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The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4 + T-cell counts. Previously, we deleted three chitin deacetylase genes from Cryptococcus neoformans to create a chitosan-deficient, avirulent strain, designated as cda1∆2∆3∆ , which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8 + T cells. In contrast, protection was lost in mice lacking α/β T cells or CD4 + T cells. Moreover, CD4 + T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4 + T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4 + T cells after vaccination but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in interferon-γ (IFNγ), tumor necrosis factor alpha (TNFα), or interleukin (IL)-23p19. A robust influx of leukocytes and IFNγ- and TNFα-expressing CD4 + T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFNγ production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8 + T cells are dispensable, IFNγ and CD4 + T cells have overlapping roles in generating protective immunity prior to cda1∆2∆3∆ vaccination. However, once vaccinated, protection becomes less dependent on CD4 + T cells, suggesting a strategy for vaccinating HIV + persons prior to loss of CD4 + T cells., Importance: The fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4 + T-cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans , designated as cda1∆2∆3∆ . When used as a vaccine, cda1∆2∆3∆ protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8 + T cells were dispensible, protection was lost in mice genetically deficient in CD4 + T cells and the cytokines IFNγ, TNFα, or IL-23. A robust influx of cytokine-producing CD4 + T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4 + T cells following vaccination, suggesting a strategy to protect persons who are at risk of future CD4 + T-cell dysfunction., Competing Interests: The authors declare no conflict of interest.

Inter-individual variation exists in response to diet and in the endpoints related to vascular diseases and cognitive impairment. Therefore, the evaluation and characterisation of responses to a dietary intervention targeting these endpoints is important. A dietary intervention with 37 participants has been performed comparing two forms of blueberry, either whole fresh blueberry (160 g), freeze-dried blueberry powder (20 g) or a placebo control (microcrystalline cellulose), in a 1-week single-blinded cross-over randomised controlled trial (RCT) in a healthy population. The response to the intervention was calculated for each endpoint using the percentage change (±%) compared to the baseline. Extensive inter-individual variation was found in vascular health parameters (-141 to +525%) and cognitive domains (-114 to +96%) post-intervention, but there was no consistent response following the two interventions between and within participants for each endpoint measured. No significant putative discriminating urinary metabolites between interventions were found using supervised multivariate analysis. Although several discriminatory metabolites were found between the responder and non-responder groups, it was not possible to identify predictors of the response using receiver operating curve analysis. To conclude, this is the first blueberry intervention applying quartile divisions to characterise individual responses in vascular and cognitive endpoints following a specific dietary intervention; however, we did not find any consistency in the individual responses to the interventions, and we could not identify a predictive urinary metabolite as a potential biomarker for differentiation between responders and non-responders. However, the overall approach of defining a metabolic signature of response could be used in the future for tailored personalised nutritional advice.

Cryptococcus neoformans and C. gattii , the etiologic agents of cryptococcosis, cause over 100,000 deaths worldwide every year, yet no cryptococcal vaccine has progressed to clinical trials. In preclinical studies, mice vaccinated with an attenuated strain of C. neoformans deleted of three cryptococcal chitin deacetylases ( Cn - cda1 Δ 2 Δ 3 Δ) were protected against a lethal challenge with C. neoformans strain KN99. While Cn-cda1 Δ 2 Δ 3 Δ extended the survival of mice infected with C. gattii strain R265 compared to unvaccinated groups, we were unable to demonstrate fungal clearance as robust as that seen following KN99 challenge. In stark contrast to vaccinated mice challenged with KN99, we also found that R265-challenged mice failed to induce the production of protection-associated cytokines and chemokines in the lungs. To investigate deficiencies in the vaccine response to R265 infection, we developed a KN99-R265 coinfection model. In unvaccinated mice, the strains behaved in a manner which mirrored single infections, wherein only KN99 disseminated to the brain and spleen. We expanded the coinfection model to Cn-cda1 Δ 2 Δ 3 Δ-vaccinated mice. Fungal burden, cytokine production, and immune cell infiltration in the lungs of vaccinated, coinfected mice were indicative of immune evasion by C. gattii R265 as the presence of R265 neither compromised the immunophenotype established in response to KN99 nor inhibited clearance of KN99. Collectively, these data indicate that R265 does not dampen a protective vaccine response, but rather suggest that R265 remains largely undetected by the immune system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hester, Carlson, Lodge, Levitz and Specht.)

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Creating a safe and effective vaccine against infection by the fungal pathogen Cryptococcus neoformans is an appealing option that complements the discovery of new small molecule antifungals. Recent animal studies have yielded promising results for a variety of vaccines that include live-attenuated and heat-killed whole-cell vaccines, as well as subunit vaccines formulated around recombinant proteins. Some of the recombinantly engineered cryptococcal mutants in the chitosan biosynthesis pathway are avirulent and very effective at conferring protective immunity. Mice vaccinated with these avirulent chitosan-deficient strains are protected from a lethal pulmonary infection with C. neoformans strain KN99. Heat-killed derivatives of the vaccination strains are likewise effective in a murine model of infection. The efficacy of these whole-cell vaccines, however, is dependent on a number of factors, including the inoculation dose, route of vaccination, frequency of vaccination, and the specific mouse strain used in the study. Here, we present detailed methods for identifying and optimizing various factors influencing vaccine potency and efficacy in various inbred mouse strains using a chitosan-deficient cda1Δcda2Δcda3Δ strain as a whole-cell vaccine candidate. This chapter describes the protocols for immunizing three different laboratory mouse strains with vaccination regimens that use intranasal, orotracheal, and subcutaneous vaccination routes after the animals were sedated using two different types of anesthesia., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryptococcus neoformans is an opportunistic human fungal pathogen capable of surviving in a wide range of environments and hosts. It has been developed as a model organism to study fungal pathogenesis due to its fully sequenced haploid genome and optimized gene deletion and mutagenesis protocols. These methods have greatly aided in determining the relationship between Cryptococcus genotype and phenotype. Furthermore, the presence of congenic mata and matα strains associated with a defined sexual cycle has helped further understand cryptococcal biology. Several in vitro stress conditions have been optimized to closely mimic the stress that yeast encounter in the environment or within the infected host. These conditions have proven to be extremely useful in elucidating the role of several genes in allowing yeast to adapt and survive in hostile external environments. This chapter describes various in vitro stress conditions that could be used to test the sensitivity of different mutant strains, as well as the protocol for preparing them. We have also included a list of mutants that could be used as a positive control strain when testing the sensitivity of the desired strain to a specific stress., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Beneficial effects of nut supplementation on cognitive function have previously been demonstrated in young and older adults. Alterations to gut microbiota have also been shown following tree nut consumption. However, no data exists on the effects of nuts on cognition and intestinal microbial communities assessed within the same study., Objectives: The study aimed to examine the effects of daily consumption of tree nuts for 4 wk on cognitive function (primary outcome), mood, metabolomics, and gut microbial species (secondary outcomes) in healthy, nonelderly adults., Methods: This randomized, placebo-controlled, double-blind, counterbalanced crossover study assessed the effects of 4 wk of supplementation with 30 g/d mixed tree nuts versus placebo on cognition and mood in 79 healthy adults aged 18-49 y. Metabolic responses, gut bacterial community structure, and the potential for these to impact cognition were explored using a multi-omic approach. Bacterial community analysis was conducted in Quantitative Insights Into Microbial Ecology 2 (QIIME2)., Results: Mixed model analysis indicated that nut consumption led to significant improvements to accuracy (placebo M = 92.2% compared with NUTS M = 94.5%; P = 0.019) and speed of response (placebo M = 788 ms compared with NUTS M = 757 ms; P = 0.004) on a picture recognition task. No significant changes to bacterial community α or β diversity were observed when comparing nut consumption to the placebo arm. However, an unclassified Lachnospiraceae amplicon sequence variant (ASV) was significantly enriched in participants when supplemented with nuts (P = 0.015). No correlations were observed between the changes to picture recognition and the changes to the unclassified Lachnospiraceae ASV. There were no significant changes to the urinary metabolome., Conclusions: These findings indicate a positive effect of nut on cognition following only 4 wk of consumption in a healthy nonelderly sample, as well as upregulation of a microbial taxa associated with gut health. The effects appear to be independent of one another, but further exploration is required in those experiencing cognitive decline and/or gut dysbiosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Objectives: The cognitive-protective effects related to the consumption of a variety of fruits are supported by several intervention studies. This systematic review and meta-analysis compared the magnitude of effects following chronic (≥1 week) consumption of frozen, freeze-dried powder including extracts and juices of fruits, covering berries, cherries and citrus, on cognition and mood in adults., Methods: PubMed, Web of Science, Scopus, and psycARTICLES were searched from inception until February, 2021. Inclusion criteria were randomised controlled trials assessing memory, executive function, psychomotor speed, mood and mini mental state examination in adult participants ≥18 years of age. Cognition was tested by global or domain specific tasks., Results: Out of 13,861 articles identified, 16 papers were included; 11 studies provided suitable data for meta-analysis. Fourteen studies reported improvement or trend for improvement in cognition, five studies assessed mood and one study supplementing grape juice found trend for mood improvement. From the meta-analysis, cherry juice supplementation was suggested to improve psychomotor speed by -0.37 of standardised mean difference (95% CI [-0.74, 0.01]) in reaction time (P = 0.05)., Conclusions: The meta-analysis did not sufficiently support a role for fruits or fruit forms to improve cognition and mood., (© 2022. The Author(s).)

