MXene-based electrochemical devices applied for healthcare applications.

The initial part of the review provides an extensive overview about MXenes as novel and exciting 2D nanomaterials describing their basic physico-chemical features, methods of their synthesis, and possible interfacial modifications and techniques, which could be applied to the characterization of MXenes. Unique physico-chemical parameters of MXenes make them attractive for many practical applications, which are shortly discussed. Use of MXenes for healthcare applications is a hot scientific discipline which is discussed in detail. The article focuses on determination of low molecular weight analytes (metabolites), high molecular weight analytes (DNA/RNA and proteins), or even cells, exosomes, and viruses detected using electrochemical sensors and biosensors. Separate chapters are provided to show the potential of MXene-based devices for determination of cancer biomarkers and as wearable sensors and biosensors for monitoring of a wide range of human activities.

How do the carbon and nitrogen sources affect the synthesis of ß-(1,3/1,6)-glucan, its structure and the susceptibility of Candida utilis yeast cells to immunolabelling with ß-(1,3)-glucan monoclonal antibodies?

BACKGROUND: The need to limit antibiotic therapy due to the spreading resistance of pathogenic microorganisms to these medicinal substances stimulates research on new therapeutic agents, including the treatment and prevention of animal diseases. This is one of the goals of the European Green Deal and the Farm-To-Fork strategy. Yeast biomass with an appropriate composition and exposure of cell wall polysaccharides could constitute a functional feed additive in precision animal nutrition, naturally stimulating the immune system to fight infections. RESULTS: The results of the research carried out in this study showed that the composition of Candida utilis ATCC 9950 yeast biomass differed depending on growth medium, considering especially the content of ß-(1,3/1,6)-glucan, α-glucan, and trehalose. The highest ß-(1,3/1,6)-glucan content was observed after cultivation in deproteinated potato juice water (DPJW) as a nitrogen source and glycerol as a carbon source. Isolation of the polysaccharide from yeast biomass confirmed the highest yield of ß-(1,3/1,6)-glucan after cultivation in indicated medium. The differences in the susceptibility of ß-(1,3)-glucan localized in cells to interaction with specific ß-(1,3)-glucan antibody was noted depending on the culture conditions. The polymer in cells from the DPJW supplemented with glycerol and galactose were labelled with monoclonal antibodies with highest intensity, interestingly being less susceptible to such an interaction after cell multiplication in medium with glycerol as carbon source and yeast extract plus peptone as a nitrogen source. CONCLUSIONS: Obtained results confirmed differences in the structure of the ß-(1,3/1,6)-glucan polymers considering side-chain length and branching frequency, as well as in quantity of ß-(1,3)- and ß-(1,6)-chains, however, no visible relationship was observed between the structural characteristics of the isolated polymers and its susceptibility to immunolabeling in whole cells. Presumably, other outer surface components and molecules can mask, shield, protect, or hide epitopes from antibodies. ß-(1,3)-Glucan was more intensely recognized by monoclonal antibody in cells with lower trehalose and glycogen content. This suggests the need to cultivate yeast biomass under appropriate conditions to fulfil possible therapeutic functions. However, our in vitro findings should be confirmed in further studies using tissue or animal models.

Amphoteric Mannan as an Immune Response Modifier. New Model Immunobiologically Active Candida albicans Mannan-Based Formula.

BACKGROUND: Currently, the incidence and prevalence of serious fungal infections is increasing, especially in immunosuppressed individuals. The co-administration of antibiotic and immunosuppressive therapies has driven the emergence of new multidrug-resistant fungal pathogens. Their significant increase and their ability to form biofilms is associated with rising morbidity and mortality. Research into novel synthetically prepared immunomodulators as potential immune response modifiers and prospective participants in drug delivery systems is of interest. Microbial polysaccharides with zwitterionic charge motifs were shown to be promising candidates. METHODS: Native and ultrasonically treated mannan from the yeast Candida albicans were chemically modified to contain both positive and negative charges in a nearly equimolar ratio mimicking the zwitterionic polysaccharides. RAW 264.7 macrophages and Balb/c mice were subjected as in vitro and in vivo models. Macrophage exposure to the set of amphoteric derivatives of mannan induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The functionality of the exposed macrophages was assayed by cell proliferation and phagocytosis. RESULTS: The Th1 and Th17 dominance was over Th2. The phagocytosis and respiratory burst, together with the viability based on cell proliferation supported the bioavailability of formulas. Mouse immunization induced humoral immune responses with high titers of the IgM isotype with the IgM/IgG shift. CONCLUSION: Our study demonstrated the immunobiological activities of amphoteric derivatives of mannan from Candida albicans. Amphoteric derivatives can be considered as bioavailable formulas with an effective immunomodulatory potency, prospectively applied as a subunit formula in the design of a mannan-based platform for drug and vaccine delivery systems.

