Amino Acids Tryptophan Research Articles

Study of resistance mechanism of Alternaria blight (Alternaria brassicicola) by biochemical markers in Indian Mustard (Brassica juncea L. Czern. &Coss.).

Indian mustard (Brassica juncea) is an important oilseed crop in India. Alternaria leaf spot (Alternaria blight) is incited by the fungus Alternaria brassicicola. It majorly affects crop production leading to a yield loss of up to 70%. To circumvent this problem, the study of the resistance mechanism and identification of biochemical markers is one of the important strategies for its management. In the present study, a total of 219 genotypes of Indian mustard with check were screened for Alternaria blight over two seasons. Based on the area under the disease progress curve (AUDPC) scores, ten consistently performing genotypes were selected for the screening of biochemical and yield attributes under artificial inoculated conditions of Alternaria brassicicola (Berk) Sacc. The result showed a negative correlation between disease and yield attributes. The catalase (CAT) activity was significantly increased in resistant genotypes compared to susceptible ones, indicating the crucial role of CAT in the resistance mechanism. Pathogen infection also increases the total protein content and the Alternaria-resistant genotype showed the highest total soluble protein while the susceptible genotype showed the lowest total soluble protein. The ten genotypes were categorized by SSI (stress susceptibility index) and Varuna was identified as a tolerant genotype and Giriraj as a susceptible genotype for Alternaria brassicicola (Berk) Sacc. Varuna and Giriraj were chosen for quantitative analysis of methionine and tryptophan amino acids from seeds using RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) and there were significant differences in the levels of methionine and tryptophan between the Varuna and Giriraj genotypes. Varuna showed higher methionine and tryptophan content compared to the Giriraj genotype. Higher protein content demonstrated an increase in biotic stress-responsive amino acids, such as methionine and tryptophan, suggesting increased resistance to Alternaria diseases in these high-protein genotypes. These amino acids could be used as biochemical markers for Alternaria resistance of mustard.

The Effects of Consumption of Cashew Nut (Anacardium Occidentale) Diet on Scopolamine Induced Depression and Anxiety in CD1 Mice.

Cashew nut (Anacardium occidentale) contain vitamin B6, essential amino acids (tryptophan) a precursor of serotonin and omega-3 fatty acid which had been known to affect mood. However, whether consumption of cashew nut diet which contains vitamin B6, tryptophan and omega-3 fatty acid will affect mood notably, anxiety and depression in mice has not been previously ascertained. Therefore, the effects of consumption of cashew nut (Anacardium occidentale) diets on scopolamine induced anxiety and depression in CD1 mice was studied. Twenty-eight (28) experimental mice were randomly assigned into 4 groups namely; control, cashew nut diet only, cashew nut diet + scopolamine and scopolamine only group. Forced swim test was used to assess for depression in the animals and open field maize, light/dark transition box and elevated plus maze was used to access anxiety. Results from force swim test showed that, depressive symptoms were decreased in scopolamine only treated group compared to control and other experimental groups (p<0.05). Anxiolytic behaviors were also decreased in scopolamine only treated group compared to control and other experimental groups (p<0.05). In conclusion, consumption of cashew nut diets improved depression and anxiety in mice. The anxiolytic and anti-depressive effects observed may be attributed to tryptophan, omega-3 fatty acid and vitamin B6 present in cashew nut diet.

Breeding strategies for quality protein maize: A Review

Zein proteins in maize seeds regulate food and feed quality and belong to several subfamilies. Most maize prolamin is α-zeins, which are rich in glutamine, leucine, and proline but lacking in critical amino acids (lysine and tryptophan). RNAi experiments show that 22kD zeins downregulate the quality protein maize (QPM) phenotype, not 19kD. The opaque-2 mutation caused a chalky, soft endosperm and many defects. QPM genotypes with opaque-2 alleles and firm kernels were improved. As opaque-2 is recessive and biochemical lysine and tryptophan analysis is costly, conventional backcrossing alone is not very useful. Markerassisted selection (MAS) allowed top inbreds to be homozygous o2o2 utilizing opaque-2 gene-specific markers. Vivek QPM-9, a mixture of two QPM introgression lines, benefits various clients and customers. This review highlights the breeding strategies for QPM.

