Fluorescence and Biochemical Assessment of the Chitin and Chitosan Content of Cryptococcus.

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.

Exploring fungal bioemulsifiers: insights into chemical composition, microbial sources, and cross-field applications.

The demand for emulsion-based products is crucial for economic development and societal well-being, spanning diverse industries such as food, cosmetics, pharmaceuticals, and oil extraction. Formulating these products relies on emulsifiers, a distinct class of surfactants. However, many conventional emulsifiers are derived from petrochemicals or synthetic sources, posing potential environmental and human health risks. In this context, fungal bioemulsifiers emerge as a compelling and sustainable alternative, demonstrating superior performance, enhanced biodegradability, and safety for human consumption. From this perspective, the present work provides the first comprehensive review of fungal bioemulsifiers, categorizing them based on their chemical nature and microbial origin. This includes polysaccharides, proteins, glycoproteins, polymeric glycolipids, and carbohydrate-lipid-protein complexes. Examples of particular interest are scleroglucan, a polysaccharide produced by Sclerotium rolfsii, and mannoproteins present in the cell walls of various yeasts, including Saccharomyces cerevisiae. Furthermore, this study examines the feasibility of incorporating fungal bioemulsifiers in the food and oil industries and their potential role in bioremediation events for oil-polluted marine environments. Finally, this exploration encourages further research on fungal bioemulsifier bioprospecting, with far-reaching implications for advancing sustainable and eco-friendly practices across various industrial sectors.

Assessment of cell wall degrading enzymes by molecular docking and dynamics simulations: Effects of novel infrared treatment.

Cell wall-degrading enzymes' activities under infrared treatment are vital for peeling; it is critical to elucidate the mechanisms of the novel infrared peeling in relation to its impact on cell wall-degrading enzymes. In this study, the activities, and gene expressions of eight degrading enzymes closely related to pectin, cellulose and hemicellulose were determined. The most influential enzyme was selected from them, and then the mechanism of its changes was revealed by molecular dynamics simulation and molecular docking. The results demonstrated that infrared had the most significant effect on ß-glucosidase among the tested enzymes (increased activity and up-regulated gene expression of 195.65 % and 7.08, respectively). It is suggested infrared crucially promotes cell wall degradation by affecting ß-glucosidase. After infrared treatment, ß-glucosidase's structure moderately transformed to a more open one and became flexible, increasing the affinity between ß-glucosidase and substrate (increasing 75 % H-bonds and shortening 15.89 % average length), thereby improving ß-glucosidase's activity. It contributed to cell wall degradation. The conclusion is that the effect of infrared on the activity, gene expression and molecular structure of ß-glucosidase causes damage to the peel, thus broadening the applicability of the new infrared dry-peeling technique, which has the potential to replace traditional wet-peeling methods.

Comparative Assessment of Different Yeast Cell Wall-Based Mycotoxin Adsorbents Using a Model- and Bioassay-Based In Vitro Approach.

Frequently reported occurrences of deoxynivalenol (DON), beauvericin (BEA), and, to a lesser extent, ochratoxin A (OTA) and citrinin (CIT) in ruminant feed or feedstuff could represent a significant concern regarding feed safety, animal health, and productivity. Inclusion of yeast cell wall-based mycotoxin adsorbents in animal feeds has been a common strategy to mitigate adverse effects of mycotoxins. In the present study, an in vitro approach combining adsorption isotherm models and bioassays was designed to assess the efficacy of yeast cell wall (YCW), yeast cell wall extract (YCWE), and a postbiotic yeast cell wall-based blend (PYCW) products at the inclusion rate of 0.5% (w/v) (ratio of adsorbent mass to buffer solution volume). The Hill's adsorption isotherm model was found to best describe the adsorption processes of DON, BEA, and CIT. Calculated binding potential for YCW and YCWE using the Hill's model exhibited the same ranking for mycotoxin adsorption, indicating that BEA had the highest adsorption rate, followed by DON and CIT, which was the least adsorbed. PYCW had the highest binding potential for BEA compared with YCW and YCWE. In contrast, the Freundlich isotherm model presented a good fit for OTA adsorption by all adsorbents and CIT adsorption by PYCW. Results indicated that YCW was the most efficacious for sequestering OTA, whereas YCWE was the least efficacious. PYCW showed greater efficacy at adsorbing OTA than CIT. All adsorbents exhibited high adsorption efficacy for BEA, with an overall percentage average of bound mycotoxin exceeding 60%, whereas moderate efficacies for the other mycotoxins were observed (up to 37%). Differences in adsorbent efficacy of each adsorbent significantly varied according to experimental concentrations tested for each given mycotoxin (p < 0.05). The cell viability results from the bioassay using a bovine mammary epithelial cell line (MAC-T) indicated that all tested adsorbents could potentially mitigate mycotoxin-related damage to bovine mammary epithelium. Results from our studies suggested that all tested adsorbents had the capacity to adsorb selected mycotoxins in vitro, which could support their use to mitigate their effects in vivo.