MXene as a novel cartridge for N-glycan enrichment.

In this study, we applied MXene as column cartridge for N-glycan enrichment from human samples with a focus on the analysis of sialic acid linkages using a derivatisation protocol followed by glycan analysis via Matrix Assisted Laser Desorption Ionisation-Time Of Flight Mass Spectrometry (MALDI-TOF-MS). The MXene-based cartridge enriches a higher number of glycans (i.e., sialylated and bisecting N-glycans) when compared to the commercial HILIC columns. We demonstrate the strong potential of MXene as a stationary phase in MS glycomic analysis.

Antimicrobial and prebiotic activity of mannoproteins isolated from conventional and nonconventional yeast species-the study on selected microorganisms.

Yeast mannoproteins are proposed as a paraprobiotics with antimicrobial and prebiotic properties. They can be used as biopreservatives in food and in diseases therapies. The knowledge about the specificity and/or capability of their influence on the growth of different microorganism is limited. The study determined the effect of mannoprotein preparations of Saccharomyces cerevisiae (S. cerevisiae) ATCC 7090 and nonconventional yeast origin [Metschnikowia reukaufii (M. reukaufii) WLP 4650 and Wickerhamomyces anomalus (W. anomalus) CCY 38-1-13] on the growth of selected bacteria of the genera: Lactobacilllus, Limosilatobacillus, Limosilatobacillus, Bifidobacterium, Staphylococcus, Enterococcus, Pseudomonas, Escherichia, Proteus and Salmonella. The degree of stimulation or growth inhibition of tested bacteria depended on the type and dose of the mannoprotein and the bacterial strain. The addition of the tested preparations in the entire range of applied concentrations had a positive effect especially on the growth of Lactobacillus arabinosus ATCC 8014 and Bifidobacterium animalis subsp. lactis B12. Mannoproteins isolated from S. cerevisiae limited the growth of the Escherichia coli (E. coli) ATCC 25922, Pseudomonas aureoginosa (P. aureoginosa) ATCC 27853, Proteus mirabilis ATCC 35659 and Salmonella Enteritidis ATCC 13076 to the greatest extent, while preparations of M. reukaufii and W. anomalus origin most effectively limited the growth of Staphylococcus aureus strains, E. coli and P. aureoginosa. The growth of Enterococcus faecalis was stimulated by the presence of all studied preparations in most of the concentrations used. Further research will determine how the purification process of studied mannoproteins or oligosaccharide fractions, its structure and composition influence on the growth of selected bacteria and what is the mechanism of its activity.

Redox features of hexaammineruthenium(III) on MXene modified interface: Three options for affinity biosensing.

