Potential use of yeast protein in terms of biorefinery, functionality, and sustainability in food industry.

A growing demand for sustainable, alternative protein sources that are nutrient-dense, such as microorganisms, and insects, has gradually evolved. When paired with effective processing techniques, yeast cells contain substantial substances that could supply the population's needs for food, medicine, and fuel. This review article explores the potential of yeast proteins as a sustainable and viable alternative to animal and plant-based protein sources. It highlights the various yeast protein extraction methods including both mechanical and non-mechanical methods. The application of nanoparticles is one example of the fast-evolving technology used to damage microbial cells. SiO2 or Al2O3 nanoparticles break yeast cell walls and disrupt membranes, releasing intracellular bioactive compounds. Succinylation of yeast protein during extraction can increase yeast protein extraction rate, lower RNA concentration, raise yeast protein solubility, increase amino acid content, and improve yeast protein emulsification and foaming capabilities. Combining physical and enzymatic extraction methods generates the most representative pool of mannose proteins from yeast cell walls. Ethanol or isoelectric precipitation purifies mannose proteins. Mannoproteins can be used as foamy replacement for animal-derived components like egg whites due to their emulsification, stability, and foaming capabilities. Yeast bioactive peptide was separated by ultrafiltration after enzymatic hydrolysis of yeast protein and has shown hypoglycemic, hypotensive, and oxidative action in vitro studies. Additionally, the review delves into the physicochemical properties and stability of yeast-derived peptides as well as their applications in the food industry. The article infers that yeast proteins are among the promising sources of sustainable protein, with a wide range of potential applications in the food industry.

Inflammatory-Targeted Lipid Carrier as a New Nanomaterial to Formulate an Inhaled Drug Delivery System.

There is a pressing need for efficacious therapies in the field of respiratory diseases and infections. Lipid nanocarriers, administered through aerosols, represent a promising tool for maximizing therapeutic concentration in targeted cells and minimizing systemic exposure. However, this approach requires the application of efficient and safe nanomaterials. Palmitoylethanolamide (PEA), an endocannabinoid-like endogenous lipid, plays a crucial role in providing protective mechanisms during inflammation, making it an interesting material for preparing inhalable lipid nanoparticles (LNPs). This report aims to preliminarily explore the in vitro behavior of LNPs prepared with PEA (PEA-LNPs), a new inhalable inflammatory-targeted nanoparticulate drug carrier. PEA-LNPs exhibited a size of about 250 nm, a rounded shape, and an marked improvement in PEA solubility in comparison to naked PEA, indicative of easily disassembled nanoparticles. A twin glass impinger instrument was used to screen the aerosol performance of PEA-LNP powders, obtained via freeze-drying in the presence of two quantities of mannose as a cryoprotectant. Results indicated that a higher amount of mannose improved the emitted dose (ED), and in particular, the fine particle fraction (FPF). A cytotoxicity assay was performed and indicated that PEA-LNPs are not toxic towards the MH-S alveolar macrophage cell line up to concentrations of 0.64 mg/mL, and using coumarin-6 labelled particles, a rapid internalization into the macrophage was confirmed. This study demonstrates that PEA could represent a suitable material for preparing inhalable lipid nanocarrier-based dry powders, which signify a promising tool for the transport of drugs employed to treat respiratory diseases and infections.

It takes two to attach - endo-1,3-ß-d-glucanase as a potential receptor of mannose-independent, FimH-dependent Salmonella Typhimurium binding to spinach leaves.

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-ß-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Engineered therapeutic antibodies with mannose 6-phosphate analogues as a tool to degrade extracellular proteins.

