Identification of the Active Principle Conferring Anti-Inflammatory and Antinociceptive Properties in Bamboo Plant.

Early plants began colonizing earth about 450 million years ago. During the process of coevolution, their metabolic cellular pathways produced a myriad of natural chemicals, many of which remain uncharacterized biologically. Popular preparations containing some of these molecules have been used medicinally for thousands of years. In Brazilian folk medicine, plant extracts from the bamboo plant Guadua paniculata Munro have been used for the treatment of infections and pain. However, the chemical basis of these therapeutic effects has not yet been identified. Here, we performed protein biochemistry and downstream pharmacological assays to determine the mechanisms underlying the anti-inflammatory and antinociceptive effects of an aqueous extract of the G. paniculata rhizome, which we termed AqGP. The anti-inflammatory and antinociceptive effects of AqGP were assessed in mice. We identified and purified a protein (AgGP), with an amino acid sequence similar to that of thaumatins (~20 kDa), capable of repressing inflammation through downregulation of neutrophil recruitment and of decreasing hyperalgesia in mice. In conclusion, we have identified the molecule and the molecular mechanism responsible for the anti-inflammatory and antinociceptive properties of a plant commonly used in Brazilian folk medicine.

Influence of a prebiotic and natural sweeteners on the sensory profile of skyr yogurt with mango pulp.

Skyr yogurts have been gaining prominence because of their different sensory characteristics. Due to their healthy appeal, the use of natural sweeteners to replace sucrose in this type of yogurt can be an alternative for incorporating a sweet taste, in addition to increasing the functionality of the product through the incorporation of prebiotics. This study aimed to determine whether the addition of fructooligosaccharide (FOS), sucrose, stevia, and thaumatin affects the sensory profile of the skyr yogurt with mango pulp and its acceptance in two Brazilian regions. Eight formulations of skyr with mango pulp were developed. The compositional parameters evaluated were moisture, protein, lipids, ash, and carbohydrate. The tests performed were ideal sweetness and mango flavor, sweetness equivalence for each sweetener used, Quantitative Descriptive Analysis (QDA), and consumer testing in the Southeast and Northeast regions of Brazil. In general, the addition of FOS did not impact the characteristics of the formulated skyr yogurt. The type of sweetener had an impact on the sensory profile and acceptance of the skyr yogurt, affected characteristics such as mango flavor, sweet taste, sweet aftertaste, bitter taste, bitter aftertaste, and metallic flavor. The results of the affective test demonstrated that, for consumers in the Southeast, mango flavor is a positive attribute in this yogurt, and for Northeastern consumers, in addition to mango flavor, sweetness must also be taken into consideration. PRACTICAL APPLICATION: This study may be useful for the dairy industry because in the literature, there is still a lack of sensory studies of skyr yogurt, especially when sucrose substitutes are used. The results of the consumer test in this work reinforce the importance of studies related to consumer preferences with cultural differences.

Plant Antimicrobial Peptides: State of the Art, In Silico Prediction and Perspectives in the Omics Era.

Even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. These guardians include small molecules as antimicrobial peptides (AMPs), generally cysteine-rich, functioning to prevent pathogen establishment. Some of these AMPs are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). When compared with other organisms, plants tend to present a higher amount of AMP isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. Therefore, plants arise as a rich resource for new AMPs. As these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. The possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated.

Characterization of Three Osmotin-Like Proteins from Plumeria rubra and Prospection for Adiponectin Peptidomimetics.

BACKGROUND: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet. OBJECTIVE AND METHODS: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2). RESULTS: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide. CONCLUSION: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis.

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris.

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/gDCW per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris.

Genome-wide analysis and evolution of plant thaumatin-like proteins: a focus on the origin and diversification of osmotins.

Osmotin is an important multifunctional protein related to plant stress responses and is classified into the thaumatin-like protein (TLP) family. Using genome-wide and phylogenetic approaches, we investigated osmotin origin and diversification across plant TLP evolution. Genomic and protein in silico analysis tools were also accessed and considered for the study conclusions. Phylogenetic analysis including a total of 722 sequences from 32 Viridiplantae species allowed the identification of an osmotin group that includes all previously characterized osmotins. Based on the phylogenetic tree results, it is evident that the osmotin group emerged from spermatophytes. Phylogenetic separation and gene expansion could be accounted for by an exclusive motif composition and organization that emerged and was maintained following tandem and block duplications as well as natural selection. The TLP family conserved residues and structures that were also identified in the sequences of the osmotin group, thus suggesting their maintenance for defense responses. The gene expression of Arabidopsis and rice putative osmotins reinforces its roles during stress response.

Comparative proteomic and metabolomic studies between Prunus persica genotypes resistant and susceptible to Taphrina deformans suggest a molecular basis of resistance.

