyright © 2003 Society of Chemical Industry     

Effect of the MMA content on the emulsion polymerization process and adhesive properties of poly(BA‐co‐MMA‐co‐AA) latexes

In the past work, the shear resistance of pure poly(n‐butyl acrylate) was low, even incorporation of inorganic filler, silica in the composition. It is well‐known that the copolymerization of n‐butyl acrylate (BA) with methyl methacrylate (MMA) will increase the glass transition temperature, and enhance the shear resistance of acrylic polymers. In the current work, the preparation of a series of acrylic water‐borne pressure‐sensitive adhesives (PSAs) with the controlled composition and structure for the copolymerization of BA and acrylic acid (AA) with different MMA contents, poly(BA‐co‐MMA‐co‐AA) was reported and its effects on adhesive properties of the latices were investigated. The latices of poly(BA‐co‐MMA‐co‐AA) were prepared at a solid content of 50% by two‐stage sequential emulsion polymerization, and this process consisted of a batch seed stage giving a particle diameter of 111 nm, which was then grown by the semicontinuous addition of monomers to final diameter of 303 nm. Dynamic light scattering (DLS) was used to monitor the particle diameters and proved that no new nucleation occurred during the growth stage. Copolymerization of BA with MMA raised the glass transition temperature (T_g) of the soft acrylic polymers, and had the effect of improving shear resistance, while the loop tack and peel adhesion kept relatively high. The relationship between pressure‐sensitive properties and molecular parameters, such as gel content and molecular weight, was evaluated. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Changes in the Cell Wall Proteome of Leaves in Response to High Temperature Stress in Brachypodium distachyon

High temperature stress leads to complex changes to plant functionality, which affects, i.a., the cell wall structure and the cell wall protein composition. In this study, the qualitative and quantitative changes in the cell wall proteome of Brachypodium distachyon leaves in response to high (40 °C) temperature stress were characterised. Using a proteomic analysis, 1533 non-redundant proteins were identified from which 338 cell wall proteins were distinguished. At a high temperature, we identified 46 differentially abundant proteins, and of these, 4 were over-accumulated and 42 were under-accumulated. The most significant changes were observed in the proteins acting on the cell wall polysaccharides, specifically, 2 over- and 12 under-accumulated proteins. Based on the qualitative analysis, one cell wall protein was identified that was uniquely present at 40 °C but was absent in the control and 24 proteins that were present in the control but were absent at 40 °C. Overall, the changes in the cell wall proteome at 40 °C suggest a lower protease activity, lignification and an expansion of the cell wall. These results offer a new insight into the changes in the cell wall proteome in response to high temperature.     

MicroRNAs at the Crossroad of the Dichotomic Pathway Cell Death vs. Stemness in Neural Somatic and Cancer Stem Cells: Implications and Therapeutic Strategies

Stemness and apoptosis may highlight the dichotomy between regeneration and demise in the complex pathway proceeding from ontogenesis to the end of life. In the last few years, the concept has emerged that the same microRNAs (miRNAs) can be concurrently implicated in both apoptosis-related mechanisms and cell differentiation. Whether the differentiation process gives rise to the architecture of brain areas, any long-lasting perturbation of miRNA expression can be related to the occurrence of neurodevelopmental/neuropathological conditions. Moreover, as a consequence of neural stem cell (NSC) transformation to cancer stem cells (CSCs), the fine modulation of distinct miRNAs becomes necessary. This event implies controlling the expression of pro/anti-apoptotic target genes, which is crucial for the management of neural/neural crest-derived CSCs in brain tumors, neuroblastoma, and melanoma. From a translational point of view, the current progress on the emerging miRNA-based neuropathology therapeutic applications and antitumor strategies will be disclosed and their advantages and shortcomings discussed.