Chitosan-glucan complex is fungal origin copolymer that finds application in medicine and cosmetics. Traditionally mycelium of Aspergillus and Penicillium is considered as industrial chitosan-glucan complex source, though utilization of Micromycetes in biotechnological productions is sometimes undesirable. The aim of the work was to study the possibility of Basidiomycete Schizophyllum commune submerged cultivation for industrial scale chitosan-glucan complex production use. Within the work there was studied effect of cultivation conditions (type and concentration of carbon sources in nutrient medium, ratio of carbon source to nitrogen source, medium initial pH and aeration intensity) on Sch. commune #127 mycelium growth, chitosan-glucan complex formation and exopolysaccharide synthesis. As the result, the method for chitosan-glucan complex production increase and exopolysaccharide synthesis suppression was suggested. Chitosan-glucan complex from Sch. commune #127 submerged mycelium was separated by successive alkali and acid treatments. Effects of alkali concentration and application technique, and type of acid on physical and chemical properties of chitosan-glucan complex were described. Analytical methods for in process control and final product characteristics were suggested.

Cell wall mannans of Candida albicans mask β-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Glucan exposures are predominantly single receptor-ligand interaction sites of nanoscale dimensions. Candida species vary in basal glucan exposure and molecular complexity of mannans. We used super-resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and elevated glucan exposure geometry. Thus, acid labile mannan structure influences the nanoscale features of glucan exposure, impacting the nature of the pathogenic surface that triggers immunoreceptor engagement, aggregation, and signaling. Graus et al. find that N-mannan structural features regulated by Candida mannosyltransfersases control glucan exposure. Loss of mannan increased the frequency and size of glucan exposures and changed multivalent receptor engagement. Changes to mannan structure in a bloodstream isolate are associated with elevated glucan exposure at the nanoscale.

Glucans are fungal cell wall PAMPs that promote survival in polymicrobial and candidal sepsis. Dectin-1 is the primary PRR for glucans. The goals of the present study were to characterize 1) the effects of fungal infection on Dectin-1; 2) the effects of polymicrobial sepsis in the presence and absence of glucan on Dectin-1; 3) the effects of systemic administration of glucans on Dectin-1; and 4) the intracellular trafficking of glucans. Mice were either systemically infected with Candida albicans, or made septic by CLP with and without glucan phosphate (GP) injection, or injected with GP. Flow cytometry was performed to assess cell surface Dectin-1 expression. C. albicans sepsis resulted in an increase in the percentage of Dectin-1 positive (Dectin+) blood and splenic leukocytes by increasing the percentage of neutrophils. C. albicans infection increased the percentage of Dectin+ splenic T cells. CLP decreased the percentage of highly Dectin-1 positive leukocytes in the blood by decreasing the percentage of Dectin+ neutrophils. GP treatment in sepsis further decreased the percentages of Dectinhigh blood leukocytes and Dectin+ neutrophils. CLP decreased the percentage of Dectin+ splenic leukocytes by decreasing the percentage of splenic macrophages. GP administration to CLP mice further decreased the percentage of Dectin+ splenocytes by decreasing the percentage of Dectin+ macrophages. Administration of GP resulted in a prolonged decrease in the percentage of Dectinhigh blood leukocytes. The changes in Dectin-1 expression with GP were because of decreases in the percentage of Dectin+ neutrophils and monocytes. In the trafficking studies, macrophages were incubated with fluorescent labeled glucans and then stained for intracellular organelles and signal transduction molecules. Cells were imaged using confocal microscopy. GP is internalized by clathrin and trafficked to the Golgi apparatus. GP internalization is regulated but not dependent on caveolin-1. GP co-localized with SRA, TLR2, and PI3K/p85. The trafficking of laminarin and particulate glucan is similar. We speculate that loss of cell surface Dectin-1 may be important in the protection conferred by glucans in sepsis. Additionally, intracellular trafficking and interaction with signaling components may be important steps in modulation of cellular function by glucan-pattern recognition receptor complexes.