In this article we describe construction of a bioreceptive interface for detection of a breast cancer biomarker carbohydrate antigen CA15-3. The conductive interface was patterned by a 2D nanomaterial MXene, to which a mixed layer containing sulfobetaine and carboxybetaine was electrochemically grafted through a diazonium moiety. Such a modified interface was then applied for covalent immobilisation of anti-CA15-3 antibody as a bioreceptive probe for detection of a breast cancer biomarker. Two different strategies were applied for final construction of an immunosensor i.e. an interface finally blocked by bovine serum albumin or an immunosensor without such modification. Finally, electrochemical reading was accomplished using a soluble redox probe Ru(NH3)63+ ion for detection of CA15-3 in a clinically relevant range up to 50 U mL-1. The results indicate that immunosensor based on non-blocked interface can be applied for biosensing using two modes of action: 1. differential pulse voltammetry (a plot of a peak current vs. analyte concentration) and 2. an electrochemical impedance spectroscopy (a plot of a charge transfer resistance vs. analyte concentration). The electrode blocked by bovine serum albumin (BSA) can be used by additional 3. mode of action: through detection of changes in the potential (a plot Epvs. c). Additionally, we reveal and explain that Ru(NH3)63+ is redox probe, which can be applied as interfacial molecular nanoscale ruler to distinguish negatively charged protein molecules present in the close proximity (≤ 6 nm) of the electrode (in our case adsorbed BSA molecules) from the negatively charged protein molecules at a larger distance (>12 nm) from the electrode (i.e. CA15-3 analyte).

Synthesis of Biotin-Tagged Chitosan Oligosaccharides and Assessment of Their Immunomodulatory Activity.

Chitin, a polymer of ß-(1→4)-linked N-acetyl-d-glucosamine, is one of the main polysaccharide components of the fungal cell wall. Its N-deacetylated form, chitosan, is enzymatically produced in the cell wall by chitin deacetylases. It exerts immunomodulative, anti-inflammatory, anti-cancer, anti-bacterial, and anti-fungal activities with various medical applications. To study the immunobiological properties of chitosan oligosaccharides, we synthesized a series of ß-(1→4)-linked N-acetyl-d-glucosamine oligomers comprising 3, 5, and 7 monosaccharide units equipped with biotin tags. The key synthetic intermediate employed for oligosaccharide chain elongation, a disaccharide thioglycoside, was prepared by orthogonal glycosylation of a 4-OH thioglycoside acceptor with a glycosyl trichloroacetimidate bearing the temporary 4-O-tert-butyldimethylsilyl group. The use of silyl protection suppressed aglycon transfer and provided a high yield for the target disaccharide donor. Using synthesized chitosan oligomers, as well as previously obtained chitin counterparts, the immunobiological relationship between these synthetic oligosaccharides and RAW 264.7 cells was studied in vitro. Evaluation of cell proliferation, phagocytosis, respiratory burst, and Th1, Th2, Th17, and Treg polarized cytokine expression demonstrated effective immune responsiveness and immunomodulation in RAW 264.7 cells exposed to chitin- and chitosan-derived oligosaccharides. Macrophage reactivity was accompanied by significant inductive dose- and structure-dependent protective Th1 and Th17 polarization, which was greater with exposure to chitosan- rather than chitin-derived oligosaccharides. Moreover, no antiproliferative or cytotoxic effects were observed, even following prolonged 48 h exposure. The obtained results demonstrate the potent immunobiological activity of these synthetically prepared chito-oligosaccharides.

Potential pathogenicity and antibiotic resistance of aquatic Vibrio isolates from freshwater in Slovakia.

This study aimed to evaluate the potential pathogenicity and antibiotic resistance of 31 environmental Vibrio isolates obtained from surface water in southern and eastern Slovakia. Isolates were identified as Vibrio cholerae non-O1/non-O139 and Vibrio metschnikovii by biochemical tests, MALDI biotyping, and 16S RNA gene sequencing. Analysis of the susceptibility to 13 antibacterial agents showed susceptibility of all isolates to ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, gentamicin, imipenem, tetracyclin, and doxycycline. We recorded high rates of resistance to ß-lactams and streptomycin. Investigation of antibiotic resistance showed five different antibiotic profiles with resistance to antibacterials from three classes, but no multidrug resistance was observed. The investigation of the pathogenic potential of V. cholerae isolates showed that neither the cholera toxin coding gene ctxA nor the genes zot (zonula occludens toxin), ace (accessory cholera toxin), and tcpA (toxin-coregulated pilus) were present in any of 31 isolated samples. Gene ompU (outer membrane protein) was confirmed in 80% and central regulatory protein-coding gene toxR in 71% of V. cholerae isolates, respectively. A high prevalence of the hemolysin coding gene hlyA in all V. cholerae was observed. The data point toward the importance of systematic monitoring and comparative studies of potentially pathogenic vibrios in European countries.

