Light-Activatable, Cell-Type Specific Labeling of the Nascent Proteome.

Elucidating the mechanisms by which protein synthesis contributes to complex biological processes has remained a challenging endeavor. This is particularly true in the field of neuroscience, where multiple, tightly regulated periods of new protein synthesis in different cell-types are thought to facilitate intricate neurological functions, such as memory formation. Current methods for labeling the de novo proteome have lacked the spatial and temporal resolution to accurately discriminate these overlapping and often competing windows of mRNA translation. To address this technological limitation, here we describe a novel, light-inducible specific method for labeling newly synthesized proteins within a targeted cell-type.By developing Opto-ANL, a photocaged version of the nonendogenous amino acid azidonorleucine (ANL), we can selectively label newly synthesized proteins in specific cell-types through the targeted expression of a mutant methionyl-tRNA synthetase (L274G-MetRS). We demonstrate that Opto-ANL can be rapidly uncaged by UV light treatment in both cell culture and mouse brain slices, with Opto-ANL labeled proteins being able to be visualized via fluorescent noncanonical amino acid tagging. We also reveal that pretreatment with Opto-ANL not only allows for the period of de novo proteomic labeling to be tightly controlled, but also improves labeling efficiency compared to regular ANL. To demonstrate the potential applications of this novel technique, we use Opto-ANL to detect insulin-induced increases in protein synthesis and to label the excitatory neuronal de novo proteome in mouse brain slices. We believe that this application of photopharmacology will allow researchers to generate novel insights into how the translational landscape is altered across cell-types during complex neurological phenomena such as memory formation.

Utilization of Free and Dipeptide-Bound Formyline and Pyrraline by Saccharomyces Yeasts.

The utilization of the glycated amino acids formyline and pyrraline as well as their peptide-bound derivatives by 14 Saccharomyces yeasts, including 6 beer yeasts (bottom and top fermenting), one wine yeast, 6 strains isolated from natural habitats and one laboratory reference yeast strain (wild type) was investigated. All yeasts were able to metabolize glycated amino acids via the Ehrlich pathway to the corresponding Ehrlich metabolites. While formyline and small amounts of pyrraline entered the yeast cells via passive diffusion, the amounts of dipeptide-bound MRPs, especially the dipeptides glycated at the C-terminus, decreased much faster, indicating an uptake into the yeast cells. Furthermore, the glycation-mediated hydrophobization in general leads to an faster degradation rate compared to the native lysine dipeptides. While the utilization of free formyline is yeast-specific, the amounts of (glycated) dipeptides decreased faster in the presence of brewer's yeasts, which also showed a higher formation rate of Ehrlich metabolites compared to naturally isolated strains. Due to rapid uptake of alanyl dipeptides, it can be assumed that the Ehrlich enzyme system of naturally isolated yeasts is overloaded and the intracellularly released MRP is primarily excreted from the cell. This indicates adaptation of technologically used yeasts to (glycated) dipeptides as a nitrogen source.

l-norleucine on high glucose-induced insulin sensitivity and mitochondrial function in skeletal muscle cells.

l-norleucine, an isomer of leucine, stimulates the anabolic process of insulin. However, it is not known if and how it improves insulin sensitivity and insulin resistance. This experiment describes the generation of an insulin resistance model using high glucose-induced cells and the administration of 1.0 mmol/L l-norleucine for 48 h, to observe the effects on metabolism and gene expression in skeletal muscle cells. The results showed that l-norleucine significantly increased mitochondrial ATP content, decreased the amount of reactive oxygen species (ROS) and promoted the expression of mitochondrial generation-related genes TFAM, AMPK, PGC-1α in cells under high glucose treatment; at the same time, l-norleucine also increased glucose uptake, suggesting that l-norleucine increased insulin sensitivity and improved insulin resistance. This study suggesting that l-norleucine improves insulin resistance by ameliorating oxidative stress damage of mitochondria, improving mitochondrial function, and improving insulin sensitivity in skeletal muscle cell caused by high glucose, rather than by altering mitochondrial efficiency.

