A Rapid Protocol for Preparing 8-Aminopyrene-1,3,6-Trisulfonate-Labeled Glycans for Capillary Electrophoresis-Based Enzyme Assays.

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene-1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, a technique amenable to equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel by simple diffusion and subsequent solid-phase extraction (SPE)-based desalting, affords a single glycan species free of excess labeling reagents and buffer components. The described protocol also offers a simple, rapid method for the simultaneous removal of excess APTS and unlabeled glycan material from reaction mixtures. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.

Cardiolipin released by microglia can act on neighboring glial cells to facilitate the uptake of amyloid-ß (1-42).

Cardiolipin is a mitochondrial phospholipid that is also detected in serum inferring its extracellular release; however, this process has not been directly demonstrated for any of the brain cell types. Nevertheless, extracellular cardiolipin has been shown to modulate several neuroimmune functions of microglia and astrocytes, including upregulation of their endocytic activity. Low cardiolipin levels are associated with brain aging, and may thus hinder uptake of amyloid-ß (Αß) in Alzheimer's disease. We hypothesized that glial cells are one of the sources of extracellular cardiolipin in the brain parenchyma where this phospholipid interacts with neighboring cells to upregulate the endocytosis of Αß. Liquid chromatography-mass spectrophotometry identified 31 different species of cardiolipin released from murine BV-2 microglial cells and revealed this process was accelerated by exposure to Aß42. Extracellular cardiolipin upregulated internalization of fluorescently-labeled Aß42 by primary murine astrocytes, human U118 MG astrocytic cells, and murine BV-2 microglia. Increased endocytic activity in the presence of extracellular cardiolipin was also demonstrated by studying uptake of Aß42 and pHrodo™ Bioparticles™ by human induced pluripotent stem cells (iPSCs)-derived microglia, as well as iPSC-derived human brain organoids containing microglia, astrocytes, oligodendrocytes and neurons. Our observations indicate that Aß42 augments the release of cardiolipin from microglia into the extracellular space, where it can act on microglia and astrocytes to enhance their endocytosis of Aß42. Our observations suggest that the reduced glial uptake of Aß due to the decreased levels of cardiolipin could be at least partially responsible for the extracellular accumulation of Aß in aging and Alzheimer's disease.

Understanding the Sensitivity of Grape Terpenes to Jasmonates Using In Vitro Culture and In Vivo Vineyard Experiments.

Terpene volatiles define the flavor of terpenic grape cultivars. However, grape terpene concentrations can vary 2- to 3-fold across seasons and vineyards, impacting vintage quality. The plant hormone methyl jasmonate (MeJA) stimulates grape terpene production but is expensive and can decrease berry weight and maturity. The synthetic jasmonate prohydrojasmon (PDJ) is cost-effective yet has not been evaluated on grape maturity and terpene production. Here, we performed in vitro (berry culture) and in vivo (vineyard) experiments using Gewürztraminer (Vitis vinifera L.) to evaluate the time- and concentration-dependent sensitivity of maturity parameters and terpene content to MeJA and PDJ. In vitro berry weight was reduced by high MeJA and PDJ concentration across timings. Terpenes were most sensitive to low MeJA concentration at veraison (increased 24-fold) in vitro. Moderate PDJ concentration applied at veraison doubled (increased twofold) terpene concentration in vivo without impacting berry weight or maturity. In conclusion, PDJ may provide a solution to mitigate seasonal variability in terpene production in terpenic grape cultivars.

The primary familial brain calcification-associated protein MYORG is an α-galactosidase with restricted substrate specificity.

Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG.

Correlating Sensory Assessment of Smoke-Tainted Wines with Inter-Laboratory Study Consensus Values for Volatile Phenols.