Assessment of the optical properties with physicochemical properties and cell wall polysaccharides of 'Korla' pear flesh during Alternaria alternata-induced disease development.

Cell wall polysaccharides and physicochemical properties are the major quality characteristics of fruit, but they are significantly affected by the postharvest disease. In this study, the influence of Alternaria alternata-induced disease on the contents of cell wall polysaccharides and physicochemical properties in 'Korla' pear flesh during storage, as well as their relationships of the optical absorption (µa) and reduced scattering (µs') were explored. The infected pear had lower individual sugars, covalent-soluble pectin, cellulose and hemicellulose contents than the healthy ones. The successive decreases of µa and increases of µs' in pears were observed while the process of pathogen infection. Path-coefficient analysis indicated the ionic-soluble pectin was the main reason responsible for the change of µs' in infected pear at 675 nm and 980 nm. This study indicated the optical properties have the possibility to present the physicochemical characteristics and cell wall polysaccharides of pears during postharvest pathogen infection.

Challenges and perspectives of green-like lignocellulose pretreatments selectable for low-cost biofuels and high-value bioproduction.

Lignocellulose represents the most abundant carbon-capturing substance that is convertible for biofuels and bioproduction. Although biomass pretreatments have been broadly applied to reduce lignocellulose recalcitrance for enhanced enzymatic saccharification, they mostly require strong conditions with potential secondary waste release. By classifying all major types of pretreatments that have been recently conducted with different sources of lignocellulose substrates, this study sorted out their distinct roles for wall polymer extraction and destruction, leading to the optimal pretreatments evaluated for cost-effective biomass enzymatic saccharification to maximize biofuel production. Notably, all undigestible lignocellulose residues are also aimed for effective conversion into value-added bioproduction. Meanwhile, desired pretreatments were proposed for the generation of highly-valuable nanomaterials such as cellulose nanocrystals, lignin nanoparticles, functional wood, carbon dots, porous and graphitic nanocarbons. Therefore, this article has proposed a novel strategy that integrates cost-effective and green-like pretreatments with desirable lignocellulose substrates for a full lignocellulose utilization with zero-biomass-waste liberation.

Are all ulvans equal? A comparative assessment of the chemical and gelling properties of ulvan from blade and filamentous Ulva.

Green seaweeds of the genus Ulva are rich in the bioactive sulfated polysaccharide ulvan. Herein we characterise ulvan from Ulva species collected from the Bay of Plenty, Aotearoa New Zealand. Using standardised procedures, we quantified, characterised, and compared ulvans from blade (U. australis, U. rigida, U. sp. B, and Ulva sp.) and filamentous (U. flexuosa, U. compressa, U. prolifera, and U. ralfsii) Ulva species. There were distinct differences in composition and structure of ulvans between morphologies. Ulvan isolated from blade species had higher yields (14.0-19.3 %) and iduronic acid content (IdoA = 7-18 mol%), and lower molecular weight (Mw = 190-254 kDa) and storage moduli (G' = 0.1-6.6 Pa) than filamentous species (yield = 7.2-14.6 %; IdoA = 4-7 mol%; Mw = 260-406 kDa; G' = 22.7-74.2 Pa). These results highlight the variability of the physicochemical properties of ulvan from different Ulva sources, and identifies a morphology-based division within the genus Ulva.

Overview of Popular Techniques of Raman Spectroscopy and Their Potential in the Study of Plant Tissues.

Raman spectroscopy is one of the main analytical techniques used in optical metrology. It is a vibration, marker-free technique that provides insight into the structure and composition of tissues and cells at the molecular level. Raman spectroscopy is an outstanding material identification technique. It provides spatial information of vibrations from complex biological samples which renders it a very accurate tool for the analysis of highly complex plant tissues. Raman spectra can be used as a fingerprint tool for a very wide range of compounds. Raman spectroscopy enables all the polymers that build the cell walls of plants to be tracked simultaneously; it facilitates the analysis of both the molecular composition and the molecular structure of cell walls. Due to its high sensitivity to even minute structural changes, this method is used for comparative tests. The introduction of new and improved Raman techniques by scientists as well as the constant technological development of the apparatus has resulted in an increased importance of Raman spectroscopy in the discovery and defining of tissues and the processes taking place in them.