Hepatopathy in Victorian dogs consuming pet meat contaminated with indospicine.

BACKGROUND: Indospicine is an arginine analogue and a natural toxin occurring only in Indigofera plant species, including Australian native species. It accumulates in the tissues of grazing animals, persisting for several months after ingestion. Dogs are particularly sensitive to indospicine toxicity and can suffer fatal liver disease after eating indospicine-contaminated pet meat. METHOD: A disease outbreak investigation was launched following notification to Agriculture Victoria of a cluster of 18 dogs displaying acute, severe, hepatopathy in the East Gippsland Shire in June 2021. RESULTS: Between June and September 2021, 24 pet dogs died, and 40 others experienced liver disease after eating commercially prepared pet meat found to contain indospicine. The investigation identified the toxin in serum and liver samples from affected dogs and at high levels in some samples of pet meat eaten by the dogs. Twenty-six horses that were moved from the Northern Territory and processed at a Pet Meat Processing facility (knackery) in eastern Victoria over a period of 14 days in late May-early June 2021 were identified as the likely source of the indospicine toxin in the pet meat. Pet meat produced by the knackery and on-sold by several retailers was determined to be the cause of the illness and death in the dogs. CONCLUSION: This is the first report of severe and frequently fatal hepatopathy in dogs in Victoria relating to consumption of pet meat contaminated with indospicine.

Natural 2-Amino-3-Methylhexanoic Acid as Plant Elicitor Inducing Resistance against Temperature Stress and Pathogen Attack.

2-Amino-3-methylhexanoic acid (AMHA) was synthetized as a non-natural amino acid more than 70 years ago; however, its possible function as an inducer of plant resistance has not been reported. Plant resistance inducers, also known as plant elicitors, are becoming a novel and important development direction in crop protection and pest management. We found that free AMHA accumulated in the mycelia but not in fermentation broths of four fungal species, Magnaporthe oryzae and three Alternaria spp. We unequivocally confirmed that AMHA is a naturally occurring endogenous (2S, 3S)-α-amino acid, based on isolation, purification and structural analyses. Further experiments demonstrated that AMHA has potent activity-enhancing resistance against extreme temperature stresses in several plant species. It is also highly active against fungal, bacterial and viral diseases by inducing plant resistance. AMHA pretreatment strongly protected wheat against powdery mildew, Arabidopsis against Pseudomonas syringae DC3000 and tobacco against Tomato spotted wilt virus. AMHA exhibits a great potential to become a unique natural elicitor protecting plants against biotic and abiotic stresses.

Investigation of salivary biomarkers as indicators of skeletal and dental maturity in children.

OBJECTIVE: Estimation of patient's skeletal maturity in orthodontics is essential for the diagnosis and treatment planning. The aim of the study was to investigate the potential use of metabolic fingerprint of saliva for bone growth and tooth development estimation. MATERIALS AND METHODS: Saliva samples from 54 young patients were analysed by an untargeted gas chromatography-mass spectrometry metabolomics-based method. The skeletal maturity was calculated with the cervical vertebrae maturation method, and the dental age was estimated with the Demirjian method. Multivariate analysis and univariate analysis were performed to investigate differences within skeletal, dental and chronological age groups. RESULTS: Metabolomic analysis identified 61 endogenous compounds. Mannose, glucose, glycerol, glyceric acid and pyroglutamic acid levels differentiated significantly with skeletal age (P = .02 to .043), while mannose, lactic acid, glycolic acid, proline, norleucine, 3-aminoisobutyric acid, threonine, cadaverine and hydrocinnamic acid levels differed within the dental age groups (P = .018 to .04); according to the chronological age, only the levels of mannose and 3-hydroxyphenylacetic acid showed variation (P = .029 and .048). The principal component analysis did not manage to highlight differences between the groups of the studied parameters. CONCLUSION: Differentiated levels of mannose, glucose, glycerol, glyceric acid and pyroglutamic acid related to skeletal maturation were identified. According to dental development, the levels of mannose, lactic acid, glycolic acid, proline, norleucine, 3-aminoisobutyric acid, threonine, cadaverine and hydrocinnamic acid differed within the groups, while regarding chronological age, only the levels of mannose and 3-hydroxyphenylacetic acid showed variations. Further studies are required to prove their relation to skeletal and dental development pathway by applying complementary analytical techniques to wider cover the metabolome.