Introduction: Cryptococcus neoformans is a basidiomycete fungus that can cause meningoencephalitis, especially in immunocompromised patients. Cryptococcus grows in many different media, although little attention has been paid to the role of growth conditions on the cryptococcal cell wall or on virulence., Objective: The purpose of this study was to determine how different media influenced the amount of chitin and chitosan in the cell wall, which in turn impacted the cell wall architecture and host response., Methods: Yeast extract, peptone, and dextrose (YPD) and yeast nitrogen base (YNB) are two commonly used media for growing Cryptococcus before use in in vitro or in vivo experiments. As a result, C. neoformans was grown in either YPD or YNB, which were either left unbuffered or buffered to pH 7 with MOPS. These cells were then labeled with cell wall-specific fluorescent probes to determine the amounts of various cell wall components. In addition, these cells were employed in animal virulence studies using the murine inhalation model of infection., Results: We observed that the growth of wild-type C. neoformans KN99 significantly changes the pH of unbuffered media during growth. It raises the pH to 8.0 when grown in unbuffered YPD but lowers the pH to 2.0 when grown in unbuffered YNB (YNB-U). Importantly, the composition of the cell wall was substantially impacted by growth in different media. Cells grown in YNB-U exhibited a 90% reduction in chitosan, the deacetylated form of chitin, compared with cells grown in YPD. The decrease in pH and chitosan in the YNB-U-grown cells was associated with a significant increase in some pathogen-associated molecular patterns on the surface of cells compared with cells grown in YPD or YNB, pH 7. This altered cell wall architecture resulted in a significant reduction in virulence when tested using a murine model of infection. Furthermore, when heat-killed cells were used as the inoculum, KN99 cells grown in YNB-U caused an aberrant hyper-inflammatory response in the lungs, resulting in rapid animal death. In contrast, heat-killed KN99 cells grown in YNB, pH 7, caused little to no inflammatory response in the host lung, but, when used as a vaccine, they conferred a robust protective response against a subsequent challenge infection with the virulent KN99 cells., Conclusion: These findings emphasize the importance of culture media and pH during growth in shaping the content and organization of the C. neoformans cell wall, as well as their impact on fungal virulence and the host response., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The fungal pathogen Cryptococcus neoformans causes up to 278 000 infections each year globally, resulting in up to 180,000 deaths annually, mostly impacting immunocompromised people. Therapeutic options for C. neoformans infections are very limited. Caspofungin, a member of the echinocandin class of antifungals, is generally well tolerated but clinically ineffective against C. neoformans. We sought to identify biological processes that can be targeted to render the cell more susceptible to echinocandins by screening the available libraries of gene deletion mutants made in the KN99α background for caspofungin sensitivity. We adapted a Candida albicans fungal biofilm assay for the growth characteristics of C. neoformans and systematically screened 4,030 individual gene deletion mutants in triplicate plate assays. We identified 25 strains that showed caspofungin sensitivity. We followed up with a dose dependence assay, and 17 of the 25 were confirmed sensitive, 5 of which were also sensitive in an agar plate assay. We made new deletion mutant strains for four of these genes: CFT1 , encoding an iron transporter; ERG4 , encoding a sterol desaturase; MYO1 , encoding a myosin heavy chain; and YSP2 , encoding a sterol transporter. All were more sensitive to membrane stress and showed significantly increased sensitivity to caspofungin at higher temperatures. Surprisingly, none showed any obvious cell wall defects such as would be expected for caspofungin-sensitive strains. Our microscopy analyses suggested that loss of membrane integrity contributed to the caspofungin sensitivity, either by allowing more caspofungin to enter or remain in the cell or by altering the location or orientation of the enzyme target to render it more susceptible to inhibition. IMPORTANCE The intrinsic resistance of Cryptococcus neoformans to the cell wall inhibitor caspofungin limits the available therapies for treating cryptococcal infections. We screened a collection of more than 4,000 gene deletion strains for altered caspofungin sensitivity to identify biological processes that could be targeted to render the cell more susceptible to caspofungin. We identified multiple genes with an effect on caspofungin susceptibility and found that they were associated with altered membrane permeability rather than the expected cell wall defects. This suggests that targeting these genes or other genes affecting membrane permeability is a viable path for developing novel therapies for treating this global fungal pathogen.