Immunogenicity Assessment of Cell Wall Carbohydrates of Group A Streptococcus via Self-Adjuvanted Glyco-lipopeptides.

Identifying the immunogenic moieties and their precise structure of carbohydrates plays an important role for developing effective carbohydrate-based subunit vaccines. This study assessed the structure-immunogenicity relationship of carbohydrate moieties of a single repeating unit of group A carbohydrate (GAC) present on the cell wall of group A Streptococcus (GAS) using a rationally designed self-adjuvanted lipid-core peptide, instead of a carrier protein. Immunological evaluation of fully synthetic glyco-lipopeptides (particle size: 300-500 nm) revealed that construct consisting of higher rhamnose moieties (trirhamnosyl-lipopeptide) was able to induce enhanced immunogenic activity in mice, and GlcNAc moiety was not found to be an essential component of immunogenic GAC mimicked epitope. Trirhamnosyl-lipopeptide also showed 75-97% opsonic activity against four different clinical isolates of GAS and was comparable to a subunit peptide vaccine (J8-lipopeptide) which illustrated 65-96% opsonic activity.

Fluoride absorption, transportation and tolerance mechanism in Camellia sinensis, and its bioavailability and health risk assessment: a systematic review.

Tea is the one of the most popular non-alcoholic caffeinated beverages in the world. Tea is produced from the tea plant (Camellia sinensis (L.) O. Kuntze), which is known to accumulate fluoride. This article systematically analyzes the literature concerning fluoride absorption, transportation and fluoride tolerance mechanisms in tea plants. Fluoride bioavailability and exposure levels in tea infusions are also reviewed. The circulation of fluoride within the tea plantation ecosystems is in a positive equilibrium, with greater amounts of fluoride introduced to tea orchards than removed. Water extractable fluoride and magnesium chloride (MgCl2 ) extractable fluoride in plantation soil are the main sources of absorption by tea plant root via active trans-membrane transport and anion channels. Most fluoride is readily transported through the xylem as F- /F-Al complexes to leaf cell walls and vacuole. The findings indicate that tea plants employ cell wall accumulation, vacuole compartmentalization, and F-Al complexes to co-detoxify fluoride and aluminum, a possible tolerance mechanism through which tea tolerates higher levels of fluoride than most plants. Furthermore, dietary and endogenous factors influence fluoride bioavailability and should be considered when exposure levels of fluoride in commercially available dried tea leaves are interpreted. The relevant current challenges and future perspectives are also discussed. © 2020 Society of Chemical Industry.

The Assessment of Diet Contaminated with Aflatoxin B1 in Juvenile Turbot (Scophthalmus maximus) and the Evaluation of the Efficacy of Mitigation of a Yeast Cell Wall Extract.

This study aimed to investigate the effects of dietary AFB1 on growth performance, health, intestinal microbiota communities and AFB1 tissue residues of turbot and evaluate the mitigation efficacy of yeast cell wall extract, Mycosorb® (YCWE) toward AFB1 contaminated dietary treatments. Nine experimental diets were formulated: Diet 1 (control): AFB1 free; Diets 2-5 or Diets 6-9: 20 µg AFB1/kg diet or 500 µg AFB1/kg diet + 0%, 0.1%, 0.2%, or 0.4% YCWE, respectively). The results showed that Diet 6 significantly decreased the concentrations of TP, GLB, C3, C4, T-CHO, TG but increased the activities of AST, ALT in serum, decreased the expressions of CAT, SOD, GPx, CYP1A but increased the expressions of CYP3A, GST-ζ1, p53 in liver. Diet 6 increased the AFB1 residues in serum and muscle, altered the intestinal microbiota composition, decreased the bacterial community diversity and the abundance of some potential probiotics. However, Diet 8 and Diet 9 restored the immune response, relieved adverse effects in liver, lowered the AFB1 residues in turbot tissues, promoted intestinal microbiota diversity and lowered the abundance of potentially pathogens. In conclusion, YCWE supplementation decreased the health effects of AFB1 on turbot, restoring biomarkers closer to the mycotoxin-free control diet.