The worldwide-distributed leaf peach curl disease is caused by the biotroph Taphrina deformans. To characterize the plant-fungus interaction, resistant and susceptible Prunus persica genotypes grown in the orchard were studied. Asymptomatic leaves were tested for fungal presence. In all resistant leaves analyzed the fungus was not detected. Conversely, leaves from the susceptible genotype were categorized according to the presence or absence of the pathogen. Comparative metabolomic analysis disclosed the metabolite composition associated with resistant and susceptible interactions, and of compounds involved in fungal growth inhibition such as chlorogenic acid, whose in vitro antifungal activity was verified in this work. Differential proteome studies revealed that chloroplasts are important site of plant defense responses against T. deformans. Members of the Bet-v1-like family protein differentially responded to the pathogen. Extracellular pathogenesis-related proteins, evaluated by qRT-PCR, and an enone oxidoreductase are constitutively present in leaves of resistant trees and could be related to fungal resistance. This study is a global view of the changes in the metabolome, proteome and transcripts related to plant defense in naturally infected leaves of susceptible plants during the asymptomatic stage. Additionally, it provides clues to the successful molecular mechanisms operating in resistant plants, which neither develop the disease nor harbor the pathogen.

Crystal structure of an antifungal osmotin-like protein from Calotropis procera and its effects on Fusarium solani spores, as revealed by atomic force microscopy: Insights into the mechanism of action.

CpOsm is an antifungal osmotin/thaumatin-like protein purified from the latex of Calotropis procera. The protein is relatively thermostable and retains its antifungal activity over a wide pH range; therefore, it may be useful in the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogenic fungi. To gain further insight into the mechanism of action of CpOsm, its three-dimensional structure was determined, and the effects of the protein on Fusarium solani spores were investigated by atomic force microscopy (AFM). The atomic structure of CpOsm was solved at a resolution of 1.61Å, and it contained 205 amino acid residues and 192 water molecules, with a final R-factor of 18.12% and an Rfree of 21.59%. The CpOsm structure belongs to the thaumatin superfamily fold and is characterized by three domains stabilized by eight disulfide bonds and a prominent charged cleft, which runs the length of the front side of the molecule. Similarly to other antifungal thaumatin-like proteins, the cleft of CpOsm is predominantly acidic. AFM images of F. solani spores treated with CpOsm resulted in striking morphological changes being induced by the protein. Spores treated with CpOsm were wrinkled, and the volume of these cells was reduced by approximately 80%. Treated cells were covered by a shell of CpOsm molecules, and the leakage of cytoplasmic content from these cells was also observed. Based on the structural features of CpOsm and the effects that the protein produces on F. solani spores, a possible mechanism of action is suggested and discussed.

Sugar reduction in probiotic chocolate-flavored milk: Impact on dynamic sensory profile and liking.

Reducing the sugar content of processed products has been claimed to be one of the most efficient strategies for decreasing sugar intake. The present work aimed at studying the influence of sugar reduction on the dynamic sensory profile and consumers' liking of probiotic chocolate-flavored milks using a novel temporal methodology, and to evaluate two alternatives (vanilla flavor and thaumatin) to attenuate the sensory changes caused by sugar reduction. Probiotic chocolate-flavored milks were formulated with different reductions in added sugar (0, 20, 40 and 60%). Vanilla flavor and thaumatin were added to the sugar-reduced samples at two concentrations. Samples were evaluated by trained assessors using Temporal check-all-that-apply (TCATA). Additionally, consumers evaluated the dynamic sensory profile of a subset of the samples using TCATA and indicated their overall liking using a 9-point hedonic scale. Results from the present work showed that the main effect of sugar reduction on the dynamic sensory profile of the probiotic chocolate-flavored milks was related to changes in sweetness, bitterness and thickness. A reduction in added sugar of 20% led to changes in sweetness intensity, which were perceived by both trained assessors and consumers. However, consumers' liking was not significantly affected by sugar reduction up to 40%. The addition of vanilla flavor at suprathreshold concentrations was not efficient in increasing sweetness perception in chocolate-flavored milks with the lowest sugar reduction percentage, suggesting that it may not be a feasible alternative for reducing sugar in this product category. These results suggest that in many situations sugar content of food products could be decreased without a relevant impact on consumers' sensory and hedonic perception.

Crystallization and X-ray diffraction analysis of an antifungal laticifer protein.

An osmotin (CpOsm) from the latex of Calotropis procera has been crystallized in both tetragonal and trigonal forms suitable for structure determination. Crystallographic studies of CpOsm are of great interest because limited information is available concerning the structure of latex proteins and CpOsm has previously been shown to interact with the spore membranes of some plant pathogenic fungi, thus impairing spore germination and hyphal growth. CpOsm crystals were grown using 0.1 M HEPES buffer pH 7.5, 26% PEG 4000, 0.2 M ammonium sulfate (space group P4(3)) or using 0.1 M HEPES buffer pH 7.5, 35% MPD, 0.7 M ammonium sulfate (space group P3(1)12). X-ray diffraction data were collected to 2.17 Å (P4(3)) and 1.80 Å (P3(1)12) resolution and molecular-replacement analyses produced initial phases for both crystal forms.