Vineyard exposure to wildfire smoke can taint grapes and wine. To understand the impact of this taint, it is imperative that the analytical methods used are accurate and precise. This study compared the variance across nine commercial and research laboratories following quantitative analysis of the same set of smoke-tainted wines. In parallel, correlations between the interlaboratory consensus values for smoke-taint markers and sensory analyses of the same smoke-tainted wines were evaluated. For free guaiacol, the mean accuracy was 94 ± 11% in model wine, while the free cresols and 4-methylguaiacol showed a negative bias and/or decreased precision relative to guaiacol. Similar trends were observed in smoke-tainted wines, with the cresols and glycosidically bound markers demonstrating high variance. Collectively, the interlaboratory results show that data from a single laboratory can be used quantitatively to understand smoke-taint. Results from different laboratories, however, should not be directly compared due to the high variance between study participants. Correlations between consensus compositional data and sensory evaluations suggest the risk of perceivable smoke-taint can be predicted from free cresol concentrations, overcoming limitations associated with the occurrence of some volatile phenols, guaiacol in particular, as natural constituents of some grape cultivars and of the oak used for barrel maturation.

Impact of hormone applications on ripening-related metabolites in Gewürztraminer grapes (Vitis vinifera L.): The key role of jasmonates in terpene modulation.

Terpenes play a formative role in grape and wine flavor, particularly for high-terpenic cultivars. Differences in terpene profiles influence grape varietal character and vintage quality. Little is known about the endogenous factors controlling terpene biosynthesis in grape. Through multiple experiments, six hormones (abscisic acid, ABA; ethylene, ETH; jasmonic acid, JA; methyl jasmonate, MeJA; indole-3-acetic acid, IAA; 1-naphthaleneacetic acid, NAA) that either promote or repress ripening were applied to Gewürztraminer clusters near veraison to gauge their effect on ripening and terpene biosynthesis. Jasmonates (JA, MeJA) increased terpene concentrations and the expression of terpene genes in grapes. Such increases were not associated to increases of other ripening-related metabolites such as sugars or anthocyanins. MeJA also affected the expression of several hormone related genes, increased IAA levels, and reduced sugar and anthocyanin concentration in grapes. This research provides novel insights into terpene regulation by ripening-related hormones and jasmonates in grapes.

Metabolism of a hybrid algal galactan by members of the human gut microbiome.

Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroides uniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients.

Kinetic and Structural Characterization of Sialidases (Kdnases) from Ascomycete Fungal Pathogens.

Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus (AtS) and Trichophyton rubrum (TrS). Unlike A. fumigatus Kdnase (AfS), hydrolysis with the Neu5Ac substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AfS but 30-fold higher by TrS. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both AtS and TrS show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of AfS in vesicles at the cell surface.

Carbohydrate Sulfation As a Mechanism for Fine-Tuning Siglec Ligands.

The immunomodulatory family of Siglecs recognizes sialic acid-containing glycans as "self", which is exploited in cancer for immune evasion. The biochemical nature of Siglec ligands remains incompletely understood, with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs─CHST1, CHST2, or CHST4─significantly enhance the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, a pharmacological blockade of N- and O-glycan maturation reveals a cell-type-specific pattern of importance for either class of glycan. Production of a highly homogeneous Siglec-3 (CD33) fragment enabled a mass-spectrometry-based binding assay to determine ≥8-fold and ≥2-fold enhanced affinity for Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc and Neu5Acα2-3Galß1-4(6-O-sulfo)GlcNAc, respectively, over Neu5Acα2-3Galß1-4GlcNAc. CD33 shows significant additivity in affinity (≥28-fold) for the disulfated ligand, Neu5Acα2-3(6-O-sulfo)Galß1-4(6-O-sulfo)GlcNAc. Moreover, joint overexpression of CHST1 with CHST2 in cells greatly enhanced the binding of CD33 and several other Siglecs. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a general mechanism for tuning Siglec ligands on cells, including in cancer.

Glycosidically-Bound Volatile Phenols Linked to Smoke Taint: Stability during Fermentation with Different Yeasts and in Finished Wine.