Rapid Assessment of Cell Wall Polymer Distribution and Surface Topology of Arabidopsis Seedlings.

The deposition and modulation of constituent polymers of plant cell walls are profoundly important events during plant development. Identification of specific polymers within assembled walls during morphogenesis and in response to stress conditions represents a major goal of plant cell biologists. Arabidopsis thaliana is a model organism that has become central to research focused on fundamental plant processes including those related to plant wall dynamics. Its fast life cycle and easy access to a variety of mutants and ecotypes of Arabidopsis have stimulated the need for rapid assessment tools to probe its wall organization at the cellular and subcellular levels. We describe two rapid assessment techniques that allow for elucidation of the cell wall polymers of root hairs and high-resolution analysis of surface features of various vegetative organs. Live organism immunolabeling of cell wall polymers employing light microscopy and confocal laser scanning microscopy can be effectively performed using a large microplate-based screening strategy (see Figs. 1 and 2). Rapid cryofixation and imaging of variable pressure scanning electron microscopy also allows for imaging of surface features of all portions of the plant as clearly seen in Fig. 3.

Review on the Biological Detoxification of Mycotoxins Using Lactic Acid Bacteria to Enhance the Sustainability of Foods Supply.

The challenges to fulfill the demand for a safe food supply are dramatically increasing. Mycotoxins produced by certain fungi cause great economic loss and negative impact on the sustainability of food supplies. Moreover, the occurrence of mycotoxins at high levels in foods poses a high health threat for the consumers. Biological detoxification has exhibited a high potential to detoxify foodstuffs on a cost-effective and large scale. Lactic acid bacteria showed a good potential as an alternative strategy for the elimination of mycotoxins. The current review describes the health and economic impacts associated with mycotoxin contamination in foodstuffs. Moreover, this review highlights the biological detoxification of common food mycotoxins by lactic acid bacteria.

In vitro assessment of the capacity of certain mycotoxin binders to adsorb some amino acids and water-soluble vitamins.

The objective of this study was to evaluate the capacity of 6 mycotoxin binders (MTB) to adsorb 3 AA and 4 water-soluble vitamins (WSV). Two experiments were conducted in in vitro conditions to simulate postruminal digestion with pepsin, malic acid, citric acid, acetic acid, and lactic acid at pH 3.0 and intestinal digestion with bile salts and pancreatin extract at pH 6.5. Experiment 1 was conducted with AA, and experiment 2 was conducted with WSV. Within experiment, main factors were the MTB (bentonite, clinoptiolite, sepiolite, montmorillonite, activated carbon, and yeast cell walls), the substrate (AA: Lys, Met, and Thr; WSV: B1, B2, B3, and B6), and the incubation strategy (substrates alone or mixed). Data were analyzed for the effects of main factors and their interactions. In experiment 1, the adsorption average for AA when incubated separately was 44.3%, ranging from 62.4% for Thr by clinoptiolite to 20.0% for Thr by activated carbon. When incubated together, the average adsorption was reduced to 19.9%, suggesting competition among substrates for adsorption. Adsorption ranged from 29.8% for Thr by yeast cell walls to 5.6% for Met by clinoptiolite, but there were significant interactions among MTB and AA. In experiment 2, the average adsorption of WSV when incubated separately or together was 34.1 and 45.1%, respectively, suggesting possible synergies among substrates. When vitamins were incubated separately, adsorption ranged from 90.5% for vitamin B1 to 4.0% for vitamin B3 by montmorillonite. Vitamins B1 (except by yeast cell walls) and B6 (except by bentonite, sepiolite, and montmorillonite) were absorbed the most, and vitamin B3 was absorbed the least (except by activated carbon and yeast cell walls, which were least together with vitamin B2). When vitamins were incubated together, adsorption ranged from 97.0% for vitamin B1 by montmorillonite to 0% for vitamin B2 by activated carbon and vitamin B3 by bentonite. Vitamins B1 by all MTB and B6 by clinoptiolite, sepiolite, and yeast cell walls were the most adsorbed, and vitamin B3 (except by activated carbon and yeast cell wall) was the least absorbed. There were significant interactions among MTB and WSV. Mycotoxin binders have a high degree of adsorption of the AA and WSV tested in in vitro conditions, which may limit their bioavailability. Results also suggest that when substrates were incubated together some interactions for adsorption occurred, which were competitive among AA and synergic among vitamins.

Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to ß-Lactam Antibiotics.

