Mining the Wheat Grain Proteome

Bread wheat is the most widely cultivated crop worldwide, used in the production of food products and a feed source for animals. Selection tools that can be applied early in the breeding cycle are needed to accelerate genetic gain for increased wheat production while maintaining or improving grain quality if demand from human population growth is to be fulfilled. Proteomics screening assays of wheat flour can assist breeders to select the best performing breeding lines and discard the worst lines. In this study, we optimised a robust LC–MS shotgun quantitative proteomics method to screen thousands of wheat genotypes. Using 6 cultivars and 4 replicates, we tested 3 resuspension ratios (50, 25, and 17 µL/mg), 2 extraction buffers (with urea or guanidine-hydrochloride), 3 sets of proteases (chymotrypsin, Glu-C, and trypsin/Lys-C), and multiple LC settings. Protein identifications by LC–MS/MS were used to select the best parameters. A total 8738 wheat proteins were identified. The best method was validated on an independent set of 96 cultivars and peptides quantities were normalised using sample weights, an internal standard, and quality controls. Data mining tools found particularly useful to explore the flour proteome are presented (UniProt Retrieve/ID mapping tool, KEGG, AgriGO, REVIGO, and Pathway Tools).     

SEC23B Loss-of-Function Suppresses Hepcidin Expression by Impairing Glycosylation Pathway in Human Hepatic Cells

Biallelic pathogenic variants in the SEC23B gene cause congenital dyserythropoietic anemia type II (CDA II), a rare hereditary disorder hallmarked by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypo-glycosylation of some red blood cell membrane proteins. Abnormalities in SEC23B, which encodes the homonymous cytoplasmic COPII (coat protein complex II) component, disturb the endoplasmic reticulum to Golgi trafficking and affect different glycosylation pathways. The most harmful complication of CDA II is the severe iron overload. Within our case series (28 CDA II patients), approximately 36% of them exhibit severe iron overload despite mild degree of anemia and slightly increased levels of ERFE (the only erythroid regulator of hepcidin suppression). Thus, we hypothesized a direct role of SEC23B loss-of-function in the pathomechanism of hepatic iron overload. We established a hepatic cell line, HuH7, stably silenced for SEC23B. In silenced cells, we observed significant alterations of the iron status, due to both the alteration in BMP/SMADs pathway effectors and a reduced capability to sense BMP6 stimulus. We demonstrated that the loss-of-function of SEC23B is responsible of the impairment in glycosylation of the membrane proteins involved in the activation of the BMP/SMADs pathway with subsequent hepcidin suppression. Most of these data were confirmed in another hepatic cell line, HepG2, stably silenced for SEC23B. Our findings suggested that the pathogenic mechanism of iron overload in CDA II is associated to both ineffective erythropoiesis and to a specific involvement of SEC23B pathogenic variants at hepatic level. Finally, we demonstrated the ability of SEC23B paralog, i.e., SEC23A, to rescue the hepcidin suppression, highlighting the functional overlap between the two SEC23 paralogs in human hepatic cells.     

Macrophage-Mediated Melanoma Reduction after HP-NAP Treatment in a Zebrafish Xenograft Model

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.     

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.     

Design and characterization of PNVCL-based nanofibers and evaluation of their potential applications as scaffolds for surface drug delivery of hydrophobic drugs

In this work, nanofiber scaffolds for surface drug delivery applications were obtained by electrospinning poly(N-vinylcaprolactam) (PNVCL) and its blends with poly(ε-caprolactone) and poly(N-vinylcaprolactam)-b-poly(ε-caprolactone). The process parameters to obtain smooth and beadless PNVCL fibers were optimized. The average fibers diameter was less than 1 μm, and it was determined by scanning electron microscopy analyses. Their affinity toward water was evaluated by measuring the contact angle with water. The ketoprofen release behavior from the fibers was analyzed using independent and model-dependent approaches. The low values of the release exponent (n < 0.5) obtained for 20 and 42 °C, indicating a Fickian diffusion mechanism for all formulations. Dissolution efficiencies (DEs) revealed the effect of polymer composition, methodology used in the electrospinning process, and temperature on the release rate of ketoprofen. PNVCL/poly(N-vinylcaprolactam)-b-poly(ε-caprolactone)-based nanofibers showed greater ability to control the in vitro release of ketoprofen, in view of reduced kinetic constant and DE, making this material promising system for controlling release of hydrophobic drugs. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48472.     

Determination of the Hydroxyl Value of Soybean Polyol by Attenuated Total Reflectance/Fourier Transform Infrared Spectroscopy

A rapid method for the quantitative determination of the hydroxyl value (OHV) of hydroxylated soybean oils by HATR/FTIR spectroscopy is described. Calibration standards were prepared by the formic acid/hydrogen peroxide method and OH values were determined by the official method of AOCS Tx 1a-66, covering an analytical range of 3.5–125 mg of KOH/g of sample. A partial least squares (PLS) calibration model for the prediction of the hydroxyl value (OHV) was developed based on eight different spectral subregions between 3,150 and 990 cm⁻¹ and combinations of them. On average, 36 samples were used for the modeling and 17 were used for external validation. The resulting calibration was linear over the analytical range and had a standard deviation of 2.334. Validation of the method was carried out by comparing the OHV of a series of hydroxylated soybean oils predicted by the PLS model to the values obtained by the AOCS standard method. A correlation coefficient of R ² = 0.9843 and RMSEC and RMSEP values of, respectively, 3.393 and 3.643 were obtained. After the calibration of the spectrometer, the OHV could be obtained in 2–3 min per sample, a major improvement over conventional wet chemical methods. The advantages of these methodologies are that they do not destroy the sample, have a lower cost, expedite the analysis and do not produce residues. Therefore, they may yield excellent results when used to quantify OHV of soybean polyols obtained by hydroxylation reaction.     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Phenolic Substances,Flavor Compounds,and Textural Properties of Three Native Romanian Wine Grape Varieties

In this work, the chemical compositions and texture characteristics of three native Romanian wine grape varieties (Feteasc? regal?, Feteasc? alba, and Feteasc? neagr?) were studied. We assessed the distinct characteristics directly linked to their phenolic compositions, volatile profiles, and mechanical properties and compared these characteristics with those of Pinot noir grapes. The effect of the growing zone was also evaluated. Various spectrophotometric indices directly related to the phenolic compositions of berry skins and seeds were determined. The detailed phenolic compositions (anthocyanins, hydroxycinnamoyl tartaric acids, and stilbenes) of the skins were determined using high-performance liquid chromatography methods. Free and bound volatile compounds in the berries were quantified by gas chromatography/mass spectrometry. The textural properties of the skins and seeds were measured by instrumental texture analysis. The results showed high diversity among the varieties and zones that affected the enological potential. Among the white varieties, Feteasc? alb? grapes could be less susceptible to browning as a consequence of their lower trans-caffeoyltartaric acid concentration, whereas Feteasc? regal? grapes from Cluj had the highest concentrations of total free and bound volatile compounds, particularly terpenes and norisoprenoids. Among the red varieties, Feteasc? neagr? was identified as a promising variety to be exploited in the future for its particular phenolic characteristics, particularly those grapes grown in Mica. Nevertheless, Feteasc? neagr? grapes grown in Cluj had the highest total glycosidically bound terpene concentrations. Finally, differences in the mechanical and/or acoustic properties of the skins and seeds could strongly influence the kinetics and completeness of phenolic compound extractions.