Preparative Fractionation of Brazilian Red Propolis Extract Using Step-Gradient Counter-Current Chromatography.

Propolis is a resinous bee product with a very complex composition, which is dependent upon the plant sources that bees visit. Due to the promising antimicrobial activities of red Brazilian propolis, it is paramount to identify the compounds responsible for it, which, in most of the cases, are not commercially available. The aim of this study was to develop a quick and clean preparative-scale methodology for preparing fractions of red propolis directly from a complex crude ethanol extract by combining the extractive capacity of counter-current chromatography (CCC) with preparative HPLC. The CCC method development included step gradient elution for the removal of waxes (which can bind to and block HPLC columns), sample injection in a single solvent to improve stationary phase stability, and a change in the mobile phase flow pattern, resulting in the loading of 2.5 g of the Brazilian red propolis crude extract on a 912.5 mL Midi CCC column. Three compounds were subsequently isolated from the concentrated fractions by preparative HPLC and identified by NMR and high-resolution MS: red pigment, retusapurpurin A; the isoflavan 3(R)-7-O-methylvestitol; and the prenylated benzophenone isomers xanthochymol/isoxanthochymol. These compounds are markers of red propolis that contribute to its therapeutic properties, and the amount isolated allows for further biological activities testing and for their use as chromatographic standards.

Metabolite Profiling of Pleurotus ostreatus Grown on Sisal Agro-Industrial Waste Supplemented with Cocoa Almond Tegument and Wheat Bran.

Pleurotus ostreatus is an edible fungus with high nutritional value that uses industrial and agricultural lignocellulosic residues as substrates for growth and reproduction. Understanding their growth metabolic dynamics on agro-industrial wastes would help to develop economically viable and eco-friendly biotechnological strategies for food production. Thus, we used UHPLC/MS/MS and GNPS as an innovative approach to investigate the chemical composition of two strains of P. ostreatus, coded as BH (Black Hirataki) and WH (White Hirataki), grown on sisal waste mixture (SW) supplemented with 20 % cocoa almond tegument (CAT) or 20 % of wheat bran (WB). Metabolite dereplication allowed the identification of 53 metabolites, which included glycerophospholipids, fatty acids, monoacylglycerols, steroids, carbohydrates, amino acids, and flavonoids. This is the first report of the identification of these compounds in P. ostreatus, except for the steroid ergosterol. Most of the metabolites described in this work possess potential biological activities, which support the nutraceutical properties of P. ostreatus. Thus, the results of this study provide essential leads to the understanding of white-rot fungi chemical plasticity aiming at developing alternative biotechnologies strategies for waste recycling.

Metabolic Disturbances in the Gut-brain Axis of a Mouse Model of MPTP-induced Parkinsonism Evaluated by Nuclear Magnetic Resonance.

Parkinson's Disease is a synucleinopathy that primarily affects the dopaminergic cells of the central nervous system, leading to motor and gastrointestinal disturbances. However, intestinal peripheral neurons undergo a similar neurodegeneration process, marked by α-synuclein (αSyn) accumulation and loss of mitochondrial homeostasis. We investigated the metabolic alterations in different biometrics that compose the gut-brain axis (blood, brain, large intestine, and feces) in an MPTP-induced mouse model of sporadic Parkinson's Disease. Animals received escalating administration of MPTP. Tissues and fecal pellets were collected, and the metabolites were identified through the untargeted Nuclear Magnetic Resonance spectroscopic (1H NMR) technique. We found differences in many metabolites from all the tissues evaluated. The differential expression of metabolites in these samples mainly reflects inflammatory aspects, cytotoxicity, and mitochondrial impairment (oxidative stress and energy metabolism) in the animal model used. The direct evaluation of fecal metabolites revealed changes in several classes of metabolites. This data reinforces previous studies showing that Parkinson's disease is associated with metabolic perturbation not only in brain-related tissues, but also in periphery structures such as the gut. In addition, the evaluation of the microbiome and metabolites from gut and feces emerge as promising sources of information for understanding the evolution and progression of sporadic Parkinson's Disease.