Recommendations for free sugar intake in the UK should be no more than 5 % of total energy due to increased health risks associated with overconsumption. It was therefore of interest to examine free sugar intakes and associations with health parameters in the UK population. The UK National Diet and Nutrition Survey rolling programme (2008-2017) was used for this study. Dietary intake, anthropometrical measurements and clinical biomarker data collated from 5121 adult respondents aged 19-64 years were statistically analysed. Compared with the average total carbohydrate intake (48 % of energy), free sugars comprised 12·5 %, with sucrose 9 % and fructose 3·5 %. Intakes of these sugars, apart from fructose, were significantly different over collection year ( P < 0·001) and significantly higher in males ( P < 0·001). Comparing those consuming above or below the UK recommendations for free sugars (5 % energy), significant differences were found for BMI ( P < 0·001), TAG ( P < 0·001), HDL ( P = 0·006) and homocysteine concentrations ( P = 0·028), and significant sex differences were observed (e.g. lower blood pressure in females). Regression analysis demonstrated that free sugar intake could predict plasma TAG, HDL and homocysteine concentrations ( P < 0·0001), consistent with the link between these parameters and CVD. We also found selected unhealthy food choices (using the UK Eatwell Guide) to be significantly higher in those that consumed above the recommendations ( P < 0·0001) and were predictors of free sugar intakes ( P < 0·0001). We have shown that adult free sugar intakes in the UK population are associated with certain negative health parameters that support the necessary reduction in free sugar intakes for the UK population.

Blueberries are rich in polyphenols, and their effect on cardiovascular health, including risk factors for endothelial dysfunction and hypertension, has been investigated in interventional studies. However, the difference between blueberry treatments in varied forms for their cardiovascular-protective effect remains poorly understood. The current study assessed the effects of whole blueberry and freeze-dried blueberry powder compared to a control on cardiovascular health in young adults. A cross-over randomised controlled trial (RCT) was implemented with 1 week of treatment for three treatment groups, each followed by 1 week of wash out period. Systolic (SBP) and diastolic blood pressure (DBP), pulse wave velocity (PWV), plasma cholesterol (low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol) and triglyceride levels (TAG), and glucose and nitrite (NO2-) concentrations were compared following fresh blueberry, freeze-dried blueberry powder, and control treatments. Thirty-seven participants with a mean age of 25.86 ± 6.81 completed the study. No significant difference was observed among fresh blueberry, blueberry powder, and the control arm. Plasma NO2- levels were improved by 68.66% and 4.34% separately following whole blueberry and blueberry powder supplementations compared to the baseline, whereas the control supplementation reported a decrease (-9.10%), although it was not statistically significant. There were no other effects shown for SBP, DBP, total cholesterol, HDL-C, LDL-C, TAG, or glucose. No difference was shown between whole blueberry and freeze-dried blueberry powder consumption for improving cardiovascular health.

Tart Montmorency cherries (MC) are a particularly rich source of anthocyanins and other polyphenols that have been shown to elicit antioxidant, anti-inflammatory and vasomodulatory actions. The current study aimed to determine the influence of chronic MC supplementation on cognitive function and mood. In a 3-month double-blinded, placebo-controlled parallel study, middle-aged adults (mean ± sd: 48 ± 6 years) were randomly assigned to either 30 ml twice daily of MC (n 25) or the same amount of an isoenergetic placebo (n 25). Cognitive function and mood were assessed before and after supplementation using a computerised cognitive task battery and visual analogue scales. Cerebral blood flow was also monitored by near-infrared spectroscopy during the task battery, and questionnaires were administered to determine subjective sleep and health status and plasma metabolomics were analysed before and after supplementation. After 3 months, the MC resulted in higher accuracy in digit vigilance (mean difference: 3·3, 95 % CI: 0·2, 6·4 %) with lower number of false alarms (mean difference: -1·2, 95 % CI: -2·0, -0·4) compared with the placebo. There was also a treatment effect for higher alertness (mean difference: 5·9, 95 % CI: 1·3, 10·5 %) and lower mental fatigue ratings (mean difference -9·5, 95 % CI: -16·5, -2·5 %) with MC. Plasma metabolomics revealed an increase in a number of amino acids in response to MC intake, but not placebo. These data suggest an anti-fatiguing effect of MC supplementation as well as the ability to improve sustained attention during times of high cognitive demand, this could be related to changes in amino acid metabolism.

Chitosan, a deacetylated form of chitin, is required for the virulence of Cryptococcus neoformans . There are three chitin deacetylase genes (CDA) that are essential for chitosan production, and deletion of all three genes results in the absence of chitosan, loss of virulence, and induction of a protective host response when used as a vaccine. Cda1 plays a major role in deacetylating chitin during pulmonary infection of CBA/J mice. Inoculation with the cda 1Δ strain did not lead to a lethal infection. However, the infection was not cleared. The persistence of the fungus in the host suggests that chitin is still being deacetylated by Cda2 and/or Cda3. To test this hypothesis, we subjected strains deleted of two CDA genes to fungal virulence in CBA/J, C57BL/6 and BALB/c and found that cda 1Δ cda 2Δ was avirulent in all mouse lines, as evidenced by its complete clearance. Consistent with the major role of Cda1 in CBA/J, we found that cda 2Δ cda 3Δ was as virulent as its wild-type progenitor KN99. On the other hand, cda 1Δ cda 3Δ displayed virulence comparable to that of cda 1Δ. The virulence of each mutant correlates with the amount of chitosan produced when grown under host-mimicking culture conditions. In addition, the avirulence of cda 1Δ cda 2Δ was followed by the induction of a protective immune response in C57BL/6 and CBA/J mice, when a live or heat-killed form of the mutant was used as a vaccine respectively. Taken together, these data imply that, in C. neoformans , coordinated activity of both Cda1 and Cda2 is essential for mediating fungal virulence., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Montmorency tart cherries (MC) have been found to modulate indices of vascular function with interventions of varying duration. The objective of this preliminary study was to identify the chronic effects of MC supplementation on vascular function and the potential for urinary metabolomics to provide mechanistic evidence. We performed a placebo-controlled, double-blind, randomised study on 23 healthy individuals (18M, 7F) that consumed 30 ml MC or a placebo twice daily for 28 days. Whole body measures of vascular function and spot urine collections were taken at baseline and after supplementation. There were no significant changes to vascular function including blood pressure and arterial stiffness. Urinary metabolite profiling highlighted significant changes ( P < 0⋅001) with putative discriminatory metabolites related to tryptophan and histidine metabolism. Overall, MC supplementation for 28 days does not improve indices of vascular function but changes to the urinary metabolome could be suggestive of potential mechanisms., (© The Author(s) 2021.)

Epidemiological and intervention studies have reported negative health effects of sucrose intake, but many of these studies were not representative of typical dietary habits. In this pilot study, we aimed to test the effect of increasing sucrose intakes for 1 week on body composition and blood pressure and explore the feasibility of consuming high intakes of sucrose in addition to a habitual diet. In a randomised crossover design study, twelve healthy participants (50 % female, age 28⋅4 ± 10 years, BMI 25 ± 3 kg/m 2 ), consumed either 40, 80 or 120 g sucrose in 500 ml water in addition to their habitual diet every day for 1 week, with a 1-week washout between treatment periods. Body composition (assessed using bioelectrical impedance) and blood pressure measurements were taken before and after each intervention phase. All participants reported no issues with consuming the sucrose dose for the intervention period. There was a significant increase in systolic blood pressure following 120 g sucrose intake ( P = 0⋅006), however there was no significant changes to blood pressure, body weight, BMI, percentage protein, fat or water ( P > 0⋅05) when comparing change from baseline values. There was also no effect of sucrose intakes on energy or macronutrient intakes during the intervention ( P > 0⋅05). We show here that varying doses of sucrose over a 1-week period have no effect on body composition or blood pressure. The amounts of sucrose used were an acceptable addition to the habitual diet and demonstrate the feasibility of larger-scale studies of chronic sucrose supplementation., (© The Author(s) 2021.)

