Discovery and Mechanistic Elucidation of NQO1-Bioactivatable Small Molecules That Overcome Resistance to Degraders.

Degraders hold the promise to efficiently inactivate previously intractable disease-relevant targets. Unlike traditional inhibitors, degraders act substoichiometrically and rely on the hijacked proteolysis machinery, which can also act as an entry point for resistance. To fully harness the potential of targeted protein degradation, it is crucial to comprehend resistance mechanisms and formulate effective strategies to overcome them. We conducted a chemical screening to identify synthetic lethal vulnerabilities of cancer cells that exhibit widespread resistance to degraders. Comparative profiling followed by tailored optimization delivered the small molecule RBS-10, which shows preferential cytotoxicity against cells pan-resistant to degraders. Multiomics deconvolution of the mechanism of action revealed that RBS-10 acts as a prodrug bioactivated by the oxidoreductase enzyme NQO1, which is highly overexpressed in our resistance models. Collectively, our work informs on NQO1 as an actionable vulnerability to overcome resistance to degraders and as a biomarker to selectively exploit bioactivatable prodrugs in cancer.

Modulation of Functional Phosphorylation Sites by Basic Residues in the Unique Domain of c-Src.

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

Time-resolved transcriptomic profiling of the developing rabbit's lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia.

BACKGROUND: Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations. METHODS: Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups. RESULTS: Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor ß, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition. CONCLUSION: These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

Improving the Phenolic Content of Tempranillo Grapes by Sustainable Strategies in the Vineyard.

Wine phenolics are of considerable interest due to their implication in the organoleptic appreciation of wines and due to their bioactive functions as antioxidants. In this work, the effects of sustainable strategies in the vineyard, regulated deficit irrigation treatments (RDI) and crop load level (CL) on Tempranillo grape phenolics over two seasons was studied. Rainfed (T), early (EDI) and late (LDI) regulated deficit irrigation was applied. Cluster thinning (TH) and control (C) without cluster removal were also applied under each irrigation treatment. The effect of CL remained independent of RDI for all compounds, except for phenolic acids. The RDI influence on the grape skin phenolic profile was higher than CL in the dry season (2009); however, in 2010, the effect of CL was greater. In 2009, a tendency to increase anthocyanin and hydroxycinnamic acid content in grape skins was registered in EDI with respect to T. However, significant decreases in hydroxycinnamic and flavanol compounds were found in LDI. In 2010, the wettest year, CL increased all phenolic families' content. Thus, it can be concluded that the effects of RDI and cluster thinning treatments depend on the family of compounds considered and the meteorological conditions of the year.

Relative impact of oenological tannins in model solutions and red wine according to phenolic, antioxidant, and sensory traits.

Oenological tannins (OETs) are winemaking processing aids used to facilitate stabilization and fining, to increase the antioxidant capacity, and to promote colour stability of grape juice and wine. A wide variability of pure or mix formulates are available for winemaking purposes, including hydrolysable tannins (gallotannins and ellagitannins), proanthocyanidins from grape skins and seeds (prodelphinidins and procyanidins), and from exotic wood (prorobinetinidins and profisetinidins). In this study, seventeen OETs pure and mix formulates were characterized in terms of polyphenolic content and antioxidant capacity in a model wine and in a red wine after one-month storage, as well as aroma, astringency, and bitterness sensory characteristics in water and red wine. Colour-related features were also analysed in the added red wine after one-month storage. For the first time, correlations among the obtained results in the different matrices were investigated to understand the most suitable OETs for winemaking applications. The results showed a great variability among the formulates studied in terms of phenolic content, which was strictly correlated to their antioxidant capacity. Regarding origin, hydrolysable tannins had the highest antioxidant ability, followed by exotic wood formulates. A strong and positive correlation was found in antioxidant capacity of OETs in model wine and red wine after one-month storage, in particular for ellagitannins, which confirmed also their ability to increase pigments polymerization. By contrast, quebracho tannins resulted the bitterest and most astringent when tasted in water (0.4 g/L), although in-mouth and aromatic descriptors of OETs tasted in water were not correlated with the ones of the added red wine. Therefore, the choice of OETs formulate and its optimal dose requires a characterization in terms of polyphenolic content and antioxidant capacity because these properties were well correlated with those of the added wines in a short storage period, whereas the sensory impact at oenological range doses is mainly dependent on wine features.

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors.

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

Effect of Early Basal Leaf Removal on Phenolic and Volatile Composition and Sensory Properties of Aglianico Red Wines.