Gram-negative bacteria have evolved an elaborate pathway to sense and respond to exposure to ß-lactam antibiotics. The ß-lactam antibiotics inhibit penicillin-binding proteins, whereby the loss of their activities alters/damages the cell-wall peptidoglycan. Bacteria sense this damage and remove the affected peptidoglycan into complex recycling pathways. As an offshoot of these pathways, muropeptide chemical signals generated from the cell-wall recycling manifest the production of a class C ß-lactamase, which hydrolytically degrades the ß-lactam antibiotic as a resistance mechanism. We disclose the use of a fluorescence probe that detects the activation of the recycling system by the formation of the key muropeptides involved in signaling. This same probe additionally detects natural-product cell-wall-active antibiotics that are produced in situ by cohabitating bacteria.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Inactivating pathogenic bacteria in greywater by biosynthesized Cu/Zn nanoparticles from secondary metabolite of Aspergillus iizukae; optimization, mechanism and techno economic analysis.

The inactivation of antibiotic resistant Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) seeded in greywater by bimetallic bio-nanoparticles was optimized by using response surface methodology (RSM). The bimetallic nanoparticles (Cu/Zn NPs) were synthesized in secondary metabolite of a novel fungal strain identified as Aspergillus iizukae EAN605 grown in pumpkin medium. Cu/Zn NPs were very effective for inhibiting growth of E. coli and S. aureus. The maximum inactivation was optimized with 0.028 mg mL-1 of Cu/Zn NPs, at pH 6 and after 60 min, at which the reduction of E. coli and S. aureus was 5.6 vs. 5.3 and 5.2 vs. 5.4 log reduction for actual and predicted values, respectively. The inactivation mechanism was described based on the analysis of untreated and treated bacterial cells by Field emission scanning electron microscopy (FESEM), Energy Dispersive X-Ray Spectroscopy (EDS), Atomic Force Microscopy (AFM) revealed a damage in the cell wall structure due to the effect of Cu/Zn NPs. Moreover, the Raman Spectroscopy showed that the Cu/Zn NPs led to degradation of carbohydrates and amino structures on the bacteria cell wall. The Fourier transform infrared spectroscopy (FTIR) analysis confirmed that the destruction take place in the C-C bond of the functional groups available in the bacterial cell wall. The techno economic analysis revealed that the biosynthesis Cu/Zn NPs is economically feasible. These findings demonstrated that Cu/Zn NPs can effectively inhibit pathogenic bacteria in the greywater.

Assessment of freeze damage in fruits and vegetables.

Freezing is an efficient and widely used method of food preservation. However, it can also cause irreversible damages at cellular level which in turn degrade the overall quality of the frozen food products. Therefore, qualitative and quantitative methods and technologies that will be able to evaluate with accuracy the freeze damage are of great importance. This review paper provides a comprehensive study of the methods that have been used to evaluate the freeze damage in fruits and vegetables. Further than the principles and the applications of those methods, the advantages and the limitations are also being discussed.

Assessment of seven protocols to prepare Cutibacterium acnes bacterial lysates to measure its immunogenic potential and review of the literature.

Seven protocols were tested to prepare cell wall extracts from live Cutibacterium acnes. Different parameters were modified: thawing/freezing and sonication/freezing cycles, to impact on mechanical degradation of the bacteria. Finally, the immunogenic potential of the extracts generated was evaluated by measuring IL-8 releases using an in vitro skin explants system. The aim of this article was to compare the existing protocols from the scientific literature, and also propose a standardized method developed in our facilities.

High-Throughput Screening for Inhibitors of Wall Teichoic Acid Biosynthesis in Staphylococcus aureus.

The world is heading toward a dangerous post-antibiotic era where antibiotics fail to treat infections. Staphylococcus aureus is the leading cause of healthcare-associated infections worldwide, and an ever-increasing percentage of them are methicillin-resistant (MRSA). New strategies are urgently needed to combat this pathogen. Wall teichoic acids (WTA) in S. aureus are polyribitol phosphate polymers that play important roles in virulence and resistance to ß-lactam antibiotics. Here, we describe a high-throughput whole-cell screening platform for inhibitors targeting WTA biosynthesis. This platform takes advantage of the unique dispensability patterns of genes encoding WTA biosynthesis. We further describe follow-up dose-response assays to identify WTA inhibitors among the primary bioactives. WTA inhibitors offer an exciting opportunity for the development of novel antibacterial leads of unique mechanism in the fight against drug-resistant staphylococcal infections.