Metformin acts in the gut and induces gut-liver crosstalk.

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin-induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor.

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Machine learning-driven serum metabolomics: insights into the transition to early-stage heart failure with preserved ejection fraction

Introduction: Metabolomics has emerged as a powerful tool in providing readouts of early disease states before clinical manifestation. Here we used the predictive power of Unsupervised Hierarchical Clustering Analysis (UHCA) and Automated Machine Learning (AutoML) algorithms to identify serum metabolic panels in a population at risk of developing HFpEF. Methods: We studied 215 subjects staged as non-HF, pre-HFpEF and early-stage HFpEF(es-HFpEF). We evaluated clinical, laboratory, echocardiographic, and NMR-based metabolomics of blood serum data. UHCA and AutoML were used to explore metabolic fingerprints potentially related to clinical features or HFpEF. We used Metabolite Set Enrichment Analysis to explore biochemical pathways. Results: The UHCA identified three major patients (P) and two metabolites (M) clusters (Figure). The P clusters were associated with HFpEF stages, cardiac remodeling, diastolic dysfunction, and sex (Pearson Chi-square, p < 0.05) and M clusters with glycine and serine metabolism and urea cycle pathways (FDR-adjusted p-value < 0.002). Considering non-HFpEF and es-HFpEF groups, AUROC mean for feature subset combinations was 0.897 and the highest AUROC (0.995) combined metabolites, clinical, laboratory and echo features. Of the 64 models trained that included metabolites as input, serine (25), uridine (17), 2-oxoglutarate (14), citrate (14), 2-aminobutyrate (13) and taurine (13) were observed more frequently with feature importance value greater than zero. The metabolites with higher sum values of feature importance were serine (0.173), uridine (0.131), 2-aminobutyrate (0.123), choline (0.098) and dimethylamine (0.087). Conclusions: This study revealed characteristic metabolite profiles in the sera of patients at risk of developing HFpEF. These metabolite panels can add information for classificatory algorithms development and contribute to the understanding of HFpEF pathophysiology.

Metabolomics by NMR Combined with Machine Learning to Predict Neoadjuvant Chemotherapy Response for Breast Cancer.

Neoadjuvant chemotherapy (NACT) is offered to patients with operable or inoperable breast cancer (BC) to downstage the disease. Clinical responses to NACT may vary depending on a few known clinical and biological features, but the diversity of responses to NACT is not fully understood. In this study, 80 women had their metabolite profiles of pre-treatment sera analyzed for potential NACT response biomarker candidates in combination with immunohistochemical parameters using Nuclear Magnetic Resonance (NMR). Sixty-four percent of the patients were resistant to chemotherapy. NMR, hormonal receptors (HR), human epidermal growth factor receptor 2 (HER2), and the nuclear protein Ki67 were combined through machine learning (ML) to predict the response to NACT. Metabolites such as leucine, formate, valine, and proline, along with hormone receptor status, were discriminants of response to NACT. The glyoxylate and dicarboxylate metabolism was found to be involved in the resistance to NACT. We obtained an accuracy in excess of 80% for the prediction of response to NACT combining metabolomic and tumor profile data. Our results suggest that NMR data can substantially enhance the prediction of response to NACT when used in combination with already known response prediction factors.

Gut microbiome of the largest living rodent harbors unprecedented enzymatic systems to degrade plant polysaccharides.

The largest living rodent, capybara, can efficiently depolymerize and utilize lignocellulosic biomass through microbial symbiotic mechanisms yet elusive. Herein, we elucidate the microbial community composition, enzymatic systems and metabolic pathways involved in the conversion of dietary fibers into short-chain fatty acids, a main energy source for the host. In this microbiota, the unconventional enzymatic machinery from Fibrobacteres seems to drive cellulose degradation, whereas a diverse set of carbohydrate-active enzymes from Bacteroidetes, organized in polysaccharide utilization loci, are accounted to tackle complex hemicelluloses typically found in gramineous and aquatic plants. Exploring the genetic potential of this community, we discover a glycoside hydrolase family of ß-galactosidases (named as GH173), and a carbohydrate-binding module family (named as CBM89) involved in xylan binding that establishes an unprecedented three-dimensional fold among associated modules to carbohydrate-active enzymes. Together, these results demonstrate how the capybara gut microbiota orchestrates the depolymerization and utilization of plant fibers, representing an untapped reservoir of enzymatic mechanisms to overcome the lignocellulose recalcitrance, a central challenge toward a sustainable and bio-based economy.