Long-term physical inactivity induces significant changes in biochemical pathways related to metabolism of proteins and glycerophospholipids in mice.

Physical inactivity affects multiple organ systems, including the musculoskeletal system, which upsets the delicate balance of several secretory factors leading to metabolic derailment. This reduces contractile recruitment of the skeletal muscle with dampening of its oxidative capacity resulting in impaired intramuscular lipid metabolism and substrate utilization. We hypothesized that this altered phenotype would also have an indispensable effect on circulatory cytokines and the level of metabolic intermediates. In this study, comparison between sedentary (SED) and exercised (EXER) animal models showed that organismal metabolic parameters (body mass, oxygen utilization and glucose tolerance) are altered based on physical activity. Our data suggest that cytokines linked to glycemic excursions (insulin, c-peptide, glucagon) and their passive regulators (leptin, BDNF, active ghrelin, and GIP) exhibit changes in the SED group. Furthermore, some of the proinflammatory cytokines and myokines were upregulated in SED. Interestingly, serum metabolite analysis showed that the levels of glucogenic amino acids (alanine, glycine, tryptophan, proline and valine), nitrogenous amino acids (ornithine, asparagine, and glutamine) and myogenic metabolites (taurine, creatine) were altered due to the level of physical activity. A pyrimidine nucleoside (uridine), lipid metabolite (glycerol) and ketone bodies (acetoacetate and acetate) were found to be altered in SED. A Spearman rank correlation study between SED and CTRL showed that cytokines build a deformed network with metabolites in SED, indicating significant modifications in amino acids, phosphatidylinositol phosphate and glycerophospholipid metabolic pathways. Overall, long-term physical inactivity reorganizes the profile of proinflammatory cytokines, glucose sensing hormones, and protein and glycerophospholipid metabolism, which might be the initial factors of metabolic diseases due to SED.

The shikimate pathway: gateway to metabolic diversity.

Covering: 1997 to 2023The shikimate pathway is the metabolic process responsible for the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Seven metabolic steps convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) into shikimate and ultimately chorismate, which serves as the branch point for dedicated aromatic amino acid biosynthesis. Bacteria, fungi, algae, and plants (yet not animals) biosynthesize chorismate and exploit its intermediates in their specialized metabolism. This review highlights the metabolic diversity derived from intermediates of the shikimate pathway along the seven steps from PEP and E4P to chorismate, as well as additional sections on compounds derived from prephenate, anthranilate and the synonymous aminoshikimate pathway. We discuss the genomic basis and biochemical support leading to shikimate-derived antibiotics, lipids, pigments, cofactors, and other metabolites across the tree of life.

Electrochemistry-enabled residue-specific modification of peptides and proteins.

Selective chemical reactions at precise amino acid residues of peptides and proteins have become an exploding field of research in the last few decades. With the emerging utility of bioconjugated peptides and proteins as drug leads and therapeutic agents, the design of smart protocols to modulate and conjugate biomolecules has become necessary. During this modification, the most important concern of biochemists is to keep intact the structural integrity of the biomolecules. Hence, a soft and selective biocompatible reaction environment is necessary. Electrochemistry, a mild and elegant tunable reaction platform to synthesize complex molecules while avoiding harsh and toxic chemicals, can provide such a reaction condition. However, this strategy is yet to be fully exploited in the field of selective modification of polypeptides. With this possibility, the use of electrochemistry as a reaction toolbox in peptide and protein chemistry is flourishing day by day. Unfortunately, there is no suitable review article summarizing the residue-specific modification of biomolecules. The present review provides a comprehensive summary of the latest manifested electrochemical approaches for the modulation of five redox-active amino acid residues, namely cysteine, tyrosine, tryptophan, histidine and methionine, found in peptides and proteins. The article also highlights the incredible potential of electrochemistry for the regio- as well as chemoselective bioconjugation strategy of biomolecules.

Investigating the regulatory effect of Shen Qi Bu Qi powder on the gastrointestinal flora and serum metabolites in calves.