Background and Aims: Historical and emerging data implicate fungi in Crohn's disease [CD] pathogenesis. However, a causal link between mycobiota, dysregulated immunity, and any impact of NOD2 variants remains elusive. This study aims to evaluate associations between NOD2 variants and faecal mycobiota in CD patients and non-CD subjects., Methods: Faecal samples were obtained from 34 CD patients [18 NOD2 mutant, 16 NOD2 wild-type] identified from the UK IBD Genetics Consortium. To avoid confounding influence of mucosal inflammation, CD patients were in clinical remission and had a faecal calprotectin <250 μg/g; 47 non-CD subjects were included as comparator groups, including 22 matched household [four NOD2 mutant] and 25 non-household subjects with known NOD2 genotype [14 NOD2 mutant] identified by the NIHR BioResource Cambridge. Faecal mycobiota composition was determined using internal transcribed spacer 1 [ITS1] sequencing and was compared with 16S rRNA gene sequences and volatile organic compounds., Results: CD was associated with higher numbers of fungal observed taxonomic units [OTUs] [p = 0.033]. Principal coordinates analysis using Jaccard index [p = 0.018] and weighted Bray-Curtis dissimilarities [p = 0.01] showed Candida spp. clustered closer to CD patients whereas Cryptococcus spp. clustered closer to non-CD. In CD, we found higher relative abundance of Ascomycota [p = 0.001] and lower relative abundance Basidiomycota [p = 0.019] phyla. An inverse relationship was found between bacterial and fungal Shannon diversity in NOD2 wild-type which was independent of CD [r = -0.349; p = 0.029]., Conclusions: This study confirms compositional changes in the gut mycobiota in CD and provides evidence that fungi may play a role in CD pathogenesis. No NOD2 genotype-specific differences were observed in the faecal mycobiota., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Water-soluble vitamins are essential dietary components with a multitude of important functions that require quantification from food sources to characterise the nutritional status of food. In this study, we have developed a hydrophilic interaction chromatography (HILIC) based method coupled to single-quadrupole mass spectrometry (MS) for the analysis of selected water-soluble vitamins. Due to their involvement in energy release from macronutrients, the quantification of thiamine (B 1 ), riboflavin (B 2 ), nicotinamide (B 3 ) and pyridoxine (B 6 ) offers significant value in food analysis. A commercially available vegetable soup was selected as the food matrix for this study and utilised to develop an efficient extraction procedure for the vitamins of interest. Vitamins were extracted using meta-phosphoric acid coupled with a reducing agent, DL-dithiothreitol (DTT) to produce the parent compound. The extracted vitamins were then analysed using an LC-MS system with electrospray - atmospheric pressure ionization (ES-API) source, operated in positive single ion monitoring (SIM) mode. The MS provided good linearity within the investigated range from 5 to 400 ng/mL with coefficient of determination (r 2 ) ranging from 0.98 to 0.99. Retention times (0.65-9.04 min) were reproducible and no coelution between vitamins was observed. Limit of detection (LOD) varied from 2.4 to 9.0 ng/mL and limit of quantification (LOQ) was from 8 to 30 ng/mL, comparable to previously published studies. The extraction method provided good intra-day (%CV 1.56-6.56) and inter-day precision (%CV 8.07-10.97). Standard injections were used as part of quality control measures and provided excellent reproducibility (%CV 0.9-3.4). The overall runtime of this method was 19 min, including column reconditioning. Using this method, the quantity of thiamine (67 ± 7 ng/g), riboflavin (423 ± 39 ng/g), nicotinamide (856 ± 77 ng/g) and pyridoxine (133 ± 11 ng/g) was determined from a complex food matrix. In conclusion, we have developed a rapid and reliable, HILIC-single quad MS method utilising SIM for the low-level quantification of four B vitamins in a vegetable soup matrix in under 20 min. This method has shown excellent linearity, intra- and inter-day reproducibility and is directly applicable to other plant-based food matrices., (Copyright © 2021. Published by Elsevier B.V.)

Background: Tart Montmorency cherries (MC) have been shown to be rich in anthocyanins and other phytochemicals known to have anti-inflammatory properties and influence pathways that might improve cardiometabolic health. However, there is limited evidence for the longer-term use of tart cherries on these indices. The aim of the current study was to investigate the influence of MC concentrate on cardiometabolic health indices following a 3-month supplementation period., Methods: Fifty middle-aged adults (34 males and 16 females; mean ± SD age: 48 ± 6 years and BMI: 27.6 ± 3.7 kg/m 2 ) completed a randomised, placebo-controlled parallel study in which they either received MC or an isocaloric placebo. Participants drank 30 mL of their allocated treatment twice per day for 3 months. Vascular function (blood pressure [BP], heart rate [HR], pulse wave velocity and analysis [PWV/A], and flow mediated dilation [FMD]) as well as indices of metabolic health (insulin, glucose, lipid profiles, and high sensitivity C reactive protein) were measured following an overnight fast before and after the 3 months., Results: No effect of the intervention between the groups was observed for vascular function or metabolic health variables following the intervention ( p > 0.05). However, MC concentrate was shown to be safe and well-tolerated and, importantly, did not have any deleterious effects on these outcomes. In conclusion, MC has no influence on cardiometabolic indices in middle-aged adults.

Purpose: This review aims to compare the magnitude of the effects of chronic consumption of fruits; specifically berries, citrus and cherries on cardiovascular disease (CVD) risk factors., Methods: PubMed, Web of Science, Scopus, and psycARTICLES were searched from inception until January 2020. Forty-five chronic (≥ 1 week) randomised controlled trials assessing CVD risk factors including endothelial (dys)function, blood pressure (BP), blood lipids and inflammatory biomarkers were included., Results: Investigated interventions reported improvements in endothelial function (n = 8), inflammatory biomarkers and lipid status (n = 14), and BP (n = 10). Berries including juice of barberry, cranberry, grape, pomegranate, powder of blueberry, grape, raspberry and freeze-dried strawberry significantly reduced SBP by 3.68 mmHg (95% CI - 6.79 to - 0.58; P = 0.02) and DBP by 1.52 mmHg (95% CI - 2.87 to - 0.18, P = 0.04). In subgroup analysis, these associations were limited to cranberry juice (SBP by 1.52 mmHg [95% CI - 2.97 to - 0.07; P = 0.05], DBP by 1.78 mmHg [95% CI - 3.43 to - 0.12, P = 0.04] and cherry juice (SBP by 3.11 mmHg [95% CI - 4.06 to - 2.15; P = 0.02]). Berries also significantly elevated sVCAM-1 levels by 14.57 ng/mL (85% CI 4.22 to 24.93; P = 0.02)., Conclusion: These findings suggest that supplementing cranberry or cherry juice might contribute to an improvement in blood pressure. No other significant improvements were observed for other specified fruits. More research is warranted comparing different classes of fruit and exploring the importance of fruit processing on their cardiovascular-protective effects.

Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human-pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan-deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan-deficient C. neoformans strain lacking three chitin deacetylases ( cda1 Δ cda2 Δ cda3 Δ) was found to confer protective immunity to a subsequent challenge with a virulent wild-type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase-mediated formation of chitosan. Mice inoculated with the chs3 Δ strain at a dose previously shown to induce protection with the cda1 Δ cda2 Δ cda3 Δ strain die within 36 h after installation of the organism. Mortality was not dependent on viable fungi, as mice inoculated with a heat-killed preparation of the chs3 Δ strain died at the same rate as mice inoculated with a live chs3 Δ strain, suggesting that the rapid onset of death was host mediated, likely caused by an overexuberant immune response. Histology, cytokine profiling, and flow cytometry indicate a massive neutrophil influx in the mice inoculated with the chs3 Δ strain. Mice depleted of neutrophils survived chs3 Δ inoculation, indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcus-induced host inflammatory responses. IMPORTANCE Cryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in ∼200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the host's immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different from the cell walls of many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study, we investigated the host responses to a chs3Δ strain, a chitosan-deficient strain, and found that mice inoculated with the chs3Δ strain all died within 36 h and that death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus ., (Copyright © 2020 Hole et al.)

Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans , the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes., (© 2020 Chrissian et al.)

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformans IMPORTANCE Cryptococcus neoformans is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents., (Copyright © 2019 Maybruck et al.)

Cryptococcus gattii R265 is a hypervirulent fungal strain responsible for the recent outbreak of cryptococcosis in Vancouver Island of British Columbia in Canada. It differs significantly from Cryptococcus neoformans in its natural environment, its preferred site in the mammalian host, and its pathogenesis. Our previous studies of C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains a two- to threefold larger amount of chitosan than that of C. neoformans The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDAs) have, however, been modified. To deduce their roles, single and double CDA deletion strains and a triple CDA deletion strain were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in strain R265, Cda3 is critical for virulence. Deletion of either CDA3 alone or in combination with another CDA ( cda1Δ3 Δ or cda2Δ3Δ ) or both ( cda1Δ2Δ3Δ ) rendered the fungus avirulent and cleared from the infected host. Moreover, the cda1 Δ 2 Δ 3 Δ strain of R265 induced a protective response to a subsequent infection with R265. These studies begin to illuminate the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence. IMPORTANCE The fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offer new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans ., (Copyright © 2019 Lam et al.)

There is a dearth of information regarding the reliability of non-invasive measures of vascular function taken in a single testing session. This study aimed to determine the test-retest reliability of a test battery of vascular function measures: automated blood pressure (BP), laser Doppler imaging with iontophoresis (LDI), digital volume pulse (DVP), pulse wave velocity (PWV), augmentation index (AIx) measured by pulse wave analysis (PWA) and flow-mediated dilation (FMD) taken within and between sessions. Measures were taken in 21 non-smoking males intra-session and again inter-session (one week apart) to determine repeatability and reproducibility, respectively. There was moderate to excellent repeatability (ICC: 0.53-0.93; CV=2.2-18.1%) and reproducibility (ICC: 0.71-0.96; CV 1.9-14.2%) for BP, DVP stiffness index, PWV, AIx, AIx normalised to heart rate (75 bpm), absolute and percentage FMD. Repeatability of the DVP reflection index was moderate (ICC: 0.64; CV=9.5%) but there was poor reproducibility (ICC: 0.17; CV=15.1%). Moreover, the repeatability and reproducibility of the LDI measures ranged from poor to good (ICC: 0.31-0.84; CV=28.4-36.7%). These data indicated that there was considerable variability in the repeatability and reproducibility of measurements of endothelial function and arterial stiffness taken in a battery of measurements, which needs careful consideration in future research designs., Competing Interests: Authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Accumulating evidence suggests flavonoid intake is associated with reduced risk of non-communicable diseases. We aimed to systematically determine and quantify the potential association between dietary anthocyanin intake and risk of cardiovascular diseases (CVD). A systematic literature search of studies reporting anthocyanin intake and risk of fatal or nonfatal CVD was performed using SCOPUS, MEDLINE, CINAHL and Cochrane Library. The relative risk (RR) or hazard ratio (HR) of highest category of anthocyanin foods were pooled in a random-effects meta-analysis. Subgroup analysis were conducted to determine possible sources of heterogeneity. The meta-analysis suggested intake of dietary anthocyanins and reduced risk of CHD (RR = 0.91, 95% CI: 0.83, 0.99; I 2  = 12.0, P h  = 0.337) and CVD mortality (RR = 0.92, 95% CI: 0.87, 0.97; I 2  = 0.0, P h  = 0.584). However, there was no relationship between the intake of these compounds and reduced risk of MI, stroke or total CVD. Subgroup analysis determined reduced risk of CHD and CVD mortality was more prominent for anthocyanidin intake, as opposed to anthocyanin or berries. Our systematic review and meta-analysis provides evidence that anthocyanins, specifically anthocyanidins, reduce the risk of CHD and CVD mortality. Further randomized controlled trials on anthocyanin intake and CVD risk factors are needed to support these findings.

Background and Aims: Worldwide, cardiovascular diseases (CVDs) remains as the main cause of mortality. Observational studies supports an association between intake of tomato products or lycopene with a reduced CVDs risk. Our aim was to undertake a systematic review and meta-analysis of the evidence on the topic., Methods: Medline, Web of Science, and Scopus were searched from inception until July 2017. We included longitudinal and cross-sectional studies reporting associations between lycopene and tomato consumption and cardiovascular morbidity and mortality among adult subjects. Random-effects models were used to determine the pooled effect sizes., Results: Twenty-eight publications met our inclusion criteria and 25 studies provided quantitative data for meta-analysis. Results showed that individuals in the highest consumption category of, or with the highest serum concentration of, lycopene had significantly lower risk of stroke (hazard ratio (HR) 0.74, 0.62-0.89, p = 0.02; I 2 = 32) and CVDs (HR 0.86, 0.77-0.95, p = 0.003; I 2 = 0). In addition, individuals categorised in the highest serum concentration of lycopene also had significantly lower risk of mortality (HR 0.63, 0.49-0.81, p<0.001; I 2 = 46). Lycopene was not significantly associated with myocardial infarction, while scarce evidence on the association of lycopene with atherosclerosis, congestive heart failure, or atrial fibrillation was evident. Evidence from three studies suggested that higher intakes of tomato were associated with non-significantly lower stroke, CVDs and CHD., Conclusions: This comprehensive meta-analysis suggests that high-intakes or high-serum concentration of lycopene are associated with significant reductions in the risk of stroke (26%), mortality (37%) and CVDs (14%).