Bacterial volatile organic compounds (VOCs) promote growth and induce metabolic changes in rice.

Plant growth-promoting bacteria (PGPB) represent an eco-friendly alternative to reduce the use of chemical products while increasing the productivity of economically important crops. The emission of small gaseous signaling molecules from PGPB named volatile organic compounds (VOCs) has emerged as a promising biotechnological tool to promote biomass accumulation in model plants (especially Arabidopsis thaliana) and a few crops, such as tomato, lettuce, and cucumber. Rice (Oryza sativa) is the most essential food crop for more than half of the world's population. However, the use of VOCs to improve this crop performance has not yet been investigated. Here, we evaluated the composition and effects of bacterial VOCs on the growth and metabolism of rice. First, we selected bacterial isolates (IAT P4F9 and E.1b) that increased rice dry shoot biomass by up to 83% in co-cultivation assays performed with different durations of time (7 and 12 days). Metabolic profiles of the plants co-cultivated with these isolates and controls (without bacteria and non-promoter bacteria-1003-S-C1) were investigated via 1H nuclear magnetic resonance. The analysis identified metabolites (e.g., amino acids, sugars, and others) with differential abundance between treatments that might play a role in metabolic pathways, such as protein synthesis, signaling, photosynthesis, energy metabolism, and nitrogen assimilation, involved in rice growth promotion. Interestingly, VOCs from IAT P4F9 displayed a more consistent promotion activity and were also able to increase rice dry shoot biomass in vivo. Molecular identification by sequencing the 16S rRNA gene of the isolates IAT P4F9 and E.1b showed a higher identity with Serratia and Achromobacter species, respectively. Lastly, volatilomes of these and two other non-promoter bacteria (1003-S-C1 and Escherichia coli DH5α) were evaluated through headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. Compounds belonging to different chemical classes, such as benzenoids, ketones, alcohols, sulfide, alkanes, and pyrazines, were identified. One of these VOCs, nonan-2-one, was validated in vitro as a bioactive compound capable of promoting rice growth. Although further analyses are necessary to properly elucidate the molecular mechanisms, our results suggest that these two bacterial isolates are potential candidates as sources for bioproducts, contributing to a more sustainable agriculture.

Air pollution and high-intensity interval exercise: Implications to anti-inflammatory balance, metabolome and cardiovascular responses.

High-intensity interval exercise (HIIE) is an effective non-pharmacological tool for improving physiological responses related to health. When HIIE is performed in urban centers, however, the exerciser is exposed to traffic-related air pollution (TRAP), which is associated with metabolic, anti-inflammatory imbalance and cardiovascular diseases. This paradoxical combination has the potential for conflicting health effects. Thus, the aim of this study was to determine the effects of HIIE performed in TRAP exposure on serum cytokines, non-target metabolomics and cardiovascular parameters. Fifteen participants performed HIIE in a chamber capable to deliver filtered air (FA condition) or non-filtered air (TRAP condition) from a polluted site adjacent to the exposure chamber. Non-target blood serum metabolomics, blood serum cytokines and blood pressure analyses were collected in both FA and TRAP conditions at baseline, 10 min after exercise, and 1 h after exercise. The TRAP increased IL-6 concentration by 1.7 times 1 h after exercise (p < 0.01) and did not change the anti-inflammatory balance (IL-10/TNF-α ratio). In contrast, FA led to an increase in IL-10 and IL-10/TNF-α ratio (p < 0.01), by 2.1 and 2.3 times, respectively. The enrichment analysis showed incomplete fatty acid metabolism under the TRAP condition (p < 0.05) 10 min after exercise. There was also an overactivity of ketone body metabolism (p < 0.05) at 10 min and at 1 h after exercise with TRAP. Exercise-induced acute decrease in systolic blood pressure (SBP) was not observed at 10 min and impaired at 1 h after exercise (p < 0.05). These findings reveal that TRAP potentially attenuates health benefits often related to HIIE. For instance, the anti-inflammatory balance was impaired, accompanied by accumulation of metabolites related to energy supply and reduction to exercise-induced decrease in SBP.