Inducing the degradation of pathological soluble antigens could be the key to greatly enhancing the efficacy of therapeutic monoclonal antibodies (mAbs), extensively used in the treatment of autoimmune and inflammatory disorders or cancer. Lysosomal targeting has gained increasing interest in recent years due to its pharmaceutical applications far beyond the treatment of lysosomal diseases, as a way to address proteins to the lysosome for eventual degradation. Mannose 6-phosphonate derivatives (M6Pn), called AMFA, are unique glycovectors that can significantly enhance the cellular internalization of the proteins conjugated to AMFA via the cation-independent mannose 6-phosphate receptor (M6PR) pathway. AMFA engineering of mAbs results in the generation of a bifunctional antibody that is designed to bind both the antigen and the M6PR. The improvement of the therapeutic potential by AMFA engineering was investigated using two antibodies directed against soluble antigens: infliximab (IFX), directed against tumor necrosis factor α (TNF-α), and bevacizumab (BVZ), directed against the vascular endothelial growth factor (VEGF). AMFA conjugations to the antibodies were performed either on the oligosaccharidic chains of the antibodies or on the lysine residues. Both conjugations were controlled and reproducible and provided a novel affinity for the M6PR without altering the affinity for the antigen. The grafting of AMFA to mAb increased their cellular uptake through an M6PR-dependent mechanism. The antigens were also 2.6 to 5.7 times more internalized by mAb-AMFA and rapidly degraded in the cells. Additional cell culture studies also proved the significantly higher efficacy of IFX-AMFA and BVZ-AMFA compared to their unconjugated counterparts in inhibiting TNF-α and VEGF activities. Finally, studies in a zebrafish embryo model of angiogenesis and in xenografted chick embryos showed that BVZ-AMFA was more effective than BVZ in reducing angiogenesis. These results demonstrate that AMFA grafting induces the degradation of soluble antigens and a significant increase in the therapeutic efficacy. Engineering with mannose 6-phosphate analogues has the potential to develop a new class of antibodies for autoimmune and inflammatory diseases.

M2-type macrophage-targeted delivery of IKKß siRNA induces M2-to-M1 repolarization for CNV gene therapy.

Choroidal Neovascularization (CNV) is capable of inciting recurrent hemorrhage in the macular region, severely impairing patients' visual acuity. During the onset of CNV, infiltrating M2 macrophages play a crucial role in promoting angiogenesis. To control this disease, our study utilizes the RNA interference (RNAi)-based gene therapy to reprogram M2 macrophages to the M1 phenotype in CNV lesions. We synthesize the mannose-modified siRNA-loaded liposome specifically targeting M2 macrophages to inhibit the inhibitory kappa B kinase ß (IKKß) gene involved in the polarization of macrophages, consequently modulating macrophage polarization state. In vitro and in vivo, the mannose-modified IKKß siRNA-loaded liposome (siIKKß-ML) has been proven to effectively target M2 macrophages to repolarize them to M1 phenotype, and inhibit the progression of CNV. Collectively, our findings elucidate that siIKKß-ML holds the potential to control CNV by reprogramming the macrophage phenotype, indicating a promising therapeutic avenue for CNV management.

Anti-diabetic properties of brewer's spent yeast peptides. In vitro, in silico and ex vivo study after simulated gastrointestinal digestion.

Brewer's spent yeast (BSY) hydrolysates are a source of antidiabetic peptides. Nevertheless, the impact of in vitro gastrointestinal digestion of BSY derived peptides on diabetes has not been assessed. In this study, two BSY hydrolysates were obtained (H1 and H2) using ß-glucanase and alkaline protease, with either 1 h or 2 h hydrolysis time for H1 and H2, respectively. These hydrolysates were then subjected to simulated gastrointestinal digestion (SGID), obtaining dialysates D1 and D2, respectively. BSY hydrolysates inhibited the activity of α-glucosidase and dipeptidyl peptidase IV (DPP-IV) enzymes. Moreover, although D2 was inactive against these enzymes, D1 IC50 value was lower than those found for the hydrolysates. Interestingly, after electrophoretic separation, D1 mannose-linked peptides showed the highest α-glucosidase inhibitory activity, while non-glycosylated peptides had the highest DPP-IV inhibitory activity. Kinetic analyses showed a non-competitive mechanism in both cases. After peptide identification, GILFVGSGVSGGEEGAR and IINEPTAAAIAYGLDK showed the highest in silico anti-diabetic activities among mannose-linked and non-glycosylated peptides, respectively (AntiDMPpred score: 0.70 and 0.77). Molecular docking also indicated that these peptides act as non-competitive inhibitors. Finally, an ex vivo model of mouse jejunum organoids was used to study the effect of D1 on the expression of intestinal epithelial genes related to diabetes. The reduction of the expression of genes that codify lactase, sucrase-isomaltase and glucose transporter 2 was observed, as well as an increase in the expression of Gip (glucose-dependent insulinotropic peptide) and Glp1 (glucagon-like peptide 1). This is the first report to evaluate the anti-diabetic effect of BSY peptides in mouse jejunum organoids.