The aim of this work was to study the influence of early basal leaf removal on Aglianico wines produced in the Apulia region (Italy). Three treatments were carried out, where 100% of fruit-zone leaves on the north (DN), south (DS) and on both sides of the canopy (DNS) were removed. A control (CT), where all basal leaves were retained, was also performed. Instrumental (HPLC-DAD-MS and GC-MS) and sensory analysis (QDA) were used to evaluate the treatment effect on the phenolic and volatile compositions and on the sensory descriptors of wines. DNS reached the highest amounts of phenolic compounds, showing a change in the phenolic pattern from flavonols and anthocyanins. Moreover, leaf removal influenced the levels of 37.8% of volatile compounds, quantified by increasing the concentration when early leaf removal was applied on the north side of the canopy (DN), with respect to the south (DS) and both sides (DNS). In the sensory analysis, Aglianico wines were defined by 16 sensory attributes with GM > 30%, where the highest values were reached for defoliation treatments vs. control. In conclusion, early leaf removal treatments allowed us to modulate the phenolic and volatile concentrations of Aglianico wines.

Production and characterization of a new distilled beverage from green coffee seed residue.

This study evaluated green coffee seed residue (GCSR) as an alternative substrate for producing distilled beverages. Two proportions of GCSR, 10% and 20% (w/v), were fermented and distilled in a copper alembic still. The spirits were characterized by GC-FID, HS-SPME GC-MS, and sensory analysis by trained panelists. Most of the 62 identified volatile compounds were affected by the GCSR concentration. Total terpenes, higher alcohols, and acetals showed the highest concentrations in the 10% GCSR spirit. Esters, acetates, and aldehydes were most abundant in the 20% GCSR. In the sensory analysis, the 10% GCSR spirit was characterized by floral, dairy, and almond aromas, while the 20% GCSR spirit was embodied coffee, vegetable, hazelnut, cooked cabbage, and nut descriptors. The results demonstrate the potential of GCSR as a substrate for producing coffee spirits with chemical and sensory qualities, with the 10% GCSR being the better option for fermentation.

Protamine Characterization by Top-Down Proteomics: Boosting Proteoform Identification with DBSCAN.

Protamines replace histones as the main nuclear protein in the sperm cells of many species and play a crucial role in compacting the paternal genome. Human spermatozoa contain protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or the P1/P2 ratio may be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteoforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down proteomics permits a more in-depth analysis of protamine PTMs and proteoforms, thereby opening up new perspectives to unravel their impact on male fertility. We report on the analysis of two normozoospermic semen samples by top-down proteomics. We discuss the difficulties encountered with the data analysis and propose solutions as this step is one of the current bottlenecks in top-down proteomics with the bioinformatics tools currently available. Our strategy for the data analysis combines two software packages, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of normozoospermic individuals represents the first step towards the future study of sperm pathological conditions opening up the potential personalized diagnosis of male infertility.

Chemical composition and sensory properties of Albariño wine: Fertigation effects.

Interannual climate variability and management practices, including fertigation, can alter volatile compound concentrations in wines from a given grapevine cultivar. These compounds are highly relevant for wine aroma. The current study aimed to assess the effect of two levels of fertigation on the volatile composition and sensory properties of wines from Albariño grown in two vineyards in NW Spain over three years. Treatments were fertigation to 60% (F-60) and 100% (F-100) of crop needs since budbreak, and a rain-fed control (F-0). Volatile compounds were determined through gas chromatography and mass spectrometry (GC-MS) and wine sensory evaluation was performed by nine experts using quantitative descriptive analysis. General chemical parameters of wines were similar among treatments; however, F-60 and F-0 slightly reduced volatile total concentrations in both vineyards. Wines from the fertigation treatments had greater concentrations of volatile fatty acids, ethyl esters, acetates and C6 compounds. However, terpene concentrations slightly decreased when fertigation was applied. Higher alcohols showed a different behavior between vineyards, mainly related with grapevine water status during the growing season. Seven sensory descriptors differed significantly between vineyards. This study indicates that fertigation could be a tool for modulating wine chemical and sensory characteristics.

Production of a Distilled Spirit Using Cassava Flour as Raw Material: Chemical Characterization and Sensory Profile.