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.

Asthma is the leading chronic illness in children in the United States. Since children in the U.S. spend a majority of their time indoors there is an increased need to understand key sources of daily asthma triggers in the home. Bacterial endotoxin, dust mite allergens and β-D-glucan have been shown to be potent inducers of asthma attacks, and high levels of these allergens in homes can trigger attacks in those with asthma. We aim to better understand the risks to those with asthma that might be associated with evaporative cooler (EC) use in low-income homes. ECs are often promoted because of their low energy consumption and decreased environmental impact compared to central air conditioning (AC). Because of their lower cost, ECs are more widely used in low-income homes. ECs use evaporation to cool the air, which leads to higher indoor relative humidity. This may create an ecological niche for house dust mites in semi-arid climates where they are normally absent. EC sump water also provides an ideal environment for bacteria and fungi to grow, possibly resulting in EC loading the air with more potential asthma triggers than central air conditioning. We sampled low-income homes around Utah county with central air and evaporative cooling and tested them for the presence of dust mite allergens, β-D-glucan and endotoxin. There were significantly higher levels of endotoxins and β-(1→3)-D-glucans in the EC homes compared to the AC homes, with increased odds of dust mite allergen prevalence but not at clinically significant levels. These findings suggest that in semi-arid environments, endotoxin and β-(1→3)-D-glucan levels in homes with evaporative coolers are more elevated than dust mite allergens.

<p dir="ltr">Dietary fibers have been observed to modulate the gut microbiome in ways that prevent and moderate human diseases and confer health benefits onto their human host. How dietary fibers do this is through their structure; gut microbes are equipped with a variety of differ- ent carbohydrate-active enzymes (CAZymes) that allow some to hydrolyze glycosidic bonds, thereby utilizing the dietary fiber. The more complex the dietary fiber, the more diverse the maintained gut microbiota may be, as specialist species may be required for complete hydrol- ysis. Therefore, increasing structural complexity of dietary fibers may increase gut microbial diversity and help prevent diseases. To understand if structural features impact the gut mi- crobiome, a set of resistant glucans varying in structures, including mixed-linkage -glucans, resistant maltodextrins (similar to type IV resistant starch) and polydextroses, which are comprised entirely of glucose, were used as substrates in an in vitro sequential batch fermen- tation using fecal microbiota form three healthy donors as inocula. I measured metabolic outputs, growth curves, and community structures by 16S rRNA amplicon sequencing, which I analyzed for through alpha and beta diversity differences and taxa that overrepresented and increased in each treatment. My results show that, depending on the donor and the resistant glucan, structure does significantly impact the concentrations of short-chain fatty acids (SCFAs) and other metabolites that are produced. Resistant glucan structure also impacts alpha and beta diversity to a degree and linear discriminant analysis (by LEfSe) results also support that specific species have preference towards substrates as well. Next, resistant glucans were supplemented into a high-fat diet, and compared these diets to a low- fat diet (LFD), high-fat diet with cellulose (HFD), and high-fat without cellulose (HWC) in a mouse study using C57BL/6J mice over 4 weeks. Increasing microbial diversity will not only increase diversity in the gut microbiome, but it will also provide protective effects in behavior such as helping to prevent anxiety. I measured weight, metabolic outputs, 16S community structure, changes in alpha and beta diversity, and differential abundances of OTUs and taxa by discriminant analysis Effect Size (LEfSe) and Metastats, and anxiety behaviors using open field and light/dark box tests. Microbial community structure was significantly different in treatment groups from controls. Anxiety for mice in tapioca dextrin 01 (TD01), tapioca dextrin 03 (TD03), and resistant maltodextrin (RMF) treatment groups were gen- erally increased, suggesting that the chemical structure of these resistant dextrins may alter the gut microbiome in ways that may influence behavior.</p><p dir="ltr">My overall results support the hypothesis that the fine structural features of dietary fibers do significantly impact the gut microbiome by selecting for specific microbiota, and may even impact cognition and behavior.</p>