When wine grapes are exposed to smoke, there is a risk that the resulting wines may possess smoky, ashy, or burnt aromas, a wine flaw known as smoke taint. Smoke taint occurs when the volatile phenols (VPs) largely responsible for the aroma of smoke are transformed in grape into a range of glycosides that are imperceptible by smell. The majority of VP-glycosides described to date are disaccharides possessing a reducing ß-d-glucopyranosyl moiety. Here, a two-part experiment was performed to (1) assess the stability of 11 synthesized VP-glycosides towards general acid-catalyzed hydrolysis during aging, and (2) to examine whether yeast strains differed in their capacity to produce free VPs both from these model glycosides as well as from grapes that had been deliberately exposed to smoke. When fortified into both model and real wine matrices at 200 ng/g, all VP-disaccharides were stable over 12 weeks, while (42-50 ng/g) increases in free 4-ethylphenol and p-cresol were detected when these were added to wine as their monoglucosides. Guaiacol and phenol were the most abundantly produced VPs during fermentation, whether originating from natural VP-precursors in smoked-exposed Pinot Noir must, or due to fortification with synthetic VP-glycosides. Significant yeast strain-specific differences in glycolytic activities were observed for phenyl-ß-d-glycopyranoside, with two strains (RC212 and BM45) being unable to hydrolyze this model VP, albeit both were active on the guaiacyl analogue. Thus, differences in Saccharomyces cerevisiae ß-glucosidase activity appear to be influenced by the VP moiety.

Large-Scale Reassessment of In-Vineyard Smoke-Taint Grapevine Protection Strategies and the Development of Predictive Off-Vine Models.

Smoke taint in wine is thought to be caused by smoke-derived volatile phenols (VPs) that are absorbed into grape tissues, trapped as conjugates that are imperceptible by smell, and subsequently released into wines as their free odor-active forms via metabolism by yeasts during fermentation. Blocking VP uptake into grapes would, therefore, be an effective way for vineyards to protect ripening grape crops exposed to smoke. Here, we re-evaluated a biofilm that had previously shown promise in pilot studies in reducing levels of smoke-derived VPs. A suite of nine free and acid-labile VPs were quantitated in Pinot Noir grapes that had been exposed to smoke after being coated with the biofilm one, seven or 14 days earlier. In contrast with earlier studies, our results demonstrated that in all cases, the biofilm treatments led to increased concentrations of both free and total VPs in smoke-exposed grapes, with earlier applications elevating concentrations of some VPs more than the later time points. Tracking VP concentrations through the grape ripening process demonstrated that some (phenol, p/m-cresol, and guaiacol) were not entirely sequestered in grapes as acid-labile conjugates, suggesting the presence of VP storage forms beyond simple glycosides. Free VPs in grapes, though a minor portion of the total, most clearly correlated with concentrations present in the resulting wines. Finally, red table grapes, available year round, were observed to replicate the effects of the biofilm treatments and were capable of transforming most VPs into acid-labile conjugates in under 24 h, indicating that they might be an effective model for rapidly assessing smoke-taint prophylactic products in the laboratory.

Analysis of the biosynthetic flux in bovine milk oligosaccharides reveals competition between sulfated and sialylated species and the existence of glucuronic acid-containing analogues.

We previously observed that sialylated bovine milk oligosaccharides (BMOs) decline in both absolute and relative abundances over the initial stages of bovine lactation, with initial evidence suggesting that this decline occurred due to increased concentrations of unique sulfated BMOs. Since both sulfated and sialylated BMOs have distinct bioactivites, a follow up study was launched in order to more clearly define relative changes in these classes of BMOs over the first week of lactation in dairy cattle. Capillary electrophoresis (CE) and several liquid chromatography mass spectrometry (LC-MS) methods, including a novel multiplexed tandem MS method, were used to profile the BMOs extracted from milk collected from the same 20 Holstein cows at milkings 1, 2, 3, 4, 8, and 14 post-partum. In addition to clearly validating that sulfated and sialylated BMOs exist in direct biosynthetic completion, our study has identified over 170 unique BMOs including 14 unique glucuronic acid-containing trisaccharides.

Maternal Intake of Dietary Fat Pre-Programs Offspring's Gut Ecosystem Altering Colonization Resistance and Immunity to Infectious Colitis in Mice.