Plasma l-indospicine and 3-nitropropionic acid in ponies fed creeping indigo: Comparison with results from an episode of presumptive creeping indigo toxicosis.

BACKGROUND: Creeping indigo (Indigofera spicata) toxicosis is an emerging problem among horses in Florida and bordering states. OBJECTIVES: To quantify the putative toxins l-indospicine (IND) and 3-nitropropionic acid (NPA) in creeping indigo collected from multiple sites and to measure plasma toxin concentrations in ponies fed creeping indigo and horses with presumptive creeping indigo toxicosis. STUDY DESIGN: Experimental descriptive study with descriptive observational field investigation. METHODS: Air-dried creeping indigo was assayed for IND and NPA content. Five ponies were fed chopped creeping indigo containing 1 mg/kg/day of IND and trace amounts of NPA for 5 days, then observed for 28 days. Blood samples from these ponies and from horses involved in a presumptive creeping indigo toxicosis were assayed for IND and NPA. RESULTS: IND in creeping indigo plants was 0.4-3.5 mg/g dry matter whereas NPA was <0.01 to 0.03 mg/g. During creeping indigo feeding, clinical and laboratory signs were unchanged except for significant weight loss (median 6%, range 2%-9%; p = .04) and significant increase from baseline plasma protein concentration (median 16 g/L, range 8-25 g/L; p < .001). These changes could not definitively be ascribed to creeping indigo ingestion. Plasma IND rose to 3.9 ± 0.52 mg/L on day 6. Pharmacokinetic modelling indicated an elimination half-life of 25 days and a steady state plasma concentration of 22 mg/L. Plasma IND concentration in sick horses during an incident of creeping indigo toxicosis was approximately twice that of clinically normal pasture mates. Plasma NPA was <0.05 mg/L in all samples. MAIN LIMITATIONS: Creeping indigo used in the feeding trial may not be representative of plants involved in creeping indigo toxicosis. There was no control group without creeping indigo in the feeding trial. CONCLUSIONS: Indospicine can be detected in blood of horses consuming creeping indigo and the toxin accumulates in tissues and clears slowly. The role of NPA in the neurological signs of this syndrome is unclear.

An Ornithine-Free Gramicidin S Analogue Using Norleucine, Cyclo(Val-Nle-Leu-D-Phe-Pro)2, Forms Helically Aligned ß-Sheets.

The structure of an ornithine (Orn)-free Gramicidin S (GS) analogue, cyclo(Val-Nle-Leu-D-Phe-Pro)2 (NGS), was studied. Its circular dichroism (CD) spectrum showed that NGS has a structure similar to GS, though the value of [θ] indicated smaller ß-turn and sheet populations. This is probably because the Nle side chain could not form intramolecular hydrogen bonds stabilizing the sheet structure. The chemical shift perturbation of αH and JNH-αH were similar in GS and NGS. Three independent NGS molecules formed intramolecular ß-sheet structures in crystal. The turn structures of D-Phe-Pro moieties were classed as type II' ß-turns, but one part was unclassed. The molecules were arranged in a twisting manner, which resulted in the formation of a helical sheet. Similar structural characteristics were observed previously in a Leu-type, Orn-free GS analogue and in GS trifluoroacetic acid salt.

Biophysical and mass spectrometry based characterization of methylglyoxal-modified myoglobin: Role of advanced glycation end products in inducing protein structural alterations.