Benchmarking glycoform-resolved affinity separation - mass spectrometry assays for studying FcγRIIIa binding.

The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb - FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.

Anticancer and Antibacterial Properties of Curcumin-Loaded Mannosylated Solid Lipid Nanoparticles for the Treatment of Lung Diseases.

Lung cancer and Mycobacterium avium complex infection are lung diseases associated with high incidence and mortality rates. Most conventional anticancer drugs and antibiotics have certain limitations, including high drug resistance rates and adverse effects. Herein, we aimed to synthesize mannose surface-modified solid lipid nanoparticles (SLNs) loaded with curcumin (Man-CUR SLN) for the effective treatment of lung disease. The synthesized Man-CUR SLNs were analyzed using various instrumental techniques for structural and physicochemical characterization. Loading curcumin into SLNs improved the encapsulation efficiency and drug release capacity, as demonstrated by high-performance liquid chromatography analysis. Furthermore, we characterized the anticancer effect of curcumin using the A549 lung cancer cell line. Cells treated with Man-CUR SLN exhibited an increased cellular uptake and cytotoxicity. Moreover, treatment with free CUR could more effectively reduce cancer migration than treatment with Man-CUR SLNs. Similarly, free curcumin elicited a stronger apoptosis-inducing effect than that of Man-CUR SLNs, as demonstrated by reverse transcription-quantitative PCR analysis. Finally, we examined the antibacterial effects of free curcumin and Man-CUR SLNs against Mycobacterium intracellulare (M.i.) and M.i.-infected macrophages, revealing that Man-CUR SLNs exerted the strongest antibacterial effect. Collectively, these findings indicate that mannose-receptor-targeted curcumin delivery using lipid nanoparticles could be effective in treating lung diseases. Accordingly, this drug delivery system can be used to target a variety of cancers and immune cells.

Mannose modified targeted immersion vaccine delivery system improves protective immunity against Infectious spleen and kidney necrosis virus in mandarin fish (Siniperca chuatsi).

Vaccination is an effective method to prevent viral diseases. However, the biological barrier prevents the immersion vaccine from achieving the best effect without adding adjuvants and carriers. Researches on the targeted presentation technology of vaccines with nanocarriers are helpful to develop immersion vaccines for fish that can break through biological barriers and play an effective role in fish defense. In our study, functionally modified single-walled carbon nanotubes (SWCNTs) were used as carriers to construct a targeted immersion vaccine (SWCNTs-M-MCP) with mannose modified major capsid protein (MCP) to target antigen-presenting cells (APCs), against iridovirus diseases. After bath immunization, our results showed that SWCNTs-M-MCP induced the presentation process and uptake of APCs, triggering a powerful immune response. Moreover, the highest relative percent survival (RPS) was 81.3% in SWCNTs-M-MCP group, which was only 41.5% in SWCNTs-MCP group. Altogether, this study indicates that the SWCNTs-based targeted immersion vaccine induces strong immune response and provided an effective protection against iridovirus diseases.

Mannose enhances intestinal immune barrier function and dextran sulfate sodium salt-induced colitis in mice by regulating intestinal microbiota.