SCOPE: The transgenerational impact of dietary fat remains unclear. Here, the role of maternal fat consumption as a modulator of gut microbial communities and infectious disease outcomes in their offspring is explored. METHODS AND RESULTS: C57BL/6 mice are fed isocaloric high-fat diets throughout breeding, gestation and lactation. Diets contained either milk fat (MF), olive oil (OO) or corn oil (CO), with or without fish oil. The pups born to maternally exposed mice are weaned on to chow and raised into adulthood. At 8 weeks, the offsprings are either euthanized for colonic 16S rRNA analysis or challenged with the enteric pathogen, Citrobacter rodentium. Maternal CO exposure resulted in unique clustering of bacterial communities in offspring compared with MF and OO. Diets rich in CO reduced survival in offspring challenged with C. rodentium. The addition of fish oil did not improve mortality caused by CO and worsened disease outcomes when combined with OO. Unlike the unsaturated diets, MF is protective with and without fish oil. CONCLUSIONS: Overall, these data reveal that maternal intake of fatty acids do have transgenerational impacts on their offspring's bacteriome and enteric infection risk. Based on this study, saturated fats should be included in maternal diets.

Host responses to Clostridium perfringens challenge in a chicken model of chronic stress.

BACKGROUND: This study utilized a chicken model of chronic physiological stress mediated by corticosterone (CORT) administration to ascertain how various host metrics are altered upon challenge with Clostridium perfringens. Necrotic enteritis (NE) is a disease of the small intestine of chickens incited by C. perfringens, which can result in elevated morbidity and mortality. The objective of the current study was to investigate how physiological stress alters host responses and predisposes birds to subclinical NE. RESULTS: Birds administered CORT exhibited higher densities of C. perfringens in their intestine, and this corresponded to altered production of intestinal mucus. Characterization of mucus showed that C. perfringens treatment altered the relative abundance of five glycans. Birds inoculated with C. perfringens did not exhibit evidence of acute morbidity. However, histopathologic changes were observed in the small intestine of infected birds. Birds administered CORT showed altered gene expression of tight junction proteins (i.e. CLDN3 and CLDN5) and toll-like receptors (i.e. TLR2 and TLR15) in the small intestine. Moreover, birds administered CORT exhibited increased expression of IL2 and G-CSF in the spleen, and IL1ß, IL2, IL18, IFNγ, and IL6 in the thymus. Body weight gain was impaired only in birds that were administered CORT and challenged with C. perfringens. CONCLUSION: CORT administration modulated a number of host functions, which corresponded to increased densities of C. perfringens in the small intestine and weight gain impairment in chickens. Importantly, results implicate physiological stress as an important predisposing factor to NE, which emphasizes the importance of managing stress to optimize chicken health.

3D biofilms: in search of the polysaccharides holding together lichen symbioses.

Stable, long-term interactions between fungi and algae or cyanobacteria, collectively known as lichens, have repeatedly evolved complex architectures with little resemblance to their component parts. Lacking any central scaffold, the shapes they assume are casts of secreted polymers that cement cells into place, determine the angle of phototropic exposure and regulate water relations. A growing body of evidence suggests that many lichen extracellular polymer matrices harbor unicellular, non-photosynthesizing organisms (UNPOs) not traditionally recognized as lichen symbionts. Understanding organismal input and uptake in this layer is key to interpreting the role UNPOs play in lichen biology. Here, we review both polysaccharide composition determined from whole, pulverized lichens and UNPOs reported from lichens to date. Most reported polysaccharides are thought to be structural cell wall components. The composition of the extracellular matrix is not definitively known. Several lines of evidence suggest some acidic polysaccharides have evaded detection in routine analysis of neutral sugars and may be involved in the extracellular matrix. UNPOs reported from lichens include diverse bacteria and yeasts for which secreted polysaccharides play important biological roles. We conclude by proposing testable hypotheses on the role that symbiont give-and-take in this layer could play in determining or modifying lichen symbiotic outcomes.

Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers.

Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5-N-glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants.

Development and Evaluation of a Vineyard-Based Strategy To Mitigate Smoke-Taint in Wine Grapes.

Smoke-taint is a wine defect that may occur when ripening grape crops absorb volatile phenols (VPs), compounds associated with the negative sensory attributes of smoke-taint, due to exposure of grapes to wildfire smoke. This study examined potential methods to reduce the impact that smoke-exposure has on wine grapes. Specifically, agricultural sprays normally used to protect grapes from fungal pathogens and a spray used to prevent cracking in soft-fleshed fruits were assessed for their capacity to inhibit increases in VP concentrations in wine grapes following on-vine smoke-exposure. The results indicated that an artificial grape cuticle applied 1 week before exposure to simulated forest fire smoke (at 1-2 weeks after veraison) can significantly hinder an increase in VP concentrations in smoke-exposed grapes at commercial maturity. This reduction in VP concentrations may mitigate crop losses experienced globally by the wine industry due to exposure of grapes on-vine (at key phenological stages) to wildfire smoke.