Women with temporomandibular disorders: Untargeted proton nuclear magnetic resonance spectroscopy-based metabolomics of saliva and psychological instruments dataset.

This article introduces the first dataset of 1H- nuclear magnetic resonance - based metabolomic spectroscopy of saliva samples from women with temporomandibular disorders (TMD) of muscular origin. Our data generated a metabolomic profile for TMD of muscular origin. The samples were separated in two groups: Experimental Group (EG) represented by women with TMD who were submitted to a conservative treatment compared with a Control group (CG) of women without TMD. These data also include information about time of onset the pain, measures of pain obtained before and after the treatment by the visual analogic scale. Information about some psychological instruments as pain catrastophizing scale, hospital anxiety and depression, and oral health impact profile-14 were also obtained in the CG and in the EG before submitted to the conservative treatment (EG-pre) and at the end of the treatment (EG-post). Those instruments help differentiate the groups, due to the psychosocial impact that TMD has on their lives perpetuating the physiological imbalance of the stomatognathic system. Raw data are available at: https://data.mendeley.com/datasets/wys5xd2vfg/1. It's published on mendeley, the DOI is DOI:10.17632/wys5xd2vfg.1. The data presented in this article are related to the research article entitled "1H-NMR-Based salivary metabolomics from female with temporomandibular disorders - a pilot study" (Lalue Sanches et al. 2020, https://doi.org/10.1016/j.cca.2020.08.006).

Electrospun Nanofibrous Architectures of Thrombin-Loaded Poly(ethylene oxide) for Faster in Vivo Wound Clotting.

Wound healing materials to prevent blood loss are crucial during emergency medical treatment because uncontrolled bleeding can lead to patient death. Herein, bioabsorbable fibrous architectures of thrombin-loaded poly(ethylene oxide)-PEO/thrombin-are conceptualized and accomplished via electrospinning for faster wound clotting. Membranes with average fiber diameters ranging from 188 to 264 nm are achieved, where the active thrombin is entrapped within the nanofibers. The results of in vitro and in vivo wound healing activity tests revealed that when the nanofibers with thrombin-loaded capacity are in contact with the wound, the presence of water in the skin or blood catalyzes the degradation of the membranes, thus releasing thrombin. Thrombin then accelerates the wound clotting process. In contrast to other hemostatic materials, PEO/thrombin nanofibers do not require mechanical removal after application, and the viscoelastic nature of such biomaterials enables their conformation to a variety of wound topographies. Remarkably, PEO/thrombin membranes are promising functional materials and their use is a powerful strategy for hemostatic treatment, ranging from simple first aid and sealing to a wound to small surgical procedures.

1H-NMR-Based salivary metabolomics from females with temporomandibular disorders - A pilot study.