Cassava plays a key role in the food production and economies of several countries worldwide. Due to its starch content, alcoholic fermentation is a promising transformation process for adding value to cassava. However, most of the existing cassava beverages are from traditional origin, with the yields and quality often poorly known or controlled due to the use of artisanal production processes. This work aims at the application of easily implementable biotechnological tools for the production of cassava spirits, in order to add value to this raw material. Cassava flour was liquefied and saccharified using enzymatic cocktails, generating a fermentable broth with ~184 g L-1 of fermentable sugars. This was then fermented into an alcoholic product with ~10% ethanol by volume and distilled for spirit production. Cassava spirits with 40% ethanol by volume, with or without application of oak wood, were produced. For further valorization, volatile fractions of cassava spirits were characterized by gas chromatography-flame ionization detection (GC-FID) and GC-MS. These showed a predominance of yeast fermentation metabolites, complemented by wood extractives where oak chips were applied. Both produced spirits showed desirable sensory traits, receiving good acceptance by experienced tasters, demonstrating the feasibility of the proposed process to add value to cassava surplus.

Validation of a LLME/GC-MS Methodology for Quantification of Volatile Compounds in Fermented Beverages.

Knowledge of composition of beverages volatile fraction is essential for understanding their sensory attributes. Analysis of volatile compounds predominantly resorts to gas chromatography coupled with mass spectrometry (GC-MS). Often a previous concentration step is required to quantify compounds found at low concentrations. This work presents a liquid-liquid microextraction method combined with GC-MS (LLME/GC-MS) for the analysis of compounds in fermented beverages and spirits. The method was validated for a set of compounds typically found in fermented beverages comprising alcohols, esters, volatile phenols, and monoterpenic alcohols. The key requirements for validity were observed, namely linearity, sensitivity in the studied range, accuracy, and precision within the required parameters. Robustness of the method was also evaluated with satisfactory results. Thus, the proposed LLME/GC-MS method may be a useful tool for the analysis of several fermented beverages, which is easily implementable in a laboratory equipped with a GC-MS.

Abiotic stress management and Verdejo wine composition: cluster thinning in different water regimes.

BACKGROUND: The effects of cluster thinning (TH) on Verdejo wine composition were studied. Cluster thinning was applied in three water regimes (WR) (R0, rainfed control; R25, drip-irrigated at 25% ETo, and R50, drip-irrigated at 50% ETo) during the 2012-2014 seasons. RESULTS: Cluster thinning advanced grape maturity, although this was only significant in 2012. A significant effect of TH on R0 was observed in 2013, the wettest season, increasing the concentration of alcohols, esters, acetates, and lactones. The same trend was shown in 2012 for all groups of volatile compounds in R0. In contrast, for all volatile families to increase was observed in 2014, the driest season, when TH was applied to R50. WR*TH interactions were found in most wine chemical parameters. CONCLUSION: The variation in wine volatile composition among the vintages that were observed shows that the capacity of TH to exert an influence depends not only on the water regime but also on annual conditions. © 2019 Society of Chemical Industry.

Characterization of Human Sperm Protamine Proteoforms through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches.

Protamine 1 (P1) and protamine 2 (P2) family are extremely basic, sperm-specific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies is not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach. By top-down MS, described and novel truncated P1 and pre-P2 proteoforms were identified. Intact P1, pre-P2, and P2 mature proteoforms and their phosphorylation pattern were also detected. Additionally, a +61 Da modification in different proteoforms was observed. By the bottom-up MS approach, phosphorylated residues for pre-P2, as well as the new P2 isoform 2, which is not annotated in the UniProtKB database, were revealed. Implementing these strategies in comparative studies of different infertile phenotypes, together with the evaluation of P1/P2 and pre-P2/P2 MS-derived ratios, would permit determining specific alterations in the protamine proteoforms and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.

Top-Down Proteomics Applied to Human Cerebrospinal Fluid.

Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.

Effect of the timing of water deficit on the must amino acid profile of Tempranillo grapes grown under the semiarid conditions of SW Spain.

Must nitrogen is very important for successful fermentation. Irrigation can influence vine nitrogen availability, and therefore must nitrogen content and wine quality. The aim of this work was to assess the effect of vine water status on the nitrogen concentration of cv. Tempranillo grown under semiarid conditions. A rainfed and two regulated deficit irrigation (RDI) strategies, late (LDI) and early deficit irrigation (EDI) were applied during pre- and post-veraison respectively periods over two seasons in Badajoz (Spain). Twenty-eight amino acids, amines and ammonia were determined by high performance liquid chromatography (HPLC) in the must. Results showed an effect of the pre-veraison vine water status on the must amino acid concentration. Higher values of nitrogenous indices were reached in musts from LDI treatments, mainly in the dry year. Significant correlations between pre-veraison stem water potential and 19 amino acid concentrations were observed. Therefore, LDI was a more suitable strategy for increasing the content of nitrogenous substances in the must.