To investigate the effects of Shen Qi Bu Qi Powder (SQBQP) on the average daily gain, blood indexes, gastrointestinal microflora, and serum metabolites of calves. A total of 105 calves were randomly assigned to three groups (n = 35 per group): the control group (C, fed with a basal diet for 21 days) and two treatment groups (SQBQP-L and SQBQP-H, fed with the basal diet supplemented with 15 and 30 g/kg of SQBQP), respectively for 21 days. The active components of SQBQP were identified using LC-MS/MS. Serum digestive enzymes and antioxidant indices were determined by ELISA kits and biochemical kits, respectively. Serum differential metabolites were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), while flora in rumen fluid and fecal were analyzed by 16S rDNA sequencing. Further correlation analysis of gastrointestinal flora and serum metabolites of SQBQP-H and C groups were performed with Spearman's correlation. The principal active components of SQBQP mainly includes polysaccharides, flavonoids, and organic acids. Compared to the control group (C), calves in the SQBQP-H (high dose) and SQBQP-L (low dose) groups showed a significant increase in serum amylase (AMS) levels (P<0.001), while lipase content significantly decreased (P<0.05). Additionally, the average daily gain, T-AOC, and cellulase content of calves in the SQBQP-H group significantly increased (P<0.05). Proteobacteria and Succinivibrio in the rumen flora of the SQBQP-H group was significantly lower than that of the C group (P<0.05). The relative abundance of Proteobacteria, Actinobacteria, Candidatus_Saccharibacteria, Deinococcus_Thermus, Cyanobacteria, and Succinivibrio in the SQBQP-H group was significantly increased (P<0.05), while the relative abundance of Tenericutes and Oscillibacter was significantly decreased (P<0.05). Serum metabolomics analysis revealed 20 differential metabolites, mainly enriched in amino acid biosynthesis, β-alanine metabolism, tyrosine, and tryptophan biosynthesis metabolic pathways (P<0.05). Correlation analysis results showed that Butyrivibrio in rumen flora and Oscillibacter_valericigenes in intestinal flora were significantly positively correlated with average daily gain, serum biochemical indexes, and differential metabolite (-)-Epigallocatechin (R>0.58, P<0.05). SQBQP can promote calves weight gain and enhance health by modulating gastrointestinal flora and metabolic processes in the body.

Photoelectron spectroscopy of the deprotonated tryptophan anion: the contribution of deprotomers to its photodetachment channels.

Photoelectron spectroscopy and electronic structure calculations are used to investigate the electronic structure of the deprotonated anionic form of the aromatic amino acid tryptophan, and its chromophore, indole. The photoelectron spectra of tryptophan, recorded at different wavelengths across the UV, consist of two direct detachment channels and thermionic emission, whereas the hν = 4.66 eV spectrum of indole consists of two direct detachment features. Electronic structure calculations indicate that two deprotomers of tryptophan are present in the ion beam; deprotonation of the carboxylic acid group (Trp(I)-) or the N atom on the indole ring (Trp(II)-). Strong similarities are observed between the direct detachment channels in the photoelectron spectra of tryptophan and indole, which in conjunction with electronic structure calculations, indicate that electron loss from Trp(II)- dominates this portion of the spectra. However, there is some evidence that direct detachment of Trp(I)- is also observed. Thermionic emission is determined to predominantly arise from the decarboxylation of Trp(I)-, mediated by the ππ* excited state near λ = 300 nm, which results in an anionic fragment with a negative electron affinity that readily autodetaches.

A Review of the Evidence for Tryptophan and the Kynurenine Pathway as a Regulator of Stem Cell Niches in Health and Disease.