Chitin is an essential component of the cell wall of Cryptococcus neoformans conferring structural rigidity and integrity under diverse environmental conditions. Chitin deacetylase genes encode the enyzmes (chitin deacetylases [Cdas]) that deacetylate chitin, converting it to chitosan. The functional role of chitosan in the fungal cell wall is not well defined, but it is an important virulence determinant of C. neoformans Mutant strains deficient in chitosan are completely avirulent in a mouse pulmonary infection model. C. neoformans carries genes that encode three Cdas (Cda1, Cda2, and Cda3) that appear to be functionally redundant in cells grown under vegetative conditions. Here we report that C. neoformans Cda1 is the principal Cda responsible for fungal pathogenesis. Point mutations were introduced in the active site of Cda1 to generate strains in which the enzyme activity of Cda1 was abolished without perturbing either its stability or localization. When used to infect CBA/J mice, Cda1 mutant strains produced less chitosan and were attenuated for virulence. We further demonstrate that C. neoformans Cda genes are transcribed differently during a murine infection from what has been measured in vitro IMPORTANCE Cryptococcus neoformans is unique among fungal pathogens that cause disease in a mammalian host, as it secretes a polysaccharide capsule that hinders recognition by the host to facilitate its survival and proliferation. Even though it causes serious infections in immunocompromised hosts, reports of infection in hosts that are immunocompetent are on the rise. The cell wall of a fungal pathogen, its synthesis, composition, and pathways of remodelling are attractive therapeutic targets for the development of fungicides. Chitosan, a polysaccharide in the cell wall of C. neoformans is one such target, as it is critical for pathogenesis and absent in the host. The results we present shed light on the importance of one of the chitin deacetylases that synthesize chitosan during infection and further implicates chitosan as being a critical factor for the pathogenesis of C. neoformans ., (Copyright © 2018 Upadhya et al.)

The consumption of berries has been linked to decreased risk of degenerative disease. Berries are regularly processed into juices. It is largely unknown how the juicing process affects the bioavailability of metabolites. As metabolomics has shown to be a valuable nutritional tool to study global metabolite differences, the aim of this study was to investigate the effect of juicing on the relative appearance of blueberry metabolites in humans using metabolomics. Nine healthy subjects consumed 250 g of fresh blueberries either as the whole fruit or after juicing, and provided blood and urine samples before and 2 h after intake in a cross-over design. Samples underwent metabolite profiling using LCMS, and data were mined with multivariate analysis. Overall, <12 % of all ions detected were significantly influenced by blueberry treatment (P<0·05). Partial least-squared discriminant analysis models of post-treatment samples revealed good discrimination. In urinary samples, whole blueberry treatment resulted in 108 ions that were significantly higher compared with juiced treatment (positive and negative mode combined), whereas only eight were significantly higher after juiced treatment. Examples of putative annotations included metabolites of ferulic and caffeic acids, several phenolic metabolites conjugated to sulphate, glycoside or glucuronide and fatty acyl derivatives, which were of higher intensity after whole blueberry treatment. In conclusion, consumption of whole blueberries resulted in a higher range of phenolic and other metabolites in plasma and urine samples 2 h after consumption. Both whole and juiced blueberries resulted in very similar metabolite profiles at 2 h, although this was the only time point measured.

Development of a vaccine to protect against cryptococcosis is a priority given the enormous global burden of disease in at-risk individuals. Using glucan particles (GPs) as a delivery system, we previously demonstrated that mice vaccinated with crude Cryptococcus -derived alkaline extracts were protected against lethal challenge with Cryptococcus neoformans and Cryptococcus gattii The goal of the present study was to identify protective protein antigens that could be used in a subunit vaccine. Using biased and unbiased approaches, six candidate antigens (Cda1, Cda2, Cda3, Fpd1, MP88, and Sod1) were selected, recombinantly expressed in Escherichia coli , purified, and loaded into GPs. Three mouse strains (C57BL/6, BALB/c, and DR4) were then vaccinated with the antigen-laden GPs, following which they received a pulmonary challenge with virulent C. neoformans and C. gattii strains. Four candidate vaccines (GP-Cda1, GP-Cda2, GP-Cda3, and GP-Sod1) afforded a significant survival advantage in at least one mouse model; some vaccine combinations provided added protection over that seen with either antigen alone. Vaccine-mediated protection against C. neoformans did not necessarily predict protection against C. gattii Vaccinated mice developed pulmonary inflammatory responses that effectively contained the infection; many surviving mice developed sterilizing immunity. Predicted T helper cell epitopes differed between mouse strains and in the degree to which they matched epitopes predicted in humans. Thus, we have discovered cryptococcal proteins that make promising candidate vaccine antigens. Protection varied depending on the mouse strain and cryptococcal species, suggesting that a successful human subunit vaccine will need to contain multiple antigens, including ones that are species specific. IMPORTANCE The encapsulated fungi Cryptococcus neoformans and Cryptococcus gattii are responsible for nearly 200,000 deaths annually, mostly in immunocompromised individuals. An effective vaccine could substantially reduce the burden of cryptococcosis. However, a major gap in cryptococcal vaccine development has been the discovery of protective antigens to use in vaccines. Here, six cryptococcal proteins with potential as vaccine antigens were expressed recombinantly and purified. Mice were then vaccinated with glucan particle preparations containing each antigen. Of the six candidate vaccines, four protected mice from a lethal cryptococcal challenge. However, the degree of protection varied as a function of mouse strain and cryptococcal species. These preclinical studies identify cryptococcal proteins that could serve as candidate vaccine antigens and provide a proof of principle regarding the feasibility of protein antigen-based vaccines to protect against cryptococcosis., (Copyright © 2017 Specht et al.)