Importance of Candida Antigenic Factors: Structure-Driven Immunomodulation Properties of Synthetically Prepared Mannooligosaccharides in RAW264.7 Macrophages.

The incidence and prevalence of serious fungal infections is rising, especially in immunosuppressed individuals. Moreover, co-administration of antibiotics and immunosuppressants has driven the emergence of new multidrug-resistant pathogens. The significant increase of multidrug-resistant pathogens, together with their ability to form biofilms, is associated with morbidity and mortality. Research on novel synthetically prepared immunomodulators as potential antifungal immunotherapeutics is of serious interest. Our study demonstrated the immunobiological activity of synthetically prepared biotinylated mannooligosaccharides mimicking Candida antigenic factors using RAW264.7 macrophages. Macrophage exposure to the set of eight structurally different mannooligosaccharides induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The observed immune responses were tightly associated with structure, dose, exposure time, and selected signature cytokines. The viability/cytotoxicity of the mannooligosaccharide formulas was assessed based on cell proliferation. The structure-based immunomodulatory activity of the formulas was evaluated with respect to the length, branching and conformation of the various formulas. Glycoconjugate formulas with terminal ß-mannosyl-units tended to be more potent in terms of Candida relevant cytokines IL-12 p70, IL-17, GM-CSF, IL-6, and TNFα induction and cell proliferation, and this tendency was associated with structural differences between the studied glycoconjugate formulas. The eight tested mannooligosaccharide conjugates can be considered potential in vitro immunomodulative agents suitable for in vitro Candida diagnostics or prospectively for subcellular anti-Candida vaccine design.

Bioimmunological activities of Candida glabrata cellular mannan.

Candida glabrata is a second most common human opportunistic pathogen which causes superficial but also life-threatening systemic candidosis. According to the localisation of mannans and mannoproteins in the outermost layer of the cell wall, mannan detection could be one of the first steps in the cell recognition of Candida cells by the host innate immune system. Mannans from the cell wall provide important immunomodulatory activities, comprising stimulation of cytokine production, induction of dendritic cells (DCs) maturation and T-cell immunity. The model of DCs represents a promising tool to study immunomodulatory interventions throughout the vaccine development. Activated DCs induce, activate and polarise T-cell responses by expression of distinct maturation markers and cytokines regulating the adaptive immune responses. In addition, they are uniquely adept at decoding the fungus-associated information and translate it in qualitatively different T helper responses. We find out, that C. glabrata mannan is able to induce proliferation of splenocytes and to increase the production of TNF-α and IL-4. Next, increased the expression of co-stimulatory molecules CD80 and CD86 and the proportion of CD4+CD25+ and CD4+CD28+ T cells during in vitro stimulation of splenocytes. Reported results provide C. glabrata mannan capability to modulate cytokine production, DCs activation and antigen presentation activity, influencing T-cell phenotype in response to stimulation.

Efficient separation of mannan-protein mixtures by ionic liquid aqueous two-phase system, comparison with lectin affinity purification.

Analysis of carbohydrates from complex biological samples often requires their isolation from proteins and other contaminants to avoid interference. An effective separation of mannan-protein mixtures by 1-butyl-3-methylimidazolium bromide/K2HPO4 ionic liquid aqueous two-phase system (IL-APTS) is reported. Extraction efficiency of bovine serum albumin (BSA) ranged from 92% to 97% while extraction efficiency of mannan reached values from 95% to about 100% depending on phase and/or model sample composition. On the contrary, lower efficiency of BSA removal (73-84%) was recorded for lectin affinity purification with concanavalin A-triazine bead cellulose (Con A-TBC); the low mannan-binding capacity was limiting factor here. The size exclusion chromatography pattern of model mannan-BSA samples after both IL-APTS and Con A-TBC treatments were consistent with the spectrophotometric component analysis. In case of biological experiment, the ionic liquid separation technique was superior in pre-purification of 2-aminobenzamide-labelled mannan from cell culture medium prior to HPLC-FLD analysis.