Background: Inflammatory bowel disease (IBD) greatly affects human quality of life. Mannose has been reported to be used to treat IBD, but the mechanism is currently unknown. Methods: C57/BL mice were used as research subjects, and the mouse acute colitis model was induced using dextran sulfate sodium salt (DSS). After oral administration of mannose, the body weights and disease activity index (DAI) scores of the mice were observed. The colon lengths, histopathological sections, fecal content microbial sequencing, colon epithelial inflammatory genes, and tight junction protein Occludin-1 expression levels were measured. We further used the feces of mice that had been orally administered mannose to perform fecal bacterial transplantation on the mice with DSS-induced colitis and detected the colitis-related indicators. Results: Oral administration of mannose increased body weights and colon lengths and reduced DAI scores in mice with DSS-induced colitis. In addition, it reduced the expression of colon inflammatory genes and the levels of serum inflammatory factors (TNF-α, IL-6, and IL-1ß), further enhancing the expression level of the colonic Occludin-1 protein and alleviating the toxic response of DSS to the intestinal epithelium of the mice. In addition, gut microbial sequencing revealed that mannose increased the abundance and diversity of intestinal flora. Additionally, after using the feces of the mannose-treated mice to perform fecal bacterial transplantation on the mice with DSS-induced colitis, they showed the same phenotype as the mannose-treated mice, and both of them alleviated the intestinal toxic reaction induced by the DSS. It also reduced the expression of intestinal inflammatory genes (TNF-α, IL-6, and IL-1ß) and enhanced the expression level of the colonic Occludin-1 protein. Conclusion: Mannose can treat DSS-induced colitis in mice, possibly by regulating intestinal microorganisms to enhance the intestinal immune barrier function and reduce the intestinal inflammatory response.

Characterization of Solitalea canadensis α-mannosidase with specific activity towards α1,3-Mannosidic linkages.

A recombinant exo-α-mannosidase from Solitalea canadensis (Sc3Man) has been characterized to exhibit strict specificity for hydrolyzing α1,3-mannosidic linkages located at the non-reducing end of glycans containing α-mannose. Enzymatic characterization revealed that Sc3Man operates optimally at a pH of 5.0 and at a temperature of 37 °C. The enzymatic activity was notably enhanced twofold in the presence of Ca2+ ions, emphasizing its potential dependency on this metal ion, while Cu2+ and Zn2+ ions notably impaired enzyme function. Sc3Man was able to efficiently cleave the terminal α1,3 mannose residue from various high-mannose N-glycan structures and from the model glycoprotein RNase B. This work not only expands the categorical scope of bacterial α-mannosidases, but also offers new insight into the glycan metabolism of S. canadensis, highlighting the enzyme's utility for glycan analysis and potential biotechnological applications.

The regulation of Cytochrome f by mannose treatment in broccoli and its relationship with programmed cell death in tobacco BY-2 cells.

It is well established that programmed cell death (PCD) occurred in broccoli during postharvest senescence, but no studies have been conducted on the regulation of broccoli cytochrome f by mannose treatment and its relationship with PCD. In this study, we treated broccoli buds with mannose to investigate the changes in color, total chlorophyll content, gene expression related to chlorophyll metabolism, chloroplast structure, and cytochrome f determination during postharvest storage. In addition, to investigate the effect of cytochrome f on PCD, we extracted cytochrome f from broccoli and treated Nicotiana tabacum L. cv Bright Yellow 2 (BY-2) cells with extracted cytochrome f from broccoli at various concentrations. The results showed that cytochrome f can induce PCD in tobacco BY-2 cells, as evidenced by altered cell morphology, nuclear chromatin disintegration, DNA degradation, decreased cell viability, and increased caspase-3-like protease production. Taken together, our study indicated that mannose could effectively delay senescence of postharvest broccoli by inhibiting the expression of gene encoding cytochrome f which could induce PCD.

Design, synthesis, biological evaluation and docking study of some new aryl and heteroaryl thiomannosides as FimH antagonists.

FimH is a mannose-recognizing lectin that is expressed by Escherichia coli guiding its ability to adhere and infect cells. It is involved in pathogenesis of urinary tract infections and Chron's disease. Several X-ray structure-guided ligand design studies were extensively utilized in the discovery and optimization of small molecule aryl mannoside FimH antagonists. These antagonists retain key specific interactions of the mannose scaffolds with the FimH carbohydrate recognition domains. Thiomannosides are attractive and stable scaffolds, and this work reports the synthesis of some of their new aryl and heteroaryl derivatives as FimH antagonists. FimH-competitive binding assays as well as biofilm inhibition of the new compounds (24-32) were determined in comparison with the reference n-heptyl α-d-mannopyranoside (HM). The affinity among these compounds was found to be governed by the structure of the aryl and heteroarylf aglycones. Two compounds 31 and 32 revealed higher activity than HM. Molecular docking and total hydrophobic to topological polar surface area ratio calculations attributed to explain the obtained biological results. Finally, the SAR study suggested that introducing an aryl or heteroaryl aglycone of sufficient hydrophobicity and of proper orientation within the tyrosine binding site considerably enhance binding affinity. The potent and synthetically feasible FimH antagonists described herein hold potential as leads for the development of sensors for detection of E. coli and treatment of its diseases.

PMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness.

Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.

GalNAc- or Mannose-PEG-Functionalized Polyplexes Enable Effective Lectin-Mediated DNA Delivery.

A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.

Synthesis of a unique mannose α-1-phosphate side chain moiety found in Candida auris cell wall mannan.

Candida auris is an emerging fungal pathogen that has become a world-wide public health threat. While there have been numerous studies into the nature, composition and structure of the cell wall of Candida albicans and other Candida species, much less is known about the C. auris cell wall. We have shown that C. auris cell wall mannan contains a unique phosphomannan structure which distinguishes C. auris mannan from the mannans found in other fungal species. Specifically, C. auris exhibits two unique acid-labile mannose α-1-phosphate (Manα1PO4) sidechains that are absent in other fungal mannans and fungal pathogens. This unique mannan structural feature presents an opportunity for the development of vaccines, therapeutics, diagnostic tools and/or research reagents that target C. auris. Herein, we describe the successful synthesis and structural characterization of a Manα1PO4-containing disaccharide moiety that mimics the phosphomannan found in C. auris. Additionally, we present evidence that the synthetic Manα1PO4 glycomimetic is specifically recognized and bound by cell surface pattern recognition receptors, i.e. rhDectin-2, rhMannose receptor and rhMincle, that are known to play important roles in the innate immune response to C. auris as well as other fungal pathogens. The synthesis of the Manα1PO4 glycomimetic may represent an important starting point in the development of vaccines, therapeutics, diagnostics and research reagents which target a number of C. auris clinical strains. In addition, these data provide new insights and understanding into the structural biology of this unique fungal pathogen.

Macrophage-mannose-receptor-targeted photoactivatable agent for in vivo imaging and treatment of atherosclerosis.

Previous studies have demonstrated the effects of theranostic agents on atherosclerotic plaques. However, there is limited information on targeted theranostics for photodynamic treatment of atherosclerosis. This study aimed to develop a macrophage-mannose-receptor-targeted photoactivatable nanoagent that regulates atherosclerosis and to evaluate its efficacy as well as safety in atherosclerotic mice. We synthesised and characterised D-mannosamine (MAN)-polyethylene glycol (PEG)-chlorin e6 (Ce6) for phototheranostic treatment of atherosclerosis. The diagnostic and therapeutic effects of MAN-PEG-Ce6 were investigated using the atherosclerotic mouse model. The hydrophobic Ce6 photosensitiser was surrounded by the hydrophilic MAN-PEG outer shell of the self-assembled nanostructure under aqueous conditions. The MAN-PEG-Ce6 was specifically internalised in macrophage-derived foam cells through receptor-mediated endocytosis. After laser irradiation, the MAN-PEG-Ce6 markedly increased singlet oxygen generation. Intravital imaging and immunohistochemistry analyses verified MAN-PEG-Ce6's specificity to plaque macrophages and its notable anti-inflammatory impact by effectively reducing mannose-receptor-positive macrophages. The toxicity assay showed that MAN-PEG-Ce6 had negligible effects on the biochemical profile and structural damage in the skin and organs. Targeted photoactivation with MAN-PEG-Ce6 thus has the potential to rapidly reduce macrophage-derived inflammatory responses in atheroma and present favourable toxicity profiles, making it a promising approach for both imaging and treatment of atherosclerosis.

Maduramycin, a novel glycosylation modulator for mammalian fed-batch and steady-state perfusion processes.