Although temporomandibular disorder (TMD) is the second most common musculoskeletal disorder in the general population, the disease is multifactorial and presents symptoms common to other conditions which misdiagnosis can lead to treatment failure. In this case-control study, we performed, for the first time, a high-resolution 1H-nuclear magnetic resonance spectroscopy metabolomic analysis of the saliva of 26 women with TMD of muscular origin (experimental group [EG]) at the beginning (EG-pre) and at the end (EG-post) of a conservative treatment, and of 27 normal women (control group [CG]) to identify a metabolic signature for TMD. One-way analysis of variance showed changes in the concentration of phenylacetate, dimethylamine, maltose, acetoin, and isovalerate. Partial least-square discriminant analysis showed that metabolite signals did not overlap in CG X EG-pre and EG-pre X EG-post, but overlapped in CG X EG-post. The area under the receiver operating characteristic curve was 1 in CG X EG-pre (95% CI, 1.000-1.000; p < 0.002), 0.993 in EG-pre X EG-post (95% CI, 0.963-1.000), and 0.832 in CG X EG-post (95% CI, 0.699-0.961). These results suggest that the metabolomic profiles of women with and without TMD differ, while after treatment there is a lower distinction and slight tendency towards overlapping between CG and EG-post compared to pre treatment. We also found that phenylacetate, dimethylamine, maltose, acetoin, and isovalerate are potential biomarkers for TMD of muscular origin.

The MAF1 Phosphoregulatory Region Controls MAF1 Interaction with the RNA Polymerase III C34 Subunit and Transcriptional Repression in Plants.

MAF1 is a phosphoprotein that plays a critical role in cell growth control as the central regulator of RNA polymerase (Pol) III activity. Citrus MAF1 (CsMAF1) was identified as a direct target of PthA4, a bacterial effector protein required to induce tumors in citrus. CsMAF1 binds to Pol III to restrict transcription; however, exactly how CsMAF1 interacts with the polymerase and how phosphorylation modulates this interaction is unknown. Moreover, how CsMAF1 binds PthA4 is also obscure. Here we show that CsMAF1 binds predominantly to the WH1 domain of the citrus Pol III subunit C34 (CsC34) and that its phosphoregulatory region, comprising loop-3 and α-helix-2, contributes to this interaction. We also show that phosphorylation of this region decreases CsMAF1 affinity to CsC34, leading to Pol III derepression, and that Ser 45, found only in plant MAF1 proteins, is critical for CsC34 interaction and is phosphorylated by a new citrus AGC1 kinase. Additionally, we show that the C-terminal region of the citrus TFIIIB component BRF1 competes with CsMAF1 for CsC34 interaction, whereas the C-terminal region of CsMAF1 is essential for PthA4 binding. Based on CsMAF1 structural data, we propose a mechanism for how CsMAF1 represses Pol III transcription and how phosphorylation controls this process.

Using a Fragment-Based Approach to Identify Alternative Chemical Scaffolds Targeting Dihydrofolate Reductase from Mycobacterium tuberculosis.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors.

Effect of particle functionalization and solution properties on the adsorption of bovine serum albumin and lysozyme onto silica nanoparticles.

Silica nanoparticles present an enormous potential as controlled drug delivery systems with high selectivity towards diseased cells. This application is directly related to the phenomenon of protein corona, characterized by the spontaneous adsorption of proteins on the nanoparticle surface, which is not fully understood. Here, we report an investigation on the influence of pH, ionic strength and temperature on the thermodynamics of interaction of bovine serum albumin protein (BSA) with non-functionalized silica nanoparticles (SiO2NPs). Complementary, we also investigated the ability of polyethylene glycol (PEG) and zwitterionic sulfobetaine (SBS) surface-modified nanoparticles to prevent the adsorption of BSA (protein negatively charged at physiological pH) and lysozyme (protein positively charged at physiological pH). We showed that BSA interaction with SiO2NPs is enthalpically governed. On the other hand, functionalization of silica nanoparticles with PEG and SBS completely prevented BSA adsorption. However, these functionalized nanoparticles presented a negative zeta potential and were not able to suppress lysozyme anchoring due to strong nanoparticle-protein electrostatic attraction. Due to the similarity of BSA with Human Serum Albumin, this investigation bears a resemblance to processes involved in the phenomenon of protein corona in human blood, producing information that is relevant for the future biomedical use of functionalized nanoparticles.

3D bioprinting of liver spheroids derived from human induced pluripotent stem cells sustain liver function and viability in vitro.

The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver's epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

Clinical Metabolomics Identifies Blood Serum Branched Chain Amino Acids as Potential Predictive Biomarkers for Chronic Graft vs. Host Disease.