Stem cells are ubiquitously found in various tissues and organs in the body, and underpin the body's ability to repair itself following injury or disease initiation, though repair can sometimes be compromised. Understanding how stem cells are produced, and functional signaling systems between different niches is critical to understanding the potential use of stem cells in regenerative medicine. In this context, this review considers kynurenine pathway (KP) metabolism in multipotent adult progenitor cells, embryonic, haematopoietic, neural, cancer, cardiac and induced pluripotent stem cells, endothelial progenitor cells, and mesenchymal stromal cells. The KP is the major enzymatic pathway for sequentially catabolising the essential amino acid tryptophan (TRP), resulting in key metabolites including kynurenine, kynurenic acid, and quinolinic acid (QUIN). QUIN metabolism transitions into the adjoining de novo pathway for nicotinamide adenine dinucleotide (NAD) production, a critical cofactor in many fundamental cellular biochemical pathways. How stem cells uptake and utilise TRP varies between different species and stem cell types, because of their expression of transporters and responses to inflammatory cytokines. Several KP metabolites are physiologically active, with either beneficial or detrimental outcomes, and evidence of this is presented relating to several stem cell types, which is important as they may exert a significant impact on surrounding differentiated cells, particularly if they metabolise or secrete metabolites differently. Interferon-gamma (IFN-γ) in mesenchymal stromal cells, for instance, highly upregulates rate-limiting enzyme indoleamine-2,3-dioxygenase (IDO-1), initiating TRP depletion and production of metabolites including kynurenine/kynurenic acid, known agonists of the Aryl hydrocarbon receptor (AhR) transcription factor. AhR transcriptionally regulates an immunosuppressive phenotype, making them attractive for regenerative therapy. We also draw attention to important gaps in knowledge for future studies, which will underpin future application for stem cell-based cellular therapies or optimising drugs which can modulate the KP in innate stem cell populations, for disease treatment.

Molecular docking and multi-spectroscopic approaches to unravel the mechanism of the interaction between thiocolchicoside and bovine serum albumin

A popular muscle relaxant for the treatment of severe, painful muscular spasms is thiocolchicoside (TCS). Although the precise mechanism underlying its ability to relax muscles is unknown, it demonstrates a specific affinity for the inhibitory gamma-aminobutyric acid and glycinergic receptors. This study used a variety of spectroscopic methods and molecular docking to examine the interaction of TCS with bovine serum albumin (BSA). UV absorption and fluorescence spectroscopic titration analysis supported the conclusion that TCS suppressed BSA's fluorescence through a blend of static and dynamic mechanisms. The thermodynamical constraints revealed that the interaction between BSA and TCS is spontaneous and that van der Waals and hydrogen bonding forces play key roles in stabilising the complex. TCS binds to the site III on BSA, as demonstrated by competitive binding assays utilising site-specific markers and molecular docking studies. By binding TCS, BSA exhibits minor microenvironmental modifications near the tryptophan amino acid residue, according to a structural study employing synchronous fluorescent.

Formation of lipid corona on Ag nanoparticles and its impact on Ag+ ion dissolution and aggregation of Ag nanoparticles against external stimuli

The application of lipid coated nanoparticles has been expanding in nanoscience over the past few years. Lipid corona formation is one of the novel ways for the lipid conjugation around the nanoparticles. Although several properties of nanoparticles and lipids are well reported in literature in last few years, the dependency of lipid corona formation on metallic core and surface ligands is poorly understood. The aggregation, surface oxidation and metal ions dissolution of nanoparticles limit their usefulness in various applications. Besides all the reported methods to overcome all of these limitations, lipid corona could be an alternative, feasible, and more efficient method. In the current manuscript, we investigate the interaction of aromatic amino acids (tyrosine and tryptophan) functionalized silver nanoparticles (Ag-Tyr and Ag-Trp NPs) with different surface charged and phase states lipid vesicles and compare them with the reported amino acid functionalized gold nanoparticles (Au-AA NPs). The effectiveness of different zwitterionic and positively charged lipid coated Ag-Tyr and Ag-Trp NPs to prevent the aggregation and Ag+ ion dissolution of Ag NPs against different external stimuli (acidic pH, high NaCl concentration, and freeze-thaw cycles) are investigated. Our results reveal that surface ligands around the NPs play crucial role for lipid corona formation irrespective of the metallic core of NPs. Although all the lipid coated Ag NPs exhibit greater stability against external stimuli than native Ag NPs, zwitterionic lipid coated Ag NPs is better choice than positively charged lipid coated Ag NPs. This study on lipid corona and its stability will help the nanoresearcher to choose the suitable lipid for lipid corona formation.