Methylglyoxal (MG) is a highly reactive α-dicarbonyl compound which reacts with proteins to form advanced glycation end products (AGEs). MG-induced AGE (MAGE) formation is particularly significant in diabetic condition. In the current study, we have undertaken a time-dependant characterization of MG-modified myoglobin following incubation of the heme protein with the α-dicarbonyl compound for different time periods. Interestingly, mass spectrometric studies indicated modifications at two specific lysine residues, Lys-87 and Lys-133. The AGE adducts identified at Lys-87 were carboxymethyllysine and carboxyethyllysine, while those detected at Lys-133 included pyrraline-carboxymethyllysine and carboxyethyllysine, respectively. Far-UV CD studies revealed a decrease in the native α-helical content of the heme protein gradually with increasing time of MG incubation. In addition, MG modification was found to induce changes in tertiary structure as well as surface hydrophobicity of the heme protein. MG-derived AGE adducts thus appear to alter the structure of Mb considerably. Considering the increased level of MG in diabetic condition, the current study appears physiologically relevant in terms of understanding AGE-mediated protein modification and subsequent structural changes.

Pyrrovobasine, hybrid alkylated pyrraline monoterpene indole alkaloid pseudodimer discovered using a combination of mass spectral and NMR-based machine learning annotations.

A new vobasine-tryptamine-based monoterpene indole alkaloid pseudodimer was isolated from the stem bark of Voacanga africana. As a minor constituent occurring in a thoroughly investigated plant, this molecule was targeted based on a molecular networking strategy and a rational MS2-guided phytochemical investigation led to its isolation. Its structure was formally established based on HRMS, 1D/2D NMR data, and the application of the tool Small Molecule Accurate Recognition Technology (SMART 2.0). Its absolute configuration was assigned by the exciton chirality method and TD-DFT ECD calculations. Besides featuring an unprecedented intermonomeric linkage in the small group of vobasine/tryptamine hybrids, pyrrovobasine also represents the first pyrraline-containing representative in the whole monoterpene indole alkaloids group. Biosynthetic hypotheses possibly underpinning these structural oddities are proposed here.

Metabolic labeling of enterovirus 71 with quantum dots for the study of virus receptor usage.

Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71.

Two Dipeptide-Bound Pyrralines with Ile or Ala: A Study on Their Synthesis, Transport across Caco-2 Cell Monolayers, and Interaction with Aminopeptidase N.

In this study, pyrralylisoleucine (Pyrr-Ile) and pyrralylalanine (Pyrr-Ala), two dipeptide-bound pyrralines with different C-termini were synthesized as the representatives of dietary advanced glycation end products (dAGEs). The structures of Pyrr-Ile and Pyrr-Ala were characterized by high-resolution mass spectrometry, nuclear magnetic resonance, and Fourier transform infrared spectroscopy. Then, the transport of Pyrr-Ile and Pyrr-Ala across intestinal epithelial cells was investigated using Caco-2 cell monolayers, and their interaction with aminopeptidase N (APN) was analyzed. The results showed that the apparent permeability coefficient (Papp) of Pyrr-Ala was (14.1 ± 2.26) × 10-7 cm·s-1 calculated by free pyrraline, while the Papp values of Pyrr-Ile were (32.4 ± 5.35) × 10-7 and (19.1 ± 1.46) × 10-7 cm·s-1 when they were, respectively, calculated according to their dipeptide-bound or free form. Both Pyrr-Ala and Pyrr-Ile were potential substrates of APN, and their hydrolysis by APN may make the intact transmembrane transport of Pyrr-Ala and Pyrr-Ile more difficult, especially for Pyrr-Ala. Besides, the occurrence of product inhibition in hydrolysis of Pyrr-Ile was possible. Pyrr-Ile and Pyrr-Ala were different in Papp values and transport forms, which suggested that the C-terminus may play an important role in their transport across the Caco-2 cell monolayers. In addition, the results highlight the intact transmembrane transport of dipeptide-bound pyrraline.