One-pot preparation of labelled mannan-peptide conjugate, model for immune cell processing.

An efficient method for preparation of fluorescently labelled mannan-peptide glycoconjugates has been developed. After selective Dess-Martin periodinane oxidation of mannan, it was conjugated to the fluorescent label alone and a peptide with the label via reductive amination. Prepared glycoconjugates were characterised by HPSEC, FTIR-ATR and UV-VIS spectroscopy. Finally, the fluorescently labelled mannan and mannan-peptide conjugate were used for microscopic visualization of their accumulation in intracellular organelles of RAW 264.7 cells.

Preparation and immunogenicity of conjugate based on hydrazine-treated lipopolysaccharide antigen of Vibrio cholerae O139.

A glycoconjugate construct was based on attachment of V. cholerae O139 hydrazine-treated lipopolysaccharide (LPS) to carboxylated bovine serum albumin (CBSA) via its amino group. The immunological properties of the glycoconjugate were tested using BALB/c mice, injected subcutaneously without any adjuvant three times at 2 weeks interval. The immunogenicity of the conjugate was estimated by enzyme-linked immunosorbent assay, testing of anti-LPS IgG, IgM, and IgA antibodies. The conjugate elicited a statistically significant increase of LPS-specific IgG levels in mice (p < 0.001). The specific anti-LPS IgG and IgA response after the second booster dose was significantly higher compared with reference and unconjugated detoxified LPS response. Antibodies elicited by the dLPS-CBSA conjugate were vibriocidal.

Comparison of EDC and DMTMM efficiency in glycoconjugate preparation.

A series of mono- or disaccharide-protein conjugates were prepared by amide bond formation. Incorporation efficiency of the aminosaccharides on bovine serum albumin was determined by colorimetry and matrix assisted laser desorption/ionization mass spectrometry. Study compares two amide bond coupling agents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. Paper demonstrates a practical usefulness of natural bovine serum albumin succinylation. Large number of its carboxyl groups results in its superior reactivity towards simple aminosaccharides.

Immunological efficacy of glycoconjugates derived from Vibrio cholerae O1 serotype Ogawa detoxified LPS in mice.

This study focused on changes in selected parameters of humoral and cellular immunity following vaccination of mice with unique Vibrio cholerae LPS-protein-complexed conjugates. The V. cholerae detoxified LPS (dLPS)-derived antigenic structures O-specific polysaccharide (O-SP) and de-O-acylated LPS (DeOAc-LPS) were used to prepare glycoconjugates by linking both dLPSs to glucan, the immunomodulating matrix, and then to BSA carrier. Animals were given a primary vaccination and boosted at 2-week intervals with a dose of 4.5 µg saccharide antigen. The last boost was given either subcutaneously or intraperitoneally (i.p.) to compare the boosting effect and to optimize the effective immunization route. Both conjugates (O-SP-BSA and DeOAc-LPS-BSA) induced significant levels of antigen-specific Ig isotypes, especially IgG and IgM. The i.p. booster route was more effective. A T helper 1 response was achieved only by immunization with O-SP-BSA conjugate administered i.p. Significant acceleration of phagocytic capacity and respiratory burst of neutrophils was demonstrated by both immunogenic formulations. Activation of T- and B-cell adaptive immunities was exhibited as specific changes in CD3 : CD19 and CD4 : CD8 ratios, B-cell low-affinity Fcγ II and III receptor expression and induction of CD45R antigen.

Preparation of synthetic polyoxazoline based carrier and Vibrio cholerae O-specific polysaccharide conjugate vaccine.

Multiple chemical attachments of carbohydrate antigens to linear polymer represent promising technique for creating biologically effective conjugates. A novel conjugate consisting of detoxified lipopolysaccharide of Vibrio cholerae O135, linear polymer (polyoxazoline copolymer, serving as a matrix) and BSA (as immunogenic protein), has been prepared. The reaction conditions were optimized for obtaining high degree of conjugation. Analytical methods were evaluated to characterize conjugates obtained. Proposed chemistry is suitable for preparation of multivalent glycoconjugates in general.