Repurposing ebselen as an inhalable dry powder to treat respiratory tract infections

Respiratory tract infections (RTIs) are one of the leading causes of death globally, lately exacerbated by the increasing prevalence of antimicrobial resistance. While antimicrobial resistance could be overcome by developing new antimicrobial agents, the use of a safe repurposed agent having potent antimicrobial activity against various RTIs can be an efficient and cost-effective alternative to overcome the long and complex process of developing and testing new drugs. Ebselen, a synthetic organoselenium drug originally developed to treat noise-inducing hearing problems, has shown promising antimicrobial activity in vitro against several respiratory pathogens including viruses (e.g., SARS-CoV-2, influenza A virus) and bacteria (e.g., Mycobacterium tuberculosis, Streptococcus pneumoniae, and Staphylococcus aureus). Inhaled drug delivery is considered a promising approach for treating RTIs, as it can ensure effective drug concentrations at a lower dose, thereby minimizing the side effects that are often encountered by using oral or injectable drugs. In this study, we developed inhalable ebselen dry powder formulations using a spray-drying technique. The amino acids leucine, methionine, and tryptophan were incorporated with ebselen to enhance the yield and aerosolization of the dry powders. The amino acid-containing ebselen dry powders showed a better yield (37–56.4 %) than the amino acid-free formulation (30.9 %). All dry powders were crystalline in nature. The mass median aerodynamic diameter (MMAD) was less than 5 µm for amino acids containing dry powders (3–4 µm) and slightly higher (5.4 µm) for amino acid free dry powder indicating their suitability for inhalation. The aerosol performance was higher when amino acids were used, and the leucine-containing ebselen dry powder showed the highest emitted dose (84 %) and fine particle fraction (68 %). All amino acid formulations had similar cytotoxicity as raw ebselen, tested in respiratory cell line (A549 cells), with half-maximal inhibitory concentrations (IC50) between 100 and 250 μg/mL. Raw ebselen and amino acid-containing dry powders showed similar potent antibacterial activity against the Gram-positive bacteria S. aureus and S. pneumoniae with minimum inhibitory concentrations of 0.31 μg/mL and 0.16 μg/mL, respectively. On the other hand, raw ebselen and the formulations showed limited antimicrobial activity against the Gram-negative pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae. In summary, in this study we were able to develop amino-acid-containing inhalable dry powders of ebselen that could be used against different respiratory pathogens, especially Gram-positive bacteria, which could ensure more drug deposition in the respiratory tract, including the lungs. DPIs are generally used to treat lung (lower respiratory tract) diseases. However, DPIs can also be used to treat both upper and lower RTIs. The deposition of the dry powder in the respiratory tract is dependent on its physicochemical properties and this properties can be modulated to target the intended site of infection (upper and/or lower respiratory tract). Further studies will allow the development of similar formulations of individual and/or combination of antimicrobials that could be used to inhibit a number of respiratory pathogens.

Solid-phase fluorescence: Reproducibility and comparison with the solution states

Solid-phase fluorescence excitation-emission matrix (SPF-EEM) spectroscopy has potential for non-extractive, non-destructive, and non-contact analytical measurements of powder and solid-state samples, as well as front-face EEM spectroscopy for suspensions of high optical density. However, as there is no unified measurement method for SPF spectroscopy, comparing samples measured in different research fields is difficult. Therefore, this study designs a cell that can be created by a 3D printer and examines reproducibility on measuring fluorescent powders. The developed cell is applied to proteins (ovalbumin, BSA, gliadin, gluten, powdered collagen, casein), amino acids (tryptophan, tyrosine, and phenylalanine), soybean ingredients (daidzein, and genistein), and fluorescent chemicals (rhodamine B, fluorescein sodium salt, pyrene, and quinine sulfate dihydrate) and their spectra are compared with those in the solution states. When samples are refilled into the cell three times, the cell exhibits high reproducibility in terms of fluorescence peak wavelength and intensity. The solid proteins exhibit peaks attributed to the fluorescent amino acid residues, and broad peaks which are not detected for the proteins in the solution states. Powdered rhodamine B and fluorescein sodium salt do not exhibit fluorescence, possibly due to the inner-filter effect (IFE). Some non-colored molecules also exhibit loss of fluorescence or a remarkable difference between the solid and solution states, possibly due to the interaction of the fluorescent structure with the surrounding local environment, similar to the solvent effect, which is possibly affected by the molecular proximity, three-dimensional structure, and moisture absorption capacity.

Application of the C3-Methyltransferase StspM1 for the Synthesis of the Natural Pyrroloindole Motif.