The allogeneic hematopoietic stem cell transplantation procedure-the only curative therapy for many types of hematological cancers-is increasing, and graft vs. host disease (GVHD) is the main cause of morbidity and mortality after transplantation. Currently, GVHD diagnosis is clinically performed. Whereas, biomarker panels have been developed for acute GVHD (aGVHD), there is a lack of information about the chronic form (cGVHD). Using nuclear magnetic resonance (NMR) and gas chromatography coupled to time-of-flight (GC-TOF) mass spectrometry, this study prospectively evaluated the serum metabolome of 18 Brazilian patients who had undergone allogeneic hematopoietic stem cell transplantation (HSCT). We identified and quantified 63 metabolites and performed the metabolomic profile on day -10, day 0, day +10 and day +100, in reference to day of transplantation. Patients did not present aGVHD or cGVHD clinical symptoms at sampling times. From 18 patients analyzed, 6 developed cGVHD. The branched-chain amino acids (BCAAs) leucine and isoleucine were reduced and the sulfur-containing metabolite (cystine) was increased at day +10 and day +100. The area under receiver operating characteristics (ROC) curves was higher than 0.79. BCAA findings were validated by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in 49 North American patients at day +100; however, cystine findings were not statistically significant in this patient set. Our results highlight the importance of multi-temporal and multivariate biomarker panels for predicting and understanding cGVHD.

Synthesis and testing of novel alternative oxidase (AOX) inhibitors with antifungal activity against Moniliophthora perniciosa (Stahel), the causal agent of witches' broom disease of cocoa, and other phytopathogens.

BACKGROUND: Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is the causal agent of witches' broom disease (WBD) of cocoa (Theobroma cacao L.) and a threat to the chocolate industry. The membrane-bound enzyme alternative oxidase (AOX) is critical for M. perniciosa virulence and resistance to fungicides, which has also been observed in other phytopathogens. Notably AOX is an escape mechanism from strobilurins and other respiration inhibitors, making AOX a promising target for controlling WBD and other fungal diseases. RESULTS: We present the first study aimed at developing novel fungal AOX inhibitors. N-Phenylbenzamide (NPD) derivatives were screened in the model yeast Pichia pastoris through oxygen consumption and growth measurements. The most promising AOX inhibitor (NPD 7j-41) was further characterized and displayed better activity than the classical AOX inhibitor SHAM in vitro against filamentous fugal phytopathogens, such as M. perniciosa, Sclerotinia sclerotiorum and Venturia pirina. We demonstrate that 7j-41 inhibits M. perniciosa spore germination and prevents WBD symptom appearance in infected plants. Finally, a structural model of P. pastoris AOX was created and used in ligand structure-activity relationships analyses. CONCLUSION: We present novel fungal AOX inhibitors with antifungal activity against relevant phytopathogens. We envisage the development of novel antifungal agents to secure food production. © 2018 Society of Chemical Industry.

Mechanistic insights revealed by a UBE2A mutation linked to intellectual disability.

Ubiquitin-conjugating enzymes (E2) enable protein ubiquitination by conjugating ubiquitin to their catalytic cysteine for subsequent transfer to a target lysine side chain. Deprotonation of the incoming lysine enables its nucleophilicity, but determinants of lysine activation remain poorly understood. We report a novel pathogenic mutation in the E2 UBE2A, identified in two brothers with mild intellectual disability. The pathogenic Q93E mutation yields UBE2A with impaired aminolysis activity but no loss of the ability to be conjugated with ubiquitin. Importantly, the low intrinsic reactivity of UBE2A Q93E was not overcome by a cognate ubiquitin E3 ligase, RAD18, with the UBE2A target PCNA. However, UBE2A Q93E was reactive at high pH or with a low-pKa amine as the nucleophile, thus providing the first evidence of reversion of a defective UBE2A mutation. We propose that Q93E substitution perturbs the UBE2A catalytic microenvironment essential for lysine deprotonation during ubiquitin transfer, thus generating an enzyme that is disabled but not dead.