Degradation of the Indospicine Toxin from Indigofera spicata by a Mixed Population of Rumen Bacteria.

The leguminous plant species, Indigofera linnaei and Indigofera spicata are distributed throughout the rangeland regions of Australia and the compound indospicine (L-2-amino-6-amidinohexanoic acid) found in these palatable forage plants acts as a hepatotoxin and can accumulate in the meat of ruminant livestock and wild camels. In this study, bovine rumen fluid was cultivated in an in vitro fermentation system provided with Indigofera spicata plant material and the ability of the resulting mixed microbial populations to degrade indospicine was determined using UPLC-MS/MS over a 14 day time period. The microbial populations of the fermentation system were determined using 16S rRNA gene amplicon sequencing and showed distinct, time-related changes occurring as the rumen-derived microbes adapted to the fermentation conditions and the nutritional substrates provided by the Indigofera plant material. Within eight days of commencement, indospicine was completely degraded by the microbes cultivated within the fermenter, forming the degradation products 2-aminopimelamic acid and 2-aminopimelic acid within a 24 h time period. The in vitro fermentation approach enabled the development of a specifically adapted, mixed microbial population which has the potential to be used as a rumen drench for reducing the toxic side-effects and toxin accumulation associated with ingestion of Indigofera plant material by grazing ruminant livestock.

Ligand-Dependent Effects of Methionine-8 Oxidation in Parathyroid Hormone Peptide Analogues.

LA-PTH is a long-acting parathyroid hormone (PTH) peptide analogue in preclinical development for hypoparathyroidism (HP). Like native PTH, LA-PTH contains a methionine at position 8 (Met8) that is predicted to be critical for function. We assessed the impact of Met oxidation on the functional properties of LA-PTH and control PTH ligands. Oxidation of PTH(1-34) resulted in marked (~20-fold) reductions in binding affinity on the PTH receptor-1 (PTHR1) in cell membranes, similarly diminished potency for 3',5'-cyclic AMP signaling in osteoblastic cell lines (SaOS-2 and UMR106), and impaired efficacy for raising blood calcium in mice. Surprisingly, oxidation of LA-PTH resulted in little or no change in these functional responses. The signaling potency of oxidized-LA-PTH was, however, reduced approximately 40-fold compared to LA-PTH in cells expressing a PTHR1 construct that lacks the N-terminal extracellular domain (ECD). Molecular modeling revealed that while Met8 of both LA-PTH and PTH(1-34) is situated within the orthosteric ligand-binding pocket of the receptor's transmembrane domain bundle (TMD), the Met8 sidechain position is shifted for the 2 ligands so that on Met8 oxidation of PTH(1-34), steric clashes occur that are not seen with oxidized LA-PTH. The findings suggest that LA-PTH and PTH(1-34) engage the receptor differently in the Met8-interaction environment of the TMD bundle, and that this interaction environment can be allosterically influenced by the ECD component of the ligand-receptor complex. The findings should be useful for the future development of novel PTH-based peptide therapeutics for diseases of bone and mineral ion metabolism.

Indospicine combined with arginine deprivation triggers cancer cell death via caspase-dependent apoptosis.

Arginine-deprivation therapy is a rapidly developing metabolic anticancer approach. To overcome the resistance of some cancer cells to this monotherapy, rationally designed combination modalities are needed. In this report, we evaluated for the first time indospicine, an arginine analogue of Indigofera plant genus origin, as potential enhancer compound for the metabolic therapy that utilizes recombinant human arginase I. We demonstrate that indospicine at low micromolar concentrations is selectively toxic for human colorectal cancer cells only in the absence of arginine. In arginine-deprived cancer cells indospicine deregulates some prosurvival pathways (PI3K-Akt and MAPK) and activates mammalian target of rapamycin, exacerbates endoplasmic reticulum stress and triggers caspase-dependent apoptosis, which is reversed by the exposure to translation inhibitors. Simultaneously, indospicine is not degraded by recombinant human arginase I and does not inhibit this arginine-degrading enzyme at its effective dose. The obtained results emphasize the potential of arginine structural analogues as efficient components for combinatorial metabolic targeting of malignant cells.

Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Blockage of O-linked GlcNAcylation induces AMPK-dependent autophagy in bladder cancer cells.

BACKGROUND: High levels of the post-translational modification O-GlcNAcylation (O-GlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of O-GlcNAcylation on autophagy in bladder cancer remains unclear. Here, we evaluate the change in autophagic activity in response to O-GlcNAcylation and explore the potential mechanisms. METHODS: O-GlcNAcylation levels in bladder cancer cells were altered through pharmacological or genetic manipulations: treating with 6-diazo-5-oxo-norleucine (DON) or thiamet-G (TG) or up- and downregulation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA). Autophagy was determined using fluorescence microscopy and western blotting. Co-immunoprecipitation (Co-IP) assays were performed to evaluate whether the autophagy regulator AMP-activated protein kinase (AMPK) was O-GlcNAc modified. RESULTS: Cellular autophagic flux was strikingly enhanced as a result of O-GlcNAcylation suppression, whereas it decreased at high O-GlcNAcylation levels. Phosphorylation of AMPK increased after the suppression of O-GlcNAcylation. We found that O-GlcNAcylation of AMPK suppressed the activity of this regulator, thereby inhibiting ULK1 activity and autophagy. CONCLUSION: We characterized a new function of O-GlcNAcylation in the suppression of autophagy via regulation of AMPK. GRAPHICAL ABSTRACT: Blockage of O-linked GlcNAcylation induces AMPK dependent autophagy in bladder cancer cells.

Kinetic study on the generation of furosine and pyrraline in a Maillard reaction model system of d-glucose and l-lysine.

A kinetic model for Maillard reaction (MR) model system of d-glucose and l-lysine was established; activation energy (Ea) of each step was calculated. Potential generation pathways of furosine and pyrraline were a combination of either 3-deoxyglucosone (3-DG) or methylglyoxal (MG) with l-lysine. Ea value for furosine generated through 3-DG pathway was 81.70 ± 14.01 kJ mol-1, which was significantly higher than that through MG pathway (52.08 ± 4.48 kJ mol-1). As for pyrraline, Ea for the 3-DG pathway (53.45 ± 4.02 kJ mol-1) was significantly lower than that through the MG pathway (110.22 ± 18.77 kJ mol-1). Results of the kinetic study indicated that furosine was preferred to be generated through the MG pathway since MG is more likely to react with each other and form a furan ring as a precursor of furosine. Pyrraline was more easily to be generated from the 3-DG pathway through cyclization of 1,4-dicarbonyl compounds to pyrrole.

Effect of protein fortification on heat damage and occurrence of ß-casomorphins in (un)digested donor human milk intended for nutrition of preterm infants.

Pasteurized donor human milk (PDHM) for preterm infant nutrition is fortified with hydrolyzates of cow's milk proteins, which have been poorly investigated in relation to heat-damage and occurrence of the bioactive peptides ß-casomorphins (BCMs). Therefore, thermal protein modifications of three commercial fortifiers were assessed by measuring well-recognized indexes of heat load. The fortifiers did not contain pyrraline, whereas furosine and lysinoalanine levels roughly overlapped the lowest values reported for liquid formulas addressed to term infant nutrition. Bovine BCMs 3 to 7 and human BCMs 3 to 9 were searched. Bovine BCMs 3, 4, 6 and 7 were found in the undigested fortifiers. Following in vitro digestion simulating the digestive conditions of premature infant, bovine BCMs still occurred in fortified PDHM; the human BCMs 3, 7, 8 and 9 formed. Overall, these results better address the nutritional features of protein fortifiers and fortified PDHM intended for nutrition of preterm infants.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.