Even though pyrroloindoles are widely present in natural products with different kinds of biological activities, their selective synthesis remains challenging with existing tools in organic chemistry, and there is furthermore a demand for stereoselective and mild methods to access this structural motif. Nature uses C3-methyltransferases to form the pyrroloindole framework, starting from the amino acid tryptophan. In the present study, the SAM-dependent methyltransferase StspM1 from Streptomyces sp. HPH0547 is used to build the pyrroloindole structural motif in tryptophan-based diketopiperazines (DKP). The substrate scope of the enzyme regarding different Trp-Trp-DKP isomers was investigated on an experimental and computational level. After further characterization and optimization of the methylation reaction with a design of experiment approach, a preparative scale reaction with the immobilized enzyme including a SAM regeneration system was performed to show the synthetic use of this biocatalytic tool to access the pyrroloindole structural motif.

Niacin - a scoping review for Nordic Nutrition Recommendations 2023.

Niacin is the precursor to pyridine nucleotides NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate). Niacin (vitamin B3) is the common term for nicotinic acid, nicotinamide and derivatives that exhibit the biological activity of nicotinamide. Furthermore, the indispensable amino acid tryptophan is the substrate for de novo synthesis of NAD. Thus, the requirements and intake of niacin are expressed as niacin equivalents (NE). The focus of interest for niacin over the last decade has primarily been on pharmacological doses of nicotinic acid as a lipid-lowering agent and other NAD precursors as potential enhancers of cellular NAD+ concentrations. None of these studies, however, makes a useful contribution to understanding dietary requirements in healthy populations. The requirement for niacin is estimated based on the relationship between intake and biochemical indices of niacin status, primarily urinary excretion of nicotinamide metabolites.

Impact of IDO activation and alterations in the kynurenine pathway on hyperserotonemia, NAD+ production, and AhR activation in autism spectrum disorder

Hyperserotonemia is the most replicated biochemical anomaly associated with autism spectrum disorder (ASD) and has been reported in 35–46% of individuals with ASD. Serotonin is synthesised from the essential amino acid tryptophan (TRP). However, the main catabolic route of TRP is the kynurenine pathway (KP), which competes with serotonin synthesis when indoleamine dioxygenase (IDO) is activated. Using the same cohort of individuals with ASD, we used to report extensive studies of the serotonin/melatonin pathway, and found increased kynurenine (KYN), suggesting IDO activation in 58.7% of individuals with ASD (159/271), supported by a strong negative correlation between KYN/TRP ratio and miR-153-3p plasma levels, which negatively regulates IDO. IDO activation was associated with normoserotonemia, suggesting that IDO activation could mask hyperserotonemia which meant that hyperserotonemia, if not masked by IDO activation, could be present in ~94% of individuals with ASD. We also identified several KP alterations, independent of IDO status. We observed a decrease in the activity of 3-hydroxyanthranilate dioxygenase which translated into the accumulation of the aryl hydrocarbon receptor (AhR) selective ligand cinnabarinic acid, itself strongly positively correlated with the AhR target stanniocalcin 2. We also found a deficit in NAD+ production, the end-product of the KP, which was strongly correlated with plasma levels of oxytocin used as a stereotypical neuropeptide, indicating that regulated neuropeptide secretion could be limiting. These results strongly suggest that individuals with ASD exhibit low-grade chronic inflammation that is mediated in most cases by chronic AhR activation that could be associated with the highly prevalent gastrointestinal disorders observed in ASD, and explained IDO activation in ~58% of the cases. Taken together, these results extend biochemical anomalies of TRP catabolism to KP and posit TRP catabolism as a possible major component of ASD pathophysiology.

Abstract B150: Targeting EWS-FLI1 with mithramycin analogues for Ewing sarcoma treatment