Les maladies cardiovasculaires représentent la principale cause de décès dans le monde et elles sont souvent causées par l’athérosclérose coronarienne caractérisée par l’accumulation de plaques dans la paroi des artères menant à leur rétrécissement et à la réduction ou l’abolition de l’apport sanguin régional au muscle cardiaque. Les anomalies du métabolisme des lipides, incluant l’élévation du cholestérol dans les lipoprotéines de basse densité (LDL-cholestérol) contribuent au processus d’athérosclérose. Plusieurs traitements existent pour réduire les taux de lipides plasmatiques chez les sujets à risque, dont le principal est les statines. Cependant, les statines ne permettent pas toujours d’atteindre les cibles thérapeutiques, ce qui justifie la recherche de nouveaux traitements pouvant modifier les lipides plasmatiques. Une avenue potentielle de traitement à explorer est le beta-glucane, une fibre soluble contenue dans l’avoine pour laquelle certaines données suggèrent un effet hypolipidémiant. L’objectif de ce mémoire était d’évaluer l’efficacité d’un supplément de beta-glucane dans la réduction des taux plasmatiques de LDL-C chez les patients atteints de dyslipidémie à travers le Canada. Pour ce faire, nous avons effectué un essai clinique randomisé en double aveugle contrôlé par placebo dans une population de 264 sujets atteints d’hyperlipidémie qui ont reçus aléatoirement des traitements de beta-glucane à doses de 1.5 g, 3 g et 6 g ou un placebo durant 12 semaines. Tout au long du traitement, des mesures du taux plasmatique de LDL-C ont été effectuées et des analyses statistiques de covariance (ANCOVA) ont été faites pour comparer ces taux entre les groupes actifs et placebos afin d’observer s’il y avait des changements significatifs. La mesure d’efficacité établie était une réduction du taux de LDL-C d’au moins 0.30 mmol/L. Nos résultats suggèrent que le supplément de beta-glucane à doses de 1.5 g, 3 g et 6 g n’est pas efficace pour réduire les taux de lipides sériques. Il n’y a pas eu de variation significative dans les taux de LDL-C lorsque les groupes actifs ont été comparé au groupe placebo. / Cardiovascular diseases are the principal cause of death in the world, and they are often caused by coronary atherosclerosis characterized by the accumulation of plaques in the wall of arteries leading to their narrowing and reduction or disappearance of regional blood supply to the cardiac muscle. Abnormalities of lipid metabolism, including elevated cholesterol in low-density lipoproteins (LDL-C), contribute to the atherosclerotic process. Several treatments exist to reduce levels of plasma lipids in patients at risk, the main one being statins. However, a substantial number of patients do not reach recommended therapeutic targets while treated with statins which creates the impetus for the search of new lipid-modifying treatments. One potential avenue of treatment worthy of investigation is beta-glucan, a soluble fiber found in oats, for which some data have suggested a lipid-lowering effect. The objective of this thesis was to evaluate the efficacy of a beta-glucan supplement in reducing plasma LDL-C levels in patients with dyslipidemia across Canada. To do this, we conducted a randomized, double-blind, placebo-controlled clinical trial in a population of 264 subjects with hyperlipidemia who randomly received beta-glucan treatments at doses of 1.5 g, 3 g and 6 g or a placebo for 12 weeks. Throughout the treatment, LDL-C plasma measurements were taken and statistical analyzes of covariance (ANCOVA) were done to compare these levels between the active and placebo groups to observe if there were any significant changes. The established measure of efficacy was a reduction in LDL-C of at least 0.30 mmol/L. Briefly, our results have shown that beta-glucan in doses of 1.5 g, 3 g and 6 g is not efficacious in reducing plasma lipid levels. There was no significant change in LDL-C levels when the active groups were compared to the placebo group.

No description available.