C. neoformans is an encapsulated fungal pathogen with defined asexual and sexual life cycles. Due to the availability of genetic and molecular tools for its manipulation, it has become a model organism for studies of fungal pathogens, even though it lacks a reliable system for maintaining DNA fragments as extrachromosomal plasmids. To compensate for this deficiency, we identified a genomic gene-free intergenic region where heterologous DNA could be inserted by homologous recombination without adverse effects on the phenotype of the recipient strain. Since such a site in the C. neoformans genome at a different location has been named previously as "safe haven", we named this locus second safe haven site (SH2). Insertion of DNA into this site in the genome of the KN99 congenic strain pair caused minimal change in the growth of the engineered strain under a variety of in vitro and in vivo conditions. We exploited this 'safe' locus to create a genetically stable highly fluorescent strain expressing mCherry protein (KN99mCH); this strain closely resembled its wild-type parent (KN99α) in growth under a variety of in vitro stress conditions and in the expression of virulence traits. The efficiency of phagocytosis and the proliferation of KN99mCH inside human monocyte-derived macrophages were comparable to those of KN99α, and the engineered strain showed the expected organ dissemination after inoculation, although there was a slight reduction in virulence. The mCherry fluorescence allowed us to measure specific association of cryptococci with leukocytes in the lungs and mediastinal lymph nodes of infected animals and, for the first-time, to assess their live/dead status in vivo. These results highlight the utility of KN99mCH for elucidation of host-pathogen interactions in vivo. Finally, we generated drug-resistant KN99 strains of both mating types that are marked at the SH2 locus with a specific drug resistant gene cassette; these strains will facilitate the generation of mutant strains by mating., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background and Aims: Epidemiological evidence suggests an association between consumption of tomato products or lycopene and lower risk for cardiovascular diseases (CVD). Our aim was to evaluate the state of the evidence from intervention trials on the effect of consuming tomato products and lycopene on markers of cardiovascular (CV) function. We undertook a systematic review and meta-analysis on the effect of supplementing tomato and lycopene on CV risk factors., Methods: Three databases including Medline, Web of science, and Scopus were searched from inception to August 2016. Inclusion criteria were: intervention trials reporting effects of tomato products and lycopene supplementation on CV risk factors among adult subjects >18 years of age. The outcomes of interest included blood lipids (total-, HDL-, LDL-cholesterol, triglycerides, oxidised-LDL), endothelial function (flow-mediated dilation (FMD), pulse wave velocity (PWV)) and blood pressure (BP) inflammatory factors (CRP, IL-6) and adhesion molecules (ICAM-1). Random-effects models were used to determine the pooled effect sizes., Results: Out of 1189 publications identified, 21 fulfilled inclusion criteria and were meta-analysed. Overall, interventions supplementing tomato were associated with significant reductions in LDL-cholesterol (-0.22 mmol/L; p = 0.006), IL-6 (standardised mean difference -0.25; p = 0.03), and improvements in FMD (2.53%; p = 0.01); while lycopene supplementation reduced systolic-BP (-5.66 mmHg; p = 0.002). No other outcome was significantly affected by these interventions., Conclusions: The available evidence on the effects of tomato products and lycopene supplementation on CV risk factors supports the view that increasing the intake of these has positive effects on blood lipids, blood pressure and endothelial function. These results support the development of promising individualised nutritional strategies involving tomatoes to tackle CVD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24 B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24 B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24 B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To investigate the phytochemical uptake following human consumption of Montmorency tart cherry (L. Prunus cerasus) and influence of selected phenolic acids on vascular smooth muscle cells in vitro., Methods: In a randomised, double-blinded, crossover design, 12 healthy males consumed either 30 or 60 mL of Montmorency tart cherry concentrate. Following analysis of the juice composition, venous blood samples were taken before and 1, 2, 3, 5 and 8 h post-consumption of the beverage. In addition to examining some aspects of the concentrate contents, plasma concentrations of protocatechuic acid (PCA), vanillic acid (VA) and chlorogenic (CHL) acid were analysed by reversed-phase high-performance liquid chromatography (HPLC) with diode array for quantitation and mass spectrometry detection (LCMS) for qualitative purposes. Vascular smooth muscle cell migration and proliferation were also assessed in vitro., Results: Both the 30 and 60 mL doses of Montmorency cherry concentrate contained high amounts of total phenolics (71.37 ± 0.11; 142.73 ± 0.22 mg/L) and total anthocyanins (62.47 ± 0.31; 31.24 ± 0.16 mg/L), as well as large quantities of CHL (0.205 ± 0.24; 0.410 ± 0.48 mg/L) and VA (0.253 ± 0.84; 0.506 ± 1.68 mg/L). HPLC/LCMS identified two dihydroxybenzoic acids (PCA and VA) in plasma following MC concentrate consumption. Both compounds were most abundant 1-2 h post-initial ingestion with traces detectable at 8 h post-ingestion. Cell migration was significantly influenced by the combination of PCA and VA, but not in isolation. There was no effect of the compounds on cell proliferation., Conclusions: These data show new information that phenolic compounds thought to exert vasoactive properties are bioavailable in vivo following MC consumption and subsequently can influence cell behaviour. These data may be useful for the design and interpretation of intervention studies investigating the health effects of Montmorency cherries.

Unlabelled: Cryptococcus neoformans is a major opportunistic fungal pathogen that causes fatal meningoencephalitis in immunocompromised individuals and is responsible for a large proportion of AIDS-related deaths. The fungal cell wall is an essential organelle which undergoes constant modification during various stages of growth and is critical for fungal pathogenesis. One critical component of the fungal cell wall is chitin, which in C. neoformans is predominantly deacetylated to chitosan. We previously reported that three chitin deacetylase (CDA) genes have to be deleted to generate a chitosan-deficient C. neoformans strain. This cda1Δ2Δ3Δ strain was avirulent in mice, as it was rapidly cleared from the lungs of infected mice. Here, we report that clearance of the cda1Δ2Δ3Δ strain was associated with sharply spiked concentrations of proinflammatory molecules that are known to be critical mediators of the orchestration of a protective Th1-type adaptive immune response. This was followed by the selective enrichment of the Th1-type T cell population in the cda1Δ2Δ3Δ strain-infected mouse lung. Importantly, this response resulted in the development of robust protective immunity to a subsequent lethal challenge with a virulent wild-type C. neoformans strain. Moreover, protective immunity was also induced in mice vaccinated with heat-killed cda1Δ2Δ3Δ cells and was effective in multiple mouse strains. The results presented here provide a strong framework to develop the cda1Δ2Δ3Δ strain as a potential vaccine candidate for C. neoformans infection., Importance: The most commonly used anticryptococcal therapies include amphotericin B, 5-fluorocytosine, and fluconazole alone or in combination. Major drawbacks of these treatment options are their limited efficacy, poor availability in limited resource areas, and potential toxicity. The development of antifungal vaccines and immune-based therapeutic interventions is promising and an attractive alternative to chemotherapeutics. Currently, there are no fungal vaccines in clinical use. This is the first report of a C. neoformans deletion strain with an avirulent phenotype in mice exhibiting protective immunity when used as a vaccine after heat inactivation, although other strains that overexpress fungal or murine proteins have recently been shown to induce a protective response. The data presented here demonstrate the potential for developing the avirulent cda1Δ2Δ3Δ strain into a vaccine-based therapy to treat C. neoformans infection., (Copyright © 2016 Upadhya et al.)

Scope: The intake of sucrose is of public health concern but limited information is available on the metabolic effects of short-term exposure. Our aim was to use metabolomics to investigate the metabolic impact of acute sucrose exposure., Methods and Results: We performed a randomized, parallel, single-dose feeding study on healthy females (n = 90, aged 29.9 ± 4.7 years, BMI 23.3 ± 2.5 kg/m(2) ) consuming either 0, 50, or 100 g sucrose in 500 mL water. Blood and urine samples were taken before and 24 h post sucrose intake. Urine and plasma samples underwent detailed metabolite profiling analysis using established protocols. Flow-injection electrospray MS fingerprinting analysis showed that 3 h after intake was the most informative time point in urine and plasma and out of 120 explanatory signals, highlighted 16 major metabolite signals in urine and 25 metabolite signals in plasma that were discriminatory and correlated with sucrose intake over time. The main confirmed metabolites positively correlated with intake were sucrose, fructose, and erythronic acid, while those negatively correlating with intake included fatty acids and derivatives, acyl-carnitines, and ketone bodies. GC-TOF-MS profiling analysis confirmed the fingerprinting data., Conclusion: Acute exposure to sucrose identified a number of metabolites correlated with sucrose intake and several compounds attributed to metabolic fasting., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: A vaccine capable of protecting at-risk persons against infections due to Cryptococcus neoformans and Cryptococcus gattii could reduce the substantial global burden of human cryptococcosis. Vaccine development has been hampered though, by lack of knowledge as to which antigens are immunoprotective and the need for an effective vaccine delivery system. We made alkaline extracts from mutant cryptococcal strains that lacked capsule or chitosan. The extracts were then packaged into glucan particles (GPs), which are purified Saccharomyces cerevisiae cell walls composed primarily of β-1,3-glucans. Subcutaneous vaccination with the GP-based vaccines provided significant protection against subsequent pulmonary infection with highly virulent strains of C. neoformans and C. gattii. The alkaline extract derived from the acapsular strain was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and the most abundant proteins were identified. Separation of the alkaline extract by size exclusion chromatography revealed fractions that conferred protection when loaded in GP-based vaccines. Robust Th1- and Th17-biased CD4(+) T cell recall responses were observed in the lungs of vaccinated and infected mice. Thus, our preclinical studies have indicated promising cryptococcal vaccine candidates in alkaline extracts delivered in GPs. Ongoing studies are directed at identifying the individual components of the extracts that confer protection and thus would be promising candidates for a human vaccine., Importance: The encapsulated yeast Cryptococcus neoformans and its closely related sister species, Cryptococcus gattii, are major causes of morbidity and mortality, particularly in immunocompromised persons. This study reports on the preclinical development of vaccines to protect at-risk populations from cryptococcosis. Antigens were extracted from Cryptococcus by treatment with an alkaline solution. The extracted antigens were then packaged into glucan particles, which are hollow yeast cell walls composed mainly of β-glucans. The glucan particle-based vaccines elicited robust T cell immune responses and protected mice from otherwise-lethal challenge with virulent strains of C. neoformans and C. gattii. The technology used for antigen extraction and subsequent loading into the glucan particle delivery system is relatively simple and can be applied to vaccine development against other pathogens., (Copyright © 2015 Specht et al.)