Abstract Ewing Sarcoma, a devastating malignancy affecting mainly pediatric and young adult populations, is characterized by the aberrant activity of the oncogenic EWS-FLI1 transcription. However, the development of treatments against EWS-FLI1 is lacking. Mithramycin analogues, exhibiting specificity towards EWS-FLI1, have been posited as a groundbreaking approach in drug discovery for the treatment of Ewing Sarcoma. A phase I/II trial and pharmacokinetic (PK) study in Ewing sarcoma patients demonstrated that mithramycin (MTM) had poor PK and dose-limiting hepatic and hematologic toxicities at subtherapeutic concentrations. Here we present a novel MTM analogue with significantly improved PK and a wider therapeutic window. MTMSA-Trp was prepared by chemical conversion of MTM to the MTMSA analogue and the subsequent conjugation of a tryptophan amino acid. The bulky amino acid substitution on the 3-side chain of the molecule appears to shift the ionization properties of the two hydroxy groups on the tricyclic core and to increase protein binding, both of which appear to lead to significantly improved pharmacokinetics. Further, based on crystallographic evaluation, the bulky hydrophobic substitution on the 3-side chain protrudes outside the DNA helix and interacts with bound FLI1. Luciferase reporter and FRET assays demonstrate a dose-dependent effect of MTMSA-Trp on the attenuation of EWS-FLI1 transcription and DNA binding, and based on CETSA evidence, there is physical interaction between MTMSA-Trp and EWS-FLI1. The cytotoxicity of MTMSA-Trp is in the order of low nM across several cell lines expressing EWS-ETS fusions, and the compound disrupts the expression of positively regulated proteins (NR0B1, ID2) and induces expression of negatively regulated ones (CD44, LOX). EWS-FLI1 modulation persists longer when cells are exposed to the GI90 concentration for a short time compared to the GI50 for longer times, suggesting that daily dosing or continuous exposure is not required. MTMSA-Trp has improved PK in athymic nu/nu mice with a greater than 10-fold reduction in clearance and significantly reduced partition in the liver as compared to MTM. Initial efficacy studies in TC32 Ewing Sarcoma cells demonstrated tumor regression and improved survival at the maximum tolerated dose (MTD) and at 2/3 of the MTD on a daily x 5 intravenous bolus injection. Significantly, a more protracted dosing schedule which allowed for higher doses every third day for six doses led to an impressive reduction of tumors whose initial size was above 15 mm in one diameter (i.e., 1200-1600 mm3). Our studies with MTMSA-Trp demonstrate that improving the pharmacologic properties of mithramycin may lead to the development of an EWS-FLI1 inhibitor. Ongoing studies are evaluating the efficacy of MTMSA-Trp in additional Ewing models, and future work will focus on pre-IND studies. Citation Format: Markos Leggas, Kumar K Niloy, Rajesh Yetijaram, Jamie Horn, Yasuda Kazuto, Thomas Prisinzano, Jon S Thorson, Oleg Tsoikov, Jurgen Rohr. Targeting EWS-FLI1 with mithramycin analogues for Ewing sarcoma treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B150.