Controlling high-mannose (HM) content of therapeutic proteins during process intensification, reformulation for subcutaneous delivery, antibody-drug conjugate or biosimilar manufacturing represents an ongoing challenge. Even though a range of glycosylation levers to increase HM content exist, modulators specially increasing M5 glycans are still scarce. Several compounds of the polyether ionophore family were screened for their ability to selectively increase M5 glycans of mAb products and compared to the well-known α-mannosidase I inhibitor kifunensine known to increase mainly M8-M9 glycans. Maduramycin, amongst other promising polyether ionophores, showed the desired effect on different cell lines. For fed-batch processes, a double bolus addition modulator feed strategy was developed maximizing the effect on glycosylation by minimizing impact on culture performance. Further, a continuous feeding strategy for steady-state perfusion processes was successfully developed, enabling consistent product quality at elevated HM glycan levels. With kifunensine and maduramycin showing inverse effects on the relative HM distribution, a combined usage of these modulators was further evaluated to fine-tune a desired HM glycan pattern. The discovered HM modulators expand the current HM modulating toolbox for biotherapeutics. Their application not only for fed-batch processes, but also steady-state perfusion processes, make them a universal tool with regards to fully continuous manufacturing processes.

Structural and functional analysis of Cyanovirin-N homologs: Carbohydrate binding affinities and antiviral potential of cyanobacterial peptides.

Cyanobacteria, a group of photosynthetic prokaryotes, can sinthesize several substances due to their secondary metabolism, with notable properties, such as Cyanovirin-N(CVN), a carbohydrate-binding lectin, that exhibits antiviral activity against several pathogens, due to its ability to bind viral surface carbohydrates such as mannose, thus interfering with the viral entry on the cell. CVN has been described in several cyanobacterial strains and shows biotechnological potential for the development of drugs of pharmaceutical interest. This study focuses on the genomic exploration and characterization of Cyanovirin-N homologs to assess the conservation of carbohydrate-binding affinity within the group. The analysis of their antiviral properties was carried out using bioinformatics tools to study protein models through an in silico pipeline, following the steps of genomic prospection on public databases, homology modeling, docking, molecular dynamics and energetic analysis. Mannose served as the reference ligand, and the lectins' binding affinity with mannose was assessed across Cyanovirin-N homologs. Genomic mining identified 33 cyanobacterial lectin sequences, which underwent structural and functional characterization. The results obtained from this work indicate strong carbohydrate affinity on several homologs, pointing to the conservation of antiviral properties alongside the group. However, this affinity was not uniformly distributed among sequences, exhibiting significant heterogeneity in binding site residues, suggesting potential multi-ligand binding capabilities on the Cyanovirin-N homologs group. Studies focused on the properties involved in these molecules and the investigation of the genetic diversity of Cyanovirin-N homologs could provide valuable insights into the discovery of new drug candidates, harvesting the potential of bioinformatics for large-scale functional and structural analysis.

Integrated application of transcriptomics and metabolomics provides insight into the mechanism of Eimeria tenella resistance to maduramycin.

Avian coccidiosis, caused by Eimeria parasites, continues to devastate the poultry industry and results in significant economic losses. Ionophore coccidiostats, such as maduramycin and monensin, are widely used for prophylaxis of coccidiosis in poultry. Nevertheless, their efficacy has been challenged by widespread drug resistance. However, the underlying mechanisms have not been revealed. Understanding the targets and resistance mechanisms to anticoccidials is critical to combat this major parasitic disease. In the present study, maduramycin-resistant (MRR) and drug-sensitive (DS) sporozoites of Eimeria tenella were purified for transcriptomic and metabolomic analysis. The transcriptome analysis revealed 5016 differentially expressed genes (DEGs) in MRR compared to DS, and KEGG pathway enrichment analysis indicated that DEGs were involved in spliceosome, carbon metabolism, glycolysis, and biosynthesis of amino acids. In the untargeted metabolomics assay, 297 differentially expressed metabolites (DEMs) were identified in MRR compared to DS, and KEGG pathway enrichment analysis indicated that these DEMs were involved in 10 pathways, including fructose and mannose metabolism, cysteine and methionine metabolism, arginine and proline metabolism, and glutathione metabolism. Targeted metabolomic analysis revealed 14 DEMs in MRR compared to DS, and KEGG pathway analysis indicated that these DEMs were involved in 20 pathways, including fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism. Compared to DS, energy homeostasis and amino acid metabolism were differentially regulated in MRR. Our results provide gene and metabolite expression landscapes of E. tenella following maduramycin induction. This study is the first work involving integrated transcriptomic and metabolomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying drug resistance to polyether ionophores in coccidia.