Background: Previous studies suggest that the beneficial health effects of a diet rich in whole grains could be a result of the individual fibres found in the grain. The present study aimed to investigate the influence of a diet high in either wheat fibre (as an example of an insoluble fibre) or inulin (a nondigestible carbohydrate) on markers of cardiovascular disease., Methods: Ten male participants classified as at higher risk of cardiovascular disease [mean (SD) body mass index 30.2 (3) kg m(-2) , mean (SD) waist circumference 106.4 (7) cm, mean (SD) age 39.8 (9) years] were recruited to a randomised, controlled, cross-over study comparing the consumption of bespoke bread rolls containing either inulin, wheat germ or refined grain (control) (15 g day(-1) ) for 4 weeks with a 4-week washout period between each regime. At the end of each regime, participants underwent an oral glucose tolerance test (OGTT), measures of pulse wave velocity (PWV), 24-h ambulatory blood pressure (AMBP), plasma lipid status and markers of glucose control., Results: There was no difference in measures of glucose control, lipid status, 24-h AMBP or PWV after the intervention periods and no changes compared to baseline. There was no significant difference between OGTT glucose and insulin time profiles; however, there was a significant difference in area under the curves between the wheat fibre and control interventions when comparing change from baseline (control +10.2%, inulin +4.3%, wheat fibre -2.5%; P = 0.03)., Conclusions: Only limited differences between the interventions were identified, perhaps as a consequence of the amount of fibre used and intervention length. The wheat germ intervention resulted in a significant reduction in glucose area under the curve, suggesting that this fibre may aid glucose control., (© 2014 The British Dietetic Association Ltd.)

Unlabelled: Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis., Importance: Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway., (Copyright © 2014 Donlin et al.)

Objective: Pulse wave velocity (PWV) is a measure of arterial stiffness and a marker for cardiovascular disease. Although commonly used, there are only a few reports investigating the intersession and intrasession variability in PWV measurements, the determination of which is important in a mixed population when using PWV as a clinical marker. Therefore, the aim of this study was to investigate the variability in PWV measurements and factors that may influence PWV variability., Methods: A male population (n=8, age 30.9 ± 9.0 years; BMI 25.1 ± 4.0 kg/m(2)) underwent measurements of PWV and blood pressure several times in a single study visit and during six study visits over a 4-6-week period. During these study visits, experiments were performed at rest and following acute exercise and feeding., Results: Intersession coefficients of variation (CVs) were 5.3 and 4.5% for radial-carotid (R-C) and carotid-femoral (C-F) PWVs, respectively, whereas intrasession CVs were 9.3 and 6.9% for R-C and C-F PWVs, respectively. Good reproducibility in PWV measurements was demonstrated by individual responses in the data; two of eight participants had significant differences in C-F PWV over time (P=0.05). There were significant increases in systolic blood pressure following acute exercise (P<0.0001) and feeding (P=0.05), but there were no consistent changes in PWV measurements., Conclusion: These data demonstrate the reproducibility of PWV and that PWV measurements are not acutely influenced by the metabolic state. This has implications for the use of PWV in human intervention trials where a mixed population is being investigated.

Water-soluble vitamins are an important class of compounds that require quantification from food sources to monitor nutritional value. In this study we have analysed six water-soluble B vitamins ([thiamine (B1), riboflavin (B2), nicotinic acid (B3, NAc), nicotinamide (B3, NAm), pyridoxal (B6), folic acid (B9)], and ascorbic acid (vit C) with hydrophilic interaction liquid chromatography (HILIC), and compared UV, fluorescent (FLD) and coulometric detection to optimise a method to quantitate the vitamins from food sources. Employing UV/diode array (DAD) and fluorimetric detection, six B vitamins were detected in a single run using gradient elution from 100% to 60% solvent B [10mM ammonium acetate, pH 5.0, in acetonitrile and water 95:5 (v:v)] over 18 min. UV detection was performed at 268 nm for B1, 260 nm for both B3 species and 284 nm for B9. FLD was employed for B2 at excitation wavelength of 268 nm, emission of 513 nm, and 284 nm/317 nm for B6. Coulometric detection can be used to detect B6 and B9, and vit C, and was performed isocratically at 75% and 85% of solvent B, respectively. B6 was analysed at a potential of 720 mV, while B9 was analysed at 600 mV, and vit C at 30 mV. Retention times (0.96 to 11.81 min), intra-day repeatability (CV 1.6 to 3.6), inter-day variability (CV 1.8 to 11.1), and linearity (R 0.9877 to 0.9995) remained good under these conditions with limits of detection varying from 6.6 to 164.6 ng mL(-1), limits of quantification between 16.8 and 548.7 ng mL(-1). The method was successfully applied for quantification of six B vitamins from a fortified food product and is, to our knowledge, the first to simultaneously determine multiple water-soluble vitamins extracted from a food matrix using HILIC., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Meningitis is a frequent manifestation of infection due to Cryptococcus neoformans and a major cause of increased morbidity in patients with AIDS. Numerous in vitro gene expression and genetic studies of the fungus have predicted a myriad of genes, pathways, and biological processes that may be critical for pathogenesis, and many studies using animal models have supported the role of these processes during infection. However, the relevance of these hypotheses based on in vitro and animal models has often been questioned. A recent study by Chen et al. [Y. Chen, D. L. Toffaletti, J. L. Tenor, A. P. Litvintseva, C. Fang, T. G. Mitchell, T. R. McDonald, K. Nielsen, D. R. Boulware, T. Bicanic, and J. R. Perfect, mBio 5(1):e01087-13, 2014] represents an important step in understanding the cryptococcal response during human infection.