Structural analysis of broiler chicken small intestinal mucin O-glycan modification by Clostridium perfringens.

Clostridium perfringens is a Gram-positive opportunistic pathogen that is the principal etiological agent of necrotic enteritis (NE) in poultry. The ability of C. perfringens to incite NE depends upon its ability to penetrate the protective mucus barrier within the small intestine, which is largely composed of heavily glycosylated proteins called mucins. Mucins are decorated by N- and O-linked glycans that serve both as a formidable gel-like barrier against invading pathogens and as a rich carbon source for mucolytic bacteria. The composition of avian O-linked glycans is markedly different from mucins in other vertebrates, being enriched in sulfated monosaccharides and N-acetyl-d-neuraminic acid (Neu5Ac, sialic acid). These modifications increase the overall negative charge of mucins and are believed to impede colonization by enteric pathogens. The mechanism by which C. perfringens penetrates the poultry intestinal mucus layer during NE is still unknown. However, the CAZome (i.e., the total collection of proteins encoded within a genome active on carbohydrates) of C. perfringens strain CP1 encodes several putative and known enzymes with activities consistent with the modification of mucin. To further investigate this relationship, O-glycans from Gallus gallus domesticus mucus were extracted from the small intestine and characterized using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Chicken mucin monosaccharides included l-fucose (Fuc), d-mannose (Man), d-galactose (Gal), N-acetyl-d-galactosamine (GalNAc), N-acetyl-d-glucosamine (GlcNAc), and Neu5Ac (sialic acid). Using these monosaccharides as sole carbon sources, we showed that C. perfringens CP1 grew on Neu5Ac, Man, Gal, and GlcNAc but not on Fuc and GalNAc. We also demonstrated C. perfringens grew on different native-state preparations of intestinal mucins and mucus including porcine mucins, chicken mucus, and chicken mucins. Finally, anaerobic incubation of chicken mucin O-glycans with C. perfringens and subsequent analysis of the glycans revealed that there was preferential removal of Neu5Ac. These observations are discussed in the context of the predicted metabolic potential of C. perfringens CP1 and the mucolytic enzymes encoded within its CAZome.

Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis, under different feeding conditions.

Trypanosoma cruzi, the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions.

Smoke from simulated forest fire alters secondary metabolites in Vitis vinifera L. berries and wine.

MAIN CONCLUSION: The exposure of Vitis vinifera L. berries to forest fire smoke changes the concentration of phenylpropanoid metabolites in berries and the resulting wine. The exposure of Vitis vinifera L. berries (i.e., wine grapes) to forest fire smoke can lead to a wine defect known as smoke taint that is characterized by unpleasant "smoky" and "ashy" aromas and flavors. The intensity of smoke taint is associated with the concentration of organoleptic volatile phenols that are produced during the combustion-mediated oxidation of lignocellulosic biomass and subsequently concentrated in berries prior to fermentation. However, these same smoke-derived volatile phenols are also produced via metabolic pathways endogenous to berries. It follows then that an influx of exogenous volatile phenols (i.e., from forest fire smoke) could alter endogenous metabolism associated with volatile phenol synthesis, which occurs via the shikimic acid/phenylpropanoid pathways. The presence of ozone and karrikins in forest fire smoke, as well as changes to stomatal conductance that can occur from exposure to forest fire smoke also have the potential to influence phenylpropanoid metabolism. This study demonstrated changes in phenylpropanoid metabolites in Pinot noir berries and wine from three vineyards following the exposure of Vitis vinifera L. vines to simulated forest fire smoke. This included changes to metabolites associated with mouth feel and color in wine, both of which are important sensorial qualities to wine producers and consumers. The results reported are critical to understanding the chemical changes associated with smoke taint beyond volatile phenols, which in turn, may aid the development of preventative and remedial strategies.