Mitochondrial Pyruvate Carrier Inhibition Mitigates Murine Chronic Graft Versus Host Disease By Attenuating the Germinal Center Reaction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potent therapy for many hematopoietic diseases and malignancies. However, the long-term efficacy of allo-HSCT is limited by the development of chronic graft versus host disease (cGVHD). During cGVHD, interactions between donor-derived T follicular helper cells (TFH) and germinal center B cells (GCB) result in the production of alloreactive class-switched affinity-matured antibodies that attack host tissues, causing multi-organ damage and tissue fibrosis. Novel treatment approaches are warranted to treat cGVHD since it is often refractory to current broad immune suppressive drugs. In this study we sought to delineate the metabolic dependencies of germinal center (GC) immune cells to discover new strategies to treat active murine cGVHD. We utilized a multi-organ mouse model of cGVHD that includes bronchiolitis obliterans (BO), a severe cGVHD manifestation seen in the clinic. Toward that end, B10.BR (H2 k) recipients received myeloablative conditioning with cyclophosphamide and total body irradiation before receiving MHC mismatched C57BL/6 (H2 b) donor T cell depleted bone marrow with (cGVHD) or without (BM only control) a low dose of splenic T cells. First, we sought to assess the metabolic dependencies of TFH during cGVHD progression through Seahorse metabolic flux assays. TFH sorted early (D28) from cGVHD mice were highly glycolytic with an increased extracellular acidification rate (ECAR) and less reliant on oxidative phosphorylation (OXPHOS) as reflected by a decreased oxygen consumption rate (OCR). However, TFH sorted later (D49) from cGVHD mice demonstrated a dramatic shift in their metabolic profile to a decreased ECAR and an increased OCR indicating a transition from glycolysis towards OXPHOS as disease progresses. To understand which substrates are driving increased OXPHOS and to identify promising metabolic targets, we performed unbiased metabolomics of sort purified TFH from BM only and cGVHD mice on D49 post-transplantation (Fig A). Among the top fifty differential metabolites, cGVHD TFH had a decreased abundance of anaplerotic amino acids (serine, glutamine) and an increased abundance of multiple breakdown products of anaplerotic amino acids (tryptophan, leucine), indicative of increased replenishment of tricarboxylic acid (TCA) cycle intermediates. Notably these metabolomics data showed a decreased abundance of glutamine which is consistent with an increase in glutaminolysis, validating our prior findings that TFH cells that cause cGVHD/BO depend on glutaminolysis ( Johnson MO, Cell 2018). Additionally, there was an increased abundance of pyruvate, which can be transported into mitochondria to fuel the TCA cycle through the mitochondrial pyruvate carrier (MPC). Based on these findings we chose to target the MPC since it has a central role in tying pyruvate to the TCA cycle and OXPHOS. We assessed the effect of an MPC inhibitor (7ACC1) on the GC reaction utilizing an ex vivo co-culture assay of TFH and B cells. 7ACC1 added to this co-culture system resulted in a significant reduction in the production of class-switched immunoglobulin compared to vehicle treatment. To assess the in vivo efficacy, we administered 7ACC1 daily through intraperitoneal injections after establishment of cGVHD (Days 28-49) in the cGVHD/BO mouse model. 7ACC1 treatment resulted in a significant reduction of the GC reaction with significantly decreased TFH and GCB frequencies. Additionally, there was a significant improvement in cGVHD disease severity as evidenced by assessing pulmonary function tests in anesthetized, intubated and mechanically ventilated mice using plethysmography. In contrast to the high resistance and low compliance seen in cGVHD/BO mice, 7ACC1 treated recipients had significantly improved lung function, comparable to the BM only controls (Fig B). In summary, inhibiting MPC mediated transport of pyruvate represents a novel approach to stifling the metabolic demands of TFH as their energy dependency shifts from glycolysis towards OXPHOS during the course of cGVHD/BO disease progression.

Biofortification to improve food security

Crop biofortification has significantly progressed in the last few decades. The first biofortification success was quality protein maize, leading to double the amount of the essential amino acids lysine and tryptophan. This was followed by biofortification of staple crops such as maize, wheat, rice, legumes and cassava for nutrients such as Fe and Zn and provitamin A. These crops have reached millions of households, especially in the developing regions of the world. The development and release of these biofortified crops through conventional breeding generally took 8–10 years. To speed up the process, molecular markers, genome-wide association studies and genomic selection have been incorporated into breeding efforts. Genetic engineering has the potential to increase the efficiency of crop biofortification through multi-nutrient biofortification in a short timespan and to combine biofortification with climate resilience. Regulatory issues still prevent the dissemination of genetically modified crops in many countries. This could be overcome by CRISPR–Cas-mediated genome editing, as it seems that many countries will regulate products of genome editing less strictly than transgenic crops. Effective policies on national or regional level are needed for the sustainable production of biofortified crops. The availability of affordable quality biofortified seed and other inputs should be ensured through local seed systems, which will increase the production and adoption of biofortified crops. There is scope to expand the crops and the range of nutrients for biofortification. Genetic engineering should be combined with conventional breeding as a approach for future improvement of multi-nutrient crops.

Fibrodysplasia ossificans progressiva caused by a different mutation in a stray cat

AbstractA 2‐year‐old, unneutered, female, short‐haired stray cat was referred for general examination. A physical examination revealed gait abnormalities characterised by short strides and stiffness in all limbs. Radiographs showed malformations with excessive mineralisation affecting the shoulders and stifle joints. To confirm the diagnosis of fibrodysplasia ossificans progressiva, total RNA was isolated from blood samples of the cats (one cat with fibrodysplasia ossificans progressiva and one healthy cat). A single missense mutation was found in the coding sequence of the ACVR1 gene of the cat with fibrodysplasia ossificans progressiva. In this mutation, a single base was changed from guanine to cytosine. After this base change, the amino acid tryptophan replaced the amino acid cysteine. This is the first report that indicates cats with fibrodysplasia ossificans progressiva that do not show signs similar to the characteristic findings in humans have a different mutation.