Phenolic Composition Influences the Effectiveness of Fining Agents in Vegan-Friendly Red Wine Production.

Plant proteins have been proposed as an alternative to animal-origin proteins in the wine industry because they are allergen-free and vegan-friendly. The aim of this study was to evaluate the effectiveness of plant proteins as fining agents on red wines with different phenolic composition. Two formulations for commercially available vegetal proteins (potato and pea origin) were assessed at two doses to modulate the fining treatment to the wine phenolic profile. The results evidenced that fining agents derived from plants have different levels of effectiveness on the removal of phenolic compounds depending on the origin, the formulation used, dose applied, and also wine characteristics. On Nebbiolo wine, the study was particularly significant due to its phenolic composition. One pea-based fining agent had an effect comparable to gelatin (animal origin) on the removal of polymeric flavanols with a minor loss of anthocyanins and therefore better preserving the wine color in terms of intensity and hue. For Primitivo, Montepulciano, and Syrah wines, even though there was a formulation-dependent effect, vegetal proteins gave more balanced reductions in terms of target phenolic compounds contributing to astringency and color perception.

On-vine withering process of 'Moscato bianco' grapes: effect of cane-cut system on volatile composition.

BACKGROUND: Cane-cut on-vine withering is a grape dehydration technique used for dry and sweet wine production. The aim of this work was to study the influence of the cane-cut technique applied at harvest to Moscato bianco grapes during the on-vine withering process to produce dehydrated berries with a different chemical composition and a volatile profile. RESULTS: After 24 days of dehydration, an on-vine withering system using the cane-cut technique induced an increase in the total volatile content compared with grapes produced with a normal on-vine withering process. This increase was greater in glycosidically bound volatile compounds than in the free fraction. Bound linalool showed a significant increase of 52% when the cane-cut withering system was applied but the grapes that were normally withered appeared to be less prone to the loss of free linalool. A significant increase in the glycosylated forms of nerol and geraniol was also observed in the two on-vine withering systems on the 24th day compared with the control (fresh grapes at harvest date). CONCLUSION: A cane-cut, on-vine withering system applied at harvest induced changes in the volatile composition of Moscato bianco grapes increasing total volatile content, consisting mainly of bound compounds, by the 24th day of dehydration. The grapes dehydrated on-vine using this new system also showed significantly greater content of most of the free volatile compounds detected. © 2018 Society of Chemical Industry.

Effect of the winemaking process on the volatile composition and aromatic profile of Tempranillo Blanco wines.

The effects of the carbonic maceration and conventional winemaking on the volatile composition and aromatic sensory characteristics of Tempranillo Blanco wines were studied for the first time, during three consecutive vintages. Relationships between instrumental (volatiles) and sensory variables were analyzed applying partial least squares regression (PLS). Carbonic macerated wines had higher contents of alcohols and carbonyl compounds, yet lower concentrations of C6 alcohols and volatile acids than wines conventionally produced. The Odor Activity Values (OAV) exhibited an increase in wines when carbonic maceration was applied. According to the geometric mean (% GM) obtained from aroma descriptors the effect of the winemaking process was significant for seed fruit, ripe fruit and floral notes. When subjected to PLS the data from the instrumental analysis yielded a satisfactory model for the prediction of aroma descriptors in this set of wines.

Assessment fertigation effects on chemical composition of Vitis vinifera L. cv. Albariño.

Vineyard management can influence the growth and yield components in the vineyards and therefore on the grape and wine quality. In this work, a chemical study was conducted (2014-2015) to examine the effect of fertigation on chemical composition of Albariño. A control (Rain-fed) and fertigation (60% and 100%) treatments were apply at same irrigation depth, where fertigation 100% is complete nutrient requirements to Albariño trellis system in this location (Rias Baixas AOC, NW Spain). Results showed that non-volatile compounds of Albariño musts were not affected by fertigation treatments. However, the effect of fertigation treatments on the volatile composition was observed. Terpenes and C13-norisoprenids were the most affected families of volatile compounds by fertigation treatments, where 60% fertigation exhibited the highest concentration, improving the wine aroma quality. Application of principal component analysis (PCA) showed a good separation of Albariño grape according to fertigation treatments and vintages.