The species of Brucella were isolated and characterized almost 120 years ago and their genomes sequenced for almost 4 years. Compared to other bacterial pathogens relatively, little is known about the factors contributing to their persistence in hosts and multiplication within phagocytic cells. Also, many aspects of the interactions between Brucella and its host remain unclear. Molecular characterization of intracellular survival processes of Brucella will provide guidance for additional prevention and control measures. One of the features that distinguishes Brucella is that they do not express classic virulence factors. Thus identification of virulence factors has been elusive and some of the identified virulence genes are putative. Disruption of putative virulence genes and studying the consequent effect on attenuation in cell lines or mouse models is a widely used method. However, in most cases it is not apparent whether the mutated genes encode virulence factors or merely affect normal metabolic or biological functions. Some mutations in Brucella can be compensated by redundancy or backup mechanisms. One method for identifying putative virulence genes involved in pathogenesis is to express these genes in a nonpathogenic host and isolate recombinants with increased virulence or survival ability either in cell culture or animal model. We hypothesize that over-expression of Brucella putative virulence genes in the non-pathogenic and close phylogenic relative Ochrobactrum anthropi should enhance its survival in infection models in vivo. O. anthropi is one of the closest Brucella relatives based on DNA, rRNA, and protein analyses but it is unable to establish chronic infection and considered as opportunistic pathogen that, under certain circumstances, may produce disease in immunocompromised humans. Therefore, we established enhanced expression system in Brucella and Ochrobactrum to identify B. suis virulence genes. We created an enhanced expression system that can be used for cloning and expression of heterologous genes in Brucella and Ochrobactrum. We studied the transcriptional activity of several promoters and created some tools to enhance the expression, detection and purification of Brucella recombinant protein in Ochrobactrum. The presumable importance of alkyl hydroperoxide reductases encoded by ahpC and ahpD genes and their contribution to intracellular survival of Brucella were studied by over-expressing them. The recombinant O. anthropi expressing B. suis ahpC and ahpD genes were able to resist in vitro killing by H2O2 and or cumene hydroperoxide and survived longer in the macrophage J774 A.1 cell line. The control O. anthropi was cleared from BALB/c mice in five days while the recombinants were recovered from spleens, livers and lungs of infected mice up to eight days post-infection. We tested the contribution of B. suis cyclic glucan synthetase gene (cgs) to virulence by over-expressing it in O. anthropi. We studied the ability of the recombinant O. anthropi to resist killing in vitro and in vivo. We generated evidence that B. suis cgs when over-expressed in O. anthropi increased the amount of cyclic glucans synthesized and accumulated in the periplasmic space. This accumulation changed the virulence of the microorganism from a soil bacterium that cleared from mice in less than five days into a pathogenic organism that could survive up to 9 days and at higher doses killed the mice. In summary, several vectors have been constructed for gene expression and protein purification in Brucella and Ochrobactrum. Novel useful tools for enhancement of heterologous gene expression were created and demonstrated to work in Brucella and Ochrobactrum. Brucella putative virulence genes were studied in Ochrobactrum using the newly constructed vectors and tools. Ochrobactrum as a gain of function model for studying putative virulence genes of intracellular pathogens in general and for Brucella in particular proved to be a very useful model. / Ph. D.

The ability of the soluble fibre (1->3)(1->4)-β-D-glucan to attenuate postprandial glycemic responses depends on its viscosity which, in turn, depends on molecular weight (MW) and dose. However, the effect of altering viscosity by changing solution volume is unknown. Furthermore, β-glucan solutions may form hard gels when left to age, but the effect of these gels on glycemic responses is unknown. Therefore, the effects of varying the MW and volume of β-glucan solutions and hard gels, on glycemic responses were determined. The results showed that glycemic responses were reduced by increasing viscosity by increasing MW but not by reducing solution volume. Although β-glucan gels reduced the rate of glucose diffusion in vitro, they had no effect on glycemic responses in vivo. Thus, changing solution viscosity through changes in volume does not alter the effect of β-glucan on glycemic response, and β-glucan gels are ineffective at attenuating in